International Journal of Biological Macromolecules 70 (2014) 450–454
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Antioxidant activity of exopolysaccharide from probiotic strain Enterococcus faecium (BDU7) from Ngari Kaja Abdhul a,b , Mohan Ganesh a , Santhanam Shanmughapriya a , Murugesan Kanagavel a , Kumarasamy Anbarasu c , Kalimuthusamy Natarajaseenivasan a,∗ a
Medical Microbiology Laboratory, Department of Microbiology, Bharathidasan University, Tiruchirappalli-, 620024, India Department of Biotechnology, Nandha Arts and Science College, Bharathiar University, Erode-, 638009, India c Microbial Technology Laboratory, Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli-, 620024, India b
a r t i c l e
i n f o
Article history: Received 29 May 2014 Received in revised form 8 July 2014 Accepted 9 July 2014 Available online 22 July 2014 Keywords: Probiotics E. faecium Fish
a b s t r a c t “Ngari” is a traditional fermented fish of Manipur and considered for its therapeutic value in healing stomach ulcers. In the present study, an attempt was made to isolate and identify an efficient antioxidant probiotic isolate from Ngari. BDU7 with potent antioxidant property was isolated and characterized. The isolate was identified by 16S rRNA genotyping as Enterococcus faecium. E. faecium showed auto aggregation and hydrophobicity of 72.7 and 54.8% respectively. The extrapolysaccharide (EPS) was extracted from the culture free supernatant and assayed for its radical scavenging activity. The EPS showed significant 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (63.5%), superoxide (77.3%) and hydroxyl (38.4%) radical scavenging ability. The structural analysis of the extracted and purified EPS was performed by FTIR and NMR analysis. From the present study E. faecium BDU7 can be claimed as a promising and an efficient probiotic candidate. The present study evidenced that EPS from E. faecium BDU7 showed strong DPPH and superoxide radical scavenging ability in vitro. Considering its potency as a potential antioxidant the extracted EPS can find wide application in functional food and pharmaceutical formulations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Oxidation is an essential process in living organisms for energy production. Any abnormal formation of Reactive Species (RS) can cause damage in proteins, mutations in DNA, oxidation of membrane phospholipids, and modification in low-density lipoproteins [1]. Excessive amount of RS (both ROS and RNS) can result in cellular damage, which, in turn, promotes chronic diseases including atherosclerosis, arthritis, diabetes, neurodegenerative diseases, cardiovascular diseases and cancer [2,3]. 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
∗ Corresponding author at: Medical Microbiology Laboratory, Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli – 620 024, India. Tel.: +91 431 2407082; fax: +91 431 2407045. E-mail address:
[email protected] (K. Natarajaseenivasan). http://dx.doi.org/10.1016/j.ijbiomac.2014.07.026 0141-8130/© 2014 Elsevier B.V. All rights reserved.
antioxidant defenses is considered important in the maintenance of human health and disease prevention. In this scenario, a novel approach representing the development of probiotics exerting antioxidant activity and counteracting the oxidative stress in the host can be promising. Probiotics are live microbes which, when administered in adequate amounts, confer a health benefit to the host [4]. 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. 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. To provide health benefits, probiotic must be capable of surviving and colonizing the intestinal tract [5]. In order to survive in and colonize the gastrointestinal tract, probiotic should express high tolerance to acid and bile [6]. Probiotics are widely consumed to modulate and improve the gut microbiota balance, to respond to specific physiological targets [7], to prevent and treat pathogen-induced diarrhea [8], or to manage autoimmune and atopic diseases [9]. Among beneficial
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effects, several studies have reported antioxidant property [10–12]. 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. Exopolysaccharides (EPS) are polymers, produced by the microbes for their survival in the increased temperature and pH conditions. In medical and pharmaceutical industries, the bacterial polysaccharides are of great interest for their immunostimulatory, immunomodulatory, antitumor, antiviral, anti-inflammatory and antioxidant properties [13]. “Ngari” is a fermented fish product, which has been traditionally consumed in Manipur. The present study aims at isolating and identifying probiotcs with potential antioxidant activity from Ngari and to extract EPS with increased antioxidant activity for the well being of humans.
2. Materials and methods 2.1. Identification of potential probiotic strains The Ngari was purchased from the local markets of Manipur and processed at the Medical Microbiology Laboratory, Bharathidasan University, India. For isolation of associated bacteria, the fish samples were homogenized in 0.1% peptone saline and serially diluted 10 fold in physiological saline (0.9%) with 0.1% peptone. The serially diluted samples were plated onto Man Rogosa and Sharpe (MRS) agar. The inoculated plates were incubated at 37 ◦ C. Bacterial colonies that exhibited clear zone on the plates were individually picked and streaked on MRS agar and repeatedly sub-cultured for clonality. The selected isolates were identified biochemically by gram staining, lactic acid producing ability, catalase, oxidase activity, growth at 45 ◦ C in 6.5% NaCl and fermentation of wide range of sugars. The genotyping was performed using 16S rRNA sequencing as described by Brosius et al. [14] and Kostinek et al. [15].
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2.4. Hydrophobicity Hydrophobicity assay was performed according to Rosenberg [18]. Two milliliters of overnight bacterial suspension was transferred into a fresh tube and 0.4 ml of xylene was added. The tubes were vortexed for 2 min and were allowed to stand for 20 min. The lower aqueous phase was removed and OD600 nm was measured. Hydrophobicity (%), was calculated as per the equation [(A0 – A)/A0 ] × 100. Where, A0 and A were the absorbance before and after xylene extraction respectively. 2.5. Production and purification of EPS Enterococcus faecium BDU7 was grown in MRS broth with 2% (w/v) glucose and 3.5% NaCl (w/v) on a rotary shaker at 37 ◦ C for 72 h. The culture was centrifuged at 10,000 rpm for 30 min. The EPS was then precipitated from the supernatant overnight by the addition of equal volumes of cold isopropanol, centrifuged at 10,000 rpm for 20 min. The precipitate was washed twice with 70–100% ethanol. The precipitated EPS thus collected was dried at 60 ◦ C and stored at room temperature. Dried EPS was dissolved in sterile distilled water and dialysed. The carbohydrate content of the EPS was estimated as per Dubois et al. [19] and protein as per Lowry et al. [20]. 2.6. Antioxidant activity of EPS from E. faecium BDU7 The antioxidant activity of EPS was measured by hydroxyl [21], superoxide [22], DPPH radical scavenging activity [23]. The percentage of hydroxyl radical scavenging ability was calculated as 1 − [(As )/(Ab )] × 100. Where, As is the absorbance of sample, and Ab is the absorbance of blank. The percentage of superoxide radical scavenging ability was calculated as 1 − [(Ao)/A] × 100. Ao and A indicate the rate of pyrogallol auto oxidation before and after addition of sample. The percentage of DPPH scavenging ability was calculated as 1 − [(As )/(Ab )] × 100.
2.2. Acid and bile tolerance of the isolates
2.7. FTIR and NMR analysis of EPS
The acid and bile tolerance of the isolates were determined as described previously by Ben Salah et al. [16]. The cultures were grown in MRS broth at 37 ◦ C overnight. The cultures were centrifuged at 2000 × g for 10 min at 4 ◦ C. The pellets were washed twice in sterile phosphate-buffered saline (PBS, pH 7·2) and resuspended in MRS broth. For each isolate the culture suspension (0.1 at OD600 nm ) was added separately into series of tubes containing 2 ml of MRS broth at various pH of 3–5. Three tests, each with a duplicate was made for each isolates at each pH value. The OD600 nm was recorded every 1 h for 8 h. Bile tolerance was estimated using the same procedure except the isolates were inoculated in MRS broth containing bovine bile (1% and 5%). The MRS broth without bile served as a control.
The infrared spectrum of EPS was recorded on Bruker RFS, Switzerland. The spectra were scanned on 400–4000 cm−1 range. The spectra were obtained using potassium bromide pellet technique. Potassium bromide (AR grade) was dried under vacuum at 100 ◦ C for 48 h and100 mg of KBr with 1 mg of sample was taken to prepare KBr pellet. The spectra were plotted as intensity versus wave number. NMR spectrum was recorded in a Bruker Avance III with a 5 mm probe. The sample was dried in vacuum and dissolved in 0.7 ml of D2 O (99.96) at a concentration of 10 and 30 mg/ml for 1 H NMR and 13 C NMR respectively. 3. Results and discussions 3.1. Identification of potential probiotic strains
2.3. Auto aggregation assay Auto aggregation assay was performed according to Del Re et al. [17]. Overnight cultures were centrifuged at 2000 × g for 10 min at 4 ◦ C. The pellets thus obtained were washed twice in sterile phosphate-buffered saline (PBS, pH 7·2) and resuspended in PBS to a final OD600 nm of 0.5. The suspension (4 ml) was mixed by gentle vortexing for 10 s. Absorbance was measured immediately and after 1 h of incubation. The percentage of auto aggregation was expressed as: 1 − (At /A0 ) × 100, where At represents the absorbance at 1 h and A0 the absorbance at 0 h. All experiments were performed in triplicates.
The isolated colonies were characterized based on their morphology and biochemical characteristics. The isolates were phenotypically characterized as Bacillus spp., Bacillus coagulans, Bacillus cereus and E. faecium. The isolate E. faecium BDU7 was characterized as gram positive cocci, negative for catalase, oxidase, indole, hydrogen sulphide production and voges proskauer test, grew in medium containing 6.5% sodium chloride, no gas production from glucose and they ferment sucrose, fructose, arabinose, glucose and lactose. Further, the strain was characterized genotypically by 16S rRNA sequencing and submitted to NCBI gene bank with the accession number JX 847611. The phylogenetic analysis showed the sequence to cluster with E. faecium with a bootstrap
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Fig. 1. Phylogenetic analysis of 16S rRNA sequence of E. faecium BDU7.
Fig. 2. Antioxidant activity of purified exopolysaccharides from E. faecium BDU7. A) The extracted EPS exhibited DPPH, superoxide and hydroxyl radical scavenging ability in a dose dependent manner. X-axis: various concentration (mg/ml) of EPS; Y-axis: scavenging ability (%). B) The DPPH, superoxide and hydroxyl scavenging ability of ascorbic acid. C) The DPPH radical scavenging ability of EPS on comparison with ascorbic acid; ns: non-significant. D) The superoxide radical scavenging ability of EPS on comparison with ascorbic acid ns: non-significant. E) The hydroxyl radical scavenging ability of EPS on comparison with ascorbic acid ns: non-significant.
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3.3. Autoaggregation and hydrophobicity
Fig. 3. FT-IR spectra analysis of the purified EPS from E. faecium BDU7.
value of 0.05 and shared 98% sequence homology with E. faecium LMG 11423 (Fig. 1).
3.2. Acid and bile tolerance of the isolates The isolate E. faecium BDU7 showed acid tolerance and survived at pH value as low as 2.0 at least for 1 h but showed tolerance at pH values of 3–5. The gastric pH in healthy humans is about 2–2.5. This pH causes the destruction of most microorganisms ingested. Hence resistance to human gastric transit is an important selection criterion for probiotic bacteria. The resistance of the strains to high bile conditions was determined. The result showed 57.6 and 8.4% survival of E. faecium in 1 and 5% bile respectively. Resistance to bile is a prerequisite for colonization and metabolic activity of probiotic bacteria in the small intestine of the host. The physiological concentration of human bile is between 0.3% and 0.5%.
Fig. 4.
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The sedimentation rate of E. faecium BDU7 was measured after 1 h. Results showed that the strain exhibited a strong auto aggregation of 72.7%. The hydrophobicity analysis showed the isolate to be strongly hydrophobic with 54.8% adhesion to xylene. Auto aggregation ability is related to the cell adherence properties of the isolates. Thus increased autoaggregation plays an important role in the adhesion of bacterial cells to intestinal epithelium and it increases the chance of bacterial maintenance in the gastrointestinal tract [17]. Difference in the aggregation abilities has been reported among species of same genera and strains of the same species [24]. Hydrophobicity is an important attribute which helps the probiotics to colonize and modulate host immune system. Further hydrophobicity contributes adhesion of bacterial cells to host tissue [25]. The isolate E. faecium BDU7 showed increased autoaggregation and hydrophobicity validating the isolate to be a potential probiotic strain.
3.4. Antioxidant activity of EPS The superoxide and hydroxyl radicals are considered to be a highly potent oxidant and can react with all biomolecules. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The scavenging activity of the extracted exopolysaccharides was determined by hydroxyl, DPPH and super oxide radical scavenging activity (Fig. 2). The free radical scavenging activity of EPS increased with increasing concentration. The scavenging ability was found to be 63.5, 77.3 and 38.4% for DPPH, superoxide and hydroxyl radicals respectively at a concentration of 8 mg/ml. The extracted EPS exhibited equal scavenging activity compared to the known standard ascorbic acid Fig. 2. The EPS of Lactobacillus. lactis subsp. lactis 12 showed antioxidant activity equal to that of the control ascorbic acid [26]. Kodali and Sen [27] reported the EPS of probiotic strain B. coagulans Rk-02 to have strong super oxide and hydroxyl radical activity in vitro.
C NMR spectrum of purified EPS recorded in D2 O (99.96) using BrukerAvance III with a 5 mm probe.
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3.5. FTIR and NMR elucidation The FTIR of the extracted EPS was analyzed (Fig. 3). The EPS shows peaks that are ranging from 3942 to 687. The peaks are not observed in the region of 260–290 cm−1 it clearly indicates the sample does not have any proteins and nucleic acids. It shows the peak at 3390 cm−1 , which is characteristic for O–H existed in hydrogen bond of polymer [28]. A weak C–H stretching band observed at 2959 cm−1 . Further an asymmetrical weak peak was noticed at 1660 cm−1 and 1646 cm−1 , which were assigned to the stretching vibration of the carboxyl group(C O) [29]. The peak at 1005 cm−1 indicates for d-Glucose in Pyranose form. The peaks in the regions of 1005–1268 cm−1 indicate the presence of carbohydrates. Peaks corresponding to the region of 925–865 cm−1 are responsible for the linkage between the carbohydrates. The peak at 1075 cm−1 confirms the presence of polysaccharide [30]. The obvious absorption peaks at 924 cm−1 and 933 cm−1 revealed the ␣ and -glycosidic bonds [31,32]. The 1 H NMR spectrum of EPS showed peak at 4␣ (ı 5.32, 5.30, 5.14 and 5.13 ppm) and 4 (ı 4.6, 4.56, 4.54 and 4.13) anomeric protons. The peak at ı 5.32 ppm and ı 4.6 ppm corresponds to ␣– glucopyranosyl reducing end and the signal at ı 5.13 ppm for ␣ fucopyranosyl unit. The 13 C NMR spectrum of EPS is shown in Fig. 4. The 13 C spectrum showed four anomeric carbon peaks in the region of ı 103–92 ppm. The signal at ı 92.6 and 95.1 ppm corresponds to the anomeric carbon region of glucopyranosyl unit reducing end. The carbon resonance at ı 19 ppm and ı 181.9 ppm was assigned to methyl and carboxyl groups. The signal at ı 76.4 ppm corresponds to C-2 substituted mannose residue. The signal at ı 61.3 and ı 64.2 ppmn was assigned for C6 substituted mannose units [33,34]. The position of the C-6 -d-mannopyranosyl unit was observed at ı 6.8 ppm and ı 68.1 ppm for -d-mannopyranosyl branched at O-6 [35–38]. 4. Conclusion The E. faecium BDU7 isolated from Ngari showed acid and bile tolerance establishing a possibility of the isolate to be considered as a probiotic strain. EPS are important extracellular bioactive molecules with potential biological and therapeutic activities. The present study evidenced that EPS from E. faecium BDU7 showed strong DPPH and superoxide radical scavenging ability in vitro. Considering its potency as a potential antioxidant the extracted EPS can find wide application in functional food and pharmaceutical formulations. Acknowledgments Authors acknowledge SAIF IIT-M for their help in NMR studies and interpretation. We also extent our thanks to the
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