Introduction

Reactive oxygen metabolites (ROM), generated through normal reactions within the body during respiration in aerobic organisms, can cause damage in proteins, mutations in DNA, oxidation of membrane phospholipids, and modification in low-density lipoproteins. Excessive amount of ROM can result in cellular damage which, in turn, promotes chronic diseases including atherosclerosis, arthritis, diabetes, neurodegenerative diseases, cardiovascular diseases, and cancer (Babbs 1990; Firuzi et al. 2011). To neutralize the oxidant molecules, the human body synthesizes antioxidant enzymes and molecules that, together with the antioxidants contained in food, form the biological antioxidant barrier. However, in certain circumstances, the defense system fails to protect the body against oxidative stress; consequently, the possibility of increasing antioxidant defenses is considered important in the maintenance of human health and disease’s prevention (Serafini and Del Rio 2004).

In this direction, a novel pioneering approach is represented by the development of probiotics exerting antioxidant activity and counteracting the oxidative stress in the host. Probiotics are defined as “live microbes which, when administered in adequate amounts, confer a health benefit to the host” (FAO/WHO working group 2001). In particular, besides the long history of consumption of lactic acid bacteria, probiotic strains belonging to the genera Lactobacillus and Bifidobacterium have been reported to have a range of health-promoting features (Rossi and Amaretti 2010). Although the molecular mechanisms of probiotics have not been completely elucidated yet, their modulation of the intestinal microbiota, antibacterial substance production, improvement of the epithelial barrier function, and reduction of the intestinal inflammation are already well established. At first, probiotics were consumed to modulate and improve the gut microbiota balance; nowadays, properly selected probiotics strains have developed, in order to respond to specific physiological targets (Pompei et al. 2007a, b), to prevent and treat pathogen-induced diarrhea (Guandalini 2011), or to manage autoimmune and atopic diseases (Finch et al. 2010).

Among probiotics beneficial effects, some authors have reported the protection against oxidative stress and the capability to decrease the risk of accumulation of ROM (Martarelli et al. 2011; Kaizu et al. 1993; Kullisar et al. 2002). Studies have shown that selected strains of lactobacilli and bifidobacteria present antioxidative properties (Shimamura et al. 1992; Lin and Yen 1999; Kullisaar et al. 2002; Wang et al. 2006; Zanoni et al. 2008; Mikelsaar and Zilmer 2009; Uskova and Kravchenko 2009; Spyropoulos et al. 2011) and can be used to prepare probiotic and fermented dairy products that improve total antioxidant status and decrease markers of oxidative stress in healthy people (Naruszewicz et al. 2002; Songisepp et al. 2005; Virtanen et al. 2007).

The antioxidant mechanisms of probiotics could be assigned to ROS scavenging, metal ion chelation, enzyme inhibition, and to the reduction activity and inhibition of ascorbate autoxidation (Lin and Yen 1999; Talwalkar and Kailasapathy 2003). Probiotic metabolic activities may have an antioxidant effect via the scavenging of oxidant compounds or the prevention of their generation in the intestine (Azcárate-Peril et al. 2011).

However, although the ability of probiotic bacteria to exert antioxidant activity has attracted some attention; so far, no rigorous comparative studies of this feature have been published. The present study wants to fill this gap by exploring in vitro the antioxidant properties of 34 lactic acid bacteria, then assessing the effect of the administration of a selected mix of potentially probiotic bacteria in Wistar rats subjected to an exogenous oxidative stress induced by doxorubicin.

Materials and methods

Materials

All the chemicals were supplied by Sigma (Stenheim, Germany), unless otherwise stated. Bacterial strains were obtained from ATCC (Manassas, VA, USA), DSMZ (Braunschweig, Germany), and our own collection (formerly the department of Chemistry, University of Modena and Reggio Emilia, Modena, Italy). They were seven strains of the genus Bifidobacterium, 11 of the genus Lactobacillus, six of the genus Lactococcus, and 10 of the species S. thermophilus (Table 1). Bifidobacteria and lactobacilli were cultured in Lactobacilli MRS broth (BD Difco, Sparks, MD, USA) containing 0.5 g/L L-cysteine · HCl, and were anaerobically incubated at 37 °C for 24 h. The strains of S. thermophilus and Lactococcus were cultured aerobically at 42 °C for 24 h in M17 broth (BD Difco).

Table 1 TAALA, TAAAA, TEAC, TGSH, and SOD of bacterial strains of Bifidobacterium, Lactobacillus, Lactococcus, and S. thermophilus
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Preparation of bacterial suspensions and cell-free extracts for antioxidant assays

Bacterial samples were prepared as follows. The biomass from 24-h cultures was harvested by centrifugation (6,000×g for 10 min at 4 °C), washed three times and suspended in a buffer solution, as appropriate for the specific assay. Phosphate-buffered saline (0.15 M NaCl, 2.68 mM KCl, 10 mM Na2HPO4, and 1.76 mM KH2PO4) was used in TAAAA, TEAC, and TGSH assays; 11.5 g/L KCl was used in TAALA assay; 0.1 mM EDTA in 50 mM potassium phosphate buffer (pH 7.8) was used in SOD assay. Cell-free extracts for TEAC, TGSH, and SOD analysis were obtained from cell suspensions containing approximately 3 × 109 cfu/ml, that were subjected ultrasonic disruption (five 1-min strokes at 0 °C, with 5-min intervals) and centrifuged (10,000×g for 5 min at 4 °C) to remove the cells debries. Protein concentration of cell-free extracts was quantified according to Lowry et al. (1951). For TGSH analysis, the cell-free extracts were treated with 50 g/L metaphosphoric acid at 4 °C for 15 min to precipitate proteins and remove interference with the assay.

Antioxidant activity in bacterial cells

The antioxidant properties of bacterial samples (intact cells and cell free-extracts) were determined by measuring the inhibition of linolenic acid peroxidation (TAALA) and ascorbate autoxidation (TAAAA), the TEAC, and the cellular content of total glutathione (TGSH, oxidized plus reduced form), and SOD.

TAALA was evaluated by measuring the antioxidant activity of cell suspensions against linolenic acid peroxidation and was referred to as the percentage inhibition of color formation (λ = 534 nm) arising from thiobarbituric acid assay (Zanoni et al. 2008; Kullisaar et al. 2002). The TAAAA of cell suspensions was referred to as the percentage inhibition against spontaneous ascorbate oxidation, which was evaluated through the measurement of UV absorbance at 265 nm (Mishra and Kovachich 1984; Zanoni et al. 2008).

To evaluate the TEAC of cell-free extracts, the inhibition of the chemiluminescent reaction between luminol and H2O2 was determined and compared to standard Trolox solutions (Girotti et al. 2004; Zanoni et al. 2008).

Glutathione was analyzed in deproteinized bacterial cell-free extracts using a commercial kit (HT Glutathione, Trevigen, Gaithersburg, MD, USA). The kit exploits the reaction between reduced glutathione and 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) giving a colored compound (5-thio-2-nitrobenzoic acid, TNB) measured at 405 nm. The reaction was carried out in presence of glutathione reductase and NADPH to determine total glutathione (TGSH, oxidized plus reduced glutathione).

Finally, to estimate SOD activity of cell-free extracts, the inhibition of epinephrine autoxidation was measured (Misra and Fridovich 1972) and compared with 0 to 10 U ml−1 standard solutions of bovine SOD (Sigma-Aldrich). Specific activity was expressed as enzymatic units per milligram of proteins, one unit being defined as the amount of SOD which inhibits the reduction of nitro blue tetrazolium by 50 % (Beuchamp and Fridovich 1971; Dellomonaco et al. 2007).

In vivo animal trial

The Animal Care Committee of the University of Bologna approved the study (Prot. 20600-X/10). The experiment was carried out with 42 male Wistar rats (90 days old; body weight, 250–300 g), individually housed in cages at 20 ± 2 °C and 60–70 % humidity, with a 12/12 h light/dark cycle. Throughout the trial, rats were fed a standard diet (Mucedola, Milano, Italy), and had free access to water and food. The animals were weighted weekly and food consumption was daily recorded. After a 7-day period of acclimatization, rats were randomly divided into four groups, three of them receiving daily via gastric gavage a 6-ml water suspension containing different concentration of a mixture of the potentially probiotic strains B. animalis subsp. lactis DSMZ 23032, Lactobacillus acidophilus DSMZ 23033, and L. brevis DSMZ 23034.

The lyophilized probiotic formulation (Probiotical Ltd, Novara, Italy), which contained equal amounts of the viable counts of each strain and potato maltodextrin as excipient, was reconstituted (10 % w/v) with water and diluted as appropriate to achieve the following doses. The groups Pro107 (n = 7), Pro108 (n = 14), and Pro109 (n = 7) received 107, 108, and 109 cfu/day, respectively; the fourth group (NS, n = 14) received 6 ml of water without any probiotic supplement, as a negative control.

After 18 days of probiotic supplementation, 20 mg doxorubicin (DOXO) per kilogram b.w. were intraperitoneally administered to all Pro107 and Pro109 rats, and to seven out of 14 of Pro108 and NS rats. Besides, the other rats from Pro108 and NS groups received intraperitoneally a similar volume of physiological solution. Twenty-four hours later, after a fasting period of 3 h, animals anesthetized and sacrificed. Blood was collected using sodium heparin as anticoagulant, plasma was obtained after centrifugation at 2,000×g at 4 °C for 15 min, and immediately frozen until analyses.

The concentrations of bifidobacteria, lactobacilli, enterococci, and coliforms in rat feces were monitored at the beginning and after the 18-day probiotic supplementation.

Assays to determine the antioxidant status in vivo

The plasma concentration of ROMs was evaluated spectrophotometrically using the d-ROM test (Diacron, Grosseto, Italy) as reported (Bordoni et al. 2008). The assay is based on the ability of reaction mixture to generate alkoxyl and peroxyl radicals from hydroperoxides by the Fenton’s reaction. The reaction produces free radicals that, trapped by an alkylamine, form a colored compound detectable at 505 nm. Results were expressed as milligram of H2O2/dl of plasma.

TAA was measured in rat plasma with the method of Re et al. (1999), based on the ability of the antioxidant molecules in the sample to reduce the radical cation of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+). TAA was determined in 10 μl plasma by evaluating the decoloration of ABTS+, measured as the quenching of the absorbance at 734 nm. Values obtained were compared to the concentration–response curve of the standard Trolox solution and expressed as millimole TEAC.

Plasma reduced GSH concentration was determined as previously described by Di Nunzio et al. (2011) with slight modification. Of plasma, 100 μl were incubated for 30 min in 100 μl reagent buffer (80 mM sodium phosphate, pH 8.0; 2 mM EDTA; 2 % SDS; 250 μM DTNB), and GSH was measured spectrophotometrically by reading at 415 nm the absorbance of TNB. Results were compared to the concentration–response curve of standard GSH solutions and expressed as microgram of GSH/ml plasma.

Analysis of fecal pH and microbiota

The pH of feces was measured with a pH meter, following a 1:10 (w/v) dilution with distilled water (Pompei et al. 2007a, b). Intestinal bacterial groups were enumerated using specific fluorescence in situ hybridization commercial kits (Microscreen, Groningen, The Netherlands) for the Lactobacillus group (Lactobacillus 10-ME-H006), the Bifidobacterium genus (Bifidobacterium 10-ME-H001), the Escherichia coli group (E. coli 10-ME-H004) and Enterococcus faecalis group (E. faecalis 10-ME-H015). Depending on the number of fluorescent cells, 30–100 microscopic fields were counted and averaged. All analysis was carried out in triplicate.

Statistical analysis

All values are expressed as means ± SD. Differences in TAAAA, TAALA, TEAC, TGSH, and SOD among the bacterial groups and differences in ROMs, GSH, and TAA among the dietary treatments were evaluated using the one-way ANOVA followed by Tukey’s post hoc comparisons. Differences in pH and bacterial concentration in feces were analyzed using two-way ANOVA with repeated measures with diet as the first factor and time as the second factor, followed by Bonferroni post hoc comparisons. Differences were considered significant at P < 0.05. Calculations were performed using commercially available statistical software packages (Statistic for Windows, StatSoft and graph GraphPad PRISM Version 2.0).

Results

Bacterial antioxidative properties

TAALA ranged between 2 and 37 % (median, 23 %; mean ± SD, 21 ± 11 %). The strains that showed values higher than the 75th percentile (31 %) belonged to S. thermophilus, Lactobacillus spp., and Lactococcus lactis (five, three, and one strains, respectively; Table 1). Due to the wide distribution of TAALA values, significant differences could not be established among S. thermophilus and the other groups (Fig. 1). Conversely, Lactobacillus and Lactococcus strains presented higher TAALA than bifidobacteria (P < 0.05). In particular, the TAALA of most Lactobacillus strains (above their 25th percentile) was higher than the median of all the tested strains (23 %), while the TAALA of all bifidobacteria was lower.

Fig. 1
figure 1

TAALA (a), TAAAA (b), TEAC (c), TGSH (d), and SOD (e) of 34 bacterial strains belonging to Bifidobacterium (n = 7), Lactobacillus (n = 11), Lactococcus (n = 6), and S. thermophilus (n = 10). Boxes indicate the median and 25th and 75th percentiles; whiskers indicate 10th and 90th percentiles. Dotted lines indicate means; means with a common symbol are not significantly different (P ≥ 0.05)

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Significant differences of TAAAA values could not be established among the groups (Fig. 1). TAAAA ranged between 0 and 82 % (median, 15 %; mean ± SD, 20 ± 17 %). Among all the tested strains, the ones presenting TAAAA higher than the 75th percentile (28 %) were Bifidobacterium breve WC 0424, Bifidobacterium animalis subsp. lactis DSMZ 23032, Bifidobacterium adolescentis DSMZ 18351, Lactobacillus reuteri ATCC 23272, Lactococcus lactis ssp. cremoris ATCC 19257, Lactobacillus coryniformis ATCC 25600, S. thermophilus ATCC 19258, Lactobacillus helveticus S 40.8, and Lactobacillus acidophilus ATCC 4356, in increasing order (Table 1).

TEAC was widely dispersed among the bacterial strains, ranging between 0 and 125 μM (median, 22 μM; mean ± SD, 32 ± 32 μM; Table 1). The strains presenting TEAC higher than the 75th percentile (49 μM) were L. reuteri ATCC 23272, L. acidophilus ATCC 4356, L. lactis MB 445, B. animalis subsp. lactis DSMZ 23032, L. lactis ssp. diacetilactis MB 447, L. lactis MB 446, L. lactis ssp. cremoris MB 461, S. thermophilus MB 418, and Lactobacillus brevis DSMZ 23034, in increasing order (Table 1). On average, TEAC values were higher (P < 0.05) in Lactococcus than in Bifidobacterium and S. thermophilus strains. In particular, the median of Lactococcus group (63 μM) was higher than the 75th percentile of all the strains, while most of S. thermophilus were below the median.

Intracellular glutathione (TGSH, oxidized plus reduced forms) ranged between 0 and 30.3 nmol/mg of proteins. With the exception of Lactococcus group and few other bacteria, most of the strains presented low levels, the median and the 75th percentile being 0.6 and 4.4 nmol/mg, respectively. The median of Lactococcus group (6.1 nmol/mg) was higher than the 75th percentile of all the tested strains. Most bifidobacteria did not produce detectable amounts of TGSH, whereas B. animalis subsp. lactis DSMZ 23032 gave 30.3 nmol/mg, the highest value obtained among all the strains. Other strains containing relevant amount of TGSH (≥90th percentile, 10.3 nmol/mg) were S. thermophilus MB 410, L. lactis ssp. diacetilactis MB 447, S. thermophilus EI-16, L. lactis ssp. cremoris ATCC 19257, B. animalis subsp. lactis DSMZ 23032, in increasing order (Table 1).

SOD activity was analyzed in the strains belonging to Lactococcus and S. thermophilus, which are expected to bear sodA or sodB genes based on nucleotide sequences database. SOD was found in all the bacteria belonging to these groups, being significantly higher (P < 0.05) in Lactococcus (median, 2.9 U/mg; mean ± SD, 2.4 ± 1.6 U/mg) than in S. thermophilus strains (median, 0.5 U/mg; mean ± SD, 0.7 ± 0.7 U/mg).

According to these results, B. animalis subsp. lactis DSMZ 23032, L. acidophilus DSMZ 23033, and L. brevis DSMZ 23034 were selected among the lactobacilli and bifidobacteria which exhibited promising antioxidative properties to prepare the probiotic formulation to be administered to rats.

In vivo effects of probiotic supplementation

In basal condition, the supplementation with the probiotic mixture at the dose of 108 cfu/day did not modify plasma ROM concentration (Fig. 2). In NS rats, DOXO treatment caused an oxidative stress, evidenced by the significant increase of plasma ROMs; conversely, no modifications in ROMs concentration were observed in probiotic supplemented rats after DOXO administration. In DOXO treated rats, plasma ROMs concentration was inversely related to the dose of the administered probiotics (r = 0.947, P < 0.05). Probiotic administration at the dose of 108 cfu/day did not modify plasma TAA in basal condition (Fig. 3). The oxidative stress induced by DOXO treatment caused a significant TAA decrease in NS and Pro107 rats with respect to NS in basal condition, while no differences were detected in the other probiotic supplemented groups. In DOXO-treated animals, TAA was significantly related to the dose of administered probiotics (r = 0.95; P < 0.05). As for the other measured parameters; in basal condition, plasma GSH concentration was not modified by probiotics supplementation (Fig. 4). DOXO administration caused a significant decrease of plasma GSH content in NS rats compared to the corresponding not-stressed counterparts, without affecting GSH level in probiotic supplemented animals. In DOXO-treated rats plasma GSH concentration was significantly related to the dose of administered probiotics (r = 0.948; P < 0.05).

Fig. 2
figure 2

Plasma ROMS concentration of not supplemented and probiotic supplemented rats, in basal condition and after DOXO administration. Values are means ± SD, n = 7. Statistical analysis was by the one-way ANOVA (ns) with Tukeys as post-test comparing NS rats to other groups (NS vs NS + DOXO; P < 0.05); the comparison among DOXO-treated rats was by one way ANOVA (ns)

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Fig. 3
figure 3

Plasma total antioxidant activity (TAA) of not supplemented and probiotic supplemented rats, in basal condition and after DOXO administration. Values are means ± SD, n = 7. Statistical analysis was by the one way ANOVA (ns) with Tukeys as post-test comparing NS rats to other groups (NS vs NS + DOXO; P < 0.001; NS vs Pro107 + DOXO; P < 0.01); the comparison among DOXO-treated rats was by one way ANOVA (P < 0.001)

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Fig. 4
figure 4

Plasma GSH concentration of not supplemented and probiotic supplemented rats, in basal condition and after DOXO administration. Values are means ± SD, n = 7. Statistical analysis was by the one way ANOVA (ns) with Tukeys as post-test comparing NS rats to other groups (NS vs NS + DOXO; P < 0.05); the comparison among DOXO-treated rats was by one-way ANOVA (ns)

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Kinetics of microbial groups and pH in feces

The fecal concentration of coliforms, enterococci, lactobacilli, and bifidobacteria was not affected by 18-day administration of NS diet (P ≥ 0.05; Fig. 5). The administration of probiotic supplements caused both coliforms and enterococci to decrease (P < 0.05), but differences among Pro107, Pro108, and Pro109 were not statistically significant (P ≥ 0.05). Compared to NS group, lactobacilli, and bifidobacteria remained unchanged (P ≥ 0.05) in Pro107 group, but increased by ∼1 magnitude order in Pro108 group and by two in Pro109 group. In this latter group, the mean counts of lactobacilli and bifidobacteria were 7.8 and 7.2 Log10 cfu/g, respectively. The pH of rat feces of NS and Pro107 groups was not affected by treatment (P ≥ 0.05), whereas it decreased from 6.8 to 6.4 in Pro108 and Pro109 groups (P < 0.05).

Fig. 5
figure 5

FISH counts of coliforms, enterococci, lactobacilli, and bifidobacteria and pH of rats feces. Bars indicate means ± SD at the beginning of the trial (dashed) and after an 18-day treatment with NS (white), Pro107 (light gray), Pro108 (dark gray), and Pro109 (black). Within each bacterial group and pH, means with a common symbol are not significantly different (P ≥ 0.05)

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Discussion

Accumulating evidence suggests that probiotics exert various biological roles through several mechanisms, one of the most debated being the antioxidant activity (Rossi and Amaretti 2010). In fact, among probiotics beneficial effects, the protection against oxidative stress in humans has been reported by several authors (Naruszewicz et al. 2002; Songisepp et al. 2005; Virtanen et al. 2007). In this light, the selection of specific strains and the evidence of their effectiveness, resulting in control of reactive radicals, can be exploited to formulate novel probiotic foods or supplements that can exert a role in the prevention of oxidative stress and related diseases.

This study indicates that the antioxidative properties of probiotic bacteria, measured according to the methods that are commonly used for lactic acid bacteria (Mishra and Kovachich 1984; Lin and Yen 1999; Kullisaar et al. 2002; Zanoni et al. 2008; Mikelsaar and Zilmer 2009), are strain-specific features. Both intact cells and cell-free extracts exhibited antioxidant properties. Within each microbial group, irrespectively by the methods used, wide dispersion of the values of antioxidative parameters was observed, indicating the strain specificity of this trait. For each strain, TAAAA, TAALA, and TEAC were unrelated to each other, and were not correlated to intracellular TGSH concentration or SOD activity. Such discrepancies may be due to the fact that these methods are based on diverse reactions which may be differently affected by specific molecular mechanisms responsible for defense against oxidative stress.

The screening resulted in a panel of strains exhibiting high values of antioxidative properties. The ones that were included in the probiotic formulation were selected within the genera Lactobacillus and Bifidobacterium, the most commonly used probiotic bacteria. This approach excluded several promising strains belonging to the genus Lactococcus and to the species S. thermophilus that presented very high levels of TGSH and SOD activity. Finally, the strains B. animalis subsp. lactis DSMZ 23032, L. acidophilus DSMZ 23033, and L. brevis DSMZ 23034 were selected, based also on their technological properties, so that they could be manufactured in adequate amount and incorporated in the lyophilized formula without loosing viability. B. animalis subsp. lactis DSMZ 23032 and L. acidophilus DSMZ 23033 contained the highest amount of TGSH among all the tested strains and lactobacilli, respectively. L. brevis DSMZ 23034 presented the highest intracellular TEAC among all the strains and high values TTALA and TAAAA within the Lactobacillus group (≥75th percentile).

The selected probiotic strains acted in concert to counteract the oxidative stress induced in the animal model. During the trial, no differences were detected in animal growth and in food consumption, and all animals appeared in a good state of health. In basal condition, the treatment with probiotics did not modify the tested markers of oxidative stress; on the contrary, the administration of probiotics protected rats against the DOXO-induced oxidative damage in a dose-related manner. In fact, in not supplemented rats DOXO administration caused an increase of ROM concentration and a decrease in TAA and GSH level, while no modification were detected in animals supplemented with the two higher probiotic concentrations compared to not supplemented, not stressed rats.

The probiotic strains colonized the colon of the rats during the trial. The marked decrease of fecal pH in the treated groups, which did not occur in the control group, indicates that the probiotic diets promoted the saccharolytic metabolism and the subsequent acidification of feces, due to the fermentation of carbohydrates to short chain fatty acids (Pompei et al. 2008). The decrease of coliforms confirmed the predominance of healthy saccharolytic microbial processes over the harmful proteolytic/putrefactive ones.

Given the colon colonization by administered probiotics, it is conceivable that their overall protective effect could be related to activities taking place at intestinal level, i.e., secretion of enzymes like SOD, metal-chelating activities, promotion of the production of antioxidant biomolecules such as exopolysaccharides showing an in vitro antioxidant and free-radical scavenging activities. Furthermore, the intestinal microflora provides additional enzymatic activities involved in the transformation of dietary compounds, thus increasing the bioavailability of dietary antioxidants (Davis and Milner 2009). Some authors hypothesize that probiotics exert their protective effects against oxidative stress by restoring gut microbiota (Nardone et al. 2010; Forsyth et al. 2009).

Notwithstanding, other mechanisms could also be at the basis of the antioxidant effect, as indicated by the maintenance of GSH level in stressed rats after probiotic supplementation. According to Spyropolous et al. (2011) probiotics may concretely enhance antioxidant defenses in the host, producing and releasing GSH and vitamins which are absorbed and distributed in the organism. Interestingly, probiotics supplementation in rats is able to induce transcription of genes involved in glutathione biosynthesis in the intestinal mucosa (Lutgendorff et al. 2009) and to increase glutathione synthesis in pancreatic cells (Lutgendorff et al. 2008). A similar effectiveness in restoring GSH concentration after an oxidative stress has been already reported (Peran et al. 2007) in rats supplemented with Lactobacillus casei, L. acidophilus, and Bifidobacterium lactis.

Although further studies are needed to elucidate the possible mechanisms involved, the herein reported study confirms the effectiveness of selected probiotic strains in the counteraction of oxidative stress, confirming previous studies in animals and humans (Hathout et al. 2011; Martarelli et al. 2011; Songisepp et al. 2005). This study supports the hypothesis that selected probiotic strains can underlay the enhancement of cellular antioxidant defenses in the host. The screening of physiological traits resulted still a powerful approach to assess the potential of probiotic bacteria for specific functions that can be subsequently verified in animal models. Acting this way, antioxidant probiotic strains can be selected and investigated as promising candidates for the prevention and control of several free radical-related disorders.