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Antioxidant Lactobacilli Could Protect Gingival FibroblastsAgainst Hydrogen Peroxide: A Preliminary In Vitro Study
Aysegul Mendi • Belma Aslım
� Springer Science+Business Media New York 2014
Abstract Oxidative stress and tissue destruction are at
the heart of periodontal diseases. The dental research area
is geared toward the prevention of free radicals by nutrient
antioxidants. Lactic acid bacteria (LAB) have recently
attracted attention in alternative dental therapies. We aimed
at highlighting the antioxidative property of Lactobacilli
and Bifidobacterium strains and at determining their pro-
tective effect on gingival fibroblasts (GFs). Two Lactoba-
cilli and 2 Bifidobacterium strains were screened for their
exopolysaccharide (EPSs) production. Antioxidative assays
were conducted by spectrophotometer analysis. Resistance
to different concentrations of hydrogen peroxide (H2O2)
was determined by the serial dilution technique. The pro-
tective effect of strains on GFs on hydrogen peroxide
exposure was also examined by a new trypan blue exclu-
sion assay method. Bifidobacterium breve A28 showed the
highest EPS production (122 mg/l) and remarkable anti-
oxidant activity, which were demonstrated by its ability to
scavenge 72 % a,a-diphenyl-1-picrylhydrazyl free radical
and chelate 88 % of iron ion, respectively. Inhibition of
lipid peroxidation was determined as 71 % for the A28
strain. We suggest that LAB with antioxidative activity
could be a good natural therapy agent for periodontal
disorders.
Keywords Antioxidant activity gingival fibroblasts �Hydrogen peroxide � Lactobacilli
Introduction
Periodontitis is a chronic bacterial infection of tooth-sup-
porting structures. The disease develops as a result of host-
mediated inflammatory response to pathogenic microflora
residing in periodontal pockets [1–3]. Oxidative stress lies at
the heart of periodontal tissue damage that results from host–
microbial interactions, possibly as a direct result of excessive
reactive oxygen species (ROS) activity, the activation of
transcription factors, and the creation of a proinflammatory
state or an antioxidant deficiency. Although a myriad of
possible mechanisms leading to periodontal destruction
exists, the influence of free radicals and antioxidants cannot
undoubtedly be overlooked [4].
Tissue injury due to free radical production has been
suggested to be enhanced in individuals with periodontal
disease due to a lack of adequate antioxidant defense.
Exaggerated neutrophil activity may be attributable to
defects of inflammatory response in some individuals, but
low antioxidant capacity may be caused by a number of
factors including smoking and poor nutritional status [5].
Moreover, both growth factors and proinflammatory cyto-
kines themselves have been shown to induce an acute
generation of hydrogen peroxide (H2O2) in various cell
types [6]. Pavlica et al. [7] investigated the total antioxi-
dant capacity of serum and concluded that the total anti-
oxidant capacity in periodontitis was lower than in health
and suggested a negative correlation between total anti-
oxidant capacity and periodontal parameters. Based on
these studies, antioxidant levels appear to be a significant
factor in weakening or bolstering host resistance to peri-
odontal disease. Considering the emerging body of evi-
dence tying periodontal disease to other serious health
problems, we think that by improving the host resistance,
we may improve the periodontal status.
A. Mendi (&)
Department of Medical Microbiology, Faculty of Dentistry,
Gazi University, Ankara, Turkey
e-mail: [email protected]
B. AslımDepartment of Biology, Faculty of Science, Gazi University,
Ankara, Turkey
123
Probiotics & Antimicro. Prot.
DOI 10.1007/s12602-014-9165-3
Recent medical and dental research in this area is geared
toward the prevention of free radical-mediated diseases using
specific nutrient antioxidants. The inhibition of oxidative
stress is considered an important therapeutic approach, and
many efforts have been made to identify the antioxidant
potential of various compounds, including medicinal plants,
vitamins, and plant polyphenols [8]. Unfortunately, all tested
antioxidants failed to confirm that they do not accumulate any
toxic compounds in the human body.
Probiotic bacteria are defined as ‘‘live microorganisms
which when administered in adequate amounts confer a
health benefit on the host’’ [9]. Recently, probiotics were
suggested to play a role in maintaining oral health [10].
In human, probiotic bacteria mainly consist of strains of
Lactobacillus and Bifidobacterium and are often part of the
intestinal ecosystem [11]. Given the widespread emergence
of bacterial resistance to antibiotics, the concept of probiotic
therapy has been considered for application in oral health
[12]. Dental caries and periodontal disease are among the
oral disorders that have been targeted [13]. However, many
questions have been raised pertaining to the benefits of
probiotic administration, as the role of probiotics in treating
oral diseases is still in its infancy. We suggest it is also
important to select promising newly isolated probiotic bac-
teria since the health benefits are varied among the strains.
Based on these issues, the aim of the study was to
investigate the appropriate strain for use in dentistry. First,
we determined the exopolysaccharide (EPSs) production of
LAB cultures since EPSs may play a role in the develop-
ment of antioxidative ability. This is one of the primary
metabolic products of LAB, and they have recently
received an increasing amount of attention because of their
health benefits [14]. Of these, antioxidant properties have
been recently reported [15]. We demonstrated the antiox-
idative potential of probiotic cultures in which hydrogen
peroxide is a strong oxidant when it is absorbed by the cells
so that probiotics may be a guard for the cells. Resistance
of bacterial cultures to different hydrogen peroxide con-
centrations was investigated, and the viability of hydrogen
peroxide exposed gingival fibroblasts cultured with bacte-
rial cultures was examined.
Materials and Methods
Bacterial Strains and Culture Conditions
We used origin strains of infant stools in this study. Isolates
were identified based on Gram’s stain, morphology, and
advanced biochemical tests. The pattern of carbohydrate
fermentation of lactobacilli was determined using the API
50 CHL kit that was analyzed by NTSYSpc software version
2.0 [16]. Bifidobacteria were identified using fructose-6-
phosphate phosphoketolase (F6PPK) activity [17]. Genomic
DNA of the isolates was extracted using the Genomic DNA
Purification Kit (Fermentas, K0512), and the isolates were
identified by primers that were specific for the 16S rRNA
gene sequences. Univ-27F 50-AGA GTT TGA TCM TGG
CTC AG-30 primers amplified a 1,492-bp gene region for
lactobacilli and P0 50-GAG AGT TTG ATC CTG GCT
CAG-30, LM3 50-CGG GTG CTT NCC CAC TTT CAT G-30
primers amplified a 1,426-bp gene region for bifidobacteria
[18–20]. The PCRs were carried out in a 50-ll VeritiTM
Thermal Cycler (Applied Biosystems). PCR conditions
were 2 min at 94 �C for the first denaturation followed by 35
cycles of 20 s at 94 �C for denaturation, 30 s for annealing
(53 �C for lactobacilli; 58 �C for bifidobacteria), 1 min at
72 �C for extension, and 5 min at 72 �C for the final exten-
sion. The sequences obtained were compared with GenBank
database using the BLAST network service (http://blast.ncbi.
nlm.nih.gov/Blast.cgi; NCBI). In all experiments, the lacto-
bacilli were incubated in Man Rogosa and Sharpe medium for
20 h at 37 �C; bifidobacteria were incubated anaerobically in
the same medium supplemented with L-cysteine HCl solution
(0.5 % w/v) for 48 h at 37 �C. The number of bacteria at the
beginning of the experiment was adjusted to *1 9 1011 cfu/
ml and expressed in cfu/ml by the serial dilution technique.
The results were calculated and shown as log cfu/ml.
Isolation and Quantification of EPSs
Incubated broth cultures in MRS and MRS-C broth were
boiled at 100 �C for 15 min. The samples were treated with
17 % (v/v) of 85 % trichloroacetic acid solution and cen-
trifuged at 4 �C at 18,0009g for 25 min [21]. EPSs were
precipitated by adding 3 volumes of chilled 100 % ethanol.
The precipitate was collected by centrifugation at 4 �C at
18,0009g for 20 min and dissolved in distilled water. Total
EPSs (expressed in mg/l) were estimated in each sample by
the phenol–sulfuric acid method [22] using glucose as a
standard [23]. Briefly, samples were mixed with 5 %
phenol. Then, after the addition of 1 ml of concentrated
sulfuric acid, tubes were allowed to stand for 10 min at
room temperature, followed by 15 s of vortexing. Absor-
bance was measured at 490 nm with a spectrophotometer
(Digilab Hitachi U-1800). Sugar concentration in samples
was quantified using a standard curve with glucose
(0–100 mg/l).
Determination of the Scavenging a,a-Diphenyl-1-Picryl
hydrazyl (DPPH) Radical
The scavenging of DPPH by Lactobacillus and Bifidobacte-
rium cultures was analyzed by a modification method utilized
by Blois [24] and Lin and Chang [25]. DPPH antioxidant
Probiotics & Antimicro. Prot.
123
assay is based on the ability of DPPH, a stable free radical, to
decolorize in the presence of antioxidants. The DPPH radical
contains an odd electron that is responsible for the absorbance
at 517 nm and also for a visible deep purple color. When
DPPH accepts an electron donated by an antioxidant com-
pound, the DPPH is decolorized, which can be quantitatively
measured from the changes in absorbance. One milliliter of
intact cells and one ml of freshly prepared DPPH solution
(0.1 mM in methanol) were mixed and allowed to react for
30 min at room temperature. Blank samples contained
phosphate-buffered solution (PBS; pH 7.4). The scavenged
DPPH was monitored by measuring the decrease in absor-
bance at 517 nm. The scavenging ability was defined as
follows: [1 - (OD2/OD1)] 9 100, where OD2 is the sample
absorbance and OD1 is the blank absorbance.
Iron Ion-Chelating Ability
The chelating ability was determined by a modification
method utilized by Decker and Welch [26]. One milliliter of
the intact cells was mixed with 2 mM FeCl2 (0.05 ml) and
5 mM ferrozine as a chelator (0.2 ml). Ferrozine can quanti-
tatively form complexes with Fe2?. However, in the presence
of a good chelator, the complex formation is disrupted with the
result that the red color of the complex is decreased. Mea-
surement of color reduction, therefore, enables the estimation
of the chelating activity of the coexisting chelator. Chelating
activity was measured by the decrease in absorbance at
562 nm. Blank samples contained PBS. The scavenging
ability was defined as follows: [1 - (OD2/OD1)] 9 100,
where OD2 is the sample absorbance and OD1 is the blank
absorbance.
Inhibition of Plasma Lipid Peroxidation
Lipid peroxidation forms malondialdehyde (MDA) as a
natural byproduct. MDA in the sample is reacted with
thiobarbituric acid (TBA) and generates the MDA–TBA
adduct. The MDA–TBA adduct can be easily quantified
colorimetrically. Plasma lipid peroxidation was analyzed
by the method developed by Rodriguez-Martinez et al.
[27]. In this method, 0.4 ml of plasma (blood center of the
Gazi University), 0.1 ml FeSO4 solution (50 lM), 0.1 ml
of H2O2 (0.5 mM), and 0.2 ml of bacterial cultures were
mixed and incubated at 37 �C in a water bath. Deionized
water was used in the blank samples. After 12 h of incu-
bation, the reaction solution was mixed with 0.375 ml of
TCA (trichloroacetic acid; 4 %) and 75 ll of BHT
(butylated hydroxytoluene; 0.5 mM) and held in an ice
bath for 5 min. The upper phase was obtained by centri-
fugation at 3,0009g for 10 min. TBA (thiobarbituric acid;
0.2 ml; 0.6 %) was then added. This mixture was incubated
at 100 �C for 30 min and allowed to cool at room tem-
perature. The absorbance was then measured at 532 nm. The
percentage of inhibition of plasma lipid peroxidation was
defined as follows: [1 - (OD2/OD1) 9 100], where OD1 is
the initial absorbance and OD2 is the absorbance measured
after the reaction.
Determination of Hydrogen Peroxide Resistance
of Bacteria
Resistance of bacterial cultures was determined as descri-
bed by Doleyres et al. [28] with some modifications. H2O2
concentrations of 10, 20, and 30 mM and no H2O2 as the
control group were prepared and exposed to 1 9 1011 cfu/ml
in 1 ml phosphate-buffered saline (PBS, pH 7). We
exposed bacterial cultures to different H2O2 concentrations
at 37 �C for 15 and 30 min. At the end of the incubation,
samples were serially diluted (tenfold, PBS) and spread
onto MRS (for lactobacilli) and MRSC (for bifidobacteria)
agar. Plates were incubated under aerobic (for lactobacilli
and 48 h) and anaerobic (for bifidobacteria and 72 h)
conditions at 37 �C, and the cell counts were enumerated.
Results were showed by log10 cfu/ml.
Cell Culture
Gingival fibroblasts (GFs) were a kind gift from the Gulhane
Military Medical Academy (GATA, Ankara, Turkey) Cancer
Research Center, in cryovials belonging to one donor. GFs
were grown in plastic bottles (25 or 75 cm2 growth area;
Sarstedt) in Dulbecco’s modified Eagle’s medium (DMEM;
Invitrogen) with 10 % heat-inactivated fetal bovine serum
(FBS; Gibco), 1 % penicillin and streptomycin (5,000 IU/ml
and 5,000 lg/ml; Gibco), and 1 % 10 mM nonessential
aminoacid solution at 5–6 passages (Gibco) [29, 30]. Incu-
bation was carried out at 37 �C in 5 % CO2 atmosphere. GFs
were subcultured for the assay on 96-well microplates with
103 cells/well and incubated till they reached confluency.
Protective Effect of Bacteria on Gingival Fibroblasts
Next, we tried to demonstrate the protective effect of
promising probiotics against hydrogen peroxide (H2O2).
Cultured GFs on 96-well microplates were treated with
1 9 1011 cfu/ml in the described culture medium without
antibiotic. GFs were treated with promising probiotics for
15 min; thereafter, 10 mM of H2O2 were added for another
15 min. Cell viability was determined by trypan blue
exclusion assay, described by Peres et al. [31]. In brief, the
GFs were stained with 50 ll of 0.4 % trypan blue solution
and incubated for 15 min. The dye was then removed by
gentle but thorough washing with ice-cold PBS. The cells
Probiotics & Antimicro. Prot.
123
were lysed with 200 ll of 1 % SDS, and the contents were
gently titrated, taking care not to introduce air bubbles. The
TB/cell suspension mixture was used to determine cell
death by measuring absorbance at 590 nm.
Statistical Analysis
All assays were expressed as mean ± SD of 3 independent
experiments. The relationship between EPS production and
antioxidant ability of strains was tested by Kruskal–Wallis
test. A value of p \ 0.05 was considered statistically
verified.
Results
Isolation and Quantification of EPSs
We have determined the incubation time of bacterial cul-
tures linked to EPS production according to other studies of
our group [14, 32]. Although the incubation time and the
growth conditions were the same, we show that EPS pro-
duction was varied among the strains (p \ 0.05) (Table 1).
Of Bifidobacterium breve strains, B. breve A28 produced
more EPSs than A10. L. rhamnosus GD11 had a higher
EPS production capacity than L. plantarum LA3.
Antioxidant Activity
The results of the antioxidant activity by various strains are
shown in Table 1. As shown in Table 1, antioxidant
activity was strain specific. EPS production identified the
antioxidant ability in B. breve A28 and L. rhamnosus LA3
(p \ 0.05). The DPPH value was affected by A28, which
has the highest EPS production. The scavenging ability
of B. breve A28, which has high-level EPS-producing
capacities, contributes to the antioxidant effect. Iron ion
chelating was varied among the strains (p \ 0.05).
L. rhamnosus LA3, which has the lowest EPS-producing
capacity, exhibited decreased chelation (2 %), whereas B.
breve A28 showed the highest chelating ability (88 %).
According to Table 1, B. breve A28 inhibits MDA–TBA
adduct production better when compared with the other
strains. B. breve A28 and B. breve A10 strains inhibited
lipid peroxidation in different degrees (Table 1).
Determining Bacterial Resistance to Hydrogen
Peroxide
To evaluate the resistance of strains to H2O2, we used
different concentrations of H2O2 (Fig. 1). Also we
detected exposure time to H2O2 affected the viability of
strains. L. plantarum LA3 could not manage to exist for
30 min on any concentration of H2O2. The viability of
L. plantarum LA3 strain decreases from log1011 cfu/ml to
3.1 and to 2.6 on 10, 20, and 30 mM H2O2 concentra-
tions, respectively. L. rhamnosus GD11 resisted 30 mM
H2O2 when compared with L. plantarum LA3 for 30 min
exposure. On the other hand, B. breve A28 and A10 were
definitely the strongest strains against high concentrations
and prolonged exposure to time. Based on these data, we
selected the appropriate concentration (10 mM) and
duration time (15 min) that the strains resisted in in vitro
studies on gingival fibroblasts.
Table 1 EPS production and antioxidative ability of strains
Strain no. Mean value p value
EPS yield (mg/l)* GD11 117 ± 0.5 0.007*
LA3 28 ± 0.7
A28 122 0.7
A10 44 2
DPPH scavenging GD11 46 ± 0.082
LA3 22 ± 0.8
A28 72 ± 1.3
A10 52 ± 1
Fe ion chelating GD11 31 ± 2 0.017*
LA3 2 ± 1
A28 89.11 ± 1.1
A10 39 ± 2
Inhibition of plasma lipid
peroxidation
GD11 65 ± 1 0.054
LA3 39 ± 1
A28 71 ± 1
A10 32 ± 1.2
* Difference in EPS production of strains is significant according to
Kruskal–Wallis test (p \ 0.05). Also, EPS production and Fe ion
chelating of B. breve A28 are significantly higher than L. plantarum
LA3. GD11: L. rhamnosus; LA3: L. plantarum; A28-A10: B. breve
Fig. 1 Hydrogen peroxide resistance of bacteria. Results given here
are expressed as log10 cfu/ml. LA3 could not resist hydrogen peroxide
for 30 min
Probiotics & Antimicro. Prot.
123
In Vitro Protective Effect of Promising Probiotics
Against Hydrogen Peroxide Exposure
In order to assess the protective effect of strains against H2O2,
we cocultured the strains with GFs. Cultured GFs on 96-well
microplates were treated with 1 9 1011 cfu/ml promising
probiotics in the described culture medium without antibiotic
for 1 h. Thereafter, GFs–bacteria coculture was treated with
10 mM H2O2 for 15 min. Untreated cells were categorized as
the control group. GFs cocultured with B. breve A28 showed
minimum cell death against H2O2 exposure (Fig. 2). It was
well marked that B. breve A28 caused 9 % cell death in the
control group.
Discussion
Given the widespread emergence of bacterial resistance to
antibiotics, the concept of probiotic therapy has been
considered for application in oral health. Dental caries,
periodontal disease, and halitosis are among the oral dis-
orders that have been targeted in clinical trials [33].
However, only a few studies are available on the preva-
lence, role, and effects of probiotic bacteria in the mouth.
A Russian study examined probiotic tablets in a complex
treatment of gingivitis and different degrees of periodon-
titis [11]. The treatment of the patients of the control group
was provided by the drug Tantum Verde (Aziende
Chimiche Riunite Angelini Francesco A.C.R.A.F. S.p.A.,
Rome). The effect of probiotics on the normalization of
microflora was found to be higher in comparison with
Tantum Verde, particularly in the cases of gingivitis and
periodontitis. Nase et al. [34] reported reduced tooth decay
incidence in children taking probiotic L. rhamnosus GG-
enriched milk versus a control group of children taking
milk without probiotic enrichment. Studies on periodontitis
and gingivitis show differing results depending on the
strains. For example, Lactobacillus reuteri can be used to
reduce gingivitis and dental plaque in patients with mod-
erate to severe gingivitis and also to reduce proinflamma-
tory cytokine in gingival crevicular fluid [35, 36]. On the
other hand, Lactobacillus salivarius WB21 in tablets does
not reduce the direct count of any specific periodontopathic
bacteria—Porphyromonas gingivalis, Prevotella intermedia,
Tannerella forsyia, Trepenoma denticola, and Aggrega-
tibacter actinomycetemcomitans [37], (even though this
probiotic improves periodontal clinical parameters probing
pocket depth, gingival index, bleeding on probing, and
plaque index) especially in smoker subjects [38]. Com-
mercially available probiotics that contain Lactobacilli
species interfere with the in vitro ability of Candida albi-
cans to form biofilms on dentures [39], yet conventional
approve intestinal probiotics surprisingly have no oral
persistence and any oral cavity health benefits seem tran-
sitory. These conflicting results point out that not all the
probiotics have beneficial effects on periodontal diseases.
Therefore, it seems necessary that to perform specific
screenings for selecting appropriate probiotic strains for
preventing gingivitis or periodontitis and other oral health
diseases. Sookhee et al. [40] verified this hypothesize by
investigating 130 volunteers in Thailand and found 3,790
lactic acid bacterial strains from healthy oral cavities. Of
these, only five species expressed the inhibitory effect
against other organisms, including oral Candida. The
authors reported that the antimicrobial potentials of the
bacteria were affected by several factors, such as pH, cat-
alase, proteolytic enzymes, and temperature. Also it cannot
be assumed that research published on one strain of pro-
biotic applies to another strain, even of the same species.
The key point is to realize that all probiotics do not have
the same efficacy [41, 42]. It is important that probiotic
strains should be well characterized before and compre-
hensive in vitro research studies should be conducted [43].
This study aimed at identifying novel strains of lactic
acid bacteria to be used in dentistry. Initially, we screened
EPSs production among different strains. Reported bene-
ficial health properties make EPSs economically and sci-
entifically important [44]. The total yield of EPSs produced
by LABs depends on the composition of the medium and
the conditions in which the organisms grow (i.e., temper-
ature and incubation time) [45]. In our study, although we
have used the same culture medium and standard incuba-
tion time and temperature, EPSs yield varied among the
strains (p \ 0.05). Most of the functions ascribed to EPSs
are of a protective nature. The ability of a microorganism
to surround itself with a highly hydrated exopolysaccharide
Fig. 2 Protective effect of promising probiotics against hydrogen
peroxide exposure. GD 11: L. rhamnosus; LA3: L. plantarım; A28-
A10: B. breve. Cell death is measured by trypan blue exclusion assay.
When used alone, hydrogen peroxide inhibits cell viability (black
bar). Gray bars indicate bacterial cultures used alone. Cell death is
9 % when A28 was used. Protective effect is shown on white bars
Probiotics & Antimicro. Prot.
123
layer may provide it with protection against desiccation
and predation. It is suggested that in terms of oral envi-
ronment in clinical studies considering the stressful con-
ditions created by saliva and teeth surface, our high EPSs
productive strains, B. breve A28 and L. rhamnosus GD11,
may survive better (in vivo environment would be different
from controlled in vitro conditions). Besides, owing to the
EPSs-specific glycosidic bond, it cannot be digested by the
digestive amylase. This makes it possible to exert the
antioxidant activity in organisms [46].
Since the antioxidant mechanisms for in vitro assay
methods were diverse, the antioxidant activity should be
determined by different ways. In this study, three indexes,
including the scavenging activities of DPPH, chelation of
iron ion, and inhibition of lipid peroxidation, were mea-
sured. Antioxidant assays showed that B. breve A28 with
high EPSs production ability exhibited a good chelating
ability (p \ 0.05), a good DPPH scavenging ability, and
strong inhibition of lipid peroxidation. Based on the results
of Liu et al. [47] and Xu et al. [48], higher content of uronic
acid than usual makes EPSs antioxidative, and the molecules
carry more negative charge. The higher charge increased the
intramolecular repulsive force and made the molecules more
extended. This reduced the steric hindrance for radical attack
[49]. Thus, the free radical became more likely to be scav-
enged, and the EPSs exerted good antioxidant activity. B.
breve A28 and L. rhamnosus GD11 have remarkable anti-
oxidative capacity compared with the study conducted by
Lin and Chang [25]. B. longum (ATCC 15708) and L. aci-
dophilus (ATCC 4356) showed antioxidative activity,
inhibiting linoleic acid peroxidation by 28–48 %, and also
showed the ability to scavenge 21–52 % of the a-diphenyl-
b-picrylhydrazyl free radical.
Reactive oxygen species include superoxides, singlet
oxygen, and hydrogen peroxide [50]. Hydrogen peroxide is
relatively weak, but it is highly diffusive and has a long
lifetime. Due to these two basic properties, hydrogen per-
oxide contributes to oxidative damage either directly or as
a precursor of hydroxyl radicals [51]. Tolerance of probi-
otic strains to hydrogen peroxide is important for realizing
antioxidative ability. B. breve A28 was able to survive in
the presence of hydrogen peroxide twice longer than other
lactobacilli strains. Resistance to H2O2 is an important
criterion for the selection of oral probiotic bacteria, as
H2O2 could seriously affect strain viability in oral micro-
bial flora that contains H2O2-producing microorganisms
(e.g., mitis streptococci) [52]. The inhibitory action of
H2O2 is attributed to the formation of highly reactive OH
free radicals in the presence of iron and copper [53]. These
free radicals primarily attack polyunsaturated fatty acids
directly in cell membranes and initiate lipid peroxidation,
which leads to alterations in membrane properties and
fluidity and disrupts membrane-bound proteins [54].
Prevention of cell death against oxidative stress by probi-
otics may inhibit tissue destruction in periodontitis. Under-
standing the interaction between oral mucosal cell and
probiotics would be a confident basis for probiotic therapies in
oral disorders. Fibroblasts are the major cellular constituents of
gingival connective tissue. They produce proteoglycans,
hyaluronate, glycoproteins, collagen, and inflammatory cyto-
kines, which play an important role in the pathogenesis of
periodontitis [54]. There are a few studies on the protective
effect of lactobacilli against oxidative damage in cell cultures.
H2O2 could get through the cell membrane easily and generate
a vast number of high cytotoxic free radicals via intracellular
metabolism. The notable radical scavenging capacity and high
EPS-producing strain B. breve A28 endowed it with antioxi-
dant protective activity to some extent. The results showed that
it could chelate iron ion, scavenge DPPH radical, and prevent
plasma lipid peroxidation, leading to an improvement of
the viability of the gingival fibroblast. Indeed, when used
alone, B. breve A28 caused 9 % cell death. On the other hand,
L. rhamnosus GD 11 and L. plantarum LA3 strains showed a
cytotoxic effect with 47 and 49 %, respectively.
The majority of tissue destruction in periodontitis is con-
sidered to be the result of an aberrant inflammatory/immune
response to microbial plaque adjacent to the gingival margin
and to involve prolonged release of neutrophil enzymes and
ROS [55]. Most published work in the periodontal literature
has focused on markers of ROS reactions with lipids [56]. On
the other hand, there are only few studies to our knowledge
that have investigated total antioxidant capacity in serum/
plasma from periodontitis patients and controls [7, 57]. The
results of these studies demonstrated significantly lower total
antioxidant capacity in serum and plasma samples from
periodontitis subjects. Panjamurthy et al. [55] found lower
plasma vitamin C, vitamin E, and glutathione (GSH) in peri-
odontitis patients even after adjusting for protein levels,
whereas antioxidant enzyme levels were raised, which might
be considered as a protective response to oxidative stress [56].
Different reports suggested adjunctive use of antioxidants
with traditional therapies to improve treatment outcome of
various surgical and non-surgical periodontal therapies [58,
59]. Several in vitro assays or animal studies are very useful in
the preselection of (probiotic) bacterial strains, and the proof
of efficacy in humans should be granted by at least one well-
designed study [60, 61]. Based on this in vitro study, we
suggest that antioxidant activity could be a criterion for
selecting strains in treating periodontal diseases.
Conclusions
We suggest lactic acid bacteria with antioxidative proper-
ties may improve periodontal disorders. Functional prop-
erties of probiotic strains are different among the strains,
Probiotics & Antimicro. Prot.
123
and they do not show the same health benefit efficacy. We
propose that B. breve A28 strain could be a candidate for
biological product researches and clinical studies since it
has a high EPS production capacity, strong antioxidative
property and is friendly with GFs. In order to develop
further preventative methods by probiotics against various
oral diseases, additional investigations regarding their
molecular interactions within gingival fibroblasts in asso-
ciation with oxidative stress are required. It is important to
note that functional properties could be changed among the
strains since it is crucial to select the best convenient strain
to use in therapies.
Acknowledgments We thank Prof. Ali Ugur Oral, from the Gulh-
ane Ministry Medicinal Academy Cancer Research Center, for pro-
viding gingival fibroblast cells in cryovials and for his valuable
collaboration. This work was supported by a grant from the Scientific
and Technological Research Council of Turkey through Project No.
TBAG 109T541 and the Gazi University through Project No. SCP
05/2009-08.
Conflict of interests The authors have no conflict of interests.
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