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A Novel Delivery System of Probiotic Drop and Its Effect on Dental Caries
Risk Factors in Cleft Lip/Palate Children

Article  in  The Cleft Palate-Craniofacial Journal · February 2011

DOI: 10.1597/10-035 · Source: PubMed

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A Novel Delivery System of Probiotic Drop and Its Effect on Dental Caries
Risk Factors in Cleft Lip/Palate Children
Sule Kavaloglu Cildir, D.D.S., M.Sc., Ph.D., Nuket Sandalli, D.D.S., Ph.D., Serpil Nazli, D.D.S., Fatos Alp, D.D.S.,
Esber Caglar, D.D.S., Ph.D.

Objective: The aim of the present study was to investigate the effect of the
probiotic bacterium Lactobacillus reuteri on the levels of salivary mutans
streptococci and lactobacilli in children with cleft lip/palate who used the novel
drop containing L. reuteri.
Material and Methods: The study group consisted of 19 operated cleft lip/
palate children aged 4 to 12 years. The study had a double-blind, randomized
crossover design, and the experimental period consisted of four consecutive
time periods. During periods 2 and 4, consisting of 25 days each, parents were
instructed that their children should consume 5 drops per day (0.15 to 0.20 g) of
probiotic or placebo drops produced by the same manufacturer. The probiotic
drop, BioGaia Reuteri drops, contained L. reuteri DSM 17938 and L. reuteri
ATCC PTA 5289 ($1 3 108 CFU/5 drops). The counts of salivary mutans
streptococci and lactobacilli were evaluated using the CRT tests. The data were
processed with NCSS 2007 software using chi-square and McNemar tests.
Results: There was no statistically significant (p . .05) reduction of salivary
mutans streptococci and lactobacilli after 25 days of consumption of both
drops.
Conclusions: The novel drop containing L. reuteri may not reduce the levels
of salivary mutans streptococci and lactobacilli in cleft lip/palate children.

KEY WORDS: children, cleft lip/palate, dental, drop, probiotic

Probiotic bacteria are live microbial food supplements
that may benefit the host by influencing the balance
between the many species of the commensal flora both in
the digestive system and oral cavity (Caglar et al., 2005a,
2005b, 2006a; Lenoir-Wijnkoop et al., 2007; Caglar et al.,
2008b; Twetman and Stecksén-Blicks, 2008; Cildir et al.,
2009). The beneficial role that Lactobacillus reuteri ATCC
55730 has in general health, immunomodulation, and
disease protection has been observed and described in a
number of studies (Casas and Dobrogosz, 2000; Valeur et
al., 2004; Weizman et al., 2005; Spinler et al., 2008). Within
dentistry, there are only a few clinical studies investigating
the effect of L. reuteri in the oral cavity of young adults
where salivary mutans streptococci levels were reduced
(Nikawa et al., 2004; Caglar et al., 2006b, 2007, 2008a).
Dr. Cildir is Assistant Professor and Dr. Caglar is Assistant Professor,
Department of Pedodontics, Faculty of Dentistry, Yeditepe University,
Istanbul, Turkey. Dr. Sandalli is Head, Dr. Nazli is Postgraduate Student,
and Dr. Alp is Postgraduate Student, Department of Pedodontics, Faculty
of Dentistry, Yeditepe University, Istanbul, Turkey.
Submitted February 2010; Accepted January 2011.
Address correspondence to: Dr. Sule Kavaloglu Cildir, Yeditepe
University, Faculty of Dentistry, Department of Paediatric Dentistry,
Bagdat Cad No. 238, 34728, Goztepe Istanbul, Turkey. E-mail
sulecildir@yahoo.com.tr.
DOI: 10.1597/10-035
In daily routines, the administration of probiotics to
children is difficult. In recent clinical trials, fruit yogurt
with with Bifidobacterium DN-173 010 (Cildir et al., 2009)
and milk with Lactobacillus rhamnosus GG (Näse et al.,
2001) were used as probiotic vehicles in children where
salivary mutans streptococci levels were reduced. However,
there are actually no data on L. reuteri’s side effects on
children’s oral health, whether healthy or handicapped.
Cleft lip and palate is the most common craniofacial
birth defect. Without therapeutic and surgical manage-
ment, these children encounter significant barriers to
proper speech, nutrition, and development (Borodkin et
al., 2008). Recently, many studies have demonstrated that
children with cleft lip/palate are at an increased risk of
being infected by mutans streptococci and lactobacilli
(Bokhout et al., 1996a, 1996b; Chapple and Nunn, 2001;
Hewson et al., 2001; Besseling and Dubois, 2004). It was
also emphasized that the children with cleft lip/palate must
be considered as a group with an increased caries risk and
should therefore be subjected to an additional preventive
program (Dahllöf et al., 1989; Enacar, 1999; Perdikogianni
et al., 2009). In the United Kingdom, Ahluwalia et al.
(2004) reported that children with cleft palates had
increased oral clearance times for food and, consequently,
higher levels of caries and caries-associated micro-organ-
isms (MO) compared with healthy control children.

369
370 Cleft Palate–Craniofacial Journal, May 2012, Vol. 49 No. 3
Regarding the statements above, there is a need for a
more aggressive approach to prevention of oral disease to
optimize clinical outcomes. At this point, probiotics may
serve as optimal vehicles to balance the oral health of this
specific group of children.
To our knowledge, the possible effect of probiotics on
the caries-related oral MO of children with cleft palate has
not been reported. The aim of the present study was to
investigate the effect of the probiotic bacterium L. reuteri
on the levels of salivary mutans streptococci and lactoba-
cilli in children with cleft lip/palate who used the novel drop
containing L. reuteri. The null hypothesis was that the use
of the present drop would not alter salivary mutans
streptococci and lactobacilli.
MATERIALS AND METHODS
The study group consisted of 19 operated children with
cleft lip/palate visiting the Department of Paediatric
Dentistry (10 girls and nine boys) aged 4 to 12 years (mean
age, 7.63 6 2.33) who volunteered after informed consent.
To be considered for invitation, children had to have no
systemic diseases, undergone cleft lip/palate surgery, good
oral health with no untreated caries lesions, and daily
tooth-brushing habits using fluoride toothpaste. The
exclusion criteria were (1) habitual consumers of probiotics
or xylitol chewing gums, (2) systemic antibiotic medication
within 6 weeks, and (3) recent topical fluoride treatments
within 4 weeks. Prior to baseline, children’s parents
received both oral and written information about the study
and signed a consent form.
The study used a double-blind, randomized crossover
design, and the experimental period consisted of four
consecutive time periods. Periods 1 and 3 were run-in and
washout periods of 1 and 3 weeks, respectively, in which the
children were asked to refrain from intake of milk-based,
probiotic, and xylitol-containing products. During periods
2 and 4, consisting of 25 days each, parents were instructed
to have their children consume 5 drops per day of probiotic
or placebo drops produced by the same manufacturer
without viable bacteria. The subjects were encouraged to
maintain their normal oral hygiene habits and continue to
brush their teeth twice a day during all four periods. The
study protocol was in accordance with the Helsinki
Declaration of Human Rights and approved by the Ethical
Committee at the School of Dentistry, University of
Yeditepe, Istanbul.
The probiotic drop, BioGaia Reuteri drops (BioGaia
AB, Stockholm, Sweden), contained freeze-dried L. reuteri
DSM 17938 ($1 3 108 CFU/5 drops) and L. reuteri ATCC
PTA 5289 ($1 3 108 CFU/5 drops) suspended in oil, while
the control drops were without viable bacteria. The daily
intake was 0.15 to 0.20 g/5 drops at a single occasion, and
the subjects were advised to consume at morning. During
the study, parents were instructed to keep the product in
the refrigerator when it was not in use. No tooth brushing
was allowed for 1 hour after intake. The test and the
control products were given in a randomized order, had a
similar taste and consistency, and were delivered in 5.0 mL
oil in an amber glass bottle with tamper-evident screw cap
and dropper insert, marked with ‘‘A’’ or ‘‘B.’’ The content
was unknown to both the children/parents and the clinician
responsible for the samplings. The code was not unveiled
until after the statistical calculations.
Samplings of paraffin-stimulated whole saliva were
carried out immediately before (baseline) and after
(follow-up) period 2 and 4. After a thorough water rinse,
the saliva was collected during 5 minutes directly into a
graded test tube. The counts of salivary mutans strepto-
cocci and lactobacilli were evaluated using the CRT test
(Ivoclar Vivadent AG, Schaan, Liechtenstein) according to
the manufacturers’s manual. In brief, saliva was inoculated
on a dip slide with selective agar media for mutans
streptococci and lactobacilli. After adding an NaHCO3
tablet, the slides were cultivated in 37uC for 48 hours. The
colonies were identified, and the density of the colony-
forming units (CFU) was visually compared with a chart
provided by the manufacturer. The scores that were used
for mutans streptococci and lactobacilli growth are shown
in Tables 1 and 2.
Statistical Method
The data were processed with NCSS 2007 software. A
chi-square test was applied for intergroup comparison, and
a p value less than .05 was considered statistically
significant. Posttreatment and pretreatment values within
each regimen were compared with the McNemar test,
and a p value less than .05 was considered statistically
significant.
RESULTS
The pretreatment and posttreatment levels of salivary
mutans streptococci and lactobacilli are shown in Tables 1
and 2. All but four subjects exhibited detectable levels of
salivary mutans streptococci at baseline, and about 55%
exhibited levels $105 CFU. There was no significant
difference between the baseline values obtained at the start
of periods 2 and 4. There were no statistically significant (p
. .05) reductions of salivary mutans streptococci after
25 days of consumption of both probiotic and placebo
drops. In the probiotic group, three subjects exhibited
decreased scores (one to two steps), 11 subjects had
unchanged scores, and five displayed a one-step increased
score. In the placebo group, two subjects displayed a one-
to two-step decrease, 10 had unchanged scores, and seven
displayed a one-step increased score.
Regarding salivary lactobacilli, no statistically significant
changes appeared between the posttreatment and pretreat-
ment samples, neither during probiotic drop usage nor
during the placebo drop usage (P . .05). In the probiotic

TABLE 1 Distribution of Salivary Mutans Streptococci at Baseline
and After Usage of Probiotic and Placebo Drop (N = 19)*
Mutans
Streptococci Score, Probiotic Placebo
CFU/mL Group Group
Baseline (pretreatment)
0 No growth 4 4
1 ,105 4 4
2 105–106 4 6
3 .106 7 5
25 days (posttreatment)
0 No growth 3 2
1 ,105 4 7
2 105–106 5 3
3 .106 7 7
p NS NS
* x2 and McNemar test. CFU 5 colony-forming units; NS 5 not significant.
group, six subjects exhibited a one-step decreased score,
eight subjects had unchanged scores, and five displayed
one- to two-step increased scores. In the placebo group, six
subjects displayed a one- to two-step decrease, eight had
unchanged scores, and five displayed a one-step increased
score.
DISCUSSION
The early intervention concept is interesting as it may be
easier to affect the caries-associated MO before their
permanent colonization compared with later in life, when
the resident oral flora is firmly established (Könönen, 2000;
Caglar et al., 2008a). Therefore, young children with newly
erupted teeth constitute the primary target groups for oral
probiotic intervention. A special subgroup, cleft-affected
children, should be referred for comprehensive and
continued preventive dental care from the first year of life
(Hewson et al., 2001; Perdikogianni et al., 2009). Therefore,
it is believed that the present study is unique as the study
population is limited to a group of children with cleft lip/
palate with increased caries risk, a complicated issue of oral
colonization of children with cleft lip/palate in infancy, and
the present multistrain and the probiotic drop have not
been used previously in the literature.
It seems likely that different probiotic strains have
different mechanisms of action in the oral cavity. Regard-
ing L. reuteri, it decreases the number of mutans
streptococci in saliva (Nikawa et al., 2004; Caglar et al.,
2006b); however, both Turkish and Finnish studies have
observed that it does not colonize the oral cavity (Caglar et
al., 2006b; Haukioja et al., 2006; Caglar et al., 2007, 2008b,
2009). L. reuteri also affects the oral ecology by specifically
preventing the adherence of other bacteria and by
modifying the protein composition of the salivary pellicle
(Haukioja et al., 2008). Currently, probiotic research
focuses on the capacity of probiotics to produce acid from
dietary sugars to rule out deleterious effects on the teeth. In
their in vitro study, Hedberg et al. investigated whether the
Cildir et al., PROBIOTIC DROP IN CLEFT LIP/PALATE CHILDREN 371
TABLE 2 Distribution of Salivary Lactobacilli at Baseline and
After Usage of Probiotic and Placebo Drops (N = 19)*
Lactobacilli, Probiotic Placebo
CFU/mL Group Group p
p
Baseline (pretreatment)
0 #103 4 5
1 104 4 8 NS, p . .05
NS, p . .05 2 105 6 4
3 $106 5 2
25 days (posttreatment)
0 #103 2 4
1 104 7 9 NS, p . .05
NS, p . .05 2 105 3 6
3 $106 7 0
p NS NS
* x2 and McNemar tests. CFU 5 colony-forming units; NS 5 not significant.
metabolic activity differs between various probiotic strains,
and L. reuteri ATCC PTA 5289 slowly generated weak
reactions with glucose, lactose, sucrose, maltose, and
melibiose when incubated in 5% CO2 (Hedberg et al., 2008).
It is common in Europe to provide dietary supplements of
vitamins A and D to infants and young babies. These
supplements are conveniently provided by a delivery system
that is based on food oils. The present probiotic drop was
developed and adapted this system to allow the delivery of
live L. reuteri and screened a series of oils and mixtures of
oils for their compatibility with L. reuteri in particular and
lactobacilli in general (Connolly, 2005). The probiotic drop
is also ideal as the formulation is found to be very stable at
refrigerated temperatures for at least 18 months. Many
hospitals in Sweden are using L. reuteri drops today, mainly
in pediatric units where the main uses are related to
gastrointestinal disorders, and recent clinical studies con-
firmed that the L. reuteri drops led to a clear colonization of
the gastrointestinal tract of the infants (Valeur et al., 2004;
Savino et al., 2007). However, in the present study, the
probiotic drop containing freeze-dried L. reuteri DSM 17938
and L. reuteri ATCC PTA 5289 did not seem to have any
effect on salivary mutans streptococci and lactobacilli in
children with cleft lip/palate. Based on previous data (Caglar
et al., 2006b), contact with oral mucosa and biofilm is
thought to be important in temporary colonization of
probiotics. At this point, the selected drop, being a multi-
strain probiotic and acting for a longer period of therapy
time (25 days), did not seem to alter the salivary caries-
related MO. It is possible that drops may not be suitable for
this purpose, as nondairy probiotic lozenges (Caglar et al.,
2008a), chewing tablets (Caglar et al., 2006b, 2009), and
gums (Caglar et al., 2007) have served effectively in our prior
studies. It should be noted that the possible effect of
probiotics and their interactions on the oral microflora of
children with cleft palate may also be complex. The possible
limitation of the present study is the limited number of
subjects, as it is hard to determine children with cleft palate
and parents who wish to be volunteers and long-term
participants in such a trial.
372 Cleft Palate–Craniofacial Journal, May 2012, Vol. 49 No. 3
In conclusion, the novel drop containing L. reuteri may
not reduce the levels of salivary mutans streptococci and
lactobacilli in children with cleft lip/palate. The null
hypothesis was verified that the usage of the present drop
would not alter salivary mutans streptococci and lactoba-
cilli. Further studies are needed to clarify whether this
vehicle is an alternative strategy for the prevention of
enamel demineralization.
Acknowledgments. We would like to thank our volunteers for their
participation, Ms. Rana Konyalıoglu (Istanbul, Turkey) for her statistical
expertise, Mr. Altay Uludamar (Ivoclar-Vivadent Marketing, Istanbul,
Turkey) for supplying chairside saliva tests, and Dr. Eamonn Connolly
and Dr. Anders Zachrisson (Biogaia, Stockholm, Sweden) for providing
probiotic drops.
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