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Asian Pac J Dent 2015; 15: 3-11

Effects of Lactobacillus crispatus as a candidate for oral probiotic
bacteria on Haemophilus influenzae
Kaoru Sogabe-Ashigaki, DDS,a Susumu Imai, PhD,b Ayako Okada, DDS, PhD,b Khairul
Matin, DDS, PhD,b Gaku Akimaru, DDS,a Tomohiko Terai, MSc,c Takekazu Okumura,
PhD,c Nobuhiro Hanada, DDS, PhD,b and Hiroshi Kawahara, DDS, PhDaab

Department of Dental Anesthesiology, Department of Translational Research, Tsurumi UniversitycSchool of Dental Medicine, Yokohama, and Yakult Central Institute, Food Research Department,
Tokyo, Japan
Purpose: To verify the effects of five candidates for oral probiotics on Haemophilus influenzae and to morecharacterize the effects of Lactobacillus crispatus YIT 12319 (L.c1).
Materials and Methods: Cell free culture supernatants (CFCSs) were acquired from cultures of the clinicalisolates (oral probiotic candidate bacteria). Two laboratory strains of H. influenzae (type b and non-typeable)
were freshly cultured in Muller-Hinton broth medium. A paired competition assay was performed using eachclinical isolate versus one of the two H. influenzae strains on culture plates. Antibacterial effects of all
unprocessed CFCSs, neutralized CFCSs, and an ammonium sulfate precipitate of the CFCSs against H.
influenzae were determined using a radial diffusion assay. The bactericidal effect of L.c1 CFCS was also
verified using a colony formation assay. Furthermore, the organic acids produced by L.c1 were analyzed using
HPLC.
Results: L.c1 displayed uninterrupted growth by suppressing the growth of both stains of H. influenzae equally,
while the results for the rest of the clinical isolates were inconsistent. Only unprocessed CFCS of L.c1 clearly
inhibited the growth of H. influenzae strains and showed potentially strong bactericidal effects. Reasonableamounts of lactic acid, acetic acid, succinic acid, and propionic acid were detected by HPLC.
Conclusion: The effects of L.c1 on H. influenzae strains observed in this study will serve as important
supporting evidence for the potential use of lactobacillus species as probiotics. In addition, the growthappeared to be inhibited by the organic acids produced by L.c1.
(Asian Pac J Dent 2015; 15: 3-11.)
Key Words: Haemophilus influenzae, Lactobacillus crispatus, oral probiotics, organic acid

Introduction
Haemophilus influenzae has been frequently detected as a commensal bacterium of the human nasopharynx.
For the past few decades, it has been highlighted as a major cause of a variety of respiratory conditions.1

H.

influenzae is responsible for a variety of infections in humans including septicemia, bronchitis, pneumonia, andacute otitis media (AOM).2

Over the last few years, pneumonia has been the third leading cause of death inJapan, where 98.9/100,000 people die of the disease per year (http://www.mhlw.go.jp/english/database/db-hw/populate/index.html), making it a particularly alarming health issue.3

Previously, it was demonstrated

that H. influenzae and anaerobes are primarily detected in patients aged 40-64 years.4

In recent years, it has

been revealed that H. influenzae plays a role in chronic lower respiratory tract inflammation.
H. influenzae is a gram-negative coccobacillus with a variable shape (pleomorphic) that grows both
aerobically and anaerobically.5,6

H. influenzae is divided into typeable and nontypeable strains based on thepresence or absence of a polysaccharide capsule.

The typeable strains, which have this capsule, are classified

into six serotypes (designated a to f) based on their ability to react with antisera against recognizedpolysaccharide capsules.5,7,8

The type b form of H. influenzae (designated as Hib) is the most prominent

typeable form; its capsuler polysaccharides, polyribosylribitol phosphate (PRP), is considered to be the mostvirulent form of the bacterium.9

Invasive disease caused by Hib mainly affects infants and children, causing

potentially life-threatening conditions such as meningitis, epiglottitis, and severe sepsis.9,10
Non-typeable H. influenzae (NTHi) lacks the capsule gene and thus does not express the polysaccharide

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capsule.

NTHi causes upper and lower respiratory-tract infections and is a significant cause of AOM inchildren.

NTHi exacerbates other respiratory diseases and can persist chronically in patients with Chronic

Obstructive Pulmonary Disease (COPD).5

H. influenzae infection is a life-threating risk factor associated withgeneral anesthesia and pre- or post-operative COPD.

COPD is a major health problem; it is the fourth leading

cause of mortality worldwide and the third leading cause in Japan.11

By virtue of anatomical positioning, theupper respiratory tract flora and the oral microbiota exist in a very short transitional range. The oral microbiota
are among the most complex of the human body, with more than 700 bacterial species having been detected in
the oral cavity.12,13

A number of research reports have shown that strains of H. influenzae have been detected in
14,15

saliva and oral flora.

Use of probiotics is a potential preventive strategy for lowering the mortality rate caused by COPD or otherrespiratory infectious diseases in Japan. Probiotics are microorganisms that have been claimed to provide healthbenefits when consumed.16,17

The 2001 World Health Organization definition of probiotics is “livemicro-organisms which, when administered in adequate amounts, confer a health beneﬁt on the host”.

Morespecifically, they are believed to modify the flora in the human body by replacing harmful microbes with usefulmicrobes.

Based on that concept, efforts have been made to identify oral probiotic bacteria.18,19

In a recently

published article, it was suggested that at least seven new isolated lactic acid bacterial strains show promisingproperties for use as probiotics, alone or as a part of a probiotic formula, to improve oral health.20

Similarly,

isolates from healthy adults are being preserved and tested in some laboratories in Japan, including ours.

These

include Lactobacillus crispatus YIT 12319 (L.c1), Lactobacillus crispatus LBS 17-11 (L.c2), Lactobacillus
fermentum YIT 12320 (L.f), Lactobacillus gasseri YIT 12321 (L.g), and Streptococcus mitis YIT 12322 (S.m)
as potential candidate probiotic bacteria for the human oral cavity.
However, the effects of these five bacteria on H. influenzae remain to be explored.

Therefore, this study

was designed to examine the growth inhibitory and bactericidal effects of L.c1, which was selected as a leadingstrain from the five candidate probiotic bacteria based on activity against H. influenzae ATCC 9795 (a Hib
laboratory strain) and H. influenzae GTC 15014 (an NTHi laboratory strain).

Materials and Methods
Bacteria and growth conditions
The five candidate oral probiotic bacteria (L.c1, L.c2, L.f, L.g, and S.m) are clinical isolates from the
Department of Translational Research, Tsurumi University School of Dental Medicine.

All Lactobacillus

species were grown in Lactobacilli MRS Broth (Bacto, Becton, Dickinson and Company, Sparks, MD, USA)and S.m was grown in Tryptic Soy (TS) broth medium (Bacto).

All bacteria were grown under anaerobic

conditions of 80% N2, 10% CO2, and 10% H2 at 37˚C for 24-48 hours.

The cultures were then centrifuged at

1,710 × g for 20 minutes to collect cell free culture supernatants (CFCSs) to evaluate antibacterial activities.
H. influenzae ATCC 9795 was purchased from American Type Culture Collection (Manassas, VA, USA)and H. influenzae GTC 15014 was purchased from GTC Collection (Gifu University Graduate School of
Medicine, Gifu, Japan).

Both strains of H. influenzae were anaerobically incubated in Muller-Hinton broth

medium (MH; Bact) supplemented with 150 µL/mL of NAD+ (Oriental Yeast Co., Ltd., Tokyo, Japan) (culture
medium named as MHN) at 37˚C for 24 hours.

The cell suspension was prepared with PBS after centrifuging

the culture medium at 1,710 × g for 20 minutes.

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Competition assay
Competition assays were performed to observe the growth suppression effects of five candidate oral
probiotic bacteria (L.c1, L.c2, L.f, L.g, and S.m) on two strains of H. influenzae (ATCC 9795 and GTC 15014)
using agar plates containing a mixture of MRS and MHN mediums at a ratio of 1:1.

Each pair of competitive

species were inoculated on the plate at the same time in close proximity (5 mm or 6 mm apart) using 5 µL ofovernight cultures after adjusting the concentrations to an OD540=0.2, as mentioned above.

The plates wereincubated at 37˚C anaerobically for 24 hours before bacterial growth was inspected and scored.
Radial diffusion assay (RDA)
A series of tests were performed by employing an RDA21 to investigate the antimicrobial activities of thefive candidate probiotic oral bacteria (L.c1, L.c2, L.f, L.g, and S.m) on the growth of two strains of H. influenzae
using two layers of agarose gels.
For the underlay gel, an agarose solution was prepared containing 210 mg MH, 1.5% agarose (UltraPur
Agarose and Reagents, Invitrogen, Carlsbad, CA, USA) supplemented with NAD+, and 100 mL of distilledwater (DW) in a conical flask, which was then boiled in a water bath for 5 minutes.
then transferred to 15 mL culture tubes (10 mL each aliquot) and autoclaved.

The agarose solution was

After removal from the autoclave,

the tubes were placed into a water bath set at 50˚C to allow the agarose solution to cool to 50˚C.
strains were grown in 4 mL MH broth overnight (as described above).

H. influenzae

Then, 300 µL of MH broth containing

6

approximately 6.1×10 bacterial cells were added to each 15 mL culture tube containing 10 mL aliquot of
agarose solution.

After dispersing the micro-organisms by gentle mixing, the mixture was poured into a 10 cm

diameter sterile Petri dish and allowed to solidify inside a clean-bench.

A series of wells (2 mm in diameter)

was punched in the agar, and 5 µL of each test solution was pipetted into a designated well.

A positive control

well containing tetracycline (TC; 16 µg/mL) and two negative control wells containing only sterile phosphatebuffered saline (PBS) and MRS broth were included on each plate.

The plates were kept inside the clean-bench

for 15 minutes at room temperature to allow the experimental solutions to diffuse.

For the overlay gel thatprovides nutrients for microbial growth, an agarose solution was prepared as described above, except that it only
contained MH (420 mg in 10 mL DW) and 1.5% agarose (150 mg) supplemented with NAD+.
was then poured over the base and allowed to solidify inside a clean bench.

This solution

The plates were then turned overand incubated under anaerobic conditions (80% N2, 10% CO2, and 10% H2) at 37˚C for 24 hours, the diametersof the clear (bacterial growth inhibition) zones around the wells were measured with a slide caliper.22,23
At this stage, as L.c1 appeared to have stronger effects, further experiments were performed that focused only
on this bacterial strain.

Culture media samples containing the bacteria were collected at five different growthstages (0, 6, 12, 24, and 48 hours), the OD at 540 nm was measured immediately, the samples were centrifuged
to collect the CFCSs, the pH was measured, and aliquots were prepared.

We then utilized aliquot filter devices(Nippon Genesis Co., Ltd., Tokyo, Japan) with molecular cutoffs of 3K, 5K, and 10K Daltons.

As molecules

below the molecular cutoff pass through the filter, CFCSs containing molecular compounds of sizes less than 3K,
5K, and 10K Daltons were collected separately and used for RDA.
In addition, one set of aliquots was incubated with carboxypeptidase Y (CPY; Oriental Yeast Co., Ltd.,Tokyo, Japan) at 37˚C for 30 minutes, with the goal of hydrolyzing any available proteins or peptides.
resulting products were used for RDA as described above.

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Bactericidal effect analyzed by Colony Forming Units
One-milliliter aliquots of the H. influenzae ATCC 9795 cell suspensions were centrifuged (1,470 × g 15
minutes 4˚C), supernatants were discarded, and bacterial pellets were suspended in 1 mL of the L.c1 CFCSs bygentle vortexing.

Immediately after 5 minutes incubation, suspensions were centrifuged, the supernatants

discarded, and cell pellets were suspended and held in 1 mL of chilled PBS to be analyzed for viability, as
described previously.24
before plating.

Each bacterial cell suspension (500 µL) was serially diluted to a 100 folds final dilution

Treated and control samples were plated in petri dishes containing MHN agar medium (Bacto)

with a spiral plating instrument (Eddy Jet, IUL, Barcelona, Spain). After 48 hours incubation under anaerobic
conditions at 37˚C, the number of CFUs was counted.
Analysis of the organic acids in CFCS
As the unprocessed CFCS of L.c1 consistently showed the strongest growth inhibition effect on both strainsof H. influenzae, with the pH falling below 5 before 12 hours and to 4 after 24 hours, it was selected for further
analysis.

Acids were extracted from the L.c1 CFCS collected at the five timepoints mentioned above and wereused as separate samples.
kept at 4˚C to settle.

Briefly, each 0.9 mL sample was added to 0.1 mL of perchloric acid, mixed, andAfter 4 hours, the mixture was filtered and centrifuged (20,600 × g, 5 minutes) and thesolution was collected after passing through a 0.45 µm membrane filter.

Five organic acids were separated

from each sample using a column of a Waters 2690 Separations Module HPLC system and the electric
conductivity of each organic acid was measured using a Waters 432 Conductive Detector (Waters Corp., Milford,
MA, USA).

Results
Competition assay
Results of the competition test are shown in Table 1 and Fig. 1.

Only L.c1 could suppress growth of bothstrains of H. influenzae (Hib and NTHi types) at equal score levels, with all test scores showing ++.

Among thefive candidate probiotic bacteria, L.c1 demonstrated the strongest effects on H. influenzae strains.

For S.m,mild growth inhibition effect was observed against H. influenzae ATCC 9795 only (score: +).

Fig. 1

Fig. 2

Fig. 1 Growth inhibition score of competition assay
Fig. 2 Photographs represent the results of radial diffusion assays (n=3 for each experiment). The CFCSs of five candidate
oral probiotic bacteria formed clear zones (inhibition zones) indicating the antibacterial activities against two strains of H.
influenzae. NC1= negative control; PBS and NC2= negative control; medium only. PC= positive control; tetracycline.

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Asian Pac J Dent 2015; 15: 3-11

Growth inhibition of H. influenzae observed on RDA
Similar results were observed on RDA: the CFCS of L.c1 produced the largest inhibition of growth(antibacterial activity) on both strains of H. influenzae compared to the other four species, as shown in Table 2
and Fig. 2.
zones.

Although L.g was a close second, followed by L.f, S.m was not able to produce any detectable clear

On the other hand, L.c2 showed a greater effect on H. influenzae GTC 15014, but a smaller effect on H.

influenzae ATCC 9795.

Interestingly, the unprocessed CFCS of L.c1 produced the largest clear zone in all

experiments (Table 3), while the neutralized CFCS or 80% saturated (NH4)2SO4 precipitate did not produce any
detectable clear zones.

The unprocessed CFCS of L.c1 showed a gradual fall in pH, which decreased to below

5 before 12 hours, reached 4 at 24 hours, and further decreased to 3.8 at 48 hours, maintaining a proportional
relationship with the growth of bacteria (Fig. 3).

It was also found that the CFCSs with molecules less than 3kD (low molecular mass compounds) were most effective compared to the CFCSs containing larger molecules(Table 3).

Moreover, CPY-treated L.c1 CFCS demonstrated the same antibacterial activity on both strains of H.

influenzae based on the size of the clear zones in RDA.

The production of low molecular weight acids appears

to correlate with the largest inhibition zones on the agar plates.

Actually, lactic acid (0.1-1 M) and acetic acid

(0.1-1 M) as well as other organic acids showed antibacterial activity against both strains of H. influenzae.
Table 1

Growth competition assay between five candidate oral probiotic bacteria and two strains of H. influenzae

L. crispatus YIT
L. crispatus LBS
L. fermentum YIT
L. gasseri YIT
12319
17-11
12320
12321
H. influenzae(L.c1)(L.c2)(L.f)(L. g)
ATCC 9795
++
++
++
++
GTC 15014++++
Growth inhibition score of competition assay is based on the score expressed in Fig. 1 (n=3 for each assay).
Strains ofTable 2

S. mitis YIT
12322
(S.m)
+
-

Antibacterial activity of culture supernatants of oral probiotic bacteria against two strains of H. influenzae
Culture supernatant of

Antibacterial activity (diameter of clear-zone expressed in ‘mm’) against
H. influenzae ATCC 9795

H. influenzae GTC 15014

L. crispatus YIT 12319 (L.c1)

4.33±0.10

4.56±0.17

L. crispatus LBS 17-11 (L.c2)

3.86±0.25

4.28±0.08

L. fermentum

3.04±0.16

2.91±0.05

L. gasseri YIT 12321 (L.g)

4.04±0.22

4.17±0.13

S. mitis YIT 12322 (S.m)

n.d.

Table 3

YIT 12320 (L.f)

n.d.
n=3 for each experiment, n.d.: Not detected

Antibacterial activity of L. crispatus YIT 12319 (L.c1) CFCS samples against H. influenzae
Samples

Unprocessed CFCS
Neutralized culture supernatant
80 % saturated (NH4)2SO4 precipitation
Unprocessed CFCS after
3 kDa ultrafiltration
5 kDa ultrafiltration
10 kDa ultrafiltration
CFCS treated with CPY
CFCS treated with CPY after 3 kDa
ultrafiltration
PBS (NC1)
Culture medium only (NC2)
Tetracycline (PC)

Antibacterial activity (diameter of clear-zone expressed in mm) against
H. influenzae ATCC 9795
H. influenzae GTC 15014
4.33±0.10
4.56±0.17
n.d.
n.d.
n.d.
n.d.
3.83±0.07
3.51±0.05
3.09±0.11
4.07±0.25

4.28±0.09
4.03±0.31
3.89±0.16
4.18±0.13

3.82±0.54

3.82±0.18

n.d.
n.d.
10.04±0.15

n.d.
n.d.
9.79±0.10

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Asian Pac J Dent 2015; 15: 3-11

Bactericidal effect detected on colony forming units (CFU)
Direct application of the unprocessed L.c1 CFCS to the H. influenzae ATCC 9795 cell suspension caused asignificant reduction in colony formation (Fig. 4).

This effect could not be detected for the other CFCSs,

indicating that a strong bactericidal effect was induced by the unprocessed L.c1 CFCS only.

Fig. 3Fig. 4

Fig. 3 Graph displays the optical density (OD) of the broth medium and pH values of CFCSs collected at different time points (0,6, 12, 24, and 48 hours) from L. crispatus YIT 12319 (L.c1) cultures. The OD value took a pick in between 6 to 12 hours with therapid growth of bacteria which didn’t change significantly after that. The pH curve fell linearly with the time of culture andreached to 3.77 at 48 hours.
Fig. 4 Graph shows the revived colony forming units (per mL) of H. influenzae ATCC 9795 after application of samples for 5
minutes (n=3 for each sample). For unprocessed L.c1 CFCS the revived colony number was remarkably low (only 1.65E+05)
which was more than 1.00E+07 for other samples indicating strong bactericidal effect on H. influenzae ATCC 9795.

Organic acids detected by HPLC
Four organic acids (lactic acid, acetic acid, succinic acid and propionic acid) were revealed to be present in
L.c1 CFCS upon HPLC analysis (Table 4).

In particular, the lactic acid concentration increased linearly withthe time of culture, reaching 118.22 mM at 48 hours, although the original medium (0 hour CFCS) had aconcentration of only 5.35 mM.

Acetic acid and succinic acid also increased in concentration considerably at

48 hours, while the increase in propionic acid concentration was minimal, even at 48 hours.
concentration did not change through the culture period.

Formic acid

As expected, lactic acid and acetic acid constituted

55% and 39% of the total organic acid solution, respectively, suggesting that these two acids play a major role in
the results acquired above.
Table 4

Organic acid concentration of L. crispatus YIT 12319 (L.c1) culture supernatant as analysed by HPLC

CFCSs
collected
at
0 hour
6 hours
12 hours
24 hours
48 hours

Lactic
acid
5.35
12.88
92.54
109.93118.22

Acetic
acid
63.43
66.07
66.78
73.1
82.96

Succinic
acid
0.56
0.65
2.05
5.15
10.86

Organic acid concentration (mM)
Propionic
FormicIsobutyric
acid
acid
acid
1.96
0.87
n.d.
2.01
0.92
n.d.
2.13
0.86
n.d.
2.25
0.87
n.d.
2.42
0.89
n.d.

Butyris
acid
n.d.
n.d.
n.d.
n.d.
n.d.

Isovaleric Valeris
acid
acid
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.; Not detected

Discussion
In this study, the effects of five candidate oral probiotic bacteria originally isolated from human cavities
were investigated for their antibacterial potential against H. influenzae.

All Lactobacillus strains showedreasonably effective antibacterial activities with variable strength: L. crispatus YIT 12319 (L.c1) had the most8

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Asian Pac J Dent 2015; 15: 3-11

promising activity on both strains of H. influenzae (ATCC 9795 and GTC 15014), while L.f and L.g had
somewhat less consistent results.

It seem to be that L.f and L.g also have similar effects on H. influenzae, if notequivalent. L.c2 was not so far behind in terms of activity, but S.m didn’t show any effects on H. influenzae inthis study.
Generally, the resistance of bacteria is due to the outer membrane, which is a constituent of the cellenvelope in all gram-negative bacteria.25

This membrane acts as an efficient permeability barrier against

macromolecules and hydrophobic substances that is largely attributed to the lipopolysaccharide (LPS) layer ofthe outer membrane.

In the present study, CFCSs containing molecules less than 3 kD in size showed greater

antibacterial activity.

That may include low molecular mass compounds; acids, alcohols, CO2, diacetyl,hydrogen peroxide, peptides, amines, bacteriocines, and other metabolites.25,26

It is now known that symbiotic(probiotic) gut microorganisms release of various soluble low molecular weight molecules of different chemical
nature (surface and exogenous proteins, nucleases, serpins, sirtuines, other enzymes, lectins, peptides, amines,
bacteriocines, fatty and amino acids, lactones, furanons, miRNA, NO, etc).27

The mechanism(s) underlying theantibacterial activity of probiotic Lactobacillus strains appears to be multifactorial, including lowering of the pH
and the production of lactic acid and antibacterial compounds such as bacteriocins and nonbacteriocin.25-28

In

this study, organic acids secreted into the MRS broth culture medium by lactic acid bacteria during growthappear to be the most effective components, with lactic acid as the most potent component.

The results of thisstudy clearly indicated that L.c1 produced lactic acid at high concentrations that linearly increased with the timeof culture.

They also suggest that lactic acid was the main component in the L.c1 CFCS that inhibited thegrowth of and killed H. influenzae.
supportive role.

In addition, acetic acid and succinic acid may provide a considerable

Interestingly, S.m produced CFCS with a somewhat higher pH value of 4.7 (data not shown)compared to L.c1 and didn’t display detectable antibacterial effects on either strain of H. influenzae.

This may

be an indication that acids with higher pH value are not as effective in the suppression of growth of H. influenzae,need to be studied further.

On the contrary, the involvement of proteins, peptides, or proteolytic enzymes was

ruled out by treating the CFCS with CPY and heat-treatment (data not shown) which produced the same results
as the untreated CFCS of L.c1. Therefore, the results in the present study suggest that organic acids with low pH(3.8) are responsible for the growth inhibition and killing of H. influenzae.
As lactic acid is thought to be one of important factors to cause dental caries, we examined the cariogenicityof L.c1 by using artificial mouth model system and low cariogenicity of L.c1 would be reported elsewhere.
There are incidences of the development or aggravation of COPD after general anesthesia during medical ordental procedures resulting from pushing the lung microbiome deep into the lung follicles.
only few preventive procedures available to reduce that risk.

At present, there are

As observed in this study, the probiotic candidate

Lactobacilli has the ability to suppress the growth of H. influenzae, and may have similar effects on othergram-negative microbes.

Therefore, they could play a vital role in reducing the risk of COPD by slowly

predominating the flora of the mouth, pharynx, and upper respiratory area, thereby delivering a potential healthbenefit.

It may serve as a supplement with the practice of improving oral hygiene and frequent professional

oral health care that reduce the progression or occurrence of respiratory diseases among high-risk elderly adults
living in nursing homes and especially those in intensive care units.11,20
However, as bacteria in the oral cavity can protect themselves from even high doses of broad-spectrum
antibiotics by forming biofilms,29 the application of probiotics would be milder and could serve as a long-term

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Sogabe-Ashigaki et al.

prophylactic oral health maintenance tool.

Asian Pac J Dent 2015; 15: 3-11

This approach wouldn’t bring about a sudden change in the oral

flora, but could interfere the growth of opportunistically pathogenic bacteria, including H. influenzae, similarly
as other lactobacilli strains that can reduce the virulence of putative opportunistic oral pathogens, and may
provide insights to future therapeutic approaches for the respective diseases.18,19,30

The study furthermore

emphasizes the need for detailed studies on candidate oral probiotic bacteria, including their effectivenessagainst other gram-negative pathogens that inhabit the mouth, thorax, nasopharynx, and upper respiratory
tract.
Acknowledgements
This study was supported in part by Reseach Fund from Yakult Central Institute Tokyo, Japan. We thank staffs of the Department ofDental Anesthesiology and Department of Translational Research, School of Dental Medicine, Tsurumi University for valuable advice andgreat assistance to complete this study.
Conflict of interests
N. Hanada and S. Imai received research fund from Yakult Central Institute (Tokyo, Japan).
potential conflicts of interest with respect to authorship and publication of this article.

All remaining authors declare no

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Correspondence to:
Dr. Susumu Imai
Department of Translational Research, School of Dental Medicine, Tsurumi University
2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
Fax: +81-45-573-2473 E-mail: imai-s@tsurumi-u.ac.jp
Copyright ©2015 by the Asian Pacific Journal of Dentistry.

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Accepted January 28, 2015.
Online ISSN 2185-3487, Print ISSN 2185-3479