Gyo et al.

Int Chin J Dent 2009; 9: 55-60.

Effects of alkali-ion water on single species Streptococcus mutans
biofilm
Mariko Gyo, DDS,a Ayako Okada, DDS, PhD,a,b Masahiro Ono, DDS, PhD,a Junji
Tagami, DDS, PhD,a,c,d and Khairul Matin, BDS, PhDa,da

Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School, Tokyob
Medical and Dental University, Department of Translational Research, School of Dental Medicine,cTsurumi University, Yokohama, Global Center of Excellence Program; International Research Centerdfor Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, and Support
Program for Improving Graduate School Education, Tokyo Medical and Dental University, Tokyo,
Japan
Purpose: The aim of this study was to assess the efficacy of alkali-ion water (AW) on removal of Streptococcus
mutans (S. mutans) single species biofilms from bovine enamel surfaces in vitro.
Materials and Methods: AW solutions were produced by electrolyzing tap water using a water electrolyzing
device. S. mutans biofilms were formed on bovine enamel blocks in an oral biofilm reactor for 8 hours and 12
hours, respectively. AW solutions, AW-H (pH 11.5) and AW-L (pH 10.5), were tested for their efficacy indissolving water insoluble glucans (WIG) from the above biofilms. Sodium hydroxide solution (NaOH pH 13.5)
and mineral water (MW pH 7.5) were also compared as controls. Biofilms were tried to disintegrate employing
two different methods; 1) assisted by a driving force (shaking) which was a simulation of gargling by an adult,
and 2) incubation without applying any driving force to observe biofilm disintegration effect on self-penetrationby the solutions on their sole chemical potentiality.
Results: One-way ANOVA and Tukey’s HSD tests indicated that amount of dissolved glucan was significantly(p<0.05) more in AW-H compared to MW and AW-H yielded significant differences (p<0.05) in all tests with orwithout application of a driving force equivalent to NaOH. AW-L also dissolved more glucan than MW.
Longer incubation period without any driving force dissolved more glucan by AW time dependently.
Conclusion: It was suggested that AW has the efficacy to remove a remarkable amount of biofilms by
disintegrating glucans from the S. mutans artificial biofilms. (Int Chin J Dent 2009; 9: 55-60.)
Key Words: alkali-ion water, cariogenic biofilm, glucan, mutans streptococci, oral biofilm reactor.

Introduction
A major human health issue is dental caries which has been reported as a transmissible infectious disease inwhich mutans streptococci (MS) play major role. Streptococcus mutans (S. mutans), the leading member ofmutans streptococci group, is the bacterial species most frequently implicated in dental caries.1,2 The organism
produces glucosyltransferases (GTFs), which catalyze to synthesize glucans from dietary carbohydrate,
especially sucrose. Glucans are one of central importance in adhesive interactions of S. mutans with the tooth
surface and other oral bacteria, and contribute to the formation of the matrix of the dental plaque biofilms.3
In recent years, several commercially available functional waters are found to be remarkably useful in daily
life and health care systems. One of those functional waters is well known as alkali-ion water (AW) because of
its high alkalinity. It is usually produced by electrolyzing tap water or drinking water using a water electrolyzing
device manufactured for household use. AW is widely used for drinking and cooking in Japan, particularly by
the persons having stomachache for acid secretions. As popularity of using AW increased rapidly without
showing any remarkable health hazard, the Japanese Ministry of Health, Labor and Welfare has officiallyrecognized it’s usefulness in 2005.4 Other than improving the health condition of digestive tract AW foundtastier as a drinking water compared to ordinary tap water.5 However, there might be several other healthsupportive properties of the AW remained unexplored, the high pH level of this functional water may contributein controlling dental caries by removing plaque biofilms similarly as other high pH chemical reagents, like
NaOH.6,7

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Therefore, the aim of this study was to investigate the efficacy of AW in removing S. mutans artificial
biofilms by dissolving the glucans and detaching bacterial cells from tooth surfaces.

Materials and Methods
SolutionsTwo different pH levels of AW (AW-H, pH 11.5 and AW-L, pH 10.5) were produced using a waterelectrolysis device (TK7705, Panasonic Electric Works Co., Ltd., Osaka, Japan). The chemical process thatinduces high concentrations of OH- ion during electrolysis is shown in Fig. 1. Mineral water (MW, Crystal
Geyser, Calistoga, CA, USA) was used as a control liquid with average pH level of 7.5. Sodium hydroxide(NaOH) 0.5 mol/L solution was used as positive control with pH level above 13.5.

Fig. 1.
Diagram represents the essential components of
the water electrolysis device and the process ofelectro-chemistry to produce AW. Two electrodes
were separated by a membrane in a water tank.
With the initiation of water electrolysis+(H2O→2H +1/2O2+2e ) at the anode electrodeacid water is produced. On reverse side (cathodeside) AW is produced as water molecules splits
and large quantity of OH ions are superadded in
the water like; 2H2O+2e →2OH +H2.

Bovine enamel preparation as biofilm substrate
Square-shaped bovine enamel blocks (approximately 4.0x4.0x1.5 mm) were prepared as substrates to growbiofilms. The surface of each block was then ground flat using silicon-carbide (SiC) paper and uniformly
polished with diamond pastes down to 3 µm.
Biofilms formation
The efficacy of AW on biofilm detachment was investigated using S. mutans MT8148 (S. mutans) a laboratory
strain of cariogenic bacteria.8 A suspension of S. mutans in phosphate buffered saline (PBS) at OD500=3.0 was
prepared from a 16-hour fresh culture in brain heart infusion broth (BHI, Becton Dickinson, Sparks, MD, USA).
The S. mutans biofilms were formed on the bovine enamel block surfaces assembled inside an oral biofilm
reactor (OBR) chambers for 8 hours and 12 hours separately as described previously.6,9 In brief, eight coupons
were placed on the same chamber-holder around a pH electrode and salivary pellicle was allowed to form using
pooled sterile human saliva. Bacterial suspensions and other solutions were persistently pumped into eachchamber so that a water-dome is formed on the specimen holder. The twin chambers of the OBR were operated
simultaneously and recorded the pH continuously.
Disintegrating biofilms and measuring the dissolved glucan
1) Assisted by a driving force (shaking)
Each enamel block with artificial biofilms (of both 8-hour and 12-hour experiments) was removed from theholder and incubated in 1 mL of cool PBS. Loosely attached biofilms were removed by shaking at 5 Hz for 3

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Int Chin J Dent 2009; 9: 55-60.

minutes using a TissueLyser (Qiagen/ Retsch, Hilden, Germany). Enamel blocks were then transferred carefully
from the PBS to 1 mL of each liquid solution in separate microtubes (n=4 for each liquid). Subsequently, they
were shaken three times with the TissueLyser at 10 Hz for 20 s with an interval of 1 minute and were transferredcarefully from each liquid solution to 1 mL of 0.5 mol/L NaOH solution in order to separate water insoluble
glucan (WIG) from the bacteria in the biofilm which was considered as the retained biofilms after shaking.
Each experimental solution containing detached biofilms was collected and centrifuged at 2,300 G for 10
minutes in order to separate the dissolved WIG from the bacteria embedded in the biofilms. The amount ofdissolved WIG was analyzed by using the phenol-H2SO4 method and finally measured in a Biotrak II Plate
reader (Biochrom, Cambridge, UK). The sum of the detached WIG and retained WIG was considered as thetotal amount of WIG for each coupon.
2) Inoculation without shaking
After S. mutans biofilm formation for 8 hours and 12 hours separately on bovine enamel blocks in OBR, theloosely attached biofilms were removed. Enamel blocks were then separately inoculated in NaOH, AW-H,
AW-L and MW for 10 minutes, 2 hours, 5 hours, and 15 hours. Each experimental solution containing detachedbiofilms was collected and the dissolved glucan was measured separately in the same manner as describedabove.
Statistics
Data were analyzed by ANOVA, Tukey’s HSD test (p<0.05) and Dunett T3 test (p<0.05). The assumption of
sphericity has been met by Mauchly's test of sphericity in case of the infiltration test. In each group four samples
were used (n=4) and all experiments were repeated three times to assure reproducibility.

Results

Fig. 2aFig. 2b

The amounts of glucan after treatment with the liquid solvents (NaOH, AW-H, AW-L, MW), each value is the mean
± SD; (n = 4); (a) when a driving force was applied on 8-hour biofilms, (b) when a driving force was applied on
212-hour biofilms. The data for the amounts of glucan (µg/mL/mm ) was analyzed by one-way analysis of variance(ANOVA), Tukey’s HSD test and Dunnett’s T3 test at 95%. Horizontal lines indicate no significant differences(p>0.05), otherwise found significantly different.

The amount of dissolved glucan in S. mutans biofilms
1) Assisted by a driving force
8-hour
The amount of dissolved glucan detected significantly more in AW-H compared to MW and the amount of57

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Int Chin J Dent 2009; 9: 55-60.

retained glucan was significantly less in AW-H compared to MW as shown in the graph (Fig. 2a). However,there were no significant differences between MW and AW-L. As indicated, NaOH dissolved most of theglucan; 87.7% of total glucan; in comparison AW-H, AW-L, and MW dissolved 69.8%, 45.0%, and 32.9%,
respectively.
12-hour
The amounts of dissolved glucan when biofilm were formed for 12 hours on the surface of the bovine enamel
are shown in Fig. 2b. Almost the same results were recognized for 12 hours in glucan detachment relative to 8
hours of biofilm growth. Here also, NaOH dissolved most of the glucan (85.2% of total), compared to AW-H
(42.2%), AW-L (18.5%), MW (16.5%). However, the percentages of dissolved glucan from 12-hour biofilms
were less than from 8-hour biofilms in all four liquid solvents.
2) Incubation without shaking
8-hour
The percentages of dissolved glucan are shown when the enamel blocks with biofilms were incubated in liquidsolvents for 10 minutes, 2 hours, 5 hours, and 15 hours (Fig. 3a). The graph indicates that glucan was dissolved
gradually in all liquid solvents and the amounts of glucan were significantly (p<0.05) more in AW-H with pH
11.5 compared to MW at each time point. AW-L, at pH 10.5, also dissolved more glucan than dissolved byMW.
12-hour
The percentages of dissolved glucan when the enamel blocks with 12-hour biofilms were incubated in liquidsolvents for 10 minutes, 2 hours, 5 hours, and 15 hours are shown in Fig. 3b. The data indicate almost a similar
relationship between the solutions in dissolving glucan as for 8 hours. AW-H dissolved significantly (p<0.05)
more glucans compared to MW, but at 15 hours time point only. Although the gaps between NaOH and AWs (Hand L) remained wide at all time points AWs evidently dissolved more glucan than MW.
In both 8-hour and 12-hour biofilms increased amounts of glucan could be measured with the increase of
incubation time in all solvents, but the difference between AW-H and MW widened further.

Fig. 3aFig. 3bFig. 3a. The amounts of dissolved glucan when the 8-hour biofilm was treated without assistance of any driving
force.
Fig. 3b. The amounts of dissolved glucan when the 12-hour biofilm was treated without assistance of any driving
force.
The data for the amounts of glucan (data expressed as % of total dissolved glucan) was analyzed by one-way
analysis of variance (ANOVA), Tukey’s HSD test and Dunnett’s T3 test at 95%. Brackets indicate no significant
differences (p>0.05), otherwise found significantly different.

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Discussion
In the present study, the efficacy of AW on S. mutans biofilm detachment was evaluated using an OBR. The
result indicated that AW with pH level above 11.0 found significantly effective in disintegrating the glucan fromthe S. mutans biofilms. It was also understood that AW with pH level 10.5 effective in removing biofilms up tocertain amount.
In any type of biofilm, whether in oral, natural or industrial environment, water and aqueous solutes represent
most volume of the matrix. The dry material present is composed of a mixture of exopolysaccharides, proteins,
salts and cell materials. Bacterial biofilms associated with surfaces are complex three-dimensional structures
where bacteria are embedded in a matrix chiefly composed of extracellular polymeric substances (EPS).10,11
S. mutans promotes tooth decay through the ability to adhere and accumulate in large numbers on the tooth
surface, as well as produce and tolerate large amounts of acid. Biofilm formation occurs through the synthesis ofwater-soluble and water-insoluble glucans via the catalyzing activity of the glucosyltransferase enzymes(GTF).12-14 Glucan is believed to be the major factor contributing to the ability of S. mutans to adhere to thetooth surface and for aggregation of the bacterial cells within a biofilm. The ability of mutans streptococcal GTF
to synthesize WIG from sucrose is an important virulence factor in initial caries development, potentially byincreasing the colonization ability of infecting S. mutans and by influencing the diffusion characteristics ofdental plaque.15,16 Since synthesis of WIG is one of the most important virulence properties of S. mutans that
contributes to the development of mature biofilm, therefore the effects of AW of WIG disintegration were
examined.
Alkali based compounds are commonly used to remove EPS produced by biofilm bacteria.17 Young et al.18
reported that treatment with NaOH can break H-bonds resulting in a partially opened triple-helix structure. Incase of WIG that are synthesis by S. mutans also dissolved by alkaline solution.19 Freedman et al.20 indicatedthat the water-insoluble, 1 N NaOH-soluble species were α-1,3-linkaged glucans and were cell associated. In
some research reports it has been demonstrated that NaOH usually been used during analysis of glucans,including WIGs synthesized by S. mutans, because the strong alkaline chemical reagent is capable of completely
dissolving all available glucans.19, 21, 22
Shaking the enamel blocks by TissueLyser at a mild stroke of 10 Hz for 20 s three times with an interval of 1
minute each was a simulation of gargling by an adult. Which found to be most effective among several test
conditions investigated so far (all data not shown). Results showed that longer incubation period without any
stimuli or driving force dissolved more glucan, indicating that plenty of OH- ions available in AW might havebroken the glucan structures by slow etching reactions in a stimulation free condition. However, 12-hourglucans were not dissolved as same as 8-hours glucans, perhaps solidification of the biofilms has began by thattime.
AW is used as drinking water that contains a high concentration of OH- ion and it is produced by theelectrolysis of ordinary tap water containing dissolved lactic acid and calcium. The mechanism of how it works
was shown in Fig. 1. The Japanese Ministry of Health and Welfare has certified that alkali-ion electrolyzed
water assists in the alleviation of gastrointestinal disorders, acidosis, chronic diarrhea, and poor digestion.23,24
In the present study, biofilm formed with S. mutans, the results were almost same and the amounts of detachedglucan were increased with a rise of pH. Furthermore, the amounts of bacteria (data not shown) were also
showed the same tendency indicating bacteria in the biofilm were also detached from the enamel surface withglucan. Based on these results, it can be considered that the biofilm with pathogenic bacteria which produce
glucan may be effectively detached by AW from enamel surfaces.

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Therefore, it was suggested that glucans produced by S. mutans MT8148 was dissolved by the electrolyzed
drinkable AW. In addition, it is expected that a similar effect may be acquired if AW is used intra-orally andwould prevent plaque-biofilm accumulation on tooth surfaces on regular gargling with AW. Further studies
from different viewpoints would help in understanding the unexplored potentials of this functional water,
especially on caries prevention.
Acknowledgment
This work was supported by the grant from the Japanese Ministry of Education, Global Center of Excellence (GCOE) Program,International Research Center for Molecular Science in Tooth and Bone Diseases.

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Correspondence to:Dr. Khairul Matin
Cariology and Operative Dentistry, Dept of Restorative Sciences,
Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University
1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
Fax: +81-3-5803-0195 E-mail: matin.ope@tmd.ac.jp
Accepted November 25, 2009.

Copyright ©2009 by the International Chinese Journal of Dentistry.

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