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: aysegulmendi@gmail.com

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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