Antibacterial Activity of Enterocins EFC21 and. Pathogens, Preservative. lactis, has been used for food preservation in a large number

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1 International Journal of Public Health Research 2015; 3(4): Published online June 10, 2015 ( Antibacterial Activity of Enterocins EFC21 and EFT194 on Food-Borne Pathogenic Bacteria Strains Olawale A. K. 1, *, Olaolu A. O. 2, Adefioye O. A. 3, Olawale A. O. 4 1 Department of Applied Sciences, Osun State Polytechnic, Iree, Nigeria 2 Department of Biomedical Sciences, College of Health Sciences, Ladoke Akintola University of Technology, Osogbo, Nigeria 3 Department of Medical Microbiology, College of Health Sciences, Ladoke Akintola University of Technology, Osogbo, Nigeria 4 Department of Statistics, Osun State Polytechnic, Iree, Nigeria address akolawale@yahoo.com (Olawale A. K.) To cite this article Olawale A. K., Olaolu A. O., Adefioye O. A., Olawale A. O.. Antibacterial Activity of Enterocins EFC21 and EFT194 on Food-Borne Pathogenic Bacteria Strains. International Journal of Public Health Research. Vol. 3, No. 4, 2015, pp Abstract Six hundred and fifty-eight Enterococcus faecalis strains of canteen origin were tested against Listeria monocytogenes and Staphylococcus aureus (as indicators) for detection of enterocin producers using stab-overlay method. The result reveals highest percentage (48.13%) in E. faecalis from bukataria. This was followed by 36.22% and 8.33% in isolates from school and fast-food canteens respectively. Two bacteriocin-producing Enterococcus faecalis strains (EFT194 and EFC21) that produced highest zone of inhibition (20.5 and 22mm respectively) were selected for enterocin purification and antibacterial screening against some Gram positive and gram negative food-borne pathogens including; Bacillus cereus, Listeria monocytogenes, Clostridium perfringens, Enterococcus faecium, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Salmonella typhi ) isolated from diary and meat products. The apparent purity of the enterocins was determined by cation exchange chromatography. Enterocin from EFC21 and EFT194 were purified at 11 and 8 fold respectively, with yield of 2.2% and 4.5% respectively. It was confirmed that both Enterocin from EFC21 and EFT194 tested positive for antibacterial effects on food-borne pathogenic bacteria, though enterocin EFC21 proved to be more potent. Higher antibacterial effects were observed on the gram-positive than the gram-negative pathogens. Further investigation on enterocins EFC21 and EFT194 is essential to confirm their suitability as food preservatives and control drugs for certain bacterial infections. Keywords Enterococcus faecalis, Enterocin, Food-Borne Pathogens, Preservative 1. Introduction Enterococci and streptococci seem to be unique in their great potential to produce peptide bacteriocins. It is tempting to speculate that bacteriocins are found more frequently in enterococci and streptococci than in many other lactic acid bacteria, such as Lactococcus and Lactobacillus [25]. The class II peptide bacteriocins are dominant in enterococci. Enterococci are widely used as starter cultures for dairy, meat, and vegetable fermentations [22]. Some enterococci strains are known to produce various types of bacteriocins, which have bactericidal effects against gram-positivincluding food-borne pathogens [21, 24]. This beneficial trait bacteria, has led to utilization of bacteriocins as food additives [12]. For example, nisin, a bacteriocin produced by Lactococcus lactis, has been used for food preservation in a large number of countries [16, 19, 20]. Bacteriocin production is common in lactic acid bacteria (LAB), and since bacteriocins produced by LAB may inhibit gram-positive spoilage bacteria and food-borne pathogens [3, 27], they are attracting considerable interest for their potential use as natural and nontoxic food preservatives [4, 26]. There are many examples of natural circular proteins in different organisms. AS-48 is a 70-residue circular peptide from Enterococcus faecalis that belongs to an heterogeneous group of bacterial antimicrobial polypeptides, also called bacteriocins [30], [31], [32], [33] and [34]. This protein shows a broad antimicrobial spectrum against both Gram-positive and Gram-negative bacteria [35] and [36]. The target of AS-48 is the bacterial cytoplasmic membrane in which it inserts in a voltageforming pores and leading to independent manner the

2 International Journal of Public Health Research 2015; 3(4): dissipation of the proton motive force [31]. The complete NMR assignment indicated that the AS-48 fold is characterised by five helices (α 1 to α 5 ) arranged to form a globular structure ( Fig. 1) [37]. This bacteriocin is a strongly basic molecule (pi 10.5) that contains a high proportion (49%) of hydrophobic and uncharged hydrophilic amino acid residues [30] and [38]. Enterocin P (EntP) is a class IIa bacteriocin produced by Enterococcus faecium P13 [1]. The EntP. structural gene (entp) encodes a 71-amino-acid prepeptide consisting of a 44-aminoacid mature bacteriocin and a 27-amino-acid signal peptide. The mature EntP is a bacteriocin with a broad antimicrobial spectrum that inhibits food-borne pathogens such as Listeria monocytogenes, Clostridium perfringens, Clostridium botulinum, and Staphylococcus aureus [1, 29]. The entp gene seems to be widely distributed in E. faecium strains of different origins [2, 7]. The broad antimicrobial spectrum of EntP suggests its potential application as a natural antimicrobial additive in the food industry. However, the use of enterococci as producers of bacteriocins should be approached with caution, since many Enterococcus isolates code for potential virulence factors and carry antibiotic resistance genes [10, 13]. Fortunately, these important features are strain- dependent. For these reasons, the selection of Enterococcus strains of interest in the food industry should be based on the absence of any possible pathogenic properties, or transferable antibiotic resistance genes. For biotechnological, hygienic, and safety reasons, the use of purified enterocin only as food preservative should be given consideration. The present study is aiming at determining the antibacterial activity of purified enterocin on foodborne pathogens. 2. Materials and Methods 2.1. Detection of Enterocin Producers Detection of enterocin producers among the 658 E. faecalis strains earlier isolated from canteens was carried out by stab-overlay method as described by Ahmad et al. (2004) [1]. Brain Heart Infusion (BHI, Oxoid) agar plates were inoculated by needle stab with fresh broth cultures and the plates were incubated at 37 o C for 48hours. The bacteria were then killed using chloroform vapors and each plate was then overlaid with a thin layer of soft agar containing 10 7 cells ml - 1 of indicator strains. The plates were then incubated at 37 o C overnight after which they were examined for zones of clearance. Listeria monocytogenes SUB635 and Staphylococcus aureus SUB511 were used as indicators Bacteriocin Purification Two bacteriocinogenic strains EFT194 and EFC21 that produced highest zone of inhibition among others (20.5 and 22 mm respectively) were grown in Mueller Hinton broth at 37 o C and crude bacteriocins solutions obtained from supernatants of their early stationary phase and were subjected to 80% Ammonium precipitation. They were centrifuged and pellets were redissolved in buffer for dialysis. Dissolved pallets were further purified by Cation-exchange chromatography on SP Sepharose column which was equilibrated with 20mM sodium phosphate (ph 4.5) and 1.0 ml was added to 500ml of the fermentation product in a centrifuged bottle. The bottle was gently agitated, held for that room temperature and centrifuged at 20 O C for 20minutess at 12000xg. The supernatant was discarded and the bottle containing the pellet and bacteriocins was filled with a washing solution consisting of 20mM Na 2 HPo 4 (ph 4.5) and centrifuged at 20 o C for 20minutes at 12000xg. The washing and centrifuging were repeated once. The supernatant was discarded and the bottle was then filled with 0.9ml NaCl and gently agitated and incubated at room temperature for 1h. The bacteriocins were now dissolved in the supernatant, which again was centrifuged at 12000xg for 15minutes at 20 o C. The supernatant was transferred into a clean container Antibacterial Activity of the Enterocins Two bacteriocinogenic E. faecalis strains (EFT194 and EFC21) that produced highest zone of inhibition (20.5 and 22mm respectively) were selected for antibacterial screening against some Gram positive and gram negative foodborne pathogens including; Bacillus cereus, Listeria monocytogenes, Clostridium perfringens, Enterococcus faecium, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Salmonella typhi, isolated from diary and meat products. The strains were grown in Mueller Hinton broth at 35 o C. The supernatant from early stationary growth phase was neutralized with 1M NaOH and filter sterilized through 0.22µm-pore-size filters (Millipore Corp., Bedford, Mass.). The antimicrobial activity of the supernatant was determined by the agar well diffusion assay, performed as described by Cintas et al. and Franz et al.[1, 13]. 50µl aliquot of supernatants were placed in wells(6-mm diameter) cut in cooled molten Mueller Hinton agar previously seeded with (10 5 CFU ml -1 ) indicator microorganisms listed above. After 2h at 4 o C, the plates were incubated at 37 o C for growth of target organism; after 24h, the diameters of the growth inhibition zones were measured Statistical Analyses The data obtained from this study were analysed using the Statistical Package for Social Sciences (SPSS), Version 16.0 Inc., Chicago, IL. 3. Results and Discussion 3.1. Enterocin Production and Purification Our results indicated that out of 658 screened Enterococcus faecalis strains, 227 (34.5%) were positive for enterocin production. This confirms the findings of others that Bacteriocin production is common among Enterococci [11, 15, 18]. Enterocin production was highest from E. faecalis strains isolated from bukataria (48.13%) followed

3 154 Olawale A. K. et al.: Antibacterial Activity of Enterocins EFC21 and EFT194 on Food-Borne Pathogenic Bacteria Strains by school and fast-food canteens (36.22% and 8.33%) respectively (Table 1). Enterocin produced from strains EFC21 and EFT194 was purified up to 11 and 8 fold, with end yield of 2.2% and 4.5% respectively. The yield was low compare to that of Guchi et al. and Gutie rrez et al. [14, 15]. Most bacteriocins in grampositive bacteria are small and heat stable (peptide bacteriocins). Their antimicrobial activities are directed against a broader spectrum of bacteria than is seen for bacteriocins produced from gram-negative bacteria [4, 9, 11]. The apparent purity of the enterocins was determined by cation exchange chromatography. Enterocin from EFC21 showed an absorbance of at 280nm, which is higher than that of EFT194 (0.014). Enterocin from EFT194 standard protein curve has a gradient of while that of EFC21 is (Figures 1&2) earlier reported. The elution profile from Cation Exchange Chromatography shows single peak of enterocin activity with an absorbance of at 280 nm. The purification result of enterocin EFT194 and EFC21 is summarized in Table (2). Sample Step Volume (ml) Table 2. Summary of enterocin EFC21 and EFT194 purification. Activity (AU/ml) Protein conc. (mg/ml) The existence of multiple chromatographic peaks after purification of EntP has been reported previously [5, 8] and is ascribed to the coexistence after reversed-phase fast protein liquid chromatography (RP-FPLC) of oxidized and non-oxidized forms of the bacteriocin. Crude extract and ammonium sulphate treated enterocin E194 has activity of 27Au/ ml and 34Au/ml respectively, protein concentration of 10.3mg/ml and 9.4mg/ml respectively. This gave a specific activity of 3.30 Au/mg with a purification fold of 8.03%. Table 1. Enterocin production in Enterococcus faecalis isolates from various ready-to-eat food outlets. Number of isolates producing enterocin Sources of bacteria isolates (%) School canteen N= (36.22) Fast-food canteen N = (8.33) Bukataria N = (48.13) Total N= (34.50) Total activity (AU) Total protein (mg) specific activity (AU/mg) EFC21 Crude Ammonium Sulphate ppt Yield (%) Ion Exchange EF194 Crude Ammonium Sulphate ppt Ion Exchange Purification fold Table 3. Enterocin activity against Gram-positive and Gram- negative pathogenic Bacteria. Gram- positive pathogenic bacteria Zone of inhibition (mm) EFC 21 EFT 194 Bacillus cereus ± ±.100 Listeria monocytogenes ± ±.058 Enterococcus faecium ± ±.115 Clostridium perfringens ± ±.000 Gram- negative pathogenic bacteria Pseudomonas aeruginosa 2.100± ±.058 Escherichia coli 6.067± ±.252 Proteus mirabilis 5.067± ±.058 Salmonella typhi 5.033± ±.058 Values are means of three replicates ± standard deviation 3.2. Antibacterial Effect of Enterocin Enterocins from the two E. faecalis strains EFC21 and EFT194 were employed to determine the antibacterial activity on gram positive and gram negative food pathogens; Bacillus cereus, Listeria monocytogenes, Clostridium perfringens, Enterococcus faecium, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Salmonella typhi. The results obtained confirm that both Enterocins from EFC21 and EFT194 have bacteriocinogenic effect on the pathogenic gram-positive and gram-negative bacteria tested. This is in line with findings of Zhang et al.; Nes et al.; O Sullivan et al. [24, 26, 28]. Meanwhile, enterocin EFC21 was more potent. More bactericidal effects were observed on the gram positive pathogens than with the gram negative ones. As revealed in tables 3, EFC21 displays more antibacterial activity than EFT194 on the all Gram negative and Gram positive bacteria, only in Pseudomonas aeruginosa and Bacillus cereus was there an exception [17]. Similarly more activity was observed with the enterocins on Gram- positive bacteria compared to the Gram- negative bacteria. It must be emphasized that a lack of detectable antimicrobial activity does not necessarily mean that genes involved in bacteriocin production are defective. First, it is of key importance to use a susceptible indicator, which can pose a problem, since some peptide bacteriocins act on only a narrow range of target bacteria. Secondly, the production of peptide bacteriocins is often regulated [25].

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