An Anti-Shigella Dysenteriae Bacteriocin from Pediococcus Pentosaceus MTCC 5151 Cheese Isolate

R. AGRAWAL, S. DHARMESH Turk J Biol 36 (2012) 177-185 © TÜBİTAK doi:10.3906/biy-1010-142 An anti-Shigella dysenteriae bacteriocin from Pediococcus pentosaceus MTCC 5151 cheese isolate Renu AGRAWAL1, Shylaja DHARMESH2 1Department of Food Microbiology, Central Food Technological Research Institute, Mysore 570020 - INDIA 2Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570020 - INDIA Received: 13.10.2010 Abstract: A cheese isolate Pediococcus pentosaceus lactic acid bacterium, which has been deposited at the Microbial Type Culture Collection Centre Chandigarh with the accession number MTCC 5151, was tested for anti-Shigella dysenteriae activity and the bacteriocin was characterized. Th e protein band was observed with tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as a single band with a molecular mass of 23 kDa. Th is is a new and novel bacteriocin that inhibits S. dysenteriae and has not yet been reported from P. pentosaceus. It was purifi ed on a Sephacryl column and the active fraction specifi c for anti-Shigella dysenteriae with 23 kDa was found and confi rmed via liquid chromatography mass spectrometry (LC-MS). Th e eff ect of various physical parameters on bacteriocin activity was also studied, with the optimum conditions being determined at a pH level of 5.5 with an 18-h-grown culture. Key words: Bacteriocin, Pediococcus pentosaceus, purifi cation, anti-Shigella dysenteriae Introduction side eff ects. Pediococci spp. are saprophytes and are Shigellosis is a disease occurring in unhygienic known to preserve products by inhibiting pathogens conditions. As Shigella has acquired multiple (5). Detailed biochemical characterization of such antimicrobial resistances, it presents a challenge for bacteriocins has not been performed to evaluate their eff ective clinical management (1). Acute shigellosis potential use as food preservatives. Other pediocins symptoms include sepsis, intestinal perforation, (6-9) have been purifi ed previously. Th e present toxic megacolon, dehydration, hyponatremia, work aims to isolate and characterize a specifi c, encephalopathy, hemolytic uremic syndrome, extracellular bacteriocin from a native culture isolate and pneumonia (2). Research into alternative (Pediococcus pentosaceus, MTCC 5151) in terms of therapy for this disease indicates that using lactic its ability to inhibit shigellosis. acid bacteria (LAB) is a good approach. Th ese bacteria are generally recognized as safe (GRAS) Materials and methods and produce bacteriocins that are used as natural food preservatives (3). According to Diekema et Microbiologic methods al. (4), the bacteriocins produced by lactic acid Microorganisms bacteria are useful for the treatment of multi-drug- A number of diff erent lactic acid bacteria such as resistant infections caused by Staphylococcus spp. Leuconostoc mesenteroides, Lactobacillus plantarum, and Enterococcus spp. Lactococcus lactis, and Pediococcus pentosaceus were Th is bacteriocin may represent a potential screened for bacteriocin activity against a Shigella substitute for antibiotics that would involve no dysenteriae sample obtained from JSS Medical College 177 An anti-Shigella dysenteriae bacteriocin from Pediococcus pentosaceus MTCC 5151 cheese isolate in Mysore, India. All of the lactic acid bacterial Optimization of colony counts of P. pentosaceus cultures were grown in MRS broth (Hi-Media Pvt. against S. dysenteriae Ltd., Mumbai, India) at 37 °C for 18 h under static In order to optimize our experiment, the culture conditions while the Shigella dysenteriae sample was supernatant was taken from varying cell numbers kept in BHI broth (Hi-Media Pvt. Ltd.). Th e cultures 1 × 108, 2 × 108, 1 × 109, and 1 × 1010 CFU/mL-1 of were subcultured every 15 days and were stored at P. pentosaceus. Th is allowed us to determine most 6 4 °C. For subculturing, a loop of bacteria (10 CFU/ effi cient count for killing S. dysenteriae colonies, as a -1 mL ) was transferred into 5 mL of MRS broth and constant number of S. dysenteriae (1 × 108 CFU/mL-1) grown until reaching the exponential phase. cells was used in all cases. Chemicals Anti-Shigella dysenteriae activity of P. pentosaceus Chemicals used in this study were reagent grade during the diff erent growth phases of S. dysenteriae and purchased from Hi-Media Pvt. Ltd. High- performance liquid chromatography-grade S. dysenteriae was grown for 48 h, during which time acetonitrile, acetone, and methanol were purchased the diff erent growth phases and colony numbers were from Sigma Aldrich Co. (Bangalore, India and counted and tested for anti-S. dysenteriae activity Missouri, USA) against a known number of P. pentosaceus colonies by well diff usion assay. Selection of the LAB strain Diff erent LAB strains such as Leu. mesenteroides, L. Anti-Shigella dysenteriae activity of P. pentosaceus plantarum, Lc. lactis, and P. pentosaceus had been with increasing colony counts of S. dysenteriae previously isolated and identifi ed by biochemical S. dysenteriae colony-forming units were gradually and physiological methods and 16S rRNA gene increased from 3 × 108 CFU/mL-1 to 18 × 108 CFU/ sequencing performed in our laboratory. Th e strains mL-1 against a constant number of P. pentosaceus were screened for anti-S. dysenteriae activity. Th e (3 × 108 CFU/mL-1) to observe the inhibition of S. greatest activity was found with P. pentosaceus and dysenteriae at greater colony numbers. this strain was therefore taken up for all further Bacteriocin extraction from the culture medium studies. Growth curve of P. pentosaceus Anti-S. dysenteriae protein was extracted from the culture strain of P. pentosaceus (18 h) in MRS broth P. pentosaceus was inoculated in MRS broth with (pH 5.5) with citrate phosphate buff er in order to 1% inoculum and incubated at 37 °C for 24 h. Th e maintain the pH of the medium. Th is is a modifi cation absorbance was measured every 2 h at 600 nm and of the method described by Daba et al. (10). the viable colony counts were checked on MRS agar plates. Th e cell-free supernatant was collected by Growth curve of S. dysenteriae centrifugation at 3000 rpm for 20 min in a refrigerated centrifuge kept at 5 °C (Sorvall RC-5B plus centrifuge, S. dysenteriae was inoculated in BHI broth with 1% Sorvall Instruments, Dupont, USA). Th e cell- inoculum and incubated at 37 °C for 24 h. Aft er every free supernatant was concentrated in a lyophilizer 2 h the absorbance was measured at 600 nm and the (Lyophilization System Inc., Poughkeepsie, NY, USA) viable cell counts were checked on BHI agar plates. to a fi nal volume of 10 mL. Ice cold methanol (5 °C) Anti-Shigella dysenteriae activity in culture was added to this in a ratio of 1:3. Th e mixture was supernatant and pellet of P. pentosaceus subject to constant stirring and kept for 12 h at 20 °C. P. pentosaceus culture (1%) was grown to its Aft er centrifugation, the supernatant was collected mid exponential phase (18 h) in MRS broth and and concentrated in a fl ash evaporator under vacuum centrifuged. Anti-Shigella dysenteriae activity was at 45 °C (Rotavapor RE 121, Buchi, Switzerland). To tested both in the culture supernatant as well as in the this, ice cold acetone (–5 °C) was added in a 1:3 ratio pellet by well diff usion assay (WDA). Th e pellet (200 with slow stirring and kept overnight at 4 °C. Th e mg) was taken in phosphate buff er (0.02 M; pH 7.0). supernatant was again centrifuged at 3000 × g for 30 178 R. AGRAWAL, S. DHARMESH min at 4 °C and concentrated in a fl ash evaporator nm) on a spectrophotometer (Shimadzu Corp., under vacuum. At each step, sample aliquots were Japan). Th e quantifi cation was done by comparison collected and assayed for anti-S. dysenteriae activity. with a standard graph of bovine serum albumin Anti-Shigella dysenteriae assay (BSA). Anti-Shigella dysenteriae activity was quantifi ed by Molecular weight determination by sodium–dodecyl well diff usion assay (WDA). An MRS agar (1.5%) plate sulphate-polyacrylamide gel electrophoresis (SDS- was taken and overlaid with 5 mL of soft agar (0.8% PAGE) w/v) containing a 3 × 108 CFU/mL-1 concentration Th e partially purifi ed (methanol-acetone) sample of the indicator strain (Shigella dysenteriae). Wells extract was mixed in a 1:1 ratio with sample buff er (3 mm size) were bored in the agar plate. Each well and heated at 100 °C for 2 min. Sample (30 μL) was was fi lled with a varying concentration of (protein) subjected to SDS-PAGE on a 18.75% (w/v) gradient bacteriocin fractions. Th e agar plates were incubated gel (12,13) using standard protein as a molecular at 30 °C for 12 h. Th e diameter of the clear zone was weight marker (MW-SDS-Genie, Bangalore, India) measured around each well. Bacteriocin activity is with a range of 2-60 kDa. expressed as an arbitrary unit (AU) defi ned as the Th e gel was run at 70 V for 4 h, removed, and then reciprocal of the highest dilution for clearance against cut into 2 parts. One was stained with Coomassie Shigella dysenteriae. Blue R-250 and destained aft er 2 h to make the Th e arbitrary units/mL (AU/mL) = Diameter of bands visible. Th e other half was fi xed (20% (v/v) the zone of clearance (mm)/volume taken in the well isopropanol and 10% (v/v) glacial acetic acid) for 2 (μL) × 1000. h and washed in deionized water for 4 h. Th e gel was Analytical Method aseptically placed in a sterile petri plate, rinsed with water, and overlaid with 60 mL of BHI soft agar with Partial purifi cation by semi-preparative RP-HPLC a culture of Shigella dysenteriae (CFU/mL-1).

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