Original Article
Int J Clin Prev Dent 2017;13(1):5-10ㆍhttps://doi.org/10.15236/ijcpd.2017.13.1.5
ISSN (Print) 1738-8546ㆍISSN (Online) 2287-6197
Investigation of Efficacy of New Electrolyzed Water
Generator for Oral Bacteria
In-Woo Cho1, Tae-Kye Nam2, Dong-Heon Baek3
1
Department of Periodontology, College of Dentistry, Dankook University, Cheonan, 2ebioteco. Co., Ltd, Seoul, 3Department of
Oral Microbiology and Immunology, College of Dentistry, Dankook University, Cheonan, Korea
Objective: Electrolyzed water has antimicrobial activity against pathogenic bacteria. However, antimicrobial efficacy of electrolyzed water (EW) generator varies depending on the configuration and for of the electrodes. This study is to investigate antibacterial activity of new electrolyzed water generator against oral bacteria and biofilm.
Methods: The EW was generated with eBio-cleaner, and the EW was treated on bacterial pellets after harvest of four oral
bacteria. The EW treated bacteria were spread on agar plate and incubated at 37°C in anaerobic condition. Next, the salivary biofilm was formed using pooled saliva from the healthy donor. The salivary biofilm was treated with tap water, the EW
and listerine, and the biofilm bacteria were spread on agar plate and were observed confocal laser microscope after
live/dead staining.
Results: The EW generated by eBio-cleaner has bactericidal activity against planktonic Aggregatibacter actinomycetemcomitans,
Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans. Listerine showed the strongest antimicrobial activity against planktonic bacteria. In case of biofilm experiment, the EW generated by eBio-cleaner showed disruptive and antimicrobial effect on the salivary biofilm. Whereas, listerine exhibited antimicrobial effect on the outside of
biofilm.
Conclusion: Take together of the results, the EW generated by eBio-cleaner may be helpful in preventing and treating dental
caries and periodontitis by oral bacteria.
Keywords: electrolyzed water generator, antimicrobial activity, oral biofilm, periodontopathogens, cariogenic bacteria
Introduction
Recently, research on antimicrobial efficacy of electrolyzed
water has increased, and the use of electrolyzed water is increasing in industry [1-4]. Electrolyzed water (EW) is generated by
an electric current that is passed through water using various
metal electrodes. EW generator is divided into two categories
according to separated or non-separated chamber of the anode
and the cathode electrode by membrane [5,6]. EW using
non-separated and separated chamber is used for medical or industrial devices and edible products or human body, respec-
Corresponding author Dong-Heon Baek
Department of Oral Microbiology and Immunology, College
of Dentistry, Dankook University, 119 Dandae-ro, Dongnam-gu,
Cheonan 31116, Korea. Tel: +82-41-550-1997, Fax: +8241-550-1859, E-mail: [email protected]
Received February 25, 2017, Revised March 9, 2017,
Accepted March 9, 2017
Copyright ⓒ 2017. Korean Academy of Preventive Dentistry. All rights reserved.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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International Journal of Clinical Preventive Dentistry
tively. Beneficial effect of EW on human body has reported.
Hydrogen-enriched EW shows antimicrobial activity against
oral bacteria [7] and acts antioxidant effect by reducing oxidative stress [8]. Furthermore, this electrolyzed water inhibits tumor progression and invasion with intracellular oxidant repression [9]. However, Efficacy of EW generator varies depending on the configuration and for of the electrodes [10].
Oral disease by bacteria can be divided into dental caries and
periodontitis. Streptococcus mutans is considered with a important cariogenic bacteria. They produce abundant lactic acid
and glucan by glucose metabolism and glucosyltransferases
(Gtfs), respectively, by which dental caries is induced [11].
Aggregatibacter actinomycetemcomitans (previously Actinobacillus
actinomycetemcomitans) are detected juvenile periodontitis,
and Porphyromonas gingivalis are mainly associated with adult
periodontitis [12]. These two bacteria are gram-negative bacteria and have lipopolysaccharide as endotoxin. Although P.
gingivalis is reported to be most relevant with induction of periodontitis, periodontitis is induced when oral bacteria form biofilm as plaque. Oral biofilm is formed by stepwise. Initial biofilm is formed by Streptococcus species as early colonizer and
developed to mature biofilm by exopolysaccharides that are
produced by Gtfs of S. mutans. Next, periodontal pathogens
bind to initial biofilm through Fusobacterium nucleatum and
form biofilm in the gingival pocket [13,14]. Therefore, oral biofilm is more related to periodontitis, and the bacteria in oral biofilm are more resistant for chemical agents compared to planktonic bacteria because of glucan layer.
The purpose of present study was to investigate antimicrobial
activity of recent developed EW generator for oral bacteria.
Materials and Methods
1. Bacterial species and cultivation
A. actinomycetemcomitans ATCC 43718 and S. mutans
ATCC 25175 were cultivated with brain heart infusion (BHI)
(BD Bioscience, Franklin Lakes, NJ, USA). F. nucleatum
ATCC 25586 and P. gingivalis ATCC 33277 were cultured using BHI supplemented with hemin (1 g/ml) and vitamin K (0.2
g/ml) at 37°C in anaerobic condition (5% H2, 10% CO2, and
85% N2).
2. Generation of hydrogen enriched electrolyzed
water
Tap water (300 ml) was subjected to electrolyze for 3 min
with 24 V of DC using eBio-Cleaner (ebiotech, Seoul) at ambient temperature. The characteristic of this device was a singlecell chamber undivided anode and cathode chamber. eBio-
6 Vol. 13, No. 1, March 2017
cleaner water was used within 10 min of its preparation in all
experiments.
3. Investigation of antimicrobial activity of
eBio-cleaner water against oral bacteria
A. actinomycetemcomitans, F. nucleatum, P. gingivalis, and
S. mutans were cultivated in each specific medium for 36 h, and
the level of the bacteria was counted with a bacterial counting
chamber (Marienfeld, Lauda-Konigshofen, Germany). The
7
bacteria level was adjusted to 1×10 cells/ml with fresh BHI
broth. Each bacterial suspension (1 ml) was harvested by centrifugation at 6,000 ×g for 5 min 4°C and washed with phosphate
buffered saline (PBS, pH7.1). The bacterial pellet was treated
with 1 ml of eBio-cleaner water, Tap water, and ListerineⓇ
(Johnson and Johnson Inc., New Brunswick, NJ, USA) for 1
min, and then sequently added to 1 ml of BHI broth. After the
suspensions were processed serial dilution 10 fold to 105, fifty
microliter of each dilute was spread on BHI agar, Mitis salivarius agar, and Blood agar supplemented with hemin (1 g/ml)
and vitamin K (0.2 g/ml). The agar plates were incubated at
37°C in anaerobic chamber until colony could be counted.
4. Formation and observation of salivary biofilm
Pooled Saliva of 10 healthy donors with non-smoking was
centrifuged to remove debris at 2,000 ×g for 10 min. The supernatant was transferred to a new tube and added fresh BHI broth
(same volume) and 2% sucrose. The prepared saliva was dispensed into 12-well polystyrene plate (SPL Biosciences,
Gyeonggi) and 8-well glass chamber (BD Biosciences,
Bedford, MA, USA), The plate and the glass chamber were incubated 37°C for 72 h in anaerobic chamber. The medium was
changed with BHI broth including 2% sucrose every day. The
salivary biofilm was reacted with 2 ml of tap water, eBio-cleaner
water, and Listerine for 1 min, and the solutions were immediately aspirated by vacuum pump. In case of 12-well plate, the
salivary biofilm was disrupted with a scraper and transferred
into 1.5 ml tube. The tube was vortexed for 1 min, and biofilm
7
suspension was diluted serially 10-fold to 10 with BHI broth.
In case of 8-well glass chamber, the salivary biofilm was washed
with PBS and stained with LIVE/DEAD BacLight kit
(Invitrogen, Eugene, OR, USA). The biofilm was observed
with LSM 700 confocal laser scanning microscope(Carl-Ziss,
Oberkochen, Germany) using Z-stack scan from 0 to 30 m.
Image of the biofilm was visualized by ZEN program from
Carl-Zeiss.
5. Statistical analysis
Significant differences were analyzed by Krusical-Wallis
and Mann-Whitney test using SPSS 23 (SPSS Inc., Chicago, IL,
In-Woo Cho, et al：Evaluation of Electrolyzed Water Generator
USA). p-value less than 0.05 was considered statistically
significant.
Listerine exhibited the strongest antimicrobial activity against
S. mutans (Fig. 1).
Results
2. Antimicrobial activity of eBio-cleaner water
against periodontal pathogens.
1. Antimicrobial activity of eBio-cleaner water
against cariogenic bacteria
S. mutans was selected to examine antimicrobial activity of
eBio-cleaner water against cariogenic bacteria. When S. mutans was treated with tap water, eBio-cleaner water, and
Listerine, eBio-cleaner water showed significantly antimicrobial activity against S. mutans compared to tap water.
When eBio-cleaner water was investigated antibacterial activity against periodontal pathogens, the levels of F. nucleatum,
P. gingivalis, and A. actinomycetemcomitans were reduced by
eBio-cleaner water (Fig. 2). In case of F. nucleatum, they are
sensitive for tap water (Fig. 2A). eBio-cleaner water reduced
the levels of P. gingivalis and A. actinomycetemcomitans by
several hundred-fold compared to tap water.
Figure 1. Antimicrobial activity of the electrolyzed water against Streptococcus mutans. S. mutans was treated with tap water, electrolyzed
water of eBio-cleaner, and listerine and inoculated on agar plate. Colony-forming unit (CFU) of S. mutans was counted after incubation.
,#
* Significant difference compared with control and with tap water, respectively (p＜0.05).
Figure 2. Antimicrobial activity of the electrolyzed water against periodontopathogens. Aggregatibacter actinomycetemcomitans,
Fusobacterium nucleatum, and Porphyromonas gingivalis were treated with tap water, the electrolyzed water of eBio-cleaner, and listerine
,#
and inoculated on agar plate. Colony-forming unit (CFU) of periodontopathogens was counted after incubation. * Significant difference
compared with control and with tap water, respectively (p＜0.05).
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International Journal of Clinical Preventive Dentistry
3. Inhibitory effect of eBio-cleaner water on
salivary biofilm
Finally, we examined effect of eBio-cleaner water on salivary biofilm using pooled saliva of healthy patients. When
eBio-cleaner water was treated on salivary biofilm, the levels
of total bacteria in the biofilm was significantly reduced by
eBio-cleaner water (p<0.05). Interestingly, the effects of
eBio-cleaner water did not differ significantly from its listerine
(Fig. 3). Therefore, the biofilm was performed observation of
confocal laser microscope after staining live/dead fluorescence.
Green and red colors indicate live and dead bacteria,
respectively. eBio-cleaner water showed disruptive and antimicrobial effect on the biofilm, and listerine exhibited antimicrobial effect on the bacteria of biofilm outside (Fig. 4).
Discussion
Figure 3. Antibiofilm effect of the electrolyzed water against the
salivary biofilm. The salivary biofilm was formed using pooled
saliva. The biofilm was treated with tap water, the electrolyzed
water of eBio-cleaner, and listerine. Live bacteria were counted.
*,#Significant difference compared with control and with tap water,
respectively (p＜0.05).
Electrolyzed water (EW) is used industrial and medical area
because of antimicrobial activity and relatively safe [15,16].
However, antimicrobial efficacy of EW generator varies depending on the configuration and for of the electrodes. In this
study, we demonstrated the antimicrobial effect of new EW generator on oral bacteria.
The features of EW generator (named eBio-cleaner) in this
study are to use platinum electrodes in non-separating chamber
and are the size to wash the mouth or to store full denture. Also,
this EW showed neutral pH. Compared to tap water, this EW
exhibited significant inhibition of growth of cariogenic bacteria
and periodontal pathogens. Furthermore, eBio-cleaner water
has bactericidal activity against A. actinomycetemcomitans, P.
gingivalis, and S. mutans. Interestingly, the EW showed dis-
Figure 4. Antibiofilm effect of the electrolyzed water against the salivary biofilm. The salivary biofilm was formed using pooled saliva. The
biofilm was treated with tap water, the electrolyzed water of eBio-cleaner, and listerine and analyzed by confocal laser microscope after
live/dead staining. Green and red colors indicate live and dead bacteria, respectively.
8 Vol. 13, No. 1, March 2017
In-Woo Cho, et al：Evaluation of Electrolyzed Water Generator
ruptive and bactericidal effect on salivary biofilm unlike
listerine. In 3D image of salivary biofilm, Listerine acted on outside bacteria of the biofilm and killed the bacteria. However,
the EW penetrated into the biofilm and killed the bacteria.
Although listerine has stronger antimicrobial activity against
planktonic bacteria than EW, in case of biofilm, EW may be
more effective than listerine. In some experiment of EW using
sodium chloride, when the electrolyzed water was generated
with sodium chloride (0.1, 0.5, 1%) by eBio-cleaner, the antimicrobial activity of the EW showed stronger than sodium
chloride free-EW (data not shown). However, when high concentration of sodium chloride was added to increase antimicrobial activity, the device did not work because of overcurrent protection.
The chemical reaction of EW using tap water is H2O→
+
−
−
2
−
1/2O2+2H +2e , 2Cl →Cl +2e , and Cl2+H2O↔HClO+HCl
−
−
2
−
−
at the anode, and H2O+e →H+OH- and OCl +H O+2e →Cl +
−
2OH at the cathode [17]. These chemical reactions generate
free chlorines, reactive oxygen, and reactive hydrogen in electrolysis of tap water, and the products has antimicrobial activity.
The antibacterial action of EW is described in three ways. The
first is the action of free chlorine. The free chlorine produced
by electrolysis of tap water damages the bacterial outer membrane and degenerates or aggregates cytoplasmic proteins such
as DNA polymerases [18]. In our study, when tap water was
electrolyzed in the presence of sodium chloride (NaCl), the bactericidal activity of EW increased. Also, chlorine concentration
contained in tap water varies from region to region in Korea.
Therefore, the antimicrobial activity of EW may differ in different localities. The second is the action of reactive oxygen or oxy−
gen radical (O2 ). Oxygen radical is generated near anode electrode and acts like action of free chlorine because of negative
charge. Most bacteria surface has positive charges. Therefore,
oxygen radical easily binds on surface molecules of bacteria and
damages it. Also, they binds on bacterial DNA, and damages
its DNA [19]. Finally, it is action of reactive hydrogen (H).
Reactive hydrogen can be replaced with hydroxyethyl group,
thereby interfering with a wide range of metabolic activities
[20].
Mouthwashes have been widely used to prevent oral diseases
by bacteria. Listerine as a used mouthwash in this study has
strong antimicrobial activity. This mouthwash contains alcohol
of high concentration. The long-tern use of mouthwash including alcohol has been reported to cause many adverse effects
[21,22], whereas EW has been not reported any adverse effects.
Rather, EW provides beneficial effects on human health. EW
reduces tumor angiogenesis via inhibition of signaling pathway
and prevents chronic depression by suppressing inflammasome
activation [23]. Taken together, the EW generated by eBio-
cleaner may be helpful in preventing and treating dental caries
and periodontitis by oral bacteria.
Conclusion
The electrolyzed water generated by eBio-cleaner reduced
the growth of periodontopathogens such as A. actinomycetemcomitans, F. nucleatum and P. gingivalis. Also, the EW
decreased the growth of S. mutans. the EW generated by
eBio-cleaner showed disruptive and antimicrobial effect on the
salivary biofilm. Furthermore, the EW has been reported to
have healing effect on oral tissues and to remove reactive oxygens in the cells. Take together of the results and previous studies, the EW generated by eBio-cleaner may be helpful in preventing and treating dental caries and periodontitis by oral
bacteria.
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