Selective Capture of Pediocin Secreted by Food-grade Lactic Acid Bacteria for Development of Bactericidal
6.3. Results and Discussion
Figure 6.2. Transmission electron microscope images of (a) citrate-stabilized gold nanoparticle (AuNP), (b) silica nanoparticle (SNP) and (c) iron oxide nanoparticle (IONP). The average particle size of the nanoparticles was: AuNP (12.0 ± 2.0 nm), SNP (40-60 nm) and IONP (20-40 nm).
Figure 6.3. FTIR analysis of IONP-pediocin composite.
A representative TEM image of the nanoparticles is indicated in Figure 6.2. The average particle size of AuNPs, SNPs and IONPs were observed to be 12.0 ± 2.0 nm, 40-60 nm and 20-40 nm, respectively. The NPs were interacted with the cell-free culture filtrate and then
the NP-P composites were separated by centrifugation. The formation of NP-P composites were studied by FTIR analysis. A representative result of the FTIR analysis is depicted in Figure 6.3. It is evident from the figure that the characteristic amide stretching frequencies (1650 cm-1 and 1420 cm-1) of cell-free supernatant was retained in the iron oxide-pediocin (IONP-P) composite, which suggested the presence of the peptide on the surface of IONPs.
Similar observations were also made for gold nanoparticle-pediocin (AuNP-P) and silica nanoparticle-pediocin (SNP-P) composites (refer to Figure A6.1 in Appendix).
6.3.2. Bactericidal activity of nanoparticle-pediocin (NP-P) composite
Antibacterial activity of the generated NP-P composites was initially ascertained by a conventional agar well diffusion assay. Representative results of the initial experiments conducted with various proportions of culture-filtrate and NPs are indicated in Figures A.6.2-A6.4 (Appendix). The minimum concentration of NPs and time of interaction which enabled efficient capture of pediocin from the culture filtrate was determined by observing for the presence of zone of inhibition in the NP-P composite samples and a corresponding lack of zone of inhibition in the respective supernatants following separation of NP-P composites. From the results indicated in Figures A.6.2-A6.4 (Appendix), it was evident that the minimum concentration of the respective NPs and minimum time of interaction required to generate the NP-P composites were as follows: (i) For AuNP-P composite: 10 mL of cell-free culture filtrate and 20 mL of 0.32 nM AuNPs interacted for 30 min, (ii) For SNP-P composite: 10 mL of cell-free culture filtrate interacted with SNPs (final concentration 0.5 mg/mL) for 30 min and (iii) For IONP-P composite: 10 mL of cell-free culture filtrate interacted with IONPs (final concentration 0.5 mg/mL) for 30 min. All the subsequent experiments to generate the respective NP-P composites were conducted with the aforementioned proportions and interaction time. A representative result which demonstrates prominent anti-listerial activity of all the NP-P composites, manifested as large zones of inhibition around the well of the tested samples is indicated in Figure 6.4, well nos. 2-4. It may be mentioned that these zones of inhibition were equivalent to that observed for the cell-free culture filtrate (Figure 6.4, well no. 1). It is also to be noted that none of NPs alone exhibited any antibacterial activity (Figure 6.4, well nos. 5-7). Time-kill curves indicated that the NP-P composites had an adverse effect on the viability
Figure 6.4. Agar well diffusion assay to test the antibacterial activity of various nanoparticle- pediocin composites against Listeria monocytogenes Scott A. (1) cell-free culture supernatant of Pediococcus pentosaceus CRA51, (2) AuNP-pediocin composite, (3) SNP-pediocin composite, (4) IONP-pediocin composite, (5) AuNP, (6) SNP and (7) IONP.
of L. monocytogenes Scott A and following 6 h of treatment with the nanocomposites (200 AU/mL pediocin in each), the viable cell number dropped drastically from an initial 6.0 Log10 CFU to around 2.0 Log10 CFU (Figure 6.5a). cFDA-SE leakage assay indicated that NP-P composites rendered membrane damage in L. monocytogenes Scott A and the magnitude of cFDA efflux from the NP-P composites was comparable to purified pediocin of equivalent concentration and the activity was dose-dependent (Figure 6.5b). Treatment of L. monocytogenes Scott A cells with NP-P composites also lead to rapid dissipation of membrane potential as evident from the DiSC35 assay (Figure 6.6). Thus, the ability to dissipate the transmembrane potential in target cells, which is a signature activity of pediocin (Singh et al., 2012). was also retained in the NP-P composites. This suggested that the present method of capture of pediocin by NPs followed by concurrent generation of NP-P composites perhaps did not cause any significant perturbation of the native structure of pediocin and hence the activity was retained. Potent bactericidal activity of NP-P composite was also observed in transmission electron microscope (TEM) analysis.
Figure 6.5. (a) Time-kill curves of purified pediocin, AuNP-pediocin composite, SNP-pediocin composite and IONP-pediocin composite against L. monocytogenes Scott A. (b) cFDA-SE leakage assay with (1) purified pediocin, (2) AuNP-pediocin composite, (3) SNP-pediocin composite and (4) IONP- pediocin composite against L. monocytogenes Scott A. Pediocin activity (AU/mL) in each of these samples is indicated.
Figure 6.6. DiSC35-based membrane depolarization assay of L. monocytogenes Scott A cells treated with nanoparticle-pediocin composites.
Figure 6.7. Transmission electron microscope images of L. monocytogenes Scott A cells treated with (a) IONP and (b) IONP-pediocin composite. Arrow indicates damaged cells.
A representative result indicated that IONP-treated cells of L. monocytogenes Scott A cells retained their characteristic morphology (Figure 6.7a) whereas the IONP-P-treated cells revealed prominent morphological perturbations (Figure 6.7b). Interestingly, all the NP-P composites exhibited antibacterial activity against model pathogens (Figure 6.8).
6.3.3. Desorption of pediocin from nanoparticle-pediocin (NP-P) composite
The one-step capture of pediocin through electrostatic interaction of the bacteriocin and anionic NPs also rendered the interesting possibility of desorption of the bacteriocin. The desorption studies with the NP-P composites were conducted at pH 2.0 so as to disrupt the charge-based interaction between the bacteriocin and the NPs. The activity obtained for desorbed pediocin from AuNP-P composite was negligible as compared to that obtained for SNPs and IONPs (Figure 6.9), suggesting negligible desorption of pediocin from AuNP-P composite. Presence of four cysteines in pediocin (Rodríguez et al., 2002) renders the possibility of strong Au-thiol interactions (Brust et al., 1994) apart from charge-based interactions and perhaps prevented desorption of pediocin by mere change of pH.
Figure 6.8. Antimicrobial spectrum of purified pediocin, AuNP-pediocin composite, SNP-pediocin composite and IONP-pediocin composite determined by an agar well diffusion assay.
Figure 6.9. Activity of pediocin desorbed from anionic nanoparticles.
6.3.4. Magnetic separation of IONP-P composite and multiple adsorption-desorption cycles
Following capture of pediocin by IONPs, efficient recovery of IONP-P composite was also achieved through magnetic separation (Figure 6.10a) in lieu of a centrifugation step and the membrane-depolarization activity typically associated with pediocin was observed for the recovered composite (Figure 6.10b).Desorption of pediocin from IONP-P composite was achieved at pH 2.0 and the recovered IONP was recycled to explore multiple cycles of pediocin adsorption-desorption to ascertain the feasibility of repeated magnetic separation and re-usability of IONPs. The activity obtained for IONP-P composite using recycled IONPs was as high as 80% even in the third cycle of adsorption and decreased thereafter to around 50% after 5 cycles of adsorption (Figure 6.11). These results indicated the benefit of using IONPs, which could be salvaged and reused as a capture probe. Adsorption studies with IONPs suggested that the steady state adsorption isotherm obtained for cell- free culture supernatant as well as purified pediocin followed a Langmuir isotherm model (refer to Figure A6.5 and Table A6.1 in Appendix) and thus suggested adsorption at specific homogeneous sites on IONPs and a monolayer adsorption process (Saha et al., 2011).
6.3.5. Purification of pediocin using IONPs and HPLC
Recovery of IONP-P composite by magnetic separation followed by desorption at pH 2.0 resulted in 16-fold purification of pediocin (refer to Table A6.2 in Appendix). A similar HPLC profile and retention time suggested that the homogeneity of desorbed pediocin from IONPs was on par with purified pediocin (Figure 6.12). The HPLC eluted fraction of desorbed pediocin from IONPs exhibited anti-listerial activity in an agar well diffusion assay (Figure 6.12). Further, the HPLC eluted fraction also exhibited the characteristic membrane-directed activity in a cFDA leakage assay (Figure 6.13a) and membrane depolarization in target bacteria (Figure 6.13b), akin to purified pediocin.
Figure 6.10. (a) Image of a vial showing separation of IONP-pediocin composite using a horse- shoe magnet. The IONP-pediocin composite is visible as a brownish-black deposit on the right side wall of the vial. (b) DiSC35-based membrane depolarization assay of L. monocytogenes Scott A cells treated with magnetically separated IONP-pediocin composite and the supernatant following magnetic separation. Inset indicates agar well diffusion assay against L. monocytogenes Scott A with (1) magnetically separated IONP-pediocin composite (corresponding to 200 AU/mL pediocin) and (2) supernatant.
Figure 6.11. Pediocin activity in IONP-P composite in successive adsorption cycles following magnetic separation of IONP-pediocin composite and desorption of pediocin.
Figure 6.12. HPLC profile for pediocin desorbed from IONP and pediocin purified by cell- adsorption method and respective anti-listerial activity of eluted fractions.
Figure 6.13. (a) cFDA-SE leakage assay with HPLC-eluted fraction of purified pediocin and pediocin desorbed from IONPs. (b) Membrane depolarization assay of L. monocytogenes Scott A cells treated with HPLC-eluted fractions of pediocin desorbed from IONP-pediocin composite and purified pediocin.