Research Article

Received: 18 March 2010; Revised: 8 June 2010; Accepted: 30 June 2010; Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/ffj.2018

Effects of Flavoured Mouth Rinses on Morning

Breath Odour: a Sensory, Analytical and
Microbial Evaluation
Myriam Troccaz,a* Olivier P. Haefliger,a Isabelle Cayeux,a Sabine Beccucci,a
Nicolas Jeckelmann,a Jérôme Barra,a Anthony J. Clark,a Jacques Schrenzelb
and Pierre Baehnic
ABSTRACT: Transient oral malodour such as morning bad breath is very common and is caused by accumulation of bacteria
and food residues, mainly at the posterior part of the tongue. The use of antimicrobial flavours may help to reduce malodours
with limited side effects. The purpose of this work was to compare the effects of five alcohol-free mouth rinses containing 0.2%
w/w antimicrobial flavour oil on 24 human subjects in a randomized, double-blind clinical study. Two mouth rinses containing
triclosan and peppermint oil were used as positive controls. Oral malodours and flavour intensities, as well as analytical and
microbial analyses of saliva samples, were performed before rinsing and 1 min and 1 h after rinsing the mouth with a 20 ml
mouth rinse for 1 min. Immediately after rinsing, oral malodour scores and salivary bacterial counts were markedly reduced
(85 ⫾ 9% and 54 ⫾ 12% decreases, respectively). At 1 h after rinsing, maximum efficacy (61 ⫾ 18% reduction in oral malodours,
40 ⫾ 11% decrease in salivary bacterial count) was observed with the mouth rinses containing the highest levels of menthol,
limonene, carvone and eugenol, and these ingredients could still be detected in the saliva of most subjects. The finding that
selected antimicrobial flavours have beneficial effects against salivary microorganisms and transient oral malodours may be
important for the design of oral care products that promote fresh breath up to 1 h after rinsing without compromising taste.
Copyright © 2010 John Wiley & Sons, Ltd.

Keywords: oral malodour; sensory analysis; mouth rinse; antimicrobial; flavour

Introduction oral cavity-associated bacteria such as Streptococcus mutans and

Transient oral malodour results from the hydrolysis of peptides
and proteins present in saliva, gingival crevicular fluid and oral
soft tissues by bacteria present in the oral cavity and more par-
ticularly on the surface of the tongue.[1–3] We may distinguish
morning bad breath, which is very common and may be attrib-
uted to reduced salivary flow during sleep and increased anaero-
bic bacterial counts, from other temporary halitosis resulting
from poor oral hygiene, tobacco use, spicy foods or alcohol,
which may have a drying effect in the mouth.[1–4] Morning breath
is often used as a model to test the clinical efficacy of different
treatment modalities, as subjects with pathological halitosis
associated with infection of oral tissues are difficult to recruit and
standardize.[4–7]
Odourous metabolic products include volatile sulfur com-
pounds (VSCs), such as hydrogen sulfide, methanethiol and dim-
ethyl disulfide,[8–12] as well as other volatiles such as cresol, indole,
skatole, diamines and short chain fatty acids that may act as
odour modifiers.[13–19] Oral Gram-negative anaerobic bacteria
such as Porphyromonas gingivalis, Prevotella intermedia, Tre-
ponema denticola, Fusobacterium nucleatum, Tannerella forsythia,
Bacteroides spp. and Fusobacterium spp., when present in high
number in periodontal pockets, interdental spaces, and dental
and tongue biofilms, may produce those malodorous com-
pounds.[2,20,21] Yet uncultivable bacteria may also play a role and
contribute to oral putrefaction and malodours.[11,16,22] In contrast,
various other oral Gram-positive bacteria are more commonly
found in individuals with low or no levels of malodours.
Within this context, different approaches have proven to be
efficient at controlling and preventing oral malodour.[23] The
biofilm responsible for bad breath may be removed by careful
brushing or scraping (mechanical effect) and possibly by the
adjunctive use of oral care products containing antimicrobials
and rehydrating agents.[5,6,24–27] Today, salty water mouth rinses
have been replaced by efficient formulations containing antimi-
crobial components such as chlorhexidine, cetylpyridinium
chloride, triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol),
essential oils or zinc salts, either alone or in combination.[25,28] The
* Correspondence to: Myriam Troccaz, Corporate R&D Division, P.O. Box 239,
CH-1211 Geneva 8, Switzerland. E-mail: myriam.troccaz@firmenich.com
a
Firmenich SA, Corporate RandD, Route des Jeunes 1, P.O. Box 239, CH-1211
Geneva 8, Switzerland
b
Geneva University Hospital, Laboratoire Central de Bactériologie (LCB),
Service des Maladies Infectieuses, Rue Micheli-du-Crest 24, CH-1211 Geneva
14, Switzerland
c
School of Dental Medicine, University of Geneva, Rue Barthélemy-Mann 19,
CH-1205 Geneva, Switzerland

Flavour Fragr. J. 2010 Copyright © 2010 John Wiley & Sons, Ltd.
 M. Troccaz et al.

addition of oral flavours provides taste benefits and refreshes the
breath mainly by sensorial masking.[6,29] Flavour materials include,
however, a wide range of compounds that may have significant
antimicrobial effects and/or may inhibit the production of VSCs
by bacteria.[29,30]
The present study was designed to evaluate the efficacy of
flavours to control morning bad breath by either masking the
malodorous gases or by killing bacteria responsible for the pro-
duction of those volatiles.[10,29] For this purpose, a collection of
several hundred flavour ingredients were first screened in vitro for
their antimicrobial properties. Three flavours with the highest
antimicrobial activity were then included in mouth rinse formu-
lations, at a 0.2% w/w concentration, and selected for a clinical
trial. Oral malodours and flavour intensities were performed
before rinsing and 1 min and 1 h after rinsing. Results were com-
pared with two controls containing the widely used active ingre-
dients triclosan or peppermint oil. Saliva concentrations of
individual flavour compounds were also determined up to 1 h
after the rinse to evaluate flavour substantivity. This information
is particularly relevant when creating pleasant and active flavours
for the formulation of oral care products that promote fresh
breath with long-lasting properties without compromising taste.
Experimental
Flavour Creation
Five hundred flavour ingredients from the Firmenich SA (Geneva, Swit-
zerland) raw materials palette were screened for their minimum inhibi-
tory concentration (MIC) against the oral bacterial strains F. nucleatum
DSMZ 20482, P. gingivalis DSMZ 20709, Prevotella melaninogenica ATCC
25845 (Bacteroides melaninogenicus) and S. mutans DSMZ 6178. Sixteen
flavouring compositions were created with those materials to maximize
the content of antimicrobial actives (MIC values under 0.25% w/w).[31–33]
Those compositions were screened in vitro to confirm their antimicrobial
activity with an automated bacterial contact time test.[34] This method is
based on standardized procedures CEN/TC 216 N59 and the EN-1276
norm. Briefly, the method consists of calculating the log reduction in
viability of oral bacteria after a contact time of 80 s between flavours and
bacterial solution (108 cfu/ml). Three flavouring compositions with the
highest antimicrobial activity were selected for the clinical trial. The com-
position of the three flavours (flavours 1, 2, 3) is described in Table 1. They
contain well-known antimicrobial compounds such as eucalyptol,
carvone and aromatic alcohols (menthol, methyl salicylate), as well as
other ingredients such as limonene and anethole to mask harsh or
medicine-like tastes and to increase flavour pleasantness.[35,36] Their anti-
bacterial activity was shown to achieve up to a log 3 reduction (⫾ 0.1)
against F. nucleatum, P. gingivalis and P. melaninogenica at a concentration
of 0.01% and up to 3.5 log (⫾ 0.1) against S. mutans at a concentration of
0.1%.
Antimicrobial Mouth Rinse Composition
Five mouth rinses (M1, M2, M3, M4, M5) were tested for malodour coun-
teraction and salivary bacterial reduction in the in vivo clinical study. The
three antimicrobial flavours (flavours 1, 2, 3) were incorporated at 0.2%
w/w in a non-alcoholic mouth rinse base (M1, M2, M3) formulated with
demineralized water (82.88% w/w), sodium monofluorophosphate
(0.05%), sodium saccharin (0.03%), sorbitol 70% (8.02%), glycerin (8.02%)
and Cremophor® CO40 (1.00%) 24 h before clinical testing. No preserva-
tives or antimicrobial substances other than the flavours were added to
the mouth rinses. The effects of products containing antimicrobial fla-
vours were compared with two positive controls, M4 and M5, formulated
with 0.18% triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) (non-
flavoured control 4) and 0.2% regular peppermint oil-type flavour (flavour
5), respectively. Compositions of non-flavoured control 4 and flavour 5
are described in Table 1.

Table 1. Antimicrobial flavour composition measured by GC/MS (percentage of mass)

Analyte Retention m/z Flavour composition (% mass)*

time (min) Flavour 1 Flavour 2 Flavour 3 Non-flavoured Flavour 5
control 4
Eucalyptol 1.86 154 3.2 — 1.3 — 1.5
(⫾)-2-Methylhexanoic acid 1.86 74 9.1 9.1 9.1 —
Limonene 1.87 136 5.1 — 2.3 — 1.7
Menthone 2.36 112 6.8 — 6.9 — 6.4
Menthol 2.40 95 44.0 47.0 41.3 — 50.4
3,4-Dimethylphenol 2.44 107 0.8 0.8 0.8 —
Methyl salicylate 2.47
120 — 10.0 — — 0.1
Carvone 2.66 82 2.5 — 14.0 — 9.2
Anethol 2.77 148 10.8 6.8 — — 11.4
Cinnamic aldehyde 2.78 131 0.8 13.4 — — —
Eugenol 2.94 164 1.0 11.5 5.7 — 0.6
Heliotropin 2.96 150 — — 3.7 — —
Ethyl vanillin 3.28 137 — — 6.0 — —
Eugenyl acetate 3.39 164 — 1.2 — — —
Triclosan 4.76 288 — — — 100.0 —
% Quantifiable 84.1 99.8 91.1 100 81.3
* Flavours are a mixture of essential oils and pure compounds. The level of the analysed compounds (GC/MS analysis of 14 flavour
compounds) was investigated on a scale of 100. Therefore, 84.1, 99.8, 91.1, 100 and 81.3% of the total formulation was quantifiable
in the five products, respectively.
m/z, mass-to-charge ratio on mass spectrum.

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Effects of flavoured mouth rinses on breath odour
Subject Population
Twenty-four healthy and non-periodontally diseased adult subjects (10
males, 14 females), aged 19–29 years (mean age 24 years), participated in
the study. This number of subjects is considered to be sufficient to dis-
criminate between products in studies that use aromas or malodour
intensities as clinical variables.[37] Statistical analysis of variance (ANOVA)
confirmed that the product effect is stronger than the subject effect for all
descriptors. Recruitment was performed by the School of Dental Medi-
cine. To be included in the panel, a subject had to be a non-smoker in
good general health with no visible tongue coating and should not have
taken antibiotics for 2 months before the start of the trial. Subjects were
required not to use perfumes or cosmetics for the duration of the study.
At each visit, subjects received tubes of toothpaste (in exchange for their
old tubes) that were free of flavours, preservatives or antimicrobial sub-
stances such as triclosan. These tubes were to be used the week preced-
ing the study, as well as during the entire study period (5 weeks). Strict
rules were reiterated to the subjects every week. On the day before the
test, volunteers were asked to refrain from consuming food or drink after
midnight. Neither breakfast nor drinks were allowed before or during the
morning of the test. One week before the beginning of the study, a
training session was organized to validate the protocol. The protocol was
approved by the Ethical Committee of the Geneva University Hospital.
Experiments were undertaken with the understanding and written
consent of each subject and according to ethical principles, including the
World Medical Association Declaration of Helsinki.
Clinical Study Design
The clinical trial was performed at the School of Dental Medicine, Univer-
sity of Geneva. Subjects were asked to come once a week at fixed hours
during five consecutive weeks to test one of the mouth rinses. All tests
were performed between 7:30 and 9:30 a.m. All products were coded,
randomized and tested in a double-blind study (i.e. neither assessors nor
subjects knew which mouth rinses were tested). Products were coded
with three-digit codes generated and randomized by Fizz Software (Bio-
systèmes, Couternon, France). The presentation order of the products was
generated on a Latin square order design. Each panelist tested the five
different mouth rinses described previously. On the day of the test, sub-
jects were first examined for oral odour. A 1 ml sample of saliva was then
collected for analytical measurements and bacterial counts.[15] Volunteers
were then asked to rinse their mouth with 20 ml of mouth rinse for 1 min.
Assessments of oral malodour and collected saliva were repeated 1 min
and 1 h after the rinse. To collect the saliva, we asked subjects to mix their
saliva with a closed mouth for 1 min and then to spit (at least 4 ml of
saliva) into a polypropylene sterile 50 ml BD Falcon® tube (VWR Interna-
tional, Fontenay Sous Bois, France). To avoid flavour absorption, we did
not use saliva stimulation with paraffin. After homogenization, aliquots of
500 mg of saliva (precise weight) were transferred into 1 ml sterile tubes
and frozen at -20°C for flavour ingredient analysis. The remaining saliva
was transferred in Portagerm® transport bottles (BioMérieux, Geneva,
Switzerland) at room temperature for no longer than 4 h before homog-
enization, dilution and culturing for bacterial counting.
Assessment of Oral Odours
Assessment of oral malodours and flavour intensities was performed by
three trained assessors on an intensity basis. Assessors were trained
during preliminary sessions under the same conditions as the tests,
where they evaluated flavour and oral malodour of three different sub-
jects and also of pure chemical compounds. They were asked to replicate
their evaluations on the intensity scales, and results were discussed after
each session. Subjects were asked to close their mouth and not to
swallow for 1 min before exhaling air. Subjects and assessors were sepa-
rated by a wall with an aperture to allow the subject to breathe directly
into the assessor’s nose. This method was used to reduce any discomfort
for both the examiner and the subject. We recorded intensities by using
an unstructured linear sensorial analytical scale with scores of 0 (no
malodour/flavour) to 10 (very intense malodour/flavour).[38] In addition,
Flavour Fragr. J. 2010 Copyright © 2010 John Wiley & Sons, Ltd.
subjects were asked to perform a self-assessment of flavour intensity and
mouth rinse appeal (overall perceived sensation) 1 h after the mouth
rinse by using a linear scale of 0 to 10 for flavour intensity and a multiple
choice answer (not at all, a little, moderately, a lot) for rating flavour
pleasantness. Recording of scores was performed with Fizz Software (Bio-
systèmes, Couternon, France).
Determination of Flavour Material Concentrations in Saliva
The flavour materials present in the 500 mg saliva samples were extracted
by using 500 ml volumes of ethyl acetate containing 1 mg/l of methyl
octanoate (Fluka, Buchs, Switzerland) as the internal standard. Homog-
enization and extraction were achieved with an automatic shaker, and
phase separation was induced by centrifugation. The organic phases
were then transferred to 400 ml microvials to be analysed by gas chroma-
tography with mass spectrometric detection. Chromatographic separa-
tion was achieved on a 180 mm ¥ 10 m (0.18 mm film) DB-XLB column
mounted in an Agilent 6890 gas chromatograph equipped with a Combi-
PAL autosampler (CTC Analytics, Zwingen, Switzerland). One microlitre
aliquots were injected in the pulsed splitless mode (1.2 bar pulse for
0.6 min) by using a 4 mm tapered liner (FocusLiner tapered, SGE, Ring-
wood, Australia). Elution, with He as the carrier gas, started at 50°C for
0.8 min. The temperature was then ramped to 250°C at 50°C/min, and the
final temperature was held for 0.4 min. The interval between two injec-
tions was about 11.5 min. The mass spectrometer used for detection
(Agilent 5973 inert; Agilent, Santa Clara CA, USA) was operated in the
single ion monitoring mode. Quantitative data was obtained by external
calibration, with three concentration levels per order of magnitude after
correction to account for the internal standard.
The quantified compounds, selected to account for more than 80% of
all investigated flavour compositions were in order of elution: eucalyptol
(1,8-cineole); (⫾)-2-methylhexanoic acid; limonene ((⫾)-8-methoxy-1-p-
menthene); menthone; menthol; 3,4-dimethylphenol; methyl salicylate;
L-carvone ((-)-(R)-1(6),8-p-menthadien-2-one); anethole ((E)-1-methoxy-
4-(1-propenyl)benzene); cinnamic aldehyde (3-phenyl-2-propenal);
eugenol (4-allyl-2-methoxyphenol); heliotropin (1,3-benzodioxole-5-
carbaldehyde); ethyl vanillin (3-ethoxy-4-hydroxybenzaldehyde); eugenyl
acetate (4-allyl-2-methoxyphenyl acetate) and triclosan (2,4,4′-trichloro-
2′-hydroxydiphenyl ether). Limits of quantification were typically about
10 ng of individual flavour raw material per gram of saliva.
Bacteriological Analysis of Saliva Samples
Saliva samples were serially diluted (10-3 and 10-4) in saline solution,
plated in duplicate on Schaedler agar plates containing 5% sheep blood
(BioMérieux) by using a Spiral Plater DS Plus (Interscience, Paris, France)
and incubated for 72 h at 37°C under anaerobic conditions (Scholzen
Microbiology Systems, Wittenbach, Switzerland). After preliminary
experiments, no significant differences were shown regarding bacterial
counts after 72 h and 7 days of incubation (data not shown). Total micro-
bial counts, including facultative anaerobes and anaerobes, were deter-
mined and the results were expressed as numbers of cfu/ml.
Statistical Analysis
Statistical treatment of the sensory data was performed with XLStat soft-
ware (Addinsoft, Paris, France). First, a three-way ANOVA with interaction
at two factors was performed on the intensity ratings to determine if
mouth rinses were discriminated by the assessors. Subjects, assessors and
mouth rinses were considered as individual factors for ANOVA to deter-
mine which factors had a significant influence on the oral malodour and
flavour intensities at different times after rinsing the mouth (after 1 min
and 1 h). The two factors assessor and subject were set to fixed. Second,
the Duncan test was then computed to determine if the differences
between each pair of products were significant; and third, a Pearson’s
correlation coefficient was calculated between the malodour intensity
and flavour intensity. The ANOVA and Duncan tests were also performed
with salivary bacterial counts to evaluate differences between mouth
rinses in reducing the bacterial count after 1 min and 1 h. Percentage
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 M. Troccaz et al.

Figure 1. Sensory ratings of mouth malodour and oral flavour intensity before treatment, 1 min and
1 h after treatment with various mouth rinses (M). M1, M2 and M3 contain antimicrobial flavours 1, 2
and 3 respectively; M4, positive non-flavoured control containing triclosan; M5, positive control
containing peppermint oil (flavour 5). *Significant differences between mouth rinses with a confi-
dence interval of 95% (Duncan test). The error bars represent 95% confidence intervals

reductions in bacterial counts were calculated for each participant indi-
vidually and as an average. For flavour intensity self-assessment scores,
subjects and mouth rinses were considered as individual factors for
ANOVA and Duncan tests. Finally, a chi-squared test was performed with
self-assessment pleasantness ratings to evaluate the possible presence of
significant differences in the distribution of the ratings between mouth
rinses. The confidence level was set to 5% (p < 0.05) for ANOVA tests,
Duncan tests and correlation coefficients.
Results and Discussion
All subjects (n = 24) completed the study and tested the five
mouth rinse compositions. Three compositions contained antimi-
crobial flavours (M1, M2, M3) and two contained controls (M4,
M5). No adverse effect was reported during the study.
Sensory Evaluation of Oral Malodours
The average oral malodour of the 24 subjects measured before
rinsing (mean ⫾ confidence interval at 95%) over the 5-week
period was judged to be 3.60 ⫾ 0.45 on a scale of 0 to 10
(Figure 1). Two subjects were found to be outliers (maximum at
5.2 and minimum at 1.5) but did not impair the final results. This
relatively low malodour intensity (in average) may be explained
by non-pathological halitosis among the subjects; however, the
evaluated morning malodour was high enough to be repulsive in
a social context and to be considered in this study. Immediately
after the rinse, all formulations containing the antimicrobial fla-
vours and the two controls containing triclosan and peppermint
oil-type flavour performed equally well for oral malodour reduc-
tion. An average decrease of 72% was observed in oral malodour
scores (Figure 1). One minute after the rinse, the malodour inten-
sity decreased to a value between 0.56 ⫾ 0.20 with mouth rinse 3
(M3) and 1.41 ⫾ 0.57 with M4. One hour after the rinse, the
intensities increased to a value between 1.12 ⫾ 0.27 (M5) and
1.97 ⫾ 0.36 (M1), but were still lower compared with baseline
(56% oral odour intensity reduction). The intensity rating scale is
considered to be the gold standard for the measurement of oral
malodour because it reflects the real-time presence of an objec-
tionable odour as detected by the assessor.[9,21,38–40] In addition,
this evaluation method is not restricted to VSCs, contrary to
evaluation with a halimeter.[9,23,39] Therefore, the decrease of oral
malodour intensity may not be solely explained by the decrease
of one class of chemicals, but rather to a decrease in a variety of
bacteria-generated volatiles that contributes to halitosis.
Statistical results of ANOVA (performed with the mouth rinse,
the assessors and the subjects as individual factors) and Duncan
tests on sensory data showed that mouth rinse M3, containing
antimicrobial flavour 3, and control mouth rinse M5, containing
peppermint oil-type flavour 5, significantly reduced oral mal-
odour more than the other flavours at both times. Subjects and
assessors were also found to have some significant effects on the
results, but at lower levels than the mouth rinse effects (data not
shown). The assessor effect is significant for all evaluations
(ANOVA by evaluation, with assessor effect). Nevertheless, similar
conclusions were obtained for each assessor independently or
for the three assessors taken together (ANOVA by assessor and by
evaluation, with mouth rinse and subject effects).
Assessment of Mouth Rinse Intensity and Appeal
Flavour intensities decreased dramatically from a value between
5.80 ⫾ 0.29 and 6.11 ⫾ 0.38 immediately after the rinse to a value
between 0.42 ⫾ 0.27 and 0.93 ⫾ 0.27 at 1 h. As expected, mouth
rinse M4, containing no flavour except triclosan, was perceived as
significantly weaker when compared with the other mouth rinses
(Duncan test at 95%) at 1 min and 1 h after the rinse. In contrast,
M1, which contains high levels of limonene, menthol and aneth-
ole (flavour 1), was perceived to be significantly stronger by the
three assessors 1 h after rinsing (Duncan test at 95%). Those
results were confirmed by the subjects themselves: M1 intensity
was scored at an average of 4.1 on an intensity scale of 0 to 10,
whereas M2, M3, M4 and M5 were rated at 2.7, 2.3, 1.8 and 2.2,
respectively. No correlation could be established between mal-
odour reduction (or malodour intensity) and oral flavour intensity
at 1 min and 1 h after the rinse (Pearson’s correlation coefficient
was not significant, data not shown).
In parallel, the assessments of flavour pleasantness by the 24
subjects themselves were intended to confirm that malodour
control and anti-microbial properties did not impair flavour
acceptance. The assessment of mouth sensation was judged to
be significantly worse with the non-flavoured mouth rinse M4,
containing triclosan (chi-squared test) (Figure 2). Twelve subjects
of the 24 did not like this mouth rinse at all. M2 and M3, contain-

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Effects of flavoured mouth rinses on breath odour
Figure 2. Self-assessment of mouth rinse pleasantness by the subjects. Overall perceived sensa-
tions were recorded 1 h after rinsing the mouth with M1, M2 and M3 containing the antimicrobial
flavours 1, 2 and 3, respectively, and the two positive controls, M4 (containing triclosan) and M5
(containing peppermint oil)
Figure 3. Average reduction in salivary bacterial counts (n = 24) 1 min
and 1 h after treatment with various mouth rinses (M). M1, M2 and M3
contain antimicrobial flavours 1, 2 and 3, respectively; M4, positive non-
flavoured control containing triclosan; M5, positive control containing
peppermint oil (flavour 5). * Significant differences between mouth rinses
with a confidence interval of 95% (Duncan test). The error bars represent
95% confidence intervals
ing antimicrobial flavours 2 and 3, respectively, were judged glo-
bally as the most pleasant (ANOVA and Duncan test).
Salivary Bacterial Counts
Before rinsing, the average number of bacteria (mean ⫾ confi-
dence interval at 95%) was 8.3 ⫾ 0.2 log cfu/ml of saliva (corre-
sponding to 3.58 ¥ 108 cfu/ml). Significant differences in bacterial
counts were found between subjects. Two subjects were outliers,
with a bacterial number of 7.7 log cfu/ml and 9.0 log cfu/ml,
respectively, but these numbers did not influence final mouth
rinse efficacies. Microbial reduction per milliliter of saliva was first
calculated individually for each subject and then averaged.
Results are summarized in Figure 3.
One minute after the rinse, no significant differences were
observed between mouth rinse antimicrobial efficacies: an
average bacterial reduction of 49.0 ⫾ 8.0% was calculated for the
Flavour Fragr. J. 2010 Copyright © 2010 John Wiley & Sons, Ltd.
five formulations. Both the mechanical/dilution effect of the
mouth rinse and the antimicrobial effects of the ingredients may
be responsible for those high active percentages. A significant
decrease in bacterial counts was observed in 11, 15, 17, 15 or 16
subjects of the 24 after rinsing with M1, M2, M3, M4 or M5,
respectively. In three subjects, an increase in bacterial counts was
found after rinsing with M1 and M4 and in one subject after
rinsing with M2 and M5.
At 1 h, bacterial counts were significantly lower with M3 than
with the other mouth rinses (Duncan test at 95%) (40% decreases
in bacterial counts on average 1 h after rinsing, Figure 3). With
M3, none of the subjects showed an increase in bacterial count
and three subjects had returned to pre-rinsing values. For the
other mouth rinses (M1, M2, M4 or M5), 9, 19, 16 or 14 subjects of
the 24 had bacterial counts similar to or higher than before the
rinse, respectively.
M3 showed the highest antimicrobial effect after 1 h (40%
reduction). Flavour 3 contained high levels of the antimicrobial
ingredients menthol, limonene, carvone, eugenol and men-
thone. Some of these compounds were reported to have addi-
tive effects.[41] In addition, in vitro testing revealed that flavour 3
was strongly bactericidal against F. nucleatum, a bacterium that
generates odorous compounds (log reduction of 3.2 or 99.9%
reduction of F. nucleatum after 80 s contact time) (data not
shown). In previous studies, similar compositions have been
used to counter plaque, inhibit anaerobic bacterial growth or
neutralize VSCs in halitosis treatments. For example, US Patent
3164524 describes oral antiseptic compositions for the preven-
tion of bad breath comprising an aqueous alcoholic solution
containing menthol, methyl salicylate, thymol and eucalyptol.[42]
US-5472684 discloses an antibacterial oral composition to
counter plaque comprising thymol and eugenol.[43] Similarly,
menthol, thymol, methyl salicylate and eucalyptol, present in
the essential oil-containing mouth rinse Listerine® antiseptic,
were shown to be active on dental plaque bacteria in situ.[44]
Polyphenols such as the epigallocatechin gallate contained in
tea extracts were shown to have antimicrobial properties and to
neutralize VSCs.[45]
By contrast, in our study, antimicrobial compounds such as
triclosan, anethole and cinnamic aldehyde had limited effects on
salivary bacteria and mouth malodour when present in mouth
rinse flavour compositions. One explanation may be that the anti-
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 M. Troccaz et al.

Figure 4. Mean concentrations of the flavour ingredients present in the saliva of the subjects before rinsing the mouth and 1 min and 1 h after
treatment (number of subjects = 24). The error bars represent 95% confidence intervals. When the investigated raw material was not detected in the
saliva of all 24 subjects, values indicate the number of subjects in which it was detected; M, mouth rinse. M1, M2 and M3 contain antimicrobial flavours
1, 2 and 3 respectively; M4, positive non-flavoured control containing triclosan; M5, positive control containing peppermint oil (flavour 5). E, eucalyptol;
L, limonene; MA, (⫾)-2-methylhexanoic acid; MN, menthone; ML, menthol; DP, 3,4-dimethylphenol; MS, methyl salicylate; C, L-carvone; A, anethol;
CA, cinnamic aldehyde; EL, eugenol; H, heliotropin; EV, ethyl vanillin; EA, eugenyl acetate; T, triclosan

microbial activity of flavours is due to their lipophilic characteris-
tics. The bacteria have lipids on and in their surfaces, which may
take up lipophilic flavours that have an antibacterial effect by
destroying the bacterial membranes. Indeed, the lower hydro-
phobicity of these compounds may explain the results. In addi-
tion, such interactions may be modulated by the presence of salt,
sweeteners, hydrophobic compounds and other additives in the
mouth rinse.[46] Flavours must be active in a complex environ-
ment, the saliva, which may change flavour retention time and
antimicrobial properties.

www.wileyonlinelibrary.com/journal/ffj Copyright © 2010 John Wiley & Sons, Ltd. Flavour Fragr. J. 2010
Effects of flavoured mouth rinses on breath odour
Chemical Analysis of Saliva
Menthol was the major flavour ingredient present in saliva 1 min
after the rinse, with a concentration of 1.1 ng/g of saliva
(Figure 4). At 1 min after the rinse, an average flavour dilution of
500 was calculated between the flavour material concentration in
20 ml of the mouth rinse (containing 0.2% flavour) and the
flavour ingredient concentration expectorated in 1 ml of saliva.
The flavour dilution was 500 000 at 1 h after the rinse. The mouth-
wash M1 was rated as significantly more intense 1 h after the
rinse by both the assessors and the subjects (as described previ-
ously). These results may be explained by the chemical ingredi-
ents of flavour 1, menthol, limonene and anethole that are still
present in saliva 1 h after rinsing at a concentration of 83.7, 30.0
and 46.3 ng/g saliva, respectively. One hour after the rinse, six of
the 15 ingredients analysed were still present in the saliva of up to
21 subjects. These ingredients were menthol, anethole, cinnamic
aldehyde, limonene, carvone and triclosan. They are generally
highly hydrophobic flavour ingredients and have low solubility.
Flavour compositions comprising limonene, menthol and aneth-
ole with an average octanol–water partition coefficient (log P) of
at least 3.3 (in logarithm form) have been previously shown to
have an increased substantivity and thus longevity in the oral
cavity.[47,48]
Other compounds, including methylhexanoic acid, menthone,
eugenol and ethyl vanillin, which have a log P less than 2.5, were
detected in only a few subjects 1 h after rinsing. Five ingredients
(eucalyptol, 3,4-dimethylphenol, methyl salicylate, heliotropin
and eugenyl acetate) were not detected in saliva after 1 h
(Figure 4). Our results are in agreement with reports of human
salivary proteins changing the partitioning behaviour of aroma
compounds in the mouth by binding hydrophobic molecules or
by salting out hydrophilic molecules.[49,50] Mucin-glycoproteins,
which have low solubility and adhesiveness properties, were
identified as the key components that affect flavour release.[51] In
addition, mucins contain cysteine-rich regions that may react
with carbonyls present in those compounds to modify antimicro-
bial flavour properties, as well as the efficacies, to reduce
malodour.[51]
After the rinse with M3, the antimicrobial compounds
limonene (32 ng/g saliva), menthol (121 ng/g saliva), carvone
(18 ng/g saliva) and eugenol (7.4 ng/g saliva) were still found in
saliva at 1 h in most subjects, whereas menthone was detected in
only two subjects in an average concentration of 2.7 ng/g saliva.
Indeed, the high antimicrobial activity of M3 at 1 h after the rinse
may be explained by the presence of limonene, menthol, carvone
and eugenol at a concentration higher than their MIC values. In
contrast, triclosan was still present at a concentration of
1752 ng/g of saliva 1 h after rinsing with M4; however, it had a
limited effect on salivary bacterial counts. This may be due to the
solubilizer used to formulate the mouth rinse. The nature of the
solubilizing agent has been shown to be critical to the plaque-
inhibiting activity of triclosan.[24] It is interesting to note that one
third of our subjects were found to have an average of 80 ⫾
51 ng/g of triclosan (mean ⫾ standard error) in their saliva in the
morning before the rinse despite the strong recommendations
given to the subjects not to use any oral care products containing
triclosan 1 week before and during the study (see Materials
and Methods). This was previously stated in an in vivo laboratory
scale analysis (data not shown). A concentration of 1200 ng
triclosan/g of saliva was detected in one of the subjects. Despite
such triclosan concentrations, those subjects had morning bad
Flavour Fragr. J. 2010 Copyright © 2010 John Wiley & Sons, Ltd.
breath and initial salivary bacterial counts that were similar to
those of the other subjects and did not modify our conclusions.
However, at this time, the exact source of triclosan still remains
undetermined.
In conclusion, the present clinical trial has shown that morning
breath odours can be reduced up to 1 h after rinsing with alcohol-
free mouth rinses containing in-vitro active antimicrobial flavour
ingredients. Maximum efficacies were obtained with mouth rinse
3 (M3) and the peppermint control M5, containing high levels of
menthol and carvone. The maximum malodour intensity reduc-
tions were 61% and 68% reduction after 1 h, respectively. In addi-
tion, flavour 3, containing high levels of the antimicrobial
ingredients menthol, limonene, carvone and eugenol, which are
still present in saliva 1 h after rinsing, may explain the antimicro-
bial effect of M3 (40% reduction in bacterial count 1 h after
rinsing). Today, formulating effective oral care products that
promote fresh breath with long-lasting properties and limited
side effects represents a challenge. In the present study, selected
flavours were found to have beneficial effects against salivary
microorganisms as well as transient oral malodours. These fla-
vours may be considered for the designing of oral care products
that promote fresh breath without compromising the taste.
Acknowledgements
The authors are grateful to Firmenich SA for funding this study.
The authors thank Hidemi Tashiro for creating the flavour com-
positions and Dr Christopher Dean for support and advice. They
are also grateful to Dr Markus Seyfried, Pauline Gourdain and José
Fernandez for the minimum inhibitory concentration assays and
microbiological technical assistance. They are indebted to
Vanessa Petitpierre, Anne Yu and Anna Filieri for recruiting the
subjects and organizing the olfactive tests.
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