Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Probiotics & Antimicro. Prot. (2016) 8:161–169 DOI 10.1007/s12602-016-9217-y Biochemical Properties and Mechanism of Action of Enterocin LD3 Purified from Enterococcus hirae LD3 Aabha Gupta1,2 • Santosh Kumar Tiwari1,2,3 • Victoria Netrebov3 Michael L. Chikindas3,4 • Published online: 5 May 2016 Ó Springer Science+Business Media New York 2016 Abstract Enterocin LD3 was purified using activity-guided multistep chromatography techniques such as cationexchange and gel-filtration chromatography. The preparation’s purity was tested using reverse-phase ultra-performance liquid chromatography. The specific activity was tested to be 187.5 AU lg-1 with 13-fold purification. Purified enterocin LD3 was heat stable up to 121 °C (at 15 psi pressure) and pH 2–6. The activity was lost in the presence of papain, reduced by proteinase K, pepsin and trypsin, but was unaffected by amylase and lipase, suggesting proteinaceous nature of the compound and no role of carbohydrate and lipid moieties in the activity. MALDITOF/MS analysis of purified enterocin LD3 resolved m/z 4114.6, and N-terminal amino acid sequence was found to be H2NQGGQANQ–COOH suggesting a new bacteriocin. Dissipation of membrane potential, loss of internal ATP and bactericidal effect were recorded when indicator strain Micrococcus luteus was treated with enterocin LD3. It inhibited Gram-positive and Gram-negative bacteria including human pathogens such as Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, & Santosh Kumar Tiwari [email protected] 1 Department of Genetics, Maharshi Dayanand University, Rohtak, Haryana 124001, India 2 Department of Bioscience and Biotechnology, Banasthali University, Tonk, Rajasthan 304022, India 3 Health Promoting Naturals Laboratory, School of Environmental and Biological Sciences, Rutgers State University, New Brunswick, NJ 08901, USA 4 Center for Digestive Health, New Jersey Institute for Food, Nutrition and Health, New Brunswick, NJ 08901, USA Salmonella typhi, Shigella flexneri, Listeria monocytogenes, Escherichia coli O157:H7, E. coli (urogenic, a clinical isolate) and Vibrio sp. These properties of purified enterocin LD3 suggest its applications as a food biopreservative and as an alternative to clinical antibiotics. Keywords Enterocin LD3 Enterococcus hirae LD3 Bacteriocin Biopreservation Membrane potential Introduction Lactic acid bacteria (LAB) are known to produce ribosomally synthesized antimicrobial proteins as a part of their defense mechanism. Many of these molecules inhibit closely related bacteria, and they are active mostly against Gram-positive microorganisms; however, there are reports that show several LAB bacteriocins inhibiting Gram-negative bacteria [1–4]. Presently, only nisin and pediocin PA1/AcH found their commercial use as food preservatives, while many more were characterized for application in food safety [5, 6]. Based on their properties such as charge, hydrophobicity and molecular weight, several methods have been utilized for purification of bacteriocins. One of the most common is the use of salt precipitation and chromatography in combination with the bioassay [7]. Most of the reported LAB bacteriocins are stable at a broad range of pH and temperatures, sensitive to some proteolytic enzymes and generally of a small molecular weight [2, 8–10]. Bacteriocins kill sensitive cells through different mechanisms. Although structure–function relationships have only been determined for certain bacteriocins, many bacteriocins produced by LAB act by pore formation or inhibition of cell wall biosynthesis [2, 11, 12]. Pore 123 162 Probiotics & Antimicro. Prot. (2016) 8:161–169 formation can lead to cell death as a consequence of proton motive force (PMF) depletion through dissipation of the pH gradient and membrane potential. Furthermore, cell death can result from efflux of phosphate, amino acids or other substances [13]. In our previous study, we have demonstrated the bacteriocin production by a putative probiotic strain, E. hirae LD3, isolated from a fermented food, Dosa [14]. Here, we report the molecular mass, partial amino acid sequence, stability under different conditions and mode of action of enterocin LD3 purified from culture-free supernatant (CFS) of E. hirae LD3. Materials and Methods Bacterial Strains, Culture Media and Growth Conditions E. hirae LD3 was grown in Lactobacillus MRS medium [15] at 37 °C in an incubator shaker with agitation at 200 rpm (Scigenics Biotech, Chennai, India). Reference LAB strains were obtained from the ARS Culture Collection (USDA) as mentioned in Table 1. E. coli (urogenic), Pseudomonas fluorescence Psd and P. aeruginosa clinical isolates were grown in Luria–Bertani Broth (LB) at 37 °C. Micrococcus luteus MTCC 106, Staphylococcus aureus, Salmonella typhi, Shigella flexneri, Listeria monocytogenes and Vibrio sp. were grown in nutrient broth (NB) medium at 37 °C. M. luteus MTCC106 was used as an indicator strain for assay of antimicrobial activity. All the media components were purchased from Hi-Media (Mumbai, India), Sisco Research Laboratory (SRL, Mumbai, India) and Sigma-Aldrich (St-Louis, USA). Bacteriocin Purification and Assay The CFS (2 L) of E. hirae LD3 was subjected to ammonium sulfate (SRL, Mumbai, India) precipitation at different saturation levels (0–30, 30–50 and 50–90 % w/v). Salt was added slowly by continuous stirring on a magnetic stirrer at 4 °C. The precipitate was collected by centrifugation at 7000g, 10 min, 4 °C (Sigma, Germany) and suspended in minimum volume of 20 mmol l-1 sodium acetate buffer, pH 4.6 (buffer A). The samples were desalted by dialysis (2.0 kDa cutoff membrane, Sigma, St. Louis, USA) against buffer A for 24 h. The dialyzed samples were filter-sterilized through 0.2-lm membrane (Axiva, New Delhi, India) and tested for antimicrobial activity. Protein concentration was determined according to the Bradford method [16]. The desalted active sample (Fraction I) was further processed using cation-exchange Table 1 List of microorganisms, their sources and reason to test against purified enterocin LD3 S. No. Microorganisms Origin (source) Reason for selection References 1 Lactobacillus curvatus NRRL B4562 Milk Target strain ARS Culture Collection, USA 2 L. delbrueckii NRRL B4525 Target strain ARS Culture Collection, USA 3 L. acidophilus NRRL B4495 Emmental cheese Human Target strain ARS Culture Collection, USA 4 L. plantarum NRRL B4496 Pickled cabbage Target strain ARS Culture Collection, USA 5 Lactococcus lactis subsp. lactis NRRL B1821 Not available Target strain ARS Culture Collection, USA 6 L. lactis subsp. cremoris NRRL B634 Not available Target strain ARS Culture Collection, USA 7 Enterobacter cloaceae NRRL B14298 Soybean Related to producer ARS Culture Collection, USA 8 Enterococcus faecium NRRL B2354 Cheese Related to producer ARS Culture Collection, USA 9 Staphylococcus aureus Human Local hospital 10 E. coli (urogenic) Human Food-borne pathogen Pathogen 11 E. coli O157:H7 Human Food-borne pathogen Dr. K. R. Matthews lab, Rutgers State University Local hospital 12 Pseudomonas fluorescens Soil Gram-negative Dr. Sheela Srivastava lab, Delhi University 13 P. aeruginosa Human Pathogen Local hospital 14 Salmonella typhi Human Pathogen Local hospital 15 Shigella flexneri Human Pathogen Dr. J. S. Virdi, Delhi University 16 Listeria monocytogenes Human Pathogen Dr. J. S. Virdi, Delhi University 17 Vibrio sp. Human Pathogen Local hospital 123 Probiotics & Antimicro. Prot. (2016) 8:161–169 chromatography (CEC) with the benchtop column filled with SP-Sepharose fast flow (Sigma, St. Louis, USA) controlled by a peristaltic pump (MacFlow, New Delhi, India). The column was equilibrated and washed with buffer A, and Fraction I was applied to the column at a flow rate of 0.5 ml min-1. The active compound was eluted with buffer B (0.2, 0.5 and 1.0 mol l-1 NaCl in buffer A). Active fractions were pooled, dialyzed against the doubledistilled (DD) water, lyophilized at -75 °C under vacuum (10-3 torr) for 24 h (Allied Frost, New Delhi, India) and resuspended in minimum amount of sterile DD water (Fraction II). Fraction II was then loaded into a gel-filtration column (GFC) Sephadex G-25 (Amberson Biosciences, USA) equilibrated and eluted with DD water. In these fractions, protein concentrations were monitored at 280 nm and antimicrobial activity was determined. The active fractions were pooled, lyophilized and resuspended in minimum amount of sterile DD water (Fraction III). The antimicrobial activity of bacteriocin at each step of purification was determined using agar well diffusion assay (AWDA) [17] and AU ml-1. One activity unit (AU) was defined as the reciprocal of the highest dilution of bacteriocin causing 50 % growth inhibition [18]. Determination of Purity of Bacteriocin To determine the level of purity in GFC-eluted sample (Fraction III), it was applied on BEH C18 column (1.7 lm, 2.1 9 50 mm) fitted in Acquity RP-UPLC (reverse-phase ultra-performance liquid chromatography) (Waters, Massachusetts, USA). The column was equilibrated with water containing 0.1 % TFA (trifluoroacetic acid, Buffer A1) (SRL, Mumbai, India), and sample was run with a step gradient of Buffer B1 (acetonitrile (SRL, Mumbai, India) containing 0.1 % TFA), initially 5 % B then 20 % B up to 3 min and 40 % B up to 6 min. The flow rate was maintained at 1.0 ml min-1 throughout the run, and absorbance was monitored at 280 nm using a UV detector (Waters, Massachusetts, USA). Molecular Mass and Amino Acid Sequence Purified enterocin LD3 was submitted for the determination of molecular mass and amino acid sequencing using custom facilities available. Molecular mass (m/z) was determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) on LTQ Orbitrap Velos LC–MS/MS at the Center for Advanced Proteomics Research, New Jersey Medical School of the Rutgers Biomedical and Health Sciences (Newark, New Jersey). The amino-terminal sequencing was carried out on an Applied Biosystems Procise Protein 163 Sequencer equipped with an online HPLC system at New Jersey Medical School Molecular Resource Facility (NJMS-MRF). The sample was processed according to the manufacturer’s recommendations; briefly, it was loaded onto a polyvinylidene fluoride membrane, dried and placed into the reaction cartridge. The instrument was operated according to the standard protocol for Edman Sequencing that initially runs a blank cycle and a standard cycle, followed by sequencing cycles. The data were collected using SequenceProTM software. Biochemical Characterization Similar to other bacteriocins, purified enterocin LD3 was characterized for stability at different temperatures, pH and in the presence of organic solvents, detergents and surfactants. In addition, its activity was tested against the selected range of related as well as pathogenic microorganisms as listed in Table 1. Purified enterocin LD3 was treated with different hydrolyzing enzymes such as amylase, lipase, trypsin, pepsin, papain (Sigma-Aldrich, USA) and proteinase K (SRL, Mumbai, India) at a final concentration of *1000 units ml-1. Following incubation at 37 °C for 2 h, enzymatic activity was terminated by heating the sample at 100 °C for 5 min. Untreated bacteriocin sample was used as a control, and the residual antimicrobial activity was measured using AWDA method [17]. Effect of Enterocin LD3 on Viability of the M. luteus MTCC 106 Indicator Strain To understand the bactericidal or bacteriostatic nature of enterocin LD3, M. luteus cells were incubated with the bacteriocin as suggested by Kumar et al. [19]. About *106 actively growing cells of the indicator strain were resuspended in 0.8 % NaCl containing 100 AU ml-1 of bacteriocin and incubated at 37 °C. The bacteriocin’s effect on actively growing cells was studied as well. For this, M. luteus cells were inoculated in NB medium containing 100 AU ml-1 of bacteriocin at A600 0.02. Samples were withdrawn at regular time intervals of 2 h up to 8 h for the determination of viable cell counts (CFU ml-1) and growth (A600). Control set was incubated without bacteriocin treatment. Detection of Intracellular ATP and Membrane Potential The effect of enterocin LD3 on the intracellular ATP content and transmembrane electrical potential (Dw) of the indicator strain M. luteus was determined using the method as previously reported [20, 21]. ATP measurements were taken using the commercially available ATP 123 164 1.2 16 (a) 14 12 A280 0.8 10 0.6 8 6 0.4 4 0.2 0.0 2 0 5 10 15 20 25 30 35 Bacteriocin activity (inhibition zone in mm) 1.0 0 Fraction numbers (b) 10 0.6 A280 8 6 0.4 4 0.2 2 0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Bacteriocin activity (inhibition zone in mm) 12 0.8 0 Fraction numbers 0.30 (c) 0.573 0.25 0.20 AU Bioluminescent Assay Kit (Sigma, St. Louis, USA). Briefly, M. luteus cells were washed with 50 mmol l-1 HEPES buffer, pH 6.5 and energized with 0.2 % glucose. For time-dependent assay, extracellular ATP level of the energized cells treated with enterocin LD3 (100 AU ml-1) was measured at 15, 30 and 45 min after bacteriocin addition. The control set was run without bacteriocin treatment. To determine total (intracellular and extracellular) levels of ATP, 20 ll of the cell suspension was mixed with 80 ll of dimethylsulfoxide (DMSO) (SigmaAldrich, St-Louis, USA) and kept at room temperature for 5 min to permeabilize the cells. Light emission was detected using a luminometer (Luminoskan TL Plus, Labsystems Oy, Helsinki, Finland). Intracellular ATP was determined by calculating the difference in the total and extracellular ATP concentrations. For determination of Dw, fluorescence measurements were initiated followed by the addition of 5 lM of the fluorescent probe, 3,30 -dipropyltyiadicarbocyanine iodide [Di-S-C3-(5)] (ThermoFisher, NY, USA) and 10 ll of the M. luteus cell concentrate. After the stabilization of the fluorescence signal, enterocin LD3 (100 AU ml-1) was added. Further, valinomycin (10 lmol l-1) was added to collapse any remaining residual Dw. A PerkinElmer fluorescence spectrometer LS50B (Fremont, CA, USA) with excitation and emission wavelengths of 643 and 666 nm, respectively, with a 10-nm slit width and an 1800-s assay duration with reading every 0.1 s was used for all assays. Probiotics & Antimicro. Prot. (2016) 8:161–169 0.15 0.10 0.05 Statistical Analysis Experiments were performed in triplicate, mean values were plotted along with standard deviation (mean ± SD), and p \ 0.05 was considered as statistically significant. The membrane potential assay was done in singlet but repeated three times to monitor the reproducibility of the result. Results Purification, Mass Spectrometry and N-Terminal Sequencing Two initial saturation levels of ammonium sulfate precipitates (0–30 and 31–50 %) did not show antimicrobial activity and were discarded. However, saturation level (51–90 %) demonstrated antimicrobial activity and was further purified. The elution profile on CEC revealed two separate peaks where only the first peak demonstrated antimicrobial activity (Fig. 1a). The fractions corresponding to the active peak were pooled and applied on gelfiltration chromatography (GFC) where bacteriocin was 123 0.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes Fig. 1 Elution profile of protein concentration (open circle) and bacteriocin activity (closed circle) of enterocin LD3 on cationexchange (a) and gel-filtration chromatography (b). Single absorbance peak was found on RP-UPLC. Absorbance unit (AU) was detected at 280 nm (c) resolved into a broader peak with shoulder appearance. The antimicrobial activity was present only in fractions (fraction no. 4 and 5) corresponding to major peak (Fig. 1b). Further, the active fractions eluted from GFC were pooled which demonstrated single sharp peak (Fig. 1c) when applied on RP-UPLC. This indicated the homogeneity of the purified bacteriocin sample. These active fractions were further collected in repeated experiments and used for characterization of the enterocin LD3. Table 2 represents the complete purification scheme of enterocin LD3. The total protein content in 2 L CFS was 2.8 9 104 lg and activity 4.0 9 105 AU. After ammonium sulfate precipitation, volume was reduced to 50 ml and specific activity was increased by 3.5-fold. Thereafter, Probiotics & Antimicro. Prot. (2016) 8:161–169 165 Table 2 Summary of purification of enterocin LD3 from culture-free supernatant of Ent. hirae LD3 Purification steps Volume (ml) Total protein (lg) Total activity (AU) Specific activitya Purification foldb Yieldc (%) Culture supernatant 2000 2.8 9 104 4.0 9 105 14.2 1 100 2 4 Ammonium sulfate precipitation 50 4.0 9 10 2.0 9 10 50 3.5 5 Cation-exchange chromatography 30 90 6.0 9 103 66.6 4.6 1.5 Gel-filtration chromatography 1.5 8 1.5 9 103 187.5 13.2 0.37 a Specific activity (SA) = Total activity/Total protein b Purification fold = SA of sample/SA of crude c Yield (%) = Total AU of 9 100/Total AU of crude subsequent increase in the specific activity and purification fold was recorded. In purified active fraction (GFC eluted), specific activity was found to be 187.5 AU lg-1 with a total yield of 0.37 % indicating about a 13-fold purification. This sample was considered to be pure and tested for further molecular properties of enterocin LD3. The MALDI-TOF/MS analysis of purified enterocin LD3 resolved to be single peak with m/z 4114.6 suggesting the homogeneity and a small peptide (Fig. 2). Using Edman degradation method, only seven amino acid residues were identified at amino-terminal group: H2N-QGGQANQ– COOH. Biochemical Characterization Purified enterocin LD3 was active in a range of pH 2.0–6.0; however, its activity decreased at pH 7.0 and was completely lost at pH 8–10. Bacteriocin activity was retained one hundred percent when the pH was adjusted back to pH 4.0. Buffers of related pH were used as a control and did not show any growth inhibition. Enterocin LD3 remained 100 % active after 15 min at 100 °C and autoclaving at 121 °C under 15 psi pressure (Table 3). Thus, enterocin LD3 was found to be highly thermostable and active at acidic to near-neutral pH range. Similar stability was observed when the sample was treated with different organic solvents, surfactants and detergents. The bacteriocin activity was not affected by the reagents tested (Table 3). When bacteriocin sample was treated with proteolytic enzymes such as pepsin, trypsin and proteinase K, the inhibitory activity was reduced and completely lost in the presence of papain; however, amylase and lipase did not influence the activity of enterocin LD3 (Table 3). Enterocin LD3 inhibited Gram-positive and Gram-negative bacteria such as M. luteus, Lactobacillus curvatus NRRL B4562, Lact. delbrueckii NRRL B4525, Lact. acidophilus NRRL B4495, Lact. plantarum NRRL B4496, Lactococcus lactis subsp. lactis NRRL B1821, L. lactis subsp. cremoris NRRL B634, Enterococcus faecium NRRL B2354 and Enterobacter cloaceae NRRL B14298. In addition, enterocin LD3 inhibited some human pathogens including those of food origin, specifically Staphylococcus aureus, E. coli (urogenic), E. coli O157:H7 Pseudomonas fluorescens Psd, Ps. aeruginosa, Salmonella typhi, Shigella flexneri, Listeria monocytogenes and Vibrio sp. (Table 3). Effect of Enterocin LD3 on Viability of the M. luteus MTCC 106 Indicator Strain Fig. 2 MALDI-TOF/MS spectrum of purified enterocin LD3 showing m/z 4114.6 M. luteus cells were treated with purified enterocin LD3 (100 AU ml-1), and turbidity at OD600 and loss of viable cell count in terms of CFU ml-1 were monitored. There was no change in CFU ml-1 of the control cells populations. However, the loss in the viable count of indicator strain was recorded after 2 h of treatment, and almost fourlog decrease in the CFU ml-1 was observed at the end of 8 h (Fig. 3). The observed loss in cell viability suggested the bactericidal mode of action of enterocin LD3. There was no growth (A600) of the indicator cells observed when treated with bacteriocin in growth medium, whereas control was growing normally (Fig. 3). 123 166 Probiotics & Antimicro. Prot. (2016) 8:161–169 Table 3 Biochemical and antimicrobial properties of enterocin LD3 purified from culture-free supernatant of E. hirae LD3 S. No. Treatments Bacteriocin activity (mm) 1. pH 2.0 14 2. 3. 4. pH 4.0 (control, no pH adjustment) 14 pH 6.0 12 pH 7.0 8 pH 8.0 nil pH 10.0 nil Control (without heat treatment) 14 80–121 °C 15 min, 15 psi 14 Control (without solvent treatment) 14 Ethanol, methanol, isopropanol, acetone, ethyl acetate, SDS, Tween 80, urea, Triton X-100 14 Control (without enzyme treatment) Amylase, lipase (1000 units ml-1) 14 14 Pepsin, trypsin (1000 units ml-1) 12 Proteinase K (1000 units ml-1) 9 -1 Papain (1000 units ml ) 5. nil Micrococcus luteus MTCC106 14 Lactobacillus curvatus NRRL B4562 8 Lact. delbrueckii NRRL B4525 8 Lact. acidophilus NRRL B4495 8 Lact. plantarum NRRL B4496 nil Lactococcus lactis subsp. lactis NRRL B1821 8 L. lactis subsp. cremoris NRRL B634 nil Enterococcus faecium NRRL B2354 8 Enterobacter cloaceae NRRL B14298 8 Staphylococcus aureus 8 E. coli (urogenic) 8 E. coli O157:H7 Pseudomonas fluorescens 11 8 Ps. aeruginosa 10 Salmonella typhi 8 Shigella flexneri 8 Listeria monocytogenes 13 Vibrio sp. 8 Bacteriocin activity was measured in terms of the diameter size of the zone growth inhibition in mm Measurement of Intracellular ATP and Dissipation of the Membrane Potential Loss of internal ATP and dissipation of the membrane potential (Dw) were studied to further elucidate the mode of action of enterocin LD3. The DMSO-treated cells were used to determine total ATP concentration. Here, external ATP concentration was measured after bacteriocin treatment. The internal ATP concentration was calculated by subtracting the external with that of total ATP concentration. There was an increase in external ATP concentration, and corresponding decrease in internal ATP was recorded. 123 The loss of internal ATP concentration in bacteriocintreated cells was noticed after 5 min of treatment, and this loss continued to increase with the incubation time (Fig. 4). The untreated cells showed almost similar internal ATP concentration without significant change throughout the incubation period. For measurement of dissipation of Dw, probe-loaded cells were treated with purified enterocin LD3. There was an increase in the fluorescence, when probe was added which was stabilized later. The addition of indicator cells in probe solution caused a decrease in fluorescence. The fluorescence was found stable after the energization of cells Probiotics & Antimicro. Prot. (2016) 8:161–169 167 1.0 8 7 6 0.6 A600 log10 CFU mL -1 0.8 5 0.4 4 0.2 3 2 0 2 4 6 8 0.0 Time (h) Fig. 3 Loss of CFU mL-1 of treated (open square) and untreated (closed square) M. luteus cells. The growth inhibition (A600) of treated (closed circle) and untreated (open circle) cells of indicator strain Micrococcus luteus by enterocin LD3 Internal ATP conc (µM) 35x10-12 30x10-12 25x10-12 20x10-12 15x10-12 10x10-12 0 15 30 45 Time (min) Fig. 4 Loss of intracellular ATP of M. luteus cells treated with enterocin LD3 (open circle). The untreated cells (closed circle) show almost similar ATP content throughout the incubation period with glucose and further ionophore (nigericin) treatment. However, an increase in fluorescence was recorded just after enterocin LD3 was added, indicating the release of probe into medium. There was no further increase in the fluorescence recorded after the addition of valinomycin, suggesting complete dissipation of Dw in the targeted cells treated with purified enterocin LD3 as shown in Fig. 5. Discussion Only two LAB bacteriocins, nisin and pediocin, are commercially utilized as food preservatives (Perez et al. [2]. Therefore, there is a need to characterize other bacteriocins of LAB and elucidate their potential applications in food safety as well as in therapeutics. The mechanism of action of these bacteriocins is known to be pore formation and/or membrane perturbation [22]. Here, we report on the purification of enterocin LD3 from the previously characterized E. hirae LD3 strain with probiotic potential [14]. In addition, for the bacteriocin’s mode of action, targeted biochemical and molecular properties were elucidated. The use of activity-guided multi-step chromatography techniques had been quite common practice and successful for purification of several bacteriocins [2, 18, 23, 24]. Therefore, these methods were used for purification of enterocin LD3. It was purified to homogeneity and confirmed by RP-UPLC and mass spectrometry showing a single peak. The presence of a single peak identified with reverse-phase column and MALDI-TOF/MS analysis shows the purity of the molecule which has also been suggested by others [4, 18, 23]. During the course of purification, increase in specific activity (up to 187.5 AU lg-1) and purification fold (13-fold) validated the feasibility of the process. Purified enterocin LD3 was characterized for targeted biochemical properties. It was active after exposure to acidic and near-neutral pH and completely lost its antimicrobial activity in alkaline pH ranges and found to be stable at high temperatures such as 100 and 121 °C at 15 psi. Similarly, bacteriocins purified from several LAB strains were also found to be stable at different pH and higher temperatures [18, 25, 26]. Temperature and pH stability are one of the most important properties for an effective food preservative. Such stability of enterocin LD3 at different pH levels, temperatures and in various organic solvents would also facilitate the purification process of the bacteriocin. MALDI-TOF/MS analysis of enterocin LD3 was found to be m/z 4114.6 which is unique. To the best of our knowledge, no other bacteriocins are reported with the same m/z ratio. Therefore, we speculate that enterocin LD3 may be a new bacteriocin. Further, N-terminal amino acid sequencing revealed first seven amino acid residues and demonstrated lack of homology with known bacteriocins when compared using pBLAST (NCBI). Therefore, mass analysis and partial amino acid sequence of enterocin LD3 support our speculation on a new bacteriocin from Ent. hirae LD3. Inhibition of Gram-negative bacteria is the unique feature of a few LAB bacteriocins [4, 17, 27, 28]. Enterocin LD3 was able to inhibit some human pathogens such as S. aureus, P. fluorescens Psd, P. aeruginosa, S. typhi, S. flexneri, L. monocytogenes, E. coli O157:H7, E. coli (urogenic) and Vibrio sp. This indicated its possible use in food preservation and as a therapeutic agent. Inhibition of food-borne pathogens would be an additional benefit for the putative probiotic strain LD3 if utilized in food products. Bactericidal nature of enterocin LD3 was confirmed by the loss of cell viability and further confirmed by the loss of internal ATP and dissipation of membrane potential of indicator cells. There was an increase in the external 123 Probiotics & Antimicro. Prot. (2016) 8:161–169 Fluorescence (au) 168 1000 950 900 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0.1 Micrococcus luteus cells Valinomycin Enterocin LD3 Probe [DiSC3(5)] Nigericin Glucose 0.0 100 200 300 400 500 600 700 800 Time (s) 900 1000 1100 1200 1300 1400 1500 Fig. 5 Dissipation of membrane potential of indicator strain Micrococcus luteus by enterocin LD3 ATP, and subsequent decrease in internal ATP concentrations suggested the formation of pores and efflux of ATP. Further, dissipation of membrane potential of M. luteus cells also indicated the pore-forming nature of enterocin LD3 which is a typical mechanism of action found in many bacteriocins [21, 29–31]. The role of some bacteriocins in therapeutics and other related areas has been recently reviewed, and these bacteriocins were suggested for further characterization and industrial scale production [9, 32]. Enterocin LD3 inhibited a range of Gram-positive and Gram-negative bacteria including food-borne and human pathogens suggesting its possible applications in food biopreservation and therapeutics as well. Conclusions Enterocin LD3 purified to homogeneity has a unique mass, and its N-terminal sequence suggests a novel compound. Enterocin LD3 has bactericidal activity against tested sensitive strains and causes dissipation of membrane potential and the efflux of ATP, suggesting pore-forming nature of its activity. The broad range of pathogen inhibition suggests its possible applications in food safety as well as in therapeutics. This is the first study that demonstrated the characterization of a new enterocin produced by E. hirae LD3 strain isolated from a fermented food, Dosa. The biochemical properties and host range of purified enterocin LD3 suggest its applications as a food biopreservative and as an alternative to clinical antibiotics. 123 Acknowledgments The present work and AG were financially supported by University Grant Commission, New Delhi, under major research project (No. 36-38/2008 SR). SKT was supported by IndoUS Science and Technology Forum, New Delhi, and was truly acknowledged. Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest. References 1. Grande Burgos MJ, Pulido RP, Del Carmen López Aguayo M, Gálvez A, Lucas R (2014) The cyclic antibacterial peptide enterocin AS-48: isolation, mode of action, and possible food applications. Int J Mol Sci 15:22706–22727. doi:10.3390/ ijms151222706 2. Perez RH, Zondo T, Sonomoto K (2014) Novel bacteriocins from lactic acid bacteria (LAB): various structures and applications. Microb Cell Fact 13:S3. doi:10.1186/1475-2859-13-S1-S3 3. Rumjuankiat K, Perez RH, Pilasombut K, Keawsompong S, Zendo T, Sonomoto K, Nitisinprasert S (2015) Purification and characterization of a novel plantaricin, KL-1Y, from Lactobacillus plantarum KL-1. World J Microbiol Biotechnol 31:983–994. doi:10.1007/s11274-015-1851-0 4. Sahoo TK, Jena PK, Patel AK, Seshadri S (2015) Purification and molecular characterization of the novel highly potent bacteriocin TSU4 produced by Lactobacillus animalis TSU4. Appl Biochem Biotechnol 177:90–104. doi:10.1007/s12010-015-1730-z 5. Arthur TD, Cavera VL, Chikindas ML (2014) On bacteriocin delivery systems and potential applications. Future Microbiol 9:235–248. doi:10.2217/fmb.13.148 6. De Arauz LJ, Jozala AF, Mazzola PG, Penna TCV (2009) Nisin biotechnological production and application: a review. Trends Food Sci Technol 20:146–154. doi:10.1016/j.tifs.2009.01.056 Probiotics & Antimicro. Prot. (2016) 8:161–169 7. Vaillancourt K, LeBel G, Frenette M, Gottschalk M, Grenier D (2015) Suicin 3908, a new lantibiotic produced by a strain of Streptococcus suis serotype 2 isolated from a healthy carrier pig. PLoS ONE 10:e0117245. doi:10.1371/journal.pone.0117245 8. Agaliya PJ, Jeevaratnam K (2013) Characterization of the bacteriocins produced by two probiotic Lactobacillus isolates from idli batter. Ann Microbiol 4:1525–1535. doi:10.1007/s13213013-0616-y 9. Bali V, Panesar PS, Bera MB (2014) Trends in utilization of agroindustrial byproducts for production of bacteriocins and their biopreservative applications. Crit Rev Biotechnol 28:1–11. doi:10.3109/07388551.2014.947916 10. Ennahar S, Sashihara T, Sonomoto K, Ishizaki A (2000) Class IIa bacteriocins: biosynthesis, structure and activity. FEMS Microbiol Rev 24:85–106. doi:10.1111/j.1574-6976.2000.tb00534.x 11. Hassan M, Kjos M, Nes IF, Diep DB, Lotfipour F (2012) Natural antimicrobial peptides from bacteria: characteristics and potential applications to fight against antibiotic resistance. J Appl Microbiol 113:723–736. doi:10.1111/j.1365-2672.2012.05338.x 12. Yount NY, Yeaman MR (2013) Peptide antimicrobials: cell wall as a bacterial target. Ann N Y Acad Sci 1277:127–138. doi:10. 1111/nyas.12005 13. Brotz H, Beirbaum G, Markus A, Moliter E, Sahl HG (1995) Mode of action of the lantibiotic mersacidin: inhibition of peptidoglycan biosynthesis via a novel mechanism? Antimicrob Agents Chemother 39:714–719. doi:10.1128/AAC.39.3.714 14. Gupta A, Tiwari SK (2015) Probiotic potential of bacteriocin producing Enterococcus hirae strain LD3 isolated from batter of Dosa. Ann Microbiol 65:2333–2342. doi:10.1007/s13213-0151075-4 15. deMan JC, Rogosa M, Sharpe M (1960) A medium for the cultivation of Lactobacilli. J Appl Bacteriol 23:130 16. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principal of protein-dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3 17. Tiwari SK, Srivastava S (2008) Purification and characterization of a novel bacteriocin from natural isolate of Lactobacillus plantarum LR/14. Appl Microbiol Biotechnol 79:759–767. doi:10.1007/s00253-008-1482-6 18. Jimnez JJ, Borrero J, Diep DB, Gutiez L, Nes IF, Herranz C, Cintas LM, Hernandez PE (2013) Cloning, production, and functional expression of the bacteriocin sakacin A (SakA) and two SakA-derived chimeras in lactic acid bacteria (LAB) and the yeasts Pichia pastoris and Kluyveromyces lactis. J Ind Microbiol Biotechnol 40:977–993. doi:10.1007/s10295-013-1302-6 19. Kumar M, Tiwari SK, Srivastava S (2010) Purification and characterization of enterocin LR/6, a new bacteriocin from Enterococcus faecium LR/6. Appl Biochem Biotechnol 160:40–49. doi:10.1007/s12010-009-8586-z 20. Murdock C, Chikindas ML, Matthews KR (2010) The pepsin hydrolysate of bovine lactoferrin causes a collapse of the 169 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. membrane potential in Escherichia coli O157:H7. Probiotics Antimicrob Proteins 2:112–119. doi:10.1007/s12602-010-9039-2 Tiwari SK, Noll SK, Cavera VL, Chikindas ML (2015) Improved antimicrobial activity of synthetic-hybrid bacteriocins designed from enterocin E50-52 and pediocin PA-1. Appl Environ Microbiol 81:1661–1667. doi:10.1128/AEM.03477-14 Gut IM, Blanke SR, van der Donk WA (2011) Mechanism of inhibition of Bacillus anthracis spore outgrowth by the lantibiotic nisin. ACS Chem Biol 6:744–752. doi:10.1021/cb1004178 Batdorj B, Dalgalarrondo M, Choiset Y, Pedroche J, Metro F, Prevost H, Chobert JM, Haertle T (2006) Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag. J Appl Microbiol 101:837–848. doi:10.1111/j.1365-2672.2006.02966.x Tiwari SK, Srivastava S (2008) Characterization of a bacteriocin from Lactobacillus plantarum strain LR/14. Food Biotechnol 22:241–267. doi:10.1080/08905430802262582 Gautam N, Sharma N, Ahlawat OP (2014) Purification and characterization of bacteriocin produced by Lactobacillus brevis UN isolated from Dhulliachar: a traditional food product of North East India. Indian J Microbiol 54:185–189. doi:10.1007/s12088013-0427-7 Rushdy AA, Gomaa EZ (2013) Antimicrobial compounds produced by probiotic Lactobacillus brevis isolated from dairy products. Ann Microbiol 63:81–90. doi:10.1007/s13213-0120447-2 Gaaloul N, Ben Braiek O, Hani K, Volski A, Chikindas ML, Ghrairi T (2015) Isolation and characterization of large spectrum and multiple bacteriocin-producing Enterococcus faecium strain from raw bovine milk. J Appl Microbiol 21:343–355. doi:10. 1111/jam.12699 Gupta A, Tiwari SK (2014) Plantaricin LD1: a bacteriocin produced by food isolate of Lactobacillus plantarum LD1. Appl Biochem Biotechnol 172:3354–3362. doi:10.1007/s12010-0140775-8 Ariel II, Budiman C, Jenie BS, Andreas E, Yuneni A (2015) Plantaricin IIA-1A5 from Lactobacillus plantarum IIA-1A5 displays bactericidal activity against Staphylococcus aureus. Benef Microbes 25:1–11. doi:10.3920/BM2014.0064 Barbour A, Philip K, Muniandy S (2013) Enhanced production, purification, characterization and mechanism of action of salivaricin 9 lantibiotic produced by Streptococcus salivarius NU10. PLoS ONE 8:e77751. doi:10.1371/journal.pone.0077751 Moll GN, Konings WN, Driessen AJ (1999) Bacteriocins: mechanism of membrane insertion and pore formation. Antonie Van Leeuwenhoek 76:185–198 Mathur H, Rea MC, Cotter PD, Ross RP, Hill C (2014) The potential for emerging therapeutic options for Clostridium difficile infection. Gut Microbes. doi:10.4161/19490976.2014.983768 123