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Muriel Subirade - One of the best experts on this subject based on the ideXlab platform.
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An improved and simplified method for the large-scale purification of Pediocin PA-1 produced by Pediococcus acidilactici.
Biotechnology and Applied Biochemistry, 2006Co-Authors: Lucie Beaulieu, Denis Groleau, Hafida Aomari, Muriel SubiradeAbstract:The bacteriocin Pediocin PA-1 produced by Pediococcus acidilactici PAC 1.0 offers significant potential as a food preservative and as an antimicrobial agent in the medical area. However, low production yields and difficulties in obtaining significant amounts of pure Pediocin PA-1 have limited, in part, its biochemical and physical characterization. In the present study, we describe a simple and more efficient purification strategy for Pediocin PA-1. A hydrophobic interaction chromatography step using an octyl-Sepharose column was introduced for final purification and polishing. The new method is a scalable one, uses only two steps and yields highly purified Pediocin PA-1 with a recovery as high as 73%, which is at least two to three times more than that of the methods reported so far. Highly purified, biologically active Pediocin PA-1 of the correct molecular mass (4624 Da, with two disulphide bridges) was obtained. Fourier-transform infrared analysis runs at p2H 6 indicated that Pediocin PA-1 was more structured than similar Pediocin PA-1 samples purified using the earlier purification scheme.
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Production of Pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material.
Protein expression and purification, 2005Co-Authors: Lucie Beaulieu, Denis Groleau, Carlos B. Miguez, Jean-françois Jettéj.-f. Jetté, Hafida Aomari, Muriel SubiradeAbstract:Abstract Expression of the pedA gene from Pediococcus acidilactici , coding for mature bacteriocin Pediocin PA-1, was investigated using the yeast Pichia pastoris to obtain larger quantities of Pediocin to support additional studies, including structure–function research. Following various cloning strategies, a KM71H (Mut s ) strain was selected. A significant concentration (74 μg/ml) of extracellular recombinant Pediocin was obtained but the Pediocin showed no biological activity. Supernatant fluids from P. pastoris cultures, harboring or not pedA , inhibited the biological activity of natural Pediocin PA-1. The recombinant Pediocin appeared as a mixture of three main fractions (7–8, 11, 20 kDa vs. 4.6 kDa for natural Pediocin PA-1). The recombinant Pediocin was also less hydrophobic and behaved differently when subjected to isoelectric focusing. Strong evidence indicated that some “collagen-like” material was tightly associated, most probably via covalent binding, to the recombinant Pediocin. The “collagen-like” material was most probably responsible for the lack of biological activity of the recombinant Pediocin and for the differences observed regarding some of the physico-chemical properties. Both the recombinant Pediocin and natural Pediocin were sensitive to collagenase, suggesting that Pediocin PA-1 may possess a somewhat “collagen-like” nature. Interestingly, recombinant Pediocin preparations showed the ability to assemble into fibrils.
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Conformational changes of Pediocin in an aqueous medium monitored by fourier transform infrared spectroscopy: a biological implication.
International Journal of Biological Macromolecules, 2003Co-Authors: Helene Gaussier, Marc C Lavoie, Muriel SubiradeAbstract:Abstract Fourier transform infrared (FTIR) spectroscopy was used to investigate the secondary structure of Pediocin PA-1 in different aqueous media in relation to its antimicrobial activity. The experiments were performed at pD (pH meter corrected for deuterium isotope effect) 6, 7, and 8 and during a heating–cooling cycle of 20–80 °C. At pD 6, (i.e. Pediocin’s most active form), the FTIR results show that Pediocin adopts an unordered structure with a small contribution of β-turn. After a heating–cooling cycle, thermally-induced changes in Pediocin are reversed and its activity is maintained. Increasing the pD to 7 and 8 leads to a more ordered secondary structure. For these two pD values, an increase in temperature induces an irreversible aggregation of protein as revealed by the amide I′ band. The analysis of the Tyr region provides more insight into the aggregation process. In fact, it appears to be a two-step process, involving first the C (carboxy)-terminus of Pediocin and then the N (amino)-terminus. This study reveals two major points: (1) the preservation of Pediocin flexibility is essential for maintaining its activity; and (2) the aggregation of its C-terminus is sufficient to induce a loss of activity, suggesting that this region plays an important role in the activity of Pediocin.
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Binding of Pediocin PA-1 with anionic lipid induces model membrane destabilization.
Applied and environmental microbiology, 2003Co-Authors: Helene Gaussier, Thierry Lefèvre, Muriel SubiradeAbstract:To obtain molecular insights into the action mode of antimicrobial activity of Pediocin PA-1, the interactions between this bacteriocin and dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylglycerol (DMPG) model membranes have been investigated in D2O at pD 6 by Fourier transform infrared spectroscopy. The interactions were monitored with respect to alteration of the secondary structure of Pediocin, as registered by the amide I′ band, and phospholipid conformation, as revealed by the methylene νs(CH2) and carbonyl ν(C=O) stretching vibrations. The results show that no interaction between Pediocin and DMPC occurs. By contrast, Pediocin undergoes a structural reorganization in the presence of DMPG. Upon heating, Pediocin self-aggregates, which is not observed for this pD in aqueous solution. The gel-to-crystalline phase transition of DMPG shifts to higher temperatures with a concomitant dehydration of the interfacial region. Our results indicate that Pediocin is an extrinsic peptide and that its action mechanism may lie in a destabilization of the cell membrane.
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Conformational changes of Pediocin in an aqueous medium monitored by fourier transform infrared spectroscopy: a biological implication.
International journal of biological macromolecules, 2003Co-Authors: Helene Gaussier, Marc Lavoie, Muriel SubiradeAbstract:Fourier transform infrared (FTIR) spectroscopy was used to investigate the secondary structure of Pediocin PA-1 in different aqueous media in relation to its antimicrobial activity. The experiments were performed at pD (pH meter corrected for deuterium isotope effect) 6, 7, and 8 and during a heating-cooling cycle of 20-80 degrees C. At pD 6, (i.e. Pediocin's most active form), the FTIR results show that Pediocin adopts an unordered structure with a small contribution of beta-turn. After a heating-cooling cycle, thermally-induced changes in Pediocin are reversed and its activity is maintained. Increasing the pD to 7 and 8 leads to a more ordered secondary structure. For these two pD values, an increase in temperature induces an irreversible aggregation of protein as revealed by the amide I' band. The analysis of the Tyr region provides more insight into the aggregation process. In fact, it appears to be a two-step process, involving first the C (carboxy)-terminus of Pediocin and then the N (amino)-terminus. This study reveals two major points: (1) the preservation of Pediocin flexibility is essential for maintaining its activity; and (2) the aggregation of its C-terminus is sufficient to induce a loss of activity, suggesting that this region plays an important role in the activity of Pediocin.
Ismail Fliss - One of the best experts on this subject based on the ideXlab platform.
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synthesis antimicrobial activity and conformational analysis of the class iia bacteriocin Pediocin pa 1 and analogs thereof
Scientific Reports, 2018Co-Authors: Francois Bedard, Ismail Fliss, Riadh Hammami, Severine Zirah, Sylvie Rebuffat, Eric BironAbstract:The antimicrobial peptide Pediocin PA-1 is a class IIa bacteriocin that inhibits several clinically relevant pathogens including Listeria spp. Here we report the synthesis and characterization of whole Pediocin PA-1 and novel analogs thereof using a combination of solid- and solution-phase strategies to overcome difficulties due to instability and undesired reactions. Pediocin PA-1 thus synthesized was a potent inhibitor of Listeria monocytogenes (MIC = 6.8 nM), similar to the bacteriocin produced naturally by Pediococcus acidilactici. Of particular interest is that linear analogs lacking both of the disulfide bridges characterizing Pediocin PA-1 were as potent. One linear analog was also a strong inhibitor of Clostridium perfringens, another important food-borne pathogen. These results are discussed in light of conformational information derived from circular dichroism, solution NMR spectroscopy and structure-activity relationship studies.
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study of the physicochemical and biological stability of Pediocin pa 1 in the upper gastrointestinal tract conditions using a dynamic in vitro model
Journal of Applied Microbiology, 2010Co-Authors: E. Kheadr, Christophe Lacroix, A. Zihler, N. Dabour, Le G Blay, Ismail FlissAbstract:Aims: To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce Pediocin PA-1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified Pediocin PA-1 under GIT conditions. Methods and Results: Skim milk culture of Ped. acidilactici UL5 was fed to a dynamic gastrointestinal (GI) model known as TIM-1, comprising four compartments connected by computer-controlled peristaltic valves and simulating the human stomach, duodenum, jejunum and ileum. This strain tolerated a pH of 2·7 in the gastric compartment, while lower pH reduced its viability. Bile salts in the duodenal compartment brought a further 4-log reduction after 180 min of digestion, while high viable counts (up to 5 × 107 CFU ml−1 fermented milk) of Ped. acidilactici were found in both the jejunal and ileal compartments. Pediococcus acidilactici recovered from all four compartments was able to produce Pediocin at the same level as unstressed cells. The activity of the purified Pediocin in the gastric compartment was slightly reduced after 90 min of gastric digestion, while no detectable activity was found in the duodenal, jejunal and ileal compartments during 5 h of digestion. HPLC analysis showed partial degradation of the Pediocin peptide in the duodenal compartment and massive breakdown in the jejunal and ileal compartments. Conclusions: Pediococcus acidilactici UL5 showed high resistance to GIT conditions, and its ability to produce Pediocin was not affected, suggesting its potential as a probiotic candidate. The physicochemical and biological stability of Pediocin was significantly poor under GIT conditions. Significance and Impact of the Study: Pediococcus acidilactici UL5 appears to be a potential probiotic candidate because its capacity to produce Pediocin PA-1 is not affected by the GI conditions as well as the strain shows an acceptable survival rate. Meanwhile, purified Pediocin PA-1 losses activity during GIT transit; microcapsules could be used to deliver it to the target site.
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Study of the physicochemical and biological stability of Pediocin PA-1 in the upper gastrointestinal tract conditions using a dynamic in vitro model
Journal of Applied Microbiology, 2010Co-Authors: E. Kheadr, Christophe Lacroix, A. Zihler, N. Dabour, Gwenaelle Le Blay, Ismail FlissAbstract:Aims: To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce Pediocin PA-1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified Pediocin PA-1 under GIT conditions. Methods and Results: Skim milk culture of Ped. acidilactici UL5 was fed to a dynamic gastrointestinal (GI) model known as TIM-1, comprising four compartments connected by computer-controlled peristaltic valves and simulating the human stomach, duodenum, jejunum and ileum. This strain tolerated a pH of 2Æ7 in the gastric compartment, while lower pH reduced its viability. Bile salts in the duodenal compartment brought a further 4-log reduction after 180 min of digestion, while high viable counts (up to 5 · 107 CFU ml)1 fermented milk) of Ped. acidilactici were found in both the jejunal and ileal compartments. Pediococcus acidilactici recovered from all four compartments was able to produce Pediocin at the same level as unstressed cells. The activity of the purified Pediocin in the gastric compartment was slightly reduced after 90 min of gastric digestion, while no detectable activity was found in the duodenal, jejunal and ileal compartments during 5 h of digestion. HPLC analysis showed partial degradation of the Pediocin peptide in the duodenal compartment and massive breakdown in the jejunal and ileal compartments. Conclusions: Pediococcus acidilactici UL5 showed high resistance to GIT conditions, and its ability to produce Pediocin was not affected, suggesting its potential as a probiotic candidate. The physicochemical and biological stability of Pediocin was significantly poor under GIT conditions. Significance and Impact of the Study: Pediococcus acidilactici UL5 appears to be a potential probiotic candidate because its capacity to produce Pediocin PA-1 is not affected by the GI conditions as well as the strain shows an acceptable survival rate. Meanwhile, purified Pediocin PA-1 losses activity during
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Purification of Pediocin PA-1 by immunoaffinity chromatography.
Journal of AOAC International, 2008Co-Authors: Karim Naghmouchi, Djamel Drider, Ismail FlissAbstract:A one-step purification procedure for purifying Pediocin PA-1, a class IIa bacteriocin produced by Pediococcus acidilactici UL5, was developed based on column immunoaffinity chromatography with specific antiPediocin PA-1 polyclonal antibodies coupled to cyanogen bromide-activated SepharoseTM. About 13.3 microg/mL purified Pediocin PA-1 was obtained from 15 mL P. acidilactici UL5 culture supernatant, as measured by enzyme-linked immunosorbent assay. The specific activity and average recovery of the eluted Pediocin PA-1 were about 6602 AU/mg and 53.3%, respectively. This is the first report of successful purification of Pediocin PA-1 by immunoaffinity using Pediocin PA-1-specific polyclonal antibodies.
Christophe Lacroix - One of the best experts on this subject based on the ideXlab platform.
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Study of the physicochemical and biological stability of Pediocin PA-1 in the upper gastrointestinal tract conditions using a dynamic in vitro model
Journal of Applied Microbiology, 2010Co-Authors: E. Kheadr, Christophe Lacroix, A. Zihler, N. Dabour, Gwenaelle Le Blay, Ismail FlissAbstract:Aims: To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce Pediocin PA-1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified Pediocin PA-1 under GIT conditions. Methods and Results: Skim milk culture of Ped. acidilactici UL5 was fed to a dynamic gastrointestinal (GI) model known as TIM-1, comprising four compartments connected by computer-controlled peristaltic valves and simulating the human stomach, duodenum, jejunum and ileum. This strain tolerated a pH of 2Æ7 in the gastric compartment, while lower pH reduced its viability. Bile salts in the duodenal compartment brought a further 4-log reduction after 180 min of digestion, while high viable counts (up to 5 · 107 CFU ml)1 fermented milk) of Ped. acidilactici were found in both the jejunal and ileal compartments. Pediococcus acidilactici recovered from all four compartments was able to produce Pediocin at the same level as unstressed cells. The activity of the purified Pediocin in the gastric compartment was slightly reduced after 90 min of gastric digestion, while no detectable activity was found in the duodenal, jejunal and ileal compartments during 5 h of digestion. HPLC analysis showed partial degradation of the Pediocin peptide in the duodenal compartment and massive breakdown in the jejunal and ileal compartments. Conclusions: Pediococcus acidilactici UL5 showed high resistance to GIT conditions, and its ability to produce Pediocin was not affected, suggesting its potential as a probiotic candidate. The physicochemical and biological stability of Pediocin was significantly poor under GIT conditions. Significance and Impact of the Study: Pediococcus acidilactici UL5 appears to be a potential probiotic candidate because its capacity to produce Pediocin PA-1 is not affected by the GI conditions as well as the strain shows an acceptable survival rate. Meanwhile, purified Pediocin PA-1 losses activity during
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study of the physicochemical and biological stability of Pediocin pa 1 in the upper gastrointestinal tract conditions using a dynamic in vitro model
Journal of Applied Microbiology, 2010Co-Authors: E. Kheadr, Christophe Lacroix, A. Zihler, N. Dabour, Le G Blay, Ismail FlissAbstract:Aims: To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce Pediocin PA-1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified Pediocin PA-1 under GIT conditions. Methods and Results: Skim milk culture of Ped. acidilactici UL5 was fed to a dynamic gastrointestinal (GI) model known as TIM-1, comprising four compartments connected by computer-controlled peristaltic valves and simulating the human stomach, duodenum, jejunum and ileum. This strain tolerated a pH of 2·7 in the gastric compartment, while lower pH reduced its viability. Bile salts in the duodenal compartment brought a further 4-log reduction after 180 min of digestion, while high viable counts (up to 5 × 107 CFU ml−1 fermented milk) of Ped. acidilactici were found in both the jejunal and ileal compartments. Pediococcus acidilactici recovered from all four compartments was able to produce Pediocin at the same level as unstressed cells. The activity of the purified Pediocin in the gastric compartment was slightly reduced after 90 min of gastric digestion, while no detectable activity was found in the duodenal, jejunal and ileal compartments during 5 h of digestion. HPLC analysis showed partial degradation of the Pediocin peptide in the duodenal compartment and massive breakdown in the jejunal and ileal compartments. Conclusions: Pediococcus acidilactici UL5 showed high resistance to GIT conditions, and its ability to produce Pediocin was not affected, suggesting its potential as a probiotic candidate. The physicochemical and biological stability of Pediocin was significantly poor under GIT conditions. Significance and Impact of the Study: Pediococcus acidilactici UL5 appears to be a potential probiotic candidate because its capacity to produce Pediocin PA-1 is not affected by the GI conditions as well as the strain shows an acceptable survival rate. Meanwhile, purified Pediocin PA-1 losses activity during GIT transit; microcapsules could be used to deliver it to the target site.
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production of a nisin z Pediocin mixture by ph controlled mixed strain batch cultures in supplemented whey permeate
Journal of Applied Microbiology, 1999Co-Authors: F Goulhen, J Meghrous, Christophe LacroixAbstract:The conditions for high production of nisin Z and Pediocin during pH-controlled, mixed-strain batch cultures in a supplemented whey permeate medium with Lactococcus lactis subsp. lactis biovar. diacetylactis UL719, a nisin Z producer strain, and variant T5 of Pediococcus acidilactici UL5, a Pediocin-producing strain resistant to high concentrations of nisin, were studied. Mixed cultures were performed at 37 °C and pH 5·5 by first inoculating with variant T5 and then with L. diacetylactis UL719 after 8 h incubation, and were compared with single-strain batch cultures. High productions of both nisin Z and Pediocin were obtained after 18 or 16 h incubation during mixed cultures, with titres of 3000 and 730 AU ml−1, or 1060 and 1360 AU ml−1, respectively, corresponding to approximately 75 and 55, or 25 and 100 mg l−1 of pure nisin Z and Pediocin, respectively. In pure cultures, nisin Z and Pediocin productions were higher than in mixed cultures, and maximum activities were obtained after 10 h incubation, with approximately 10 000 AU ml−1 (250 mg l−1 pure nisin Z) and 2500 AU ml−1 (190 mg l−1 pure Pediocin). During mixed cultures, significant Pediocin degradation was observed in the culture supernatant fluid after 16 h incubation, together with a sharp drop in Ped. acidilactici UL5 cell viability. In the test conditions, the feasibility of producing a nisin/Pediocin mixture by mixed-strain fermentation was demonstrated. The bacteriocin mixture produced in a supplemented whey permeate medium could be used as a natural food-grade biopreservative with a broad activity spectrum.
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Growth of Listeria monocytogenes in milk and its control by Pediocin 5 produced by Pediococcus acidilactici UL5
International Dairy Journal, 1994Co-Authors: J. Huang, Christophe Lacroix, H. Daba, Ronald E. SimardAbstract:Abstract Pediocin 5, a bacteriocin produced by Pediococcus acidilactici UL5 in MRS medium, displayed a bactericidal effect on three Listeria monocytogenes strains grown in partly skimmed milk containing 1% milk-fat (MF) and 3·25% MF homogenized milk at 4°C. Viable counts of all Listeria inoculated into 1% MF milk without Pediocin 5 gradually increased from 10 7 to 10 8 –10 9 cfu/ml after 13 days storage. Addition of Pediocin 5 to the milk led to a sharp reduction in viable Listeria by approximately 3·0 log cycles during the first day, followed by a slow growth of the survivors to reach 5 × 10 5 –10 6 cfu/ml after 13 days. However, at an inoculum level of about 10 3 cfu/ml, survival of the three Listeria was below the detection limit of the method (2–3 cfu/ml) in 1% MF milk containing Pediocin 5 during 8 days storage; populations reached approximately 10 cfu/ml after 13 days, whereas they reached 10 4 –10 5 cfu/ml in the absence of Pediocin 5. Growth behavior in 3·25% MF milk was similar to that in 1% MF milk at the two inoculum levels with or without Pediocin 5. Occasional repeated contamination by Listeria was simulated by five successive inoculations of 1% MF milk every 48 h after the first inoculation (each corresponding to approximately 2 × 10 2 cfu/ml of milk). After 11 days storage, only about 50 viable Listeria per ml were found in milk containing Pediocin 5, although control without Pediocin 5 reached 10 3 –10 4 cfu/ml. Increased Pediocin 5 concentration in milk led to decreased survival of L. monocytogenes Lm21.
Helene Gaussier - One of the best experts on this subject based on the ideXlab platform.
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Conformational changes of Pediocin in an aqueous medium monitored by fourier transform infrared spectroscopy: a biological implication.
International Journal of Biological Macromolecules, 2003Co-Authors: Helene Gaussier, Marc C Lavoie, Muriel SubiradeAbstract:Abstract Fourier transform infrared (FTIR) spectroscopy was used to investigate the secondary structure of Pediocin PA-1 in different aqueous media in relation to its antimicrobial activity. The experiments were performed at pD (pH meter corrected for deuterium isotope effect) 6, 7, and 8 and during a heating–cooling cycle of 20–80 °C. At pD 6, (i.e. Pediocin’s most active form), the FTIR results show that Pediocin adopts an unordered structure with a small contribution of β-turn. After a heating–cooling cycle, thermally-induced changes in Pediocin are reversed and its activity is maintained. Increasing the pD to 7 and 8 leads to a more ordered secondary structure. For these two pD values, an increase in temperature induces an irreversible aggregation of protein as revealed by the amide I′ band. The analysis of the Tyr region provides more insight into the aggregation process. In fact, it appears to be a two-step process, involving first the C (carboxy)-terminus of Pediocin and then the N (amino)-terminus. This study reveals two major points: (1) the preservation of Pediocin flexibility is essential for maintaining its activity; and (2) the aggregation of its C-terminus is sufficient to induce a loss of activity, suggesting that this region plays an important role in the activity of Pediocin.
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Conformational changes of Pediocin in an aqueous medium monitored by fourier transform infrared spectroscopy: a biological implication.
International journal of biological macromolecules, 2003Co-Authors: Helene Gaussier, Marc Lavoie, Muriel SubiradeAbstract:Fourier transform infrared (FTIR) spectroscopy was used to investigate the secondary structure of Pediocin PA-1 in different aqueous media in relation to its antimicrobial activity. The experiments were performed at pD (pH meter corrected for deuterium isotope effect) 6, 7, and 8 and during a heating-cooling cycle of 20-80 degrees C. At pD 6, (i.e. Pediocin's most active form), the FTIR results show that Pediocin adopts an unordered structure with a small contribution of beta-turn. After a heating-cooling cycle, thermally-induced changes in Pediocin are reversed and its activity is maintained. Increasing the pD to 7 and 8 leads to a more ordered secondary structure. For these two pD values, an increase in temperature induces an irreversible aggregation of protein as revealed by the amide I' band. The analysis of the Tyr region provides more insight into the aggregation process. In fact, it appears to be a two-step process, involving first the C (carboxy)-terminus of Pediocin and then the N (amino)-terminus. This study reveals two major points: (1) the preservation of Pediocin flexibility is essential for maintaining its activity; and (2) the aggregation of its C-terminus is sufficient to induce a loss of activity, suggesting that this region plays an important role in the activity of Pediocin.
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Binding of Pediocin PA-1 with anionic lipid induces model membrane destabilization.
Applied and environmental microbiology, 2003Co-Authors: Helene Gaussier, Thierry Lefèvre, Muriel SubiradeAbstract:To obtain molecular insights into the action mode of antimicrobial activity of Pediocin PA-1, the interactions between this bacteriocin and dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylglycerol (DMPG) model membranes have been investigated in D2O at pD 6 by Fourier transform infrared spectroscopy. The interactions were monitored with respect to alteration of the secondary structure of Pediocin, as registered by the amide I′ band, and phospholipid conformation, as revealed by the methylene νs(CH2) and carbonyl ν(C=O) stretching vibrations. The results show that no interaction between Pediocin and DMPC occurs. By contrast, Pediocin undergoes a structural reorganization in the presence of DMPG. Upon heating, Pediocin self-aggregates, which is not observed for this pD in aqueous solution. The gel-to-crystalline phase transition of DMPG shifts to higher temperatures with a concomitant dehydration of the interfacial region. Our results indicate that Pediocin is an extrinsic peptide and that its action mechanism may lie in a destabilization of the cell membrane.
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replacement of trifluoroacetic acid with hcl in the hydrophobic purification steps of Pediocin pa 1 a structural effect
Applied and Environmental Microbiology, 2002Co-Authors: Helene Gaussier, Helene Morency, Marc C Lavoie, Muriel SubiradeAbstract:Trifluoroacetic acid (TFA) is a purification contaminant associated with Pediocin PA-1 that interferes with Fourier transform infrared spectroscopy structural analysis. As revealed by circular dichroism, its presence affects the structural folding of Pediocin. Consequently, we propose a new Pediocin PA-1 purification procedure using HCl instead of TFA in all of the hydrophobic steps. This procedural change does not affect the purification yield or the amount of Pediocin PA-1 purified. Furthermore, removing HCl, as opposed to TFA, after purification is an easier procedure to carry out. In fact, the removal of TFA requires more experimentation and results in protein loss. Thus, HCl is a good alternative to TFA in Pediocin PA-1 purification and can be extended to the purification of other proteins. We also show that TFA-induced structural modifications do not significantly affect the antimicrobial activity of Pediocin PA-1.
E. Kheadr - One of the best experts on this subject based on the ideXlab platform.
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Study of the physicochemical and biological stability of Pediocin PA-1 in the upper gastrointestinal tract conditions using a dynamic in vitro model
Journal of Applied Microbiology, 2010Co-Authors: E. Kheadr, Christophe Lacroix, A. Zihler, N. Dabour, Gwenaelle Le Blay, Ismail FlissAbstract:Aims: To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce Pediocin PA-1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified Pediocin PA-1 under GIT conditions. Methods and Results: Skim milk culture of Ped. acidilactici UL5 was fed to a dynamic gastrointestinal (GI) model known as TIM-1, comprising four compartments connected by computer-controlled peristaltic valves and simulating the human stomach, duodenum, jejunum and ileum. This strain tolerated a pH of 2Æ7 in the gastric compartment, while lower pH reduced its viability. Bile salts in the duodenal compartment brought a further 4-log reduction after 180 min of digestion, while high viable counts (up to 5 · 107 CFU ml)1 fermented milk) of Ped. acidilactici were found in both the jejunal and ileal compartments. Pediococcus acidilactici recovered from all four compartments was able to produce Pediocin at the same level as unstressed cells. The activity of the purified Pediocin in the gastric compartment was slightly reduced after 90 min of gastric digestion, while no detectable activity was found in the duodenal, jejunal and ileal compartments during 5 h of digestion. HPLC analysis showed partial degradation of the Pediocin peptide in the duodenal compartment and massive breakdown in the jejunal and ileal compartments. Conclusions: Pediococcus acidilactici UL5 showed high resistance to GIT conditions, and its ability to produce Pediocin was not affected, suggesting its potential as a probiotic candidate. The physicochemical and biological stability of Pediocin was significantly poor under GIT conditions. Significance and Impact of the Study: Pediococcus acidilactici UL5 appears to be a potential probiotic candidate because its capacity to produce Pediocin PA-1 is not affected by the GI conditions as well as the strain shows an acceptable survival rate. Meanwhile, purified Pediocin PA-1 losses activity during
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study of the physicochemical and biological stability of Pediocin pa 1 in the upper gastrointestinal tract conditions using a dynamic in vitro model
Journal of Applied Microbiology, 2010Co-Authors: E. Kheadr, Christophe Lacroix, A. Zihler, N. Dabour, Le G Blay, Ismail FlissAbstract:Aims: To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce Pediocin PA-1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified Pediocin PA-1 under GIT conditions. Methods and Results: Skim milk culture of Ped. acidilactici UL5 was fed to a dynamic gastrointestinal (GI) model known as TIM-1, comprising four compartments connected by computer-controlled peristaltic valves and simulating the human stomach, duodenum, jejunum and ileum. This strain tolerated a pH of 2·7 in the gastric compartment, while lower pH reduced its viability. Bile salts in the duodenal compartment brought a further 4-log reduction after 180 min of digestion, while high viable counts (up to 5 × 107 CFU ml−1 fermented milk) of Ped. acidilactici were found in both the jejunal and ileal compartments. Pediococcus acidilactici recovered from all four compartments was able to produce Pediocin at the same level as unstressed cells. The activity of the purified Pediocin in the gastric compartment was slightly reduced after 90 min of gastric digestion, while no detectable activity was found in the duodenal, jejunal and ileal compartments during 5 h of digestion. HPLC analysis showed partial degradation of the Pediocin peptide in the duodenal compartment and massive breakdown in the jejunal and ileal compartments. Conclusions: Pediococcus acidilactici UL5 showed high resistance to GIT conditions, and its ability to produce Pediocin was not affected, suggesting its potential as a probiotic candidate. The physicochemical and biological stability of Pediocin was significantly poor under GIT conditions. Significance and Impact of the Study: Pediococcus acidilactici UL5 appears to be a potential probiotic candidate because its capacity to produce Pediocin PA-1 is not affected by the GI conditions as well as the strain shows an acceptable survival rate. Meanwhile, purified Pediocin PA-1 losses activity during GIT transit; microcapsules could be used to deliver it to the target site.