ANTIOXIDANT ACTIVITY OF THE PROBIOTIC CONSORTIUM IN VITRO

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1 International Journal of Probiotics and Prebiotics Vol. 9, No. 1/2, pp , 2014 ISSN print, Copyright 2014 by New Century Health Publishers, LLC All rights of reproduction in any form reserved ANTIOXIDANT ACTIVITY OF THE PROBIOTIC CONSORTIUM IN VITRO A. Kushugulova, S. Saduakhasova, S. Kozhakhmetov, G. Shakhabayeva, I.Tynybayeva, T. Nurgozhin, F.Marotta and Zh. Zhumadilov The Center for Life Sciences Nazarbayev University, 53, Kabanbay batyr Ave., Astana, Republic of Kazakhstan, [Received May 23, 2012; Accepted June 25, 2012] ABSTRACT: The antioxidant and antigenotoxic properties of the probiotic consortium, including the strains Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum, were investigated. According to the results of our research the total antioxidant capacity (TAC) of the intact cells in the probiotic consortium was high 15.3 mmol/ml, glutathione reductase activity was U/ml, Superoxide dismutase activity was not revealed. The total antioxidant capacity of the cell lysates in the probiotic consortium is 11.1 mm/ml, glutathione reductase activity 0,008 units/ml, superoxide dismutase activity 0.24 U/mg. Co-incubation of the epithelial cells with the probiotic consortium reduces the percentage of damaged cells (DI - 0.6). KEY WORDS: probiotic, total antioxidant capacity, Trolox, superoxide dismutase, glutathione reductase, DNA comet assay Corresponding Author: Almagul Kushugulova, DMSc, Translational medicine and Longevity Department, Nazarbayev University 53, Kabanbay batyr Ave., Astana, Republic of Kazakhstan ; fax: ; akushugulova@nu.edu.kz INTRODUCTION Reactive oxygen species (ROS) play an important role in biological system. Accumulation of the free radicals cause damage of the cells and eventually leads to such diseases as cancer, cardiovascular diseases (CVD), allergies, atherosclerosis, and are also the major cause of aging (Cao et al., 1995; Agerholm-Larsen et al., 2000). Free radicals reactions are based on the peroxidation of lipids in cell membranes (oxidative degradation of lipids), denaturation of proteins and nucleic acids. Free radical oxidation is a constant process in human tissues and organs, but it does not lead to excessive formation of the ROS and cell damage. It is important to maintain this process on a baseline level. The system of the antioxidants and chelator of metal ions with mixed valence maintain this homeostasis. However, in case of a huge influx of ROS the system is not able to prevent its detrimental effect (Halliwell and Chirico, 1993). Therefore, the native individual antioxidant system requires constant intake (though the diet) of the antioxidants to maintain important physiological processes and enable organism functions. According to the previous research in this area, probiotics affect different biological processes though the different pathways including the antioxidant activity (Rossi and Amaretti, 2010). Several authors reported positive protective effect of probiotics from the oxidative stress in humans (Naruszewicz et al., 2002; Songisepp et al., 2005; Virtanen et al., 2007). Most lactic acid bacteria have special systems that can cooperate with the reactive oxygen species. Stecchini (Stecchini et al., 2000) suggests that these systems include enzyme superoxide dismutase and elevated level of intracellular Mn +2. Recent studies have shown that antioxidant properties are strain- specific (Kushugulova et al., 2013). The ability of the starter cultures in dairy products to reduce the level of free radicals is a useful feature in functional foods. The range of the antioxidant products for consumers can be expanded at the expense of probiotics. They provide antioxidants to the organism throughout the entire period of their presence and colonization of the gastrointestinal tract. The genome of the living organism is exposed to constant attacks of the variety of the physical and chemical factors such as environment and products of its own metabolism, that cause DNA damage of the cell. From the other side, DNA damage initiates the cascade of the biological reactions at the cellular and organ levels. Increase in the environmental pollution with the genotoxic agents and treatment with toxic DNA-damaging chemotherapy drugs lead to accumulation of the DNA damage, suppression of the DNA repair systems that cause mutations and subsequent oncogenesis. It is well known that the probiotic cultures have DNA -

2 56 Antioxidant activity of the probiotic consortium in vitro protective effect. Probiotic bacteria prevent the effect of dietary carcinogens. In controlled clinical trial Lactobacillus casei strain Shirota was used as a probiotic agent administered orally in patients with superficial bladder cancer. It was shown not only to reduce the risk of cancer development, but also its recurrence after transurethral resection (Seiji Naito, 2008). The main goal of this research is to investigate the total antioxidant capacity of the consortium of probiotic bacteria isolated from the traditional Kazakh milk products, determine the activity of superoxide dismutase and glutathione reductase and evaluate DNA - protective effect. MATERIALS AND METHODS Bacterial strain and culture preparation The object of this research is a probiotic consortium that includes bacterial cultures such as Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum. The components of the consortium are isolated from the traditional Kazakh national dairy products - aryan, kumys, shubat and healthy clinical material. Probiotic consortium was cultured in broth medium MRS (Fluka) at 37 C for 20 hours under microaerophilic conditions. The antioxidant properties of the intact cells of the consortium and their lysates obtained by destroying the cell wall were studied. 20- hour inoculum probiotic Consortium (10 9 cfu / ml) was centrifuged at 5000-x g for 5 min at 4 C. The supernatant was removed, the precipitate was washed twice with distilled water and resuspended in buffer. Half of the homogenized sample was collected in a separate vial and sonicated to obtain lysate. Cell walls were destroyed using ultrasonic disintegrator (Ultrasonic processor UP 100 H, Heilscher) on ice under the mode of 10 cycles, 30 seconds each, with a pause between each cycle of 1 min. Further the solution was centrifuged at x g for 5 min at 4 C. The supernatant was used to determine the antioxidant activity. The rest of the suspension containing intact cells was adjusted to the concentration of 10 9 CFU / ml and was also used to determine the antioxidant activity. Total antioxidant capacity (TAC) in vitro using Trolox Antioxidant Assay Kit (Sigma-Aldrich) was used to determine the TAC. Water-soluble vitamin E analogue - Trolox was used as a reference. The following concentrations were prepared for the Trolox standard curve: 0.0 mm, mm, mm, mm, 0.21 mm, 0.42 mm. Then 10 µl of each sample were incubated with ABTS solution, metmyoglobin and hydrogen peroxide for 5 minutes. Then the reaction was terminated by Stop Reagent and absorbance was measured at 405 nm (Spectrophotometer). Prepared samples were added to experimental samples instead of Trolox in the same volume and analyzed according to the procedure described above. The concentrations of antioxidants in each sample were defined using standard curve linear regression equation: X (mm) = y (A405) - Intercept Slope * dilution factor X (mm) - the concentration of antioxidants y (A405) - mean optical density of the sample at 405 nm Intercept - intersection point of the standard curve with Y axis (0.6219) Slope - the slope of the calibration curve, a negative value ( ) Dilution factor- fold dilution of the original sample Superoxide dismutase activity (SOD) SOD determination kit (Sigma) was used to determine the superoxide dismutase activity. Required solutions were prepared according to the instructions and added to each well. Then plates were incubated at 37 C for 20 minutes and the absorbance was measured at 450 nm. SOD activity (% inhibition) was calculated using the following equation: SOD activity (% inhibition) = {[(A blank 1 - A blank 3 ) - A sample - A blank 2 )] / (A blank1 - A blank 3 )} x 100 Glutathione reductase activity Glutathione Reductase Assay Kit (Sigma) was used to determine glutathione reductase activity. The spectrophotometer kinetic program for UV-analysis was set up according to the intsructions: wavelength nm, the initial delay - 10 seconds, interval - 10 seconds, the number of readings Further, the following solutions were introduced step by step to the cuvette: 500 µl of 2 mm Oxidized Glutathione, 350 µl Assay Buffer, 100 µl of sample, 50 µl of 2 mm NADP. Positive control was prepared by adding µl of Glutathione Reductase Positive Control Solution for the control reaction. Assay Buffer instead of the enzyme sample solution was used as blank. The spectrophotometer detects the kinetic reaction rate automatically. The concentration of the glutathione reductase was calculated using following equation: U / ml = (ΔA sample - ΔA blank ) x (dilution) / e mm x ( sample volume in ml). The extinction coefficient of NADPH (340 nm) - e mm = 6.22 cm 2 /mkmol Comet assay in vitro Comet assay in vitro was used to determine the antigenotoxic effect of the consortium. This method is based on the electrophoresis of the lysed cells with damaged DNA. Human fibroblasts were used as a sample. Fibroblast cell suspension ( cells) was centrifuged at x g for 5 min and resuspended in 1 phosphate- buffered saline (without Ca 2+, Mg 2+ ) to the final concentration of cells. Then, 200µl of fibroblasts were incubated with the test samples of the consortium and treated with peroxidase (0.8 mm). After centrifuging, fibroblasts with test samples were resuspended in a Buffer solution, mixed with molten agarose gel and applied to gel-slides (OxiSelect Comet Assay Slides 3-Well). Fixed

3 Antioxidant activity of the probiotic consortium in vitro 57 cells were treated with Lysis Buffer and alkali solution for DNA denaturation. Electrophoresis was performed in a horizontal chamber at the voltage 1 volt / cm for 10 min. Slides was immersed in 70% ethanol and air-dried to remove the residuals of the TBE buffer. Samples were stained with SYBR Green dye to visualize DNA and assessed under the fluorescence microscope. The image analysis was performed using special software (Comet Score) and the percentage of DNA in the comet tail was determined. Statistical analysis Statistical analysis was performed using software STATISTICA (Statsoft). The significance of differences between the assessed groups was determined by Student's t-test after normal distribution evaluation. Data with P-value < 0.05 was considered statistically significant. RESULTS and DISCUSSION The antioxidant properties of the probiotic consortium consisting of the bacteria strains Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum were investigated. According to the obtained data, a new milk product for daily consumption was developed. The product was proved to have therapeutic and prophylactic effect. Total antioxidant capacity and activity of the antioxidant enzymes - superoxide dismutase and glutathione reductase were determined and antigenotoxic effect was evaluated. Total antioxidant capacity (TAC) in vitro using Trolox Assay relies on the property of antioxidants to inhibit oxidation of the stable radical 2,2- azinobis (3- ethylbenzthiazoline -6- sulfonic acid) ABTS after incubation with peroxidase (metmyoglobin) and oxidase (hydrogen peroxide). During the oxidation process the stable ABTS + cation radicals (bluish-green color) are produced. The intensity of the staining is inversely proportional to the amount of antioxidants in the sample. The lower the staining the higher level of antioxidants is present in the sample (Belyakov&Semesko, 2005).Using this method the results are easily reproducible over the time (Rice-Evans, 2000). Data from the colorimetric analysis was obtained. The concentration of the antioxidants in a sample was calculated in respect to the standard Trolox concentration in mm. The total antioxidant capacity of the intact cells in the probiotic consortium is 15,3 mmol / ml, TAC of the lysates 11,1 mmol / ml. According to the obtained data, the following conclusions have been made: first, the investigated consortium possesses antioxidant activity, and second, the total antioxidant capacity of whole cells of the consortium is higher than the activity of their lysates. The samples were examined for SOD and glutathione reductase activity to define the mechanism of the antioxidant effect of the consortium. Superoxide dismutase activity Superoxide dismutase (SOD) is one of the most important antioxidative enzymes. It catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen. In order to determine the SOD activity, nitroblue tetrazolium (NBT) catalyzed by superoxide dismutase is reduced to the formazan by superoxide radicals that are generated by the reaction of enzyme oxidation of xanthine to the uric acid in the presence of xanthine oxidase. The level of the inhibition of this reaction is determined by measuring the decrease in color at 450 nm. We found that the superoxide dismutase activity of the probiotic lysates is 0.24 units per mg of protein. The intact cells revealed no SOD activity. Glutathione reductase activity In this study, the ability of the consortium components to express the glutathione reductase was evaluated and data are expressed as units per ml (Table 1). Glutathione reductase activity in lysates was higher than in intact cells. Level of the glutathione reductase correlates with the degree of resistance to oxidative stress (Table 1). TABLE 1. Indicators of kinetic rate in determination of glutathione reductase activity Sample Glutathione reductase, Units/ml Consortium cells Consortium lysate Positive control DNA - protective effect One of the key indicators of the functional efficiency of the consortium is DNA-protective effect. Gel-slides images from in vitro comet assay are shown on Figure 1. Treatment with consortium (Figure 1A) resulted into significant decrease in comet formation compared to the control group (Figure 1B). The calculated index of damage in the consortium group was 0.6 suggesting a pronounced DNA - protective effect. FIGURE 1. Comet Assay to evaluate effect of consortium treatment on DNA damage. A: Consrtium-treated group; B: Control group.

4 58 Antioxidant activity of the probiotic consortium in vitro DISCUSSION It is well known that some strains of Lactobacilli and Bifidobacteria possess antioxidant activity (Kushugulova, 2010; Kaizu et al, 1993; Korpela et al, 1997; Kullisaar et al., 2002) and may increase the protective properties of mucosa. This in turn improves the stress resistance of the body and enhances the immune response. The efficiency of the antioxidant activity varies among the cultures, and is strain-specific as described in the study of many authors such as Kullisaar et al., 2002; Lin and Yen, 1999; Mikelsaar and Zilmer, As demonstrated in the various studies the antioxidant activity of the bifidobacteria is higher than that of the Lactobacilli. Similar elevated activity was also found in dairy streptococci. Therefore, the presence of the active streptococci, lactobacilli and bifidobacteria in the probiotic consortium guarantees high antioxidant activity. The results of our studies defined a high level of total antioxidant capacity in the samples with the intact cells of the consortium. TAC was determined in respect to Trolox standard and is equal to 15.3 mmol / ml. According to the work of Virtanen et al (2007), a combination of the following cultures: Leuconstoc cremoris, Lactococcus lactis ATCC19435 and Lactobacillus acidophilus ATCC4356 showed a higher antioxidant activity in comparison with the combinations of Leuconstoc cremoris B and L.lactis ATCC 1943, TAC value was equal to 0,86 mmolxl - one and 0,16 mmolxl -, respectively. Thus, the total antioxidant capacity of our probiotic consortium is much higher than in consortium of the Leuconstoc cremoris, Lactococcus lactis ATCC and Lactobacillus acidophilus ATCC This difference is due to the presence of bifidobacteria as the antioxidant activity of some strains amplifies in the presence of others. This is called a pronounced synergistic effect. Most of the studies in this area are based on the assessment of the antioxidant properties of the probiotics cell lysates. Saide and Gilliland (2005) studied the antioxidant activity of the 19 cultures of lactic acid bacteria. They found out that the cell lysates had higher activity than the intact cells. They also proposed that the substances resulting from bacterial cell lysis by the bile acids would affect the walls of gastrointestinal tract directly and thus, provide more pronounced beneficial effect. Intracellular extracts of the intestinal strains Bifidobacterium longum and Lact.acidophilus were shown to have by 15% more inhibitory effect on the lipid peroxidation than the intact cells (Lin and Chang, 2000). TAC of the cell lysate in our probiotic consortium was equal to 11.1 mmol/ml. The antioxidant activity of the lysates was slightly lower than the activity of the probiotic intact cells. The data obtained is not consistent with the previous research in this field. Overall, the probiotic consortium of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum has been shown to have high antioxidant capacity. Superoxide dismutase, catalase and glutathione redox cycle enzymes are the major antioxidant enzymes. They prevent the oxidative stress and excess production of the free radicals that cause cell damage and cell death. Antioxidant capacity of the probiotic bacteria has not been found until recently and the mechanisms underlying this process are still under investigation. One of the mechanisms for the inactivation of the free radicals by the bacteria is SOD production. SOD prevents the formation of the toxic superoxide anion and catalyzes the dismutation reaction of the superoxide anions to hydrogen peroxide. Previous research data shows that Lactobacillus produce SOD. In our study, the superoxide dismutase activity in the probiotic consortium lysates was shown to be 0.24 U/ mg protein. In comparison to the L. fernentum E- 3 and L. fernentum E- 18 (Kullisaar et al., 2002), where these values are 0.85 units/mg and U/mg protein, respectively, the superoxide dismutase activity of the proposed consortium is low. No SOD activity was observed in the intact cells of the probiotic consortium. This is typical for the lactic acid bacteria. Another component of the antioxidant defense system is represented by tripeptide - glutathione. Glutathione is involved in the maintenance of the cell membrane integrity and removal of the toxic substances. It also protects the cell from the hydrogen peroxide and hydroxyl radicals (Bruno- Barcena et al., 2004). One of the important components of the glutathione system is glutathione reductase. Its main biological role is to maintain high intracellular concentration of the reduced glutathione. In contrast to the extensive information associated with the glutathione in eukaryotic cells, relatively little is known about the glutathione in prokaryotes (Kullisaar et al., 2010). Glutathione reductase activity was detected in the L. lactis (Li et al., 2003) and Lactobacillus fermentum ME 3 (Kullisaar et al., 2010). Strains of the Streptococcus thermophiles and Enterococcus faecalis showed increased glutathione reductase activity at high oxygen concentrations (Patel., 1998). Therefore glutathione reductase plays an important role in the effective response against oxidative stress. Glutathione reductase activity of the studied probiotic consortium was low. Its activity in the probiotic lysates was two times higher than in the intact cells and reached 0,008 units/ml. However, Li Y. et al (2003) showed that even low concentrations of the intracellular glutathione (1 to 10 μm) led to a significant protection of the L. lactis from the hydrogen peroxide. A number of studies have demonstrated that several lactic acid bacteria and bifidobacteria possess DNA-protective effect. It was also shown that consumption of yoghurt with the Lactobacillus acidophillus 145 and Bifidobacterium longum 913 reduced the DNA-damage in the cells of the human colon (Oberreuther-Moschner et al., 2004). According to the studies of Anthony J. the strains of the Lact. plantarum and Bifidobacterium Bb12 have antigenotoxic potential and may have protective effect against the colorectal cancer in the early stages (Anthony et al., 2004). In the present

5 Antioxidant activity of the probiotic consortium in vitro 59 study, we investigated the DNA protective effect of probiotic consortium using DNA comets assay. The results indicated that co-incubation of the epithelial cells with the probiotic bacteria reduced the percentage of the damaged cells. Damage index of the consortium was 0.6 that proves its antigenotoxic effect. CONCLUSION The results of this research demonstrated a high level of the total antioxidant capacity of the intact cells of the probiotic consortium that includes Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium bifidum (15,3 mmol/ml). Total antioxidant capacity in the probiotic lysates was 11.1 mmol/ml. Superoxide dismutase activity of the probiotic lysates (0.24 U/mg protein) and glutathione reductase activity (0,008 U/ml) were determined. SOD activity of the intact cells of probiotic consortium was not observed, that is typical for lactic acid bacteria. Glutathione reductase activity of the intact cells of probiotic consortium was 0,004 U / ml. Based on the obtained data the co- incubation of the epithelial cells with the probiotic bacteria reduces the percentage of the damaged cells (DI - 0.6). Thus, the studied probiotic consortium has an important antigenotoxic and antioxidant effect. Supplements and daily use products based on this probiotic consortium can be a useful protective component in the bacterial flora of the gastrointestinal tract. ACKNOWLEDGEMENTS ffthis work was supported by funding from the Ministry of Education and Science of the Republic of Kazakhstan under the subproject 0111PK00516 «Development of moleculargenetic predictors of anti-aging medicine». REFERENCES Agerholm-Larsen L., Raben A., Haulrik N., Hansen, A.S., Manders M. and Astrup A. (2000). Effect of 8 week intake of probiotic milk products on risk factors for cardiovascular diseases. European Journal of Clinical Nutrition 54: Anthony J., Burns Ian, Rowland R. (2004). Antigenotoxicity of probiotics and prebiotics on faecal water-induced DNA damage in human colon adenocarcinoma cells. Mutation Research 551: Bruno-Barcena J.M., Andrus J.M., Libby S.L., Klaenhammer T.R. and Hassan H.M. (2004). Expression of a heterologous manganese superoxide dismutase gene in intestinal lactobacilli provides protection against the toxicity of hydrogen peroxide. Applied and Environmental Microbiology 70: Belyakov N.A., Semesko S.G. (2005). Antioxidant activity of human biological fluids: methodology and clinical significance. Efferent therapy. 1: Cao G., Verdon C.P., Wu A.H.B., Wang H., Prior R. L. (1995). Automated oxygen radical absorbance capacity assay using the COBAS FARA II. Clinical Chemistry 41: Halliwell B, Chirico S. (1993). Lipid peroxidation: its mechanism, measurement, and significance. The American Journal of Clinical Nutrition 57: Kaizu H., Sasaki M., Nakajima H. and Suzuki Y. (1993) Effect of antioxidative lactic acid bacteria on rats fed a diet deficient in vitamin. Journal of Dairy Science 76: Korpela R., Peuhkuri K., Lähteenmäki T., Sievi E., Saxelin M. and Vapaatalo H. (1997). Lactobacillus rhamnosus GG shows antioxidative properties in vascular endothelial cell culture. Milchwissenschaft. 52: Kullisaar T., Songisepp E., Aunapuu M., Kilk K., Arend A., Mikelsaar M., Rehema A. and Zilmer M. (2010). Complete Glutathione System in Probiotic Lactobacillus fermentum ME 3. Applied Biochemistry and Microbiology 46:5: Kullisaar T., Zilmer M., Mikelsaar M., Vihalemma T., Annuk H., Kairanea C., Kilkclisaar A. (2002). Two antioxidative lactobacilli strains as promising probiotics. International Journal of Food Microbiology 72: Kushugulova A., Kozhakhmetov S., Supiyev A., Shakhabayeva G., Saduakhasova S., Sabitkyzy S., Gulayev A., Nurgozhin T., Zhumadilov Zh. and Sharman A. (2013) Isolation and characterization of lactobacilli from traditional kazakh dairy products. International Journal of Probiotics and Prebiotics 8: 2-3. Kushugulova A.R. (2010) The antioxidant properties of bacteria of the genus Lactobacillus spp. Health and disease Kazakh Academy of Nutrition 2:79-83 Li Y., Hugenholtz J., Abee T., and Molenaar D. (2003) Glutathione protects Lactococcus lactis against oxidative stress. Applied and Environmental Microbiology 69: Lin M.Y., Chang F.Y. (2000). Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC15708 and Lactobacillus acidophilus ATCC4356. Digestive Diseases and Sciences 45: Lin M.Y., Yen C.L. (1999). Antioxidative ability of lactic acid bacteria. Journal of Agricultural and Food Chemistry 47: Mikelsaar M., Zilmer M. (2009) Lactobacillus fermentum ME-3 - an antimicrobial and antioxidative probiotic. Microbial

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