November 2016⎪Vol. 26⎪No. 11

J. Microbiol. Biotechnol. (2016), 26(11), 1829–1835http://dx.doi.org/10.4014/jmb.1604.04008 Research Article jmbReview

Inhibitory Effect of Lactococcus lactis HY 449 on Cariogenic BiofilmYoung-Jae Kim1 and Sung-Hoon Lee2*

1Department of Pediatric Dentistry, School of Dentistry, Seoul National University, Seoul 03080, Republic of Korea2Department of Oral Microbiology and Immunology, College of Dentistry, Dankook University, Cheonan 31116, Republic of Korea

Introduction

Streptococcus mutans plays a potent role in the induction

of dental caries. This bacterium strongly produces lactic acid

and tolerates a low acidic environment [10]. Furthermore,

S. mutans produces glucan from sucrose by glucosyltransferases

such as GtfB, GtfC, and GtfD [9, 14] and uses sucrose as a

substrate for growth [8]. Oral biofilm consists of oral

bacteria, glucan, and debris. Among these components,

glucan is a key contributor to the development of biofilm

via formation of a thick barrier [11]. The biofilm of a

healthy person maintains a balanced composition of

bacterial species. However, when the conditions of the oral

biofilm are changed by a sugar-rich diet, low pH, and low

saliva flow, the proportion of S. mutans in the oral biofilm

increases compared with other streptococci [12]. Moreover,

continuous production of glucan and acid by S. mutans

reduces the pH level and leads to formation of mature

biofilm that ultimately induces dental caries. Therefore,

the glucosyltransferases (Gtfs) and acid production are

virulence factors of S. mutans. Cariogenic biofilm as oral

biofilm including S. mutans is considered to be a greater

risk factor for induction of dental caries than planktonic

S. mutans.

Probiotics have been widely used in the food industry

and in dairy products because of their beneficial effects.

These microorganisms have antibacterial activity against

pathogenic bacteria through production of bacteriocins [5].

Furthermore, probiotics slightly stimulate the immune

systems of hosts and inhibit the toxin activity of pathogenic

Received: April 5, 2016

Revised: June 4, 2016

Accepted: July 13, 2016

First published online

July 19, 2016

*Corresponding author

Phone: +82-41-550-1867;

Fax: +82-41-550-1859;

E-mail: dennisyi@dankook.ac.kr

pISSN 1017-7825, eISSN 1738-8872

Copyright© 2016 by

The Korean Society for Microbiology

and Biotechnology

Dental caries is caused by cariogenic biofilm, an oral biofilm including Streptococcus mutans.

Recently, the prevention of dental caries using various probiotics has been attempted.

Lactococcus lactis HY 449 is a probiotic bacterium. The aim of this study was to investigate the

effect of L. lactis HY 449 on cariogenic biofilm and to analyze its inhibitory mechanisms.

Cariogenic biofilm was formed in the presence or absence of L. lactis HY 449 and L. lactis

ATCC 19435, and analyzed with a confocal laser microscope. The formation of cariogenic

biofilm was reduced in cultures spiked with both L. lactis strains, and L. lactis HY 449 exhibited

more inhibitory effects than L. lactis ATCC 19435. In order to analyze and to compare the

inhibitory mechanisms, the antibacterial activity of the spent culture medium from both

L. lactis strains against S. mutans was investigated, and the expression of glucosyltransferases

(gtfs) of S. mutans was then analyzed by real-time RT-PCR. In addition, the sucrose

fermentation ability of both L. lactis strains was examined. Both L. lactis strains showed

antibacterial activity and inhibited the expression of gtfs, and the difference between both

strains did not show. In the case of sucrose-fermenting ability, L. lactis HY 449 fermented

sucrose but L. lactis ATCC 19435 did not. L. lactis HY 449 inhibited the uptake of sucrose and

the gtfs expression of S. mutans, whereby the development of cariogenic biofilm may be

inhibited. In conclusion, L. lactis HY 449 may be a useful probiotic for the prevention of dental

caries.

Keywords: Lactococcus lactis, probiotics, Streptococcus mutans, cariogenic biofilm, antibiofilm

effects

1830 Kim and Lee

J. Microbiol. Biotechnol.

bacteria [3, 17, 24]. Most studies of probiotics have been

focused on the bacterial ecology of the gut and gut immune

systems. Recently, probiotics have been investigated for

their effects on oral health. Lactobacillus rhamnosus GG

inhibits the growth and bioactivity of oral pathogens via

the production of small-molecular antimicrobial substances,

whereby it reduces the risks of dental caries and periodontitis

[13, 16, 21]. Moreover, L. reuteri and Bifidobacterium animalis

reduced the S. mutans amount in saliva [2, 20]. However,

the prevention of dental caries by these probiotics remains

controversial because they are aciduric bacteria, like

S. mutans [18]. Lactococcus lactis is a probiotic that has

antibacterial activity. It produces various bacteriocins such

as nisin and lactococcin, and diacetin [1, 7]. For these

reasons, L. lactis is widely used in fermented foods and

dairy products. Studies of L. lactis in oral health have

examined the inhibition of planktonic S. mutans growth

and mucosal immune stimulation [22, 23]. L. lactis HY 449

was isolated from contaminated milk products and

contained the same fatty acid profiles as L. lactis ATCC

19435 as a type strain. Moreover, their characteristics are to

ferment sucrose and salicine, unlike L. lactis ATCC 19435

[6]. The growth of L. lactis is inhibited at below pH 5 [4, 19].

Therefore, the current study investigated the effect of

L. lactis HY 449 on cariogenic biofilm and analyzed the

inhibitory mechanism of L. lactis HY 449 on the formation

of cariogenic biofilm.

Materials and Methods

Bacterial Strains and Culture Conditions

L. lactis HY449 was donated from Korea Yakult (Korea Yakult,

Korea), and L. lactis ATCC 19435 and S. mutans ATCC 25175 were

purchased from American Type Culture Collection (ATCC).

L. lactis ATCC 19435 and HY 449 were cultured in brain heart

infusion (BHI) broth (BD Bioscience, USA) at 37oC. S. mutans ATCC

25175 was cultivated in BHI broth including 1% sucrose at 37oC.

Preparation of Conditioning Plate for Biofilm Formation

Pooled saliva of six healthy donors was centrifuged at 7,000 ×g

for 10 min at 4oC. The supernatant was filtered through a

polyvinylidene fluoride (PVDF) membrane and diluted to 2-fold

with phosphate-buffered saline (PBS, pH 7.2). The prepared saliva

was added to wells on a 12-well polystyrene plate. The 12-well

plate was dried at 40oC in a drying oven and sterilized in an UV

sterilizer. These procedures were repeated three times.

Inhibitory Effect of L. lactis on Cariogenic Biofilm

Cariogenic biofilm was formed according to the method

described by Lee and Kim [9]. Unstimulated saliva was collected

from 10 healthy donors and pooled in equal proportions. The

pooled saliva was mixed with BHI broth containing 1% sucrose

and 1% mannose and centrifuged at 2,000 ×g for 10 min at 4oC to

remove debris. The supernatant was transferred into new tubes,

and S. mutans (1 × 106 cells) was added to form the cariogenic

biofilm. L. lactis HY 449 and L. lactis ATCC 19435 (5 × 105 and 1 ×

106 cells) were inoculated into each tube containing salivary

bacteria. The preparation was vortexed for 10 sec, and 1 ml or

400 µl of the mixtures was then inoculated into a conditioned 12-

well plate or 8-well glass chamber, respectively. The plates were

incubated at 37oC for 72 h, and the media were changed daily by

replacing with fresh BHI containing 1% sucrose and 1% mannose.

The biofilm-formed 12-well plate was washed three times with

PBS to remove planktonic bacteria, and the biofilm was disrupted

mechanically with a scraper. The suspensions were diluted

serially and plated on BHI agar plate and mitis-salivarius

bacitracin (MSB) agar plates to count total bacteria and S. mutans

in the biofilm, respectively. For the analysis of biofilm formation,

an 8-well glass chamber was washed three times with PBS and

stained with SYTO 9 dye (Invitrogen, USA) according to the

manufacturer’s instructions. The biofilm was visualized by a

confocal laser scanning microscope (Carl-Zeiss, Germany) using

z-stack scans from 0 to 30 µm.

Comparison of Antibacterial Activity of L. lactis against S. mutans

In order to analyze the inhibitory mechanism of L. lactis HY 449

on formation of cariogenic biofilm, a susceptibility assay of

S. mutans for the spent culture medium of L. lactis was performed

according to the methods recommended by the Clinical and

Laboratory Standards Institute (CLSI) [15]. The spent culture

medium of L. lactis HY 449 and L. lactis ATCC 19435 was collected

by centrifugation at 7,000 ×g for 10 min at 4oC and filtered

through a PVDF membrane with a pore size of 0.22 µm. The spent

culture medium was dispensed from 20 to 180 µl into the column

wells of a 96-well polystyrene plate (SPL Life Sciences, Korea),

and fresh BHI broth was added to bring the final volume up to

180 µl in the dispensed well of the spent culture medium.

S. mutans was counted in a Petroff-Hasser bacteria counting

chamber (Hausser Scientific, USA) and diluted to 1 × 106 cells/ml

with BHI broth. The diluted suspension of S. mutans (20 µl) was

inoculated into each well. The plate was incubated at 37oC for

36 h, and the optical density was measured at 600 nm by a

spectrophotometer.

Real-Time RT-PCR for the Analysis of Glucan Expression of

S. mutans

Glucan production by S. mutans was indirectly analyzed by

comparison of the expression levels of gtfs. S. mutans was cultured

in BHI including 1% sucrose with or without the spent medium of

both L. lactis at a non-killing concentration (20%) for S. mutans for

6 h. Total RNA was extracted with a TRIzol Max bacterial RNA

isolation kit (Invitrogen Life Tech, USA) according to the

L. lactis HY 449 as a Probiotic for Dental Caries 1831

November 2016⎪Vol. 26⎪No. 11

manufacturer’s instruction. cDNA was synthesized by Maxime

RT Premix (random primer; iNtRON, Korea) in a 20 µl reaction

volume, and the mixture was incubated at 45oC for 1 h. cDNA was

mixed with 10 µl of SYBR Premix Ex Taq and 0.4 µM of each

primer pair in a 50 µl final volume and was then subjected to 40

PCR cycles (94oC for 15 sec, 60oC for 10 sec, and 72oC for 33 sec) by

ABI 7500 real-time PCR (Applied Biosystems, USA). 16S rRNA,

which is a housekeeping gene, was used as a reference to

normalize the expression levels and to quantify changes in

expression levels of gtfs between cultures that were untreated and

treated with the spent culture medium. The sequences of primers

for real-time RT-PCR were as follows : 5’-AGC AAT GCA GCC

AAT CTA CAA AT-3’ and 5’-ACG AAC TTT GCC GTT ATT GTC

A-3’ for the gtfB gene; 5’-CTC AAC CAA CCG CCA CTG TT-3’

and 5’-GGT TAA CGT CAA AAT TAG CTG TAT TAG C-3’ for the

gtfC gene; 5’-CAC AGG CAA AAG CTG AAT TAA CA-3’ and 5’-

AAT GGC CGC TAA GTC AAC AG-3’ for the gftD gene; and 5’-

GAA AGT CTG GAG TAA AAG GCT A-3’ and 5’-GTT AGC TCC

GGC ACT AAG CC-3’ for the 16S rRNA gene.

Analysis of Sucrose Fermentation by L. lactis

In order to investigate the sucrose-fermenting ability of L. lactis

HY 449 and L. lactis ATCC 19435, the bacteria were harvested by

centrifugation at 4,000 ×g for 10 min at 4oC and washed with fresh

BHI broth. The bacteria were resuspended with fresh BHI broth,

and the concentration was adjusted to 1 × 107 cells/ml by using a

bacterial counting chamber. Ten milliliters of BHI was dispensed

into new conical tubes with or without sucrose and prewarmed at

37oC until inoculation. The bacterial suspension (1 ml) was inoculated

into 15 tubes and the level of pH was measured every hour.

Fig. 1. Inhibition of formation of cariogenic biofilm by L. lactis.

The cariogenic biofilm was formed using salivary bacteria and S. mutans in the presence or absence of L. lactis HY 449 and L. lactis ATCC 19435.

The biofilms were then washed with PBS, stained with SYTO 9, and analyzed by a confocal laser scanning microscope (A-C). To count bacteria in

the biofilm, the biofilm was disrupted by a scraper and resuspended with BHI. After plating on BHI agar plates for total bacteria (D) and MSB

agar plates for S. mutans (E), the agar plates were incubated for 36 and 48 h, respectively. The colonies on the agar plates were then counted. Data

are represented as the mean ± SD from six total experiments. * Statistically significant difference compared with untreated L. lactis (p < 0.001).

1832 Kim and Lee

J. Microbiol. Biotechnol.

Statistical Analysis

Statistically significant differences were analyzed by the

Kruskal-Wallis and Mann-Whitney tests using IBM SPSS Statistics

21 software (IBM, USA). P-values less than 0.05 were considered

statistically significant.

Results

Inhibitory Effect of L. lactis on Formation of Cariogenic

Biofilm

Salivary biofilm containing S. mutans as cariogenic biofilm

is more related to dental caries than S. mutans biofilm.

Therefore, the effect of L. lactis on cariogenic biofilm was

investigated. L. lactis HY 449 and L. lactis ATCC 19435

inhibited formation of cariogenic biofilm (Figs. 1A-1C).

The level of total bacteria in cariogenic biofilm was

decreased when the growth medium was spiked with

L. lactis (Fig. 1D). The level of S. mutans in the biofilm was

also decreased in the presence of L. lactis in a dose-

dependent manner (Fig. 1E). The peculiar point is that the

anti-biofilm activity of L. lactis HY 449 and L. lactis ATCC

19435 was different. L. lactis HY 449 showed more

inhibitory effect on formation of cariogenic biofilm than

did L. lactis ATCC 19435.

Antimicrobial Activity of L. lactis against S. mutans

In order to investigate the difference in the inhibitory

effects, the antibacterial activity of both L. lactis strains was

compared. Although the growth of S. mutans was

significantly decreased in media containing a greater than

40% concentration of the spent culture medium of both

L. lactis strains, there was no difference in the antimicrobial

activity of L. lactis HY 449 and L. lactis ATCC 19435 (Fig. 2).

Reduction of Glucosyltransferase Expression by the Spent

Culture Medium of L. lactis

The formation and development of S. mutans biofilm are

related to the presence of soluble and insoluble glucan, and

glucan is a key contributor to the development of biofilm

[9]. When S. mutans was cultivated in the presence or

absence of the spent culture medium of both L. lactis strains

at a non-killing concentration for S. mutans, the spent

medium of both L. lactis strains was found to reduce

expression of the three gtf genes (Fig. 3). However, there

was no difference in the inhibitory effect of L. lactis HY 449

and L. lactis ATCC 19435.

Sucrose Fermentation Ability of L. lactis

Sucrose is a substrate for synthesis of glucan, and the

Fig. 2. Susceptibility of S. mutans to the spent culture medium

of L. lactis.

S. mutans was cultivated with or without the spent culture medium of

L. lactis HY 449 or L. lactis ATCC 19435 at various concentrations on

96-well polystyrene plates. The growth of S. mutans was measured by

a microplate reader at 600 nm. The experiments were carried out

three times in duplicates, and data are presented as the mean ± SD. *

Statistically significant difference compared with cultures not treated

with the spent culture medium (p < 0.05).

Fig. 3. Effect of the spent culture medium of L. lactis on

expression of glucosyltransferases of S. mutans.

S. mutans was cultivated in the presence or absence of the spent

culture medium of L. lactis HY 449 or L. lactis ATCC 19435 at a non-

killing concentration for S. mutans for 12 h. Total RNA was isolated,

and cDNA was synthesized. The expression levels of gtfB, gtfC, and

gtfD were analyzed by real-time PCR. Data are presented as the

mean ± SD from six total experiments. * Statistically significant

difference compared with cultures not treated with the spent culture

medium (p < 0.001).

L. lactis HY 449 as a Probiotic for Dental Caries 1833

November 2016⎪Vol. 26⎪No. 11

glucan synthesis of S. mutans depends on the concentration

of sucrose [8]. Therefore, the sucrose-fermenting ability of

L. lactis was investigated. The acid production of L. lactis

HY 449 and L. lactis ATCC 19435 with or without sucrose

was compared. The pH level of L. lactis culture media was

determined to be near pH 5.5, even after 5 h in the presence

or absence of sucrose. Interestingly, L. lactis HY 449 was

found to produce a higher level of acid in the presence of

sucrose than in the absence of sucrose, and the pH level of

L. lactis ATCC 19435 culture media was not significantly

different between BHI broth with or without sucrose

(Fig. 4).

Discussion

Dental caries is associated with oral biofilm including

S. mutans. When the proportion of S. mutans in the oral

biofilm increases compared with other streptococci by

changing oral conditions such as sugar-rich diet, low pH,

and low saliva flow, the biofilm is called cariogenic biofilm

and induces caries lesions on the tooth surface. Therefore,

S. mutans is considered to be a greater risk factor for

induction of dental caries than planktonic S. mutans.

L. rhamnosus GG interferes with the growth and bioactivity

of cariogenic bacteria, whereby it may reduce the risks of

dental caries [13, 16]. Moreover, L. reuteri and B. animalis

reduced the S. mutans amount in saliva [2, 20]. However,

the prevention of dental caries by these probiotics remains

controversial because they are aciduric bacteria, like

S. mutans [18]. However, L. lactis is not an aciduric

bacterium because its growth is inhibited at below pH 5 [4,

19]. Therefore, the current study investigated the effects of

L. lactis HY 449 on cariogenic biofilm.

L. lactis significantly inhibited formation of cariogenic

biofilm as well as the growth of total bacteria and S. mutans

in biofilm. This report is the first to investigate the effect of

L. lactis on a cariogenic biofilm model using salivary

bacteria and S. mutans. The peculiar point is that L. lactis

HY 449 strongly inhibited formation of cariogenic biofilm

compared with L. lactis ATCC 19435. Therefore, the

characteristics of both L. lactis strains were compared and

analyzed. When the susceptibility test of S. mutans was

examined by the spent culture medium of L. lactis HY 449

and L. lactis ATCC 19435, the growth of S. mutans was

significantly decreased in media containing a greater than

40% concentration of the spent culture medium of both

L. lactis strains. However, there were no differences of the

antimicrobial activity between L. lactis HY 449 and L. lactis

ATCC 19435. Therefore, the correlation between the

antibacterial activity and inhibitory difference of the

biofilm was excluded.

Next, since glucan plays a key role in the development

and formation of cariogenic biofilm [8], we investigated the

effect of the spent culture medium of both L. lactis strains

on the expression of glucosyltransferases. When S. mutans

was cultivated with the spent culture medium of both

L. lactis strains at a non-killing concentration for S. mutans,

the expression levels of the three glycosyltransferases gtfB,

gtfC, and gtfD were significantly reduced compared with

single-cultured S. mutans. However, this experiment did

not exhibit the difference between L. lactis HY 449 and

L. lactis ATCC 19435 like the antibacterial experiment.

Finally, sucrose as a biofilm formation-related factor was

investigated. The difference between L. lactis HY 449 and

L. lactis ATCC 19435 is in their sucrose-fermenting ability.

L. lactis HY 449 ferments sucrose, unlike L. lactis ATCC

19435 [6]. Both L. lactis strains were cultivated in BHI broth

with or without sucrose, and the pH level of the culture

medium of each bacterium was measured. The pH level of

culture medium of L. lactis HY 449 quickly decreased in the

presence of sucrose compared with in the absence of

sucrose. However, the decrease of pH level of L. lactis

ATCC 19435 culture medium was not different in the two

conditions. From the S. mutans standpoint, L. lactis HY 449

is a sucrose competitor. Kreth et al. [8] reported that when

Fig. 4. pH curve of bacterial growth culture media.

L. lactis HY 449 and L. lactis ATCC 19435 were cultivated in BHI broth

with or without sucrose. Initial pH was measured immediately after

inoculating both bacteria cultures, and the pH level of the culture

medium was measured at hourly intervals. The experiments were

carried out three times in duplicates. * Statistically significant

difference compared with sucrose-free condition (p < 0.05).

1834 Kim and Lee

J. Microbiol. Biotechnol.

S. mutans is with other streptococcal competitors during

oral biofilm formation and uses sucrose for a substrate,

the antimicrobial susceptibility of S. mutans decreases.

Moreover, the glucan synthesis of S. mutans depends on the

concentration of sucrose [8]. Tong et al. [22] showed an

antagonizing effect of L. lactis on S. mutans biofilm through

production of a bacteriocin, nisin. On the basis of these

studies, L. lactis HY 449 consumes sucrose for glucan

synthesis and metabolism, whereby S. mutans may be

inhibited to take up sucrose for glucan synthesis, and the

resistance of oral bacteria in cariogenic biofilm may

decrease for the antimicrobial peptide of L. lactis HY 449.

Recently, various groups have studied to apply probiotics

for oral health, and sought candidate probiotics. However,

most proposed probiotics are controversial because of their

aciduric characteristic. In the current study, the culture

medium of L. lactis was determined to be near pH 5.5, like

oral commensal bacteria. S. mutans is a key contributor to

the development of biofilm via glucan synthesis. L. lactis

HY 449 is a competitor to S. mutans in the uptake of sucrose

and inhibited glucan synthesis by S. mutans. Therefore,

L. lactis HY 449 inhibited the formation of cariogenic

biofilm. Furthermore, the antibacterial activity of L. lactis

HY 449 inhibited the growth of S. mutans in immature

biofilm. Eventually, L. lactis HY 449 might effectively

inhibit the formation of cariogenic biofilm compared with

L. lactis ATCC 19435.

In this study, we showed that L. lactis has an inhibitory

effect on the formation of cariogenic biofilm by antimicrobial

activity, inhibition of expression of Gtfs, and consumption

of sucrose. Moreover, this bacterium is not aciduric. For

these reasons, L. lactis HY 449 may be a potential probiotic

for use in the prevention of dental caries.

Acknowledgments

The present research was supported by a research fund

of Dankook University in 2016.

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