Appl Biochem Biotechnol DOI 10.1007/s12010-014-0757-x
Characterization of a New Bacteriocin from Lactobacillus plantarum LE5 and LE27 Isolated from Ensiled Corn Jairo Amortegui & Alexander Rodríguez-López & Deicy Rodríguez & Ana K. Carrascal & Carlos J. Alméciga-Díaz & Adelina del P. Melendez & Oscar F. Sánchez
Received: 4 October 2013 / Accepted: 22 January 2014 # Springer Science+Business Media New York 2014
Abstract Bacteriocins are low molecular peptides with antimicrobial activity, which are of great interest as food bio-preservatives and for treating diseases caused by pathogenic bacteria. In this study, we present the characterization of bacteriocins produced by Lactobacillus plantarum LE5 and LE27 isolated from ensiled corn. Bacteriocins were purified through ammonium sulfate precipitation and double dialysis by using 12- and 1-kDa membranes. Bacteriocins showed activity against Listeria innocua, Listeria monocytogenes, and Enteroccocus faecalis. Molecular weight was estimated through Tricine-SDS-PAGE and overloading the gel onto Mueller-Hinton agar seeded with L. monocytogenes, showing an inhibition zone between 5 and 10 kDa. NanoLC-MS/MS analysis allowed the identification of UPF0291 protein (UniProtKB/SwissProt Q88VI7), which is also presented in other lactic acid bacteria without assigned function. Ab initio modeling showed it has an α-helix-rich structure and a large positive-charged region. J. Amortegui : A. Rodríguez-López : C. J. Alméciga-Díaz Institute for the Study of Inborn Errors of Metabolism, School of Sciences, Pontificia Universidad Javeriana, Bogotá, Colombia D. Rodríguez : A. K. Carrascal Laboratorio de Microbiología de alimentos, Grupo de Biotecnología Ambiental e Industrial, Departamento de Microbiología, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia A. d. P. Melendez (*) : O. F. Sánchez (*) Pharmacy Department, Universidad Nacional de Colombia, Carrera 30 No. 45-03, Building 450, Bogotá, Colombia e-mail: [email protected]
.edu.co e-mail: [email protected]
C. J. Alméciga-Díaz (*) Protein Expression and Purification Laboratory, Institute for the Study of Inborn Errors of Metabolism School of Science, Pontificia Universidad Javeriana, Cra 7 No. 43 E 82, Building 54, Room 303A, Bogotá, Colombia e-mail: [email protected]
Present Address: O. F. Sánchez School of Chemical Engineering, Purdue University, West Lafayette, IN, USA
Appl Biochem Biotechnol
Bacteriocins were stable between 4 and 121 °C and pH 2 and 12, and the activity was inhibited by SDS and proteases. Mode of action assay suggests that the bacteriocin causes of target microorganism. Taken together, these results describe a possible new class IIa bacteriocin produced by L. plantarum, which has a wide stability to physicochemical conditions, and that could be used as an alternative for the control of foodborne diseases. Keywords Bacteriocins . Lactobacillus plantarum . Listeria monocytogenes antimicrobial activity
Introduction Several studies have addressed the distribution of human gastrointestinal microbiota, reporting a large diversity of prokaryotic phylum in which lactic acid bacteria (LAB) belonging to the Clostridiaceae, Lactobacillaceae, and Bifidobacteriaceae families are largely presented [1, 2]. Many of these LAB have been considered to have a probiotic effect, able to help in reducing cholesterol levels, and modulate the immune system, with anticariogenic and antitumoral activity, among other activities that have been widely reviewed [3, 4]. One important and widely studied probiotic effect is based on their ability to suppress the growth of potential pathogen microorganisms in the gut , which is closely related to the ability of several Gram-positive and Gram-negative microorganisms to protect themselves and exclude competitive bacteria in ecological environments via production of antimicrobial substances. This ability has been exploited since ancient times for food preservation by using different Lactobacillus strains, allowing some of them to have the generally recognized as safe status . The antimicrobial activity in Lactobacillus strains has been attributed to the production of antimicrobial substances, like oxygen peroxide, organic acids, biosurfactants, and bacteriocins . Due to the antimicrobial activity of bacteriocins, they are of great interest as food biopreservative and for the treatment of diseases caused by pathogenic bacteria [7, 8]. Bacteriocins are low molecular peptides with antimicrobial activity against a limited range of bacteria closely related to the producer strain . The antimicrobial mechanism of bacteriocins includes pore formation in the cell membrane and impartment of gene expression or protein biosynthesis . A general classification of bacteriocins has been suggested based on the presence or not of posttranslational modifications. Class I bacteriocins or lantibiotics contain lanthionine or β-methyl-lanthionine, while unmodified antimicrobial peptides are grouped in class II bacteriocins . This class II bacteriocins could be further subdivided in five groups: antilisterial one-peptide bacteriocins with a YGNGV motif (class IIa); two-peptide bacteriocins (class IIb); circular bacteriocins (class IIc); unmodified, linear, non-pediocin-like, single-peptide bacteriocins (class IId); and the microcin E492-like bacteriocins (class IIe) . A third class corresponding to nonbacteriocin lytic proteins or bacteriolysins has been proposed by Cotter et al. . This class III is characterized for not having always the specific immunity genes that escort the bacteriocin structural genes that are presented in the “true” bacteriocins. In addition, a classification based on genetic and biochemical characteristics has been suggested . The emergence of pathogen microorganisms that present resistance to antibiotic treatments and no desirable side effects on natural human microbiota when broad-spectrum antibiotics are used and health concerns with regard food preservation in a more natural way rather than chemical have promoted the search of new antimicrobial compounds like bacteriocins. In order to address all these issues, several bacteriocins like weissellin, sakacin, pediocin, enterocin, and plantaricin, among others from different sources, have been characterized and purified [12–16].
Appl Biochem Biotechnol
Previously, we isolated Lactobacillus strains from corn ensilage and molasses and evaluated the effect of fructooligosaccharide (FOS) supplementation on their growth and bacteriocin production, from which the strains Lactobacillus plantarum LE5 and LE27 showed the highest antimicrobial activity . This activity was associated with a protein-based molecule, and the activity spectrum was different within them. The bacteriocin from L. plantarum LE5 presented activity against Listeria innocua, Listeria monocytogenes, and Enteroccocus faecalis, while the bacteriocin from L. plantarum LE27 presented antimicrobial effect against L. innocua, L. monocytogenes, E. faecalis, Escherichia coli, and Salmonella enteritidis . In this study, we present the partial purification and chemical and molecular characterization of bacteriocin UPF0291 from L. plantarum LE5 and LE27 isolated from ensiled corn. The obtained fractions describe a possible new class IIa bacteriocin produced by L. plantarum, which has a wide stability to physicochemical conditions, and that could be used as an alternative for the control of foodborne diseases.
Materials and Methods Cell-Free Protein Extract Production L. plantarum LE5 and LE27 were previously isolated from corn ensilage  and conserved in 20 % glycerol at −80 °C. Protein extracts were obtained by growing L. plantarum LE5 or LE27 in sealed screw-cap flasks with 100 mL of glucose-free Man–Rogosa–Sharpes (MRS) medium supplemented with 2 % FOS (33 % w/w, 1-kestose; 33% w/w, nystose; 33% w/w, 1fructofuranosyl nystose, Wako Pure Chemical Industries, Osaka, Japan), at pH 6.4±0.2 and 37 °C during 48 h under micro-aerophilic conditions . After cultivation, the exhausted culture medium was centrifuged at 3,200×g and 4 °C for 30 min, and the supernatant was sequentially filtered through Whatman filter paper no. 1 and polyether sulphone membranes of 0.45 and 0.22 μm (Pall Corp, Port Washington, NY, USA). Finally, this cell-free extract was adjusted to pH 6.0±0.2 with 10 N NaOH. Partial Bacteriocin Purification Bacteriocins from L. plantarum LE5 and LE27 cell-free extracts were purified through ammonium sulfate precipitation and double dialysis. Briefly, cell-free extracts were precipitated with a 70 % saturated solution of ammonium sulfate . The obtained pellet, after centrifugation at 3,200×g and 4 °C for 40 min, was resuspended in 25 mM Tris–HCl, pH 7.0. Preliminary results suggested that bacteriocins produced by L. plantarum LE5 and LE27 have a molecular mass lower than 10 kDa. In this sense, bacteriocins were purified through a double dialysis by using 12and 1-kDa membranes (BioLynx Inc.). Finally, fractions were concentrated through lyophilisation and resuspended in 25 mM Tris–HCl, pH 7.0. Protein concentration of cell-free crude extract and fractions retained at 12- and 1-kDa membranes, hereafter named 12- and 1-kDa fractions, were determined by using bicinchoninic acid protein assay kit under manufacturer instructions (Thermo Scientific, IL, USA). In addition, samples were analyzed through Tricine-sodium dodecyl sulfate (SDS)-PAGE [18, 19], and electrophoresis gels were revealed with silver staining. Antimicrobial Activity Antimicrobial activity of the cell-free extract and 12- and 1-kDa fractions was evaluated by the agar well diffusion method , using the following indicator strains for the test: L. innocua
Appl Biochem Biotechnol
(CMPUJ 290), L. monocytogenes (CMPUJ 296/ATCC 7644), E. faecalis (CMPUJ 286HUSI), Staphylococcus aureus (CMPUJ 080/ATCC 6538), S. enteritidis (CMPUJ 302/ATCC 13076), E. coli O157:H7 (CMPUJ 215), Bacillus subtilis (CMPUJ 075/ATCC 6633), Pseudomonas aeruginosa (CMPUJ 055/ATCC 9027), and Leuconostoc citreum (CMPUJ 328). All microorganisms were obtained from the bacteria collection of the Microbiology Department of the School of Science at Pontificia Universidad Javeriana (CMPUJ, Bogota, Colombia). Indicator strains were activated in brain–heart infusion (BHI) broth at their optimal temperature, 30 or 37 °C, for 16 h. For antimicrobial activity, Mueller–Hinton agar (MHA, Oxoid, UK) plates with 7-mm wells were seeded with the selected microorganism at a final concentration of 105 colony forming units (CFU)mL−1, and the wells were filled with 80 μL of either filter-sterilized cell-free extract or the fractions obtained along the purification. Then, the plates were pre-incubated at 4 °C during 18 h and later incubated at 30 or 37 °C, depending on the optimal temperature of the used microorganism. Inhibition zones were examined after 24 and 48 h incubation. Antimicrobial activity is reported as the diameter difference between the inhibition zone and the diameter of the well. The described culture medium for Lactobacillus growth and buffer 25 mM Tris–HCl, pH 6.0, were used as negative controls, while a 100mg mL−1 nisin (Sigma-Aldrich, Saint Louis, MO, USA) was used as positive control. Each assay was performed in triplicate. Proteomic Identification and Structure Modeling of Bacteriocin Bacteriocin identification was carried out by NanoLC-MS/MS. For this purpose, two different gels for Tricine-SDS-PAGE electrophoresis were loaded with the same amount of the cell-free extracts and fractions and run under the same conditions. Loaded samples were neither reduced nor heat denatured. After running the gels, one gel was rinsed with deionized autoclaved water and overlaid onto a MHA agar plate seeded with L. monocytogenes at a final concentration of 105 CFU mL−1. The MHA agar plate was pre-incubated and incubated as previously described for antimicrobial activity. The other gel was stained by an aldehydefree silver ammonia staining method . Gels were side to side compared for detecting the protein band that generated the inhibition zone on the L. monocytogenes plate culture. This protein band was cut out for further analysis by NanoLC-MS/MS (Applied Biomics, Hayward, CA, USA). Ab initio model of the identified protein (UPF0291 protein lp_2062, UniProtKB/ Swiss-Prot Q88VI7) was done by QUARK Server . Structures were analyzed with PDBsum , YASARA View v11.4.18 (YASARA Biosciences GmbH, Vienna, Austria), and Molegro Virtual Docker v5.5 (CLC bio, Aarhus N, Denmark). Effect of pH, Temperature, Chemical Agents, and Proteases The effect of pH, temperature, chemical agents, and proteases on antimicrobial activity of cellfree protein extracts and 12- and 1-kDa fractions was conducted by the agar well diffusion method, as previously described, using L. monocytogenes as the indicator strain. To evaluate the effect of pH on antimicrobial activity, aliquots of the protein extracts and fractions were pH adjusted using sterile solutions of 1 M NaOH or 1 M HCl to pH 2, 4, 6, 8, 10, or 12 and incubated at 37 °C for 1 h. After incubation, pH was adjusted to 6.0, and the antimicrobial activity was evaluated. To evaluate the effect of temperature on antimicrobial activity, aliquots of the protein extracts and fractions were pH adjusted to 6.0 and incubated for 1 h at 4, 25, 37, 80, or 100 °C. In addition, a sample set was treated by steam sterilization (at 121 °C and 15 atm for 15 min). After incubation, antimicrobial activity was evaluated.
Appl Biochem Biotechnol
To evaluate the effect of chemical compounds on antimicrobial activity, aliquots of the protein extracts and fractions were pH adjusted to 6.0 and enriched to a final concentration of 1 % SDS, 1 % Tween 80, 1 % Triton X-100, 6 M urea, 25 mM ethylenediaminetetraacetic acid (EDTA), or 6.5 % NaCl [18, 24, 25]. Samples were incubated at 37 °C for 1 h. After incubation, antimicrobial activity was evaluated. Solutions at the selected concentration of each chemical compound free of any protein extract or fraction were used as controls. To evaluate the effect of proteinase K, trypsin, and catalase (Sigma-Aldrich, Saint Louis, MO, USA) on antimicrobial activity, aliquots of the protein extracts and fractions were treated with each enzyme at a final concentration of 1 mg mL−1. Samples were incubated at 37 °C and pH 6.0 for 1 h. After incubation, samples were heated at 90 °C for 5 min to inactivate the enzymes and then tested for antimicrobial activity. Solutions of each enzyme free of any protein extract or fraction were used as control. All assays were performed in triplicate, and results are reported as relative antimicrobial activity. Minimum Inhibitory Concentration (MIC) MIC was estimated for the 12-kDa fraction from L. plantarum LE5 and LE27, due to sample volume restriction for the 1-kDa fraction. Briefly, serial dilutions between 1:2 and 1:1,000 of the 12-kDa fraction were evaluated by the agar well diffusion method as described for antimicrobial activity, using L. monocytogenes at a final concentration of 105 CFU mL−1. MIC was defined at the lowest protein concentration (in micrograms per milliliter) producing inhibition following 24 and 48 h at 37 °C. All assays were performed in triplicate. Mode of Action of L. plantarum LE5 and LE27 Bacteriocins The effect of L. plantarum LE5 and LE27 bacteriocins on L. monocytogenes growth was evaluated as reported previously [18, 24]. Briefly, L. monocytogenes was grown in BHI medium and incubated at 30 °C, without agitation . Once the microorganism reached the exponential growth phase (~4 h), 12-kDa fraction (initial protein concentration of 21.5 and 27.4 mg mL−1 for L. plantarum LE5 and LE27, respectively) was added to a final concentration of 20 % v/v to a final volume of 20 mL. Aliquots of 200 μL were taken every 2 h, for up to 14 h, to monitor the microorganism growth. Optical density was determined at 620 nm, and the number of CFU was determined by seeding dilutions of the microorganism on BHI agar, followed by incubation at 30 °C for 24 h. As control, a L. monocytogenes culture under described conditions without any bacteriocin was used.
Results and Discussion Partial Purification of L. plantarum LE5 and LE27 Bacteriocins Partial purification of bacteriocins was done through ammonium sulfate precipitation and double dialysis from which two fractions were obtained. Figure 1 shows the results of antimicrobial activity against L. innocua for the cell-free extracts and 12- and 1-kDa fractions from L. plantarum LE5 and LE27. Preliminary results suggested that the bacteriocins from these strains have a molecular weight lower than 10 kDa. In this sense, bacteriocin activity after double dialysis was expected only within the 1-kDa fraction. However, antimicrobial activity was observed in both the 12- and 1-kDa fractions (Fig. 1a), which could be owed to a
Appl Biochem Biotechnol
Fig. 1 Partial purification of L. plantarum LE5 and LE27 bacteriocins. Partial purification of bacteriocins from L. plantarum LE5 (white) and LE27 (gray) was done through ammonium sulfate precipitation and double dialysis. Antimicrobial activity, against L. innocua, is reported as diameter of the inhibition zone (in millimeter) (a) and diameter of the inhibition zone normalized against the protein concentration (in millimeters per milligram of protein) (b)
saturation of the dialysis membrane with the proteins presented into the culture media (e.g., MRS medium). The highest antimicrobial activity was observed for the 12-kDa fraction (Fig. 1a). However, when the diameter of the inhibition zones was normalized by the protein concentration of each sample, the 1-kDa fraction showed the highest antimicrobial activity (Fig. 1b), though these fractions had about 20 and 5 % of the protein concentration of the cell-free extracts and the 12kDa fractions, respectively. The obtained antimicrobial activity for the 1-kDa fraction, when compared to the corresponding cell-free extract, was 1.5- and 2.8-fold higher for L. plantarum LE5 and LE27, respectively, which suggests that the purification process removed antimicrobial activity inhibitors. Bacteriocin Identification To confirm the molecular mass of bacteriocins and discard the presence of an active peptide higher than 12 kDa, samples were analyzed by Tricine-SDS-PAGE and the electrophoresis gel was overlaid onto a MHA agar plate seeded with L. monocytogenes. The cell-free extract and the 1-kDa fraction from L. plantarum LE5 and LE27 showed an inhibition zone between 4.5 and 10 kDa (Fig. 2). This result confirms that the bacteriocin produced by these strains presents a molecular weight lower than 10 kDa, which agrees with the molecular mass reported for other bacteriocins produced by Lactobacillus spp. [9, 26]. Nevertheless, these bacteriocins are bigger than most of bacteriocins produced by L. plantarum strains, which show a molecular mass of up to 5 kDa [18, 27–29], while bacteriocins of 10 and 14 kDa have been reported for L. plantarum strains, but accompanied by smaller bacteriocins (~3.0 kDa) [28, 30]. The protein band between 4.5 and 10 kDa was cut out from the electrophoresis gel and analyzed by NanoLC-MS/MS. The peptide INELAHK was identified within the 1-kDa fraction from L. plantarum LE5 and LE27. This peptide belongs to the UPF0291 protein lp_2062 (UniProtKB/Swiss-Prot Q88VI7) of L. plantarum ATCC BAA-793/NCIMB 8826/WCFS1, which has 79 amino acids and a molecular weight of 9.3 kDa. This protein was presented in other LAB especially L. plantarum strains, without an assigned function (Fig. 3). In addition, similar proteins were not identified within the bacteriocins available at BACTIBASE . These results suggest that L. plantarum LE5 and LE27 produce a nonpreviously described bacteriocin, hereinafter named plantaricin UPF0291, which could represent a new group of antimicrobial peptides .
Appl Biochem Biotechnol
Fig. 2 Estimation of molecular weight of L. plantarum LE5 and LE27 bacteriocins. Cell-free extract and 1-kDa fraction from L. plantarum LE5 and LE27 were analyzed by Tricine-SDS-PAGE. The electrophoresis gel was overlaid onto a MHA agar plate inoculated with L. monocytogenes
Ab initio modeling of plantaricin UPF0291 suggested that it has three α-helices and a βturn (Fig. 4a), and a large positive-charged region (Fig. 4b), without the presence of disulfide bonds. Similar results have been observed for other class IIa bacteriocins, which show a hairpin/α-helix-rich structure without disulfide bonds [11, 31].
Fig. 3 Phylogenetic tree of the top 20 proteins with high identity to UPF0291 protein lp_2062 (UniProtKB/ Swiss-Prot Q88VI7). The region between 4.5 and 10 kDa was cut from the electrophoresis gel and analyzed by NanoLC-MS/MS which allowed the identification of the protein UPF0291 protein lp_2062 (UniProtKB/SwissProt Q88VI7, indicated with the arrow) of L. plantarum strain ATCC BAA-793/NCIMB 8826/WCFS1. Protein sequence was used to identify similar proteins within Uniprot database by using Blast tool. Sequences were aligned by Muscle, and neighbor joining phylogenetic tree was generated by using Mega 5.2. UniProtKB/SwissProt accession numbers are presented in parenthesis
Appl Biochem Biotechnol
Fig. 4 Ab initio model of bacteriocin UPF0291. Ab initio model was done by QUARK Server (a), while the electrostatic potential was computed by using Molegro Virtual Docker v5.5. Blue and red surfaces indicate positive and negative electrostatic potential, respectively
Antimicrobial Activity Antimicrobial activity of cell-free extracts and 12- and 1-kDa fractions was evaluated against different Gram-positive and Gram-negative microorganisms (see “Materials and Methods” section). The cell-free extracts and mentioned fractions from L. plantarum LE5 and LE27 showed antimicrobial activity against L. innocua (Fig. 1a), L. monocytogenes (Fig. 5a), and E. faecalis (Fig. 5b). After activity normalization by protein concentration, it was observed for the three microorganisms that the bacteriocin from L. plantarum LE27 has higher antimicrobial activity than that of L. plantarum LE5 (Figs. 1b and 5c, d). These results agree with those reported by Muñoz et al. , who reported an antimicrobial activity of crude protein extracts
Fig. 5 Antimicrobial activity. Cell-free extracts and dialysis fractions (12 and 1 kDa) from L. plantarum LE5 (white) and LE27 (gray) were evaluated against L. monocytogenes (a, c) and E. faecalis (b, d). Antimicrobial activity is presented as the diameter of the inhibition zone (in millimeter) (a, b) and normalized against the protein concentration (in millimeters per milligram of protein) (c, d)
Appl Biochem Biotechnol
of L. plantarum LE5 and LE27 against L. innocua ATCC 33090, L. monocytogenes ATCC 35152, and E. faecalis ATCC 29212. However, they reported antimicrobial activity of these extracts against E. coli ATCC 25922 and S. enteritidis. This discrepancy may be owed to differences in the genotype of the used strains for evaluating the antimicrobial activity (e.g., E. coli ATCC 25922 vs. E. coli O157:H7). Nevertheless, the antimicrobial activity of the bacteriocin from L. plantarum LE5 and LE27 agrees with previous reports of Lactobacillus spp. bacteriocins, in which a preferred activity against Gram-positive bacteria has been observed [26, 32, 33]. In the case of L. plantarum, antimicrobial activity of the bacteriocins ST28MS and ST26MS against Lactobacillus casei, Lactobacillus sakei, S. aureus, and E. faecalis was reported , while for the bacteriocin ST71KS, antimicrobial activity against L. monocytogenes, Enterococcus faecium, Lactobacillus delbrueckii, and Lactobacillus paracasei was reported . Effect of pH, Temperature, Chemical Agents, and Proteases The effect of pH, temperature, chemical agents, and proteases on the antimicrobial activity of cell-free extracts and 12- and 1-kDa fractions from L. plantarum LE5 and LE27 was evaluated. For both L. plantarum LE5 and LE27, the maximum antimicrobial activity for the cell-free extracts and 12- and 1-kDa fractions was obtained at pH 6.0 (Fig. 6). Overall, dialysis fractions showed higher antimicrobial activity than that of cell-free extracts, though behavior depended on the evaluated pH. At pH 2.0 and 4.0, similar antimicrobial activities were observed for cellfree extracts and dialysis fractions, with levels between 82 and 96 % of those observed at pH 6.0 (Fig. 6). On the other hand, at basic pH, it was observed that there is a marked reduction of antimicrobial activity for the cell-free extract, reaching 3 % of pH 6.0 levels, while for the dialysis fractions, the activity has reduced up to 24 % (Fig. 6). Similar results have been reported for bacteriocins ST23LD, ST341LD, bacST202Ch, bacST216Ch, and ST71KS, from L. plantarum, which have shown stability between pH 2.0 and 12.0 [18, 24, 27, 34]. Nevertheless, these results differ from the ones for the L. plantarum LPBM10 bacteriocin that has a maximum activity at pH 4.0 and presented a marked reduction at pH values higher than 5.0  and the ones for the bacteriocins ST28MS and ST26MS from L. plantarum that showed a 50 % reduction in the antimicrobial activity at pH values lower than 4.0 . The cell-free extracts and the dialysis fractions showed to be highly stable between 4 and 37 °C, and a reduction of up to 30 % in the antimicrobial activity was observed at 80 and 100 °C (Fig. 7a). On the other hand, the antimicrobial activity obtained under steam sterilization conditions, at 121 °C and 15 atm for 15 min, was between 56 and 89 % of the ones observed at 37 °C (Fig. 7b). At the evaluated temperature range, the purification of the bacteriocins showed the most significant effect on the antimicrobial activity at 121 °C, where the 12- and 1-kDa fractions exhibited an antimicrobial activity about 5 and 30 % higher, respectively, than that of the cell-free protein extracts. Nonetheless, for both strains, the most stable fraction was the 1-kDa fraction. Similar results of temperature stability have been reported for bacteriocins LPBM10, bacST202Ch, bacST216Ch, enterocin AS-48, and plantaricin OL15 [18, 35, 36]. Although other bacteriocins have shown to be highly stable up to 60 °C [24, 25, 37], they presented a significant reduction in the antimicrobial activity above 80 °C. For example, lactocin NK24 showed a reduction of 87 % at 100 °C and was completely inactivated after steam sterilization conditions ; lactocin MMFII, from L. lactis, showed 25 and 8.3 % of the antimicrobial activity at 80 and 110 °C, respectively ; and other bacteriocins isolated from Lactobacillus spp. showed an antimicrobial activity reduction of the 20 and 70 % at 100 °C and at steam sterilization conditions, respectively . The effect of several chemical agents was evaluated on the antimicrobial activity of cell-free extracts and dialyzed fractions from L. plantarum LE5 and LE27 (Fig. 8). Antimicrobial
Appl Biochem Biotechnol
Fig. 6 Effect of pH on antimicrobial activity. Cell-free extracts (white) and 12- (gray) and 1-kDa (black) fractions from L. plantarum LE5 (a) and LE27 (b) were incubated 1 h at 30 °C and pH 2, 4, 6, 8, 10, or 12. Antimicrobial activity was determined by the diameter of the inhibition zone and normalized against the condition that presented the largest inhibition zone. This is reported as a percentage of the diameter of the inhibition zone observed at pH 6.0
activity was notably affected by SDS, and an activity reduction between 70 and 95 % of the levels without SDS was observed. Exempting the cell-free extract assay with EDTA, which produced a 40 % reduction in the antimicrobial activity, the other evaluated compounds induced a reduction of about 20 % of the antimicrobial activity (Fig. 8). Similar results have been reported for L. plantarum bacteriocins, although in those cases, the antimicrobial activity is not affected by SDS addition . Furthermore, similar results have been reported for bacteriocin J49, enterocin EJ97, bozacin B14, pediocin ST18, and bacteriocin ST15 [33, 40, 41]. As mentioned before, bacteriocins can be classified in three classes: class I, which includes heat-stable peptides with several posttranslational modifications and has low molecular weight (