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Background:In the last years, Antimicrobial Peptides (AMPs) and lipopeptides have received attention as promising candidates to treat infections caused by resistant microorganisms. Objective: The main objective of this study was to investigate the effect of repetitive KLFK motifs and the attachment of aliphatic acids to the N-terminus of (KLFK)n peptides on therapeutic properties.Methods:Minimal inhibitory concentration against Gram (+) and (-) bacteria and yeast of synthetic compounds were determined by broth microtiter dilution method, and the toxicity was evaluated by hemolysis assay. Membrane-peptide interaction studies were performed with model phospholipid membranes mimicking those of bacterial and mammalian cells by Fluorescence Spectroscopy. The secondary structure in solution and membranes was determined by Circular Dichroism.Results:Our results showed that the resulting compounds have inhibitory activity against bacteria and fungi. The (KLFK)3 peptide showed the highest therapeutic index against bacterial and yeast strains, and the (KLFK)2 peptide conjugated with octanoic acid was the most active against yeasts. All the lipopeptides containing long-chain fatty acids (C14 or longer) were highly hemolytic at low concentrations. The antimicrobial activity of (KLFK)2 and (KLFK)3 lipopeptides was mainly associated with improved stability of the amphipathic secondary structure, which showed high contributions of α-helix in dipalmitoylphosphatidylglycerol (DPPG) vesicles.Conclusion:The repetition of the KLFK sequence and the conjugation with lipid tails allowed obtained compounds with high antimicrobial activity and low toxicity, becoming good candidates for treating infectious diseases.

The increasing bacterial resistance against conventional antibiotics has led to the search for new antimicrobial drugs with different modes of action. Cationic antimicrobial peptides (AMPs) and lipopeptides are promising candidates to treat infections because they act on bacterial membranes causing rapid destruction of sensitive bacteria. In this study, a decapeptide named A2 (IKQVKKLFKK) was conjugated at the N-terminus with saturated, unsaturated, methoxylated and methyl -branched fatty acids of different chain lengths (C8 - C20), the antimicrobial and structural properties of the lipopeptides being then investigated. The attachment of the fatty acid chain significantly improved the antimicrobial activity of A2 against bacteria, and so, endowed it with moderated antifungal activity against yeast strains belonging to genus Candida. Lipopeptides containing hydrocarbon chain lengths between C8 and C14 were the best antibacterial compounds (MIC = 0.7 to 5.8 μM), while the most active compounds against yeast were A2 conjugated with methoxylated and enoic fatty acids (11.1 to 83.3 μM). The improvement in antimicrobial activity was mainly related to the amphipathic secondary structure adopted by A2 lipopeptides in the presence of vesicles that mimic bacterial membranes. Peptide conjugation with long hydrocarbon chains (C12 or more), regardless of their structure, significantly increased toxicity towards eukaryotic cells, resulting in a loss of selectivity. These findings suggest that A2-derived lipopeptides are potential good candidates for the treatment of infectious diseases caused by bacteria and opportunistic pathogenic yeast belonging to genus Candida. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Amphibians&acute, skin produces a diverse array of antimicrobial peptides that play a crucial role as the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, current knowledge about the presence of peptides with antimicrobial properties is limited to a only few species. Here we used LC-MS-MS to identify antimicrobial peptides with masses ranging from 1000 to 4000 Da from samples of skin secretions of Leptodactylus latrans (Anura: Leptodactylidae). Three novel amino acid sequences were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Ll-1577, P2-Ll-1298, and P3-Ll-2085, inhibited the growth of two ATCC strains, namely Escherichia coli and Staphylococcus aureus. P3-Ll-2085 was the most active peptide. In the presence of trifluoroethanol (TFE) and anionic liposomes, it adopted an amphipathic &alpha, helical structure. P2-Ll-1298 showed slightly lower activity than P3-Ll-2085. Comparison of the MIC values of these two peptides revealed that the addition of seven amino acid residues (GLLDFLK) on the N-terminal of P2-Ll-1298 significantly improved activity against both strains. P1-Ll-1577, which remarkably is an anionic peptide, showed interesting antimicrobial activity against E. coli and S. aureus strain, showing marked membrane selectivity and non-hemolysis. Due to this, P1-L1-1577 emerges as a potential candidate for the development of new antibacterial drugs.

In vitro inhibitory activity against acetylcholinesterase (AChE) of peptides obtained by enzymatic hydrolysis of mannoproteins extracted from strains of yeasts was investigated. Yeast mannoproteins were extracted from strains belonging to the genera Brettanomyces, Candida, Pichia and Saccharomyces isolated from dairy products. They were obtained by heat treatment in citrate buffer and purified by affinity chromatography with concanavalin A. Each purified extract was subsequently hydrolysed with proteolytic enzymes (trypsin, pepsin, chymotrypsin and proteinase K) applied individually or in combination, thus generating smaller peptides. Inhibitory activity of the latter against AChE was determined. The molecular weight of mannoproteins, determined by SDS-PAGE, was between 6.5 and 30 kDa. As regards AChE inhibition, a preliminary screening of all hydrolysed extracts was performed, yielding variable results with 59% maximum inhibition. Subsequently, when inhibitory concentration 50 (IC50) was determined, the extracts showed higher inhibitory activity (between 6.75 and 12.3 mg mL-1). Results showed that the mannoproteins separated from yeast strains of food origin generated bioactive peptides by enzymatic hydrolysis, which can be of interest to the manufacturing of food with potential functional properties. Different peptide extracts were obtained by enzymatic hydrolysis of yeast mannoproteins and their in vitro inhibitory activity against acetylcholinesterase (AChE) was investigated. The most active extracts showed IC50 between 6.75 and 12.3 mg mL-1. Fil: Spontón, Pablo Gabriel. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Química Organica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Spinelli, Roque. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Química Organica; Argentina Fil: Drago, Silvina Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Tecnología de los Alimentos; Argentina Fil: Tonarelli, Georgina Guadalupe. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Química Organica; Argentina Fil: Simonetta, Arturo Carlos. Universidad Nacional del Litoral. Facultad de Ingeniería Química; Argentina

The synthesis and biodegradation of polyurethane foams obtained from environmentally benign processes were studied.Flexible polyurethane foams based on castor oil modified with maleic anhydride (MACO) were synthesized. The synthesis involved a single-stage process by mixing castor oil/MACO (weight ratios 75:25 and 25:75) and 2-4 toluene diisocyanate (TDI) in stoichiometric amount of OH:NCO. The biodegradability studies with cultures of a Pseudomonas sp. strain (DBFIQ-P36) involved incubation periods of 2 months at 37°C. Polymers were characterized before and after biodegradation by Fourier Transform Infrared Spectroscopy (FT-IR), INSTRON mechanical tester, and Scanning Electron Microscopy (SEM). The results showed that the addition of MACO produces a considerable increase in the rate of degradation and an important change in the chemical and morphological structures. This is due to the presence of ester groups that are vulnerable to chemical hydrolysis and enzymatic attack. The eco-toxicity after the biodegradation was evaluated. Toxic compounds such as primary amines were identified by Gas Chromatography-Mass Spectrometry (GC-MS) in combination with Nuclear Magnetic Resonance (NMR) as degradation products. Fil: Spontón, Marisa Elisabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Casis, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Mazo, Paula. Universidad de Antioquia; Colombia Fil: Raud, B.. Universidad Nacional del Litoral; Argentina Fil: Simonetta, Arturo Carlos. Universidad Nacional del Litoral; Argentina Fil: Ríos, L.. Universidad de Antioquia; Colombia Fil: Estenoz, Diana Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina

The increasing bacterial resistance against conventional antibiotics has led to the search for new antimicrobial drugs with different modes of action. Cationic antimicrobial peptides (AMPs) and lipopeptides are promising candidates to treat infections because they act on bacterial membranes causing rapid destruction of sensitive bacteria. In this study, a decapeptide named A2 (IKQVKKLFKK) was conjugated at the N-terminus with saturated, unsaturated, methoxylated and methyl -branched fatty acids of different chain lengths (C8 - C20), the antimicrobial and structural properties of the lipopeptides being then investigated. The attachment of the fatty acid chain significantly improved the antimicrobial activity of A2 against bacteria, and so, endowed it with moderated antifungal activity against yeast strains belonging to genus Candida. Lipopeptides containing hydrocarbon chain lengths between C8 and C14 were the best antibacterial compounds (MIC = 0.7 to 5.8 μM), while the most active compounds against yeast were A2 conjugated with methoxylated and enoic fatty acids (11.1 to 83.3 μM). The improvement in antimicrobial activity was mainly related to the amphipathic secondary structure adopted by A2 lipopeptides in the presence of vesicles that mimic bacterial membranes. Peptide conjugation with long hydrocarbon chains (C12 or more), regardless of their structure, significantly increased toxicity towards eukaryotic cells, resulting in a loss of selectivity. These findings suggest that A2-derived lipopeptides are potential good candidates for the treatment of infectious diseases caused by bacteria and opportunistic pathogenic yeast belonging to genus Candida. Copyright © 2016 European Peptide Society and John WileySons, Ltd.

Enterococcus faecalis DBFIQ E24 was formerly characterised as a producer of antibacterial and antifungal substances. This strain is vancomycin sensitive, nonhaemolytic and gelatinase negative. SDS-PAGE revealed the presence of an active band located between 1060 and 3500 Da. After the application of a three-step purification procedure, two antimicrobial peptides were isolated. One of them is mostly active against Bacillus cereus with a molecular mass of 1364 Da, and the other one with a molecular mass of 1686 Da is mainly active against Escherichia coli. These results confirm the technological potential of E. faecalis DBFIQ E24 strain as a GRAS food biopreservative. Fil: Manzo, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Cardoso, Maria de Las Mercedes. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Tonarelli, Georgina Guadalupe. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Simonetta, Arturo Carlos. Universidad Nacional del Litoral. Facultad de Ingeniería Química; Argentina

A series of peptide analogs based on region 6-22 of Plantaricin 149 sequence were synthesized. The interaction between these analogs and phospholipid-polydiacetylene vesicles was investigated to evaluate the ability of the bioassay to detect differences in the interaction of the peptides with dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine vesicles, associated with amino acid substitution and N-terminal conjugation of the sequences with short fatty acids (8 and 12 carbon atoms). Fatty acid conjugation of peptides with low antimicrobial activity resulted in lipopeptides with improved activity against strains of Staphylococcus aureus and Listeria monocytogenes. The length of the fatty acid determined the bacterial specificity, and the conjugation with n-octanoic acid yielded the most active analog (C8-CT) against Staphylococcus aureus strain (MIC: 1.0 μm) while the conjugation with n-dodecanoic acid (C12-CT) was optimal for Listeria monocytogenes strain (MIC: 2.0 μm). In contrast, the substitution of Phe by Trp had an unfavorable effect on the antimicrobial activity. Hemolysis tests and membrane interaction studies with dipalmitoylphosphatidylcholine-polydiacetylene vesicles showed that lipopeptides interact to a greater extent with both biological and biomimetic membranes. Also, a good correlation was found between antimicrobial activity against Staphylococcus aureus strain and % colorimetric response values with dipalmitoylphosphatidylglycerol-polydiacetylene vesicles.

The skin of many amphibians produces a large repertoire of antimicrobial peptides that are crucial in the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, knowledge about peptides with antimicrobial properties is limited to a few species. Here we used LC-MS-MS to analyze samples of Hypsiboas pulchellus skin with the aim to identify antimicrobial peptides in the mass range of 1000 to 2000 Da. Twenty-three novel sequences were identified by MS, three of which were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Hp-1971, P2-Hp-1935, and P3-Hp-1891, inhibited the growth of two ATCC strains: Escherichia coli (MIC: 16, 33, and 17 μM, respectively) and Staphylococcus aureus (MIC: 8, 66, and 17 μM, respectively). P1-Hp-1971 and P3-Hp-1891 were the most active peptides. P1-Hp-1971, which showed the highest therapeutic indices (40 for E. coli and 80 for S. aureus), is a proline-glycine-rich peptide with a highly unordered structure, while P3-Hp-1891 adopts an amphipathic α-helical structure in the presence of 2,2,2-trifluoroethanol and anionic liposomes. This is the first peptidomic study of Hypsiboas pulchellus skin secretions to allow the identification of antimicrobial peptides. Fil: Siano, Alvaro Sebastían. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Química Organica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Húmpola, Maria Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Química Organica; Argentina Fil: Olivera, Eliandre de. Parc Científic de Barcelona; España Fil: Fernando, Albericio. Universidad de Barcelona; España. University of KwaZulu-Natal; Sudáfrica. Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina; España. Institut de Recerca Biomèdica; España Fil: Simonetta, Arturo Carlos. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Departamento de Ingeniería en Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Lajmanovich, Rafael Carlos. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Escuela Superior de Sanidad; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Tonarelli, Georgina Guadalupe. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Química Organica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

The majority of L-arabinose isomerases have been isolated by recombinant techniques, but this methodology implies a reduced technological application. For this reason, 29 bacterial strains, some of them previously characterized as L-arabinose isomerase producers, were assayed as L-arabinose fermenting strains by employing conveniently designed culture media with 0.5% (w/v) L-arabinose as main carbon source. From all evaluated bacterial strains, Enterococcus faecium DBFIQ ID: E36, Enterococcus faecium DBFIQ ID: ETW4 and Pediococcus acidilactici ATCC ID: 8042 were, in this order, the best L-arabinose fermenting strains. Afterwards, to assay L-arabinose metabolization and L-arabinose isomerase activity, cell-free extract and saline precipitated cell-free extract of the three bacterial cultures were obtained and the production of ketoses was determined by the cysteine carbazole sulfuric acid method. Results showed that the greater the L-arabinose metabolization ability, the higher the enzymatic activity achieved, so Enterococcus faecium DBFIQ ID: E36 was selected to continue with production, purification and characterization studies. This work thus describes a simple microbiological method for the selection of L-arabinose fermenting bacteria for the potential production of the enzyme L-arabinose isomerase. Fil: Manzo, Ricardo Martín. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Departamento de Ingeniería en Alimento. Area de Biotecnología; Argentina; Fil: Simonetta, Arturo Carlos. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Departamento de Ingeniería en Alimento. Area de Biotecnología; Argentina; Fil: Rubiolo, Amelia Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET- Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina; Fil: Mammarella, Enrique José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET- Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina

Enterococcus faecalis DBFIQ E24 strain produces antimicrobial substances that inhibit the growth offood-borne pathogenic bacteria, yeasts and moulds. This broad antimicrobial spectrum was determined by obtaining the cell-free supernatant (CFS) and employing the agar-well-diffusion method. Its antibacterial activity was completely inactivated by the action of proteinase K and not affected by catalase action. Besides, it was stable to thermal treatments, showing maximum antibacterial activity at pH 7.0. Moreover, CFS showed a bactericidal effect against Escherichia coli DBFIQ Ec9 strain. All these interesting physicochemical properties allow that either CFS or E. faecalis DBFIQ E24 strain to be considered as potential food biopreservatives. Fil: Cardoso, Maria de Las Mercedes. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Manzo, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Tonarelli, Georgina Guadalupe. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Simonetta, Arturo Carlos. Universidad Nacional del Litoral. Facultad de Ingeniería Química; Argentina

1 Dpto. de Quimica Organica, Facultad de Bioquimica y Cs. Biologicas, Universidad Nacional del Litoral (U.N.L). Ciudad Universitaria Paraje El Pozo, C.C. 242Santa Fe. Argentina. 2 Cat. de Microbiologia y Biotecnologia, Dpto. de Ingenieria en Alimentos, Facultad de Ingenieria Quimica, Universidad Nacional del Litoral (U.N.L). Santiago del Estero 2829. Santa Fe. Argentina. (*) Correspondencia a: Georgina G. Tonarelli, Departamento de Quimica Organica, Facultad de Bioquimica y Cs. Biologicas, U.N.L., C.C 242, C.P. 3000. Ciudad Universitaria, Santa Fe, Argentina. E-mail: tonareli@fbcb.unl.edu.ar, Tel: 54-342-4575206 int.224, Fax: 54-0342-4575221

Aims: Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage. Methods and Results: Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5AE 0t o 6AE0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558AE85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes. Conclusions: The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition. Significance and Impact of the Study: In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.

Plantaricin-149 is a bacteriocin produced by Lactobacillus plantarum NRIC 149 (a LAB isolated from pineapple), which consists of a peptidic chain made up of 22 amino acid residues [Kato et al. J. Ferment. Bioeng. 1994; 77: 277–282]. In this work, a synthetic C-terminal amidated peptide analog denoted Pln149a was prepared by SPPS-Fmoc chemistry and the antagonistic activity against gram-positive and gram-negative bacteria was tested. The secondary structure was studied by circular dichroism (CD) and the vicinity of the tyrosine residue by fluorescence spectroscopy under different conditions. We report the results of the interaction of Pln149a with reverse micelles prepared from the amphiphilic AOT in cyclohexane. Synthetic plantaricin was active against one strain of Staphylococcus aureus and four strains of Listeria genus at pH 5.5 and 7.4 and, like its natural variant, inhibited L. plantarum ATCC 8014. The data derived from spectroscopic measurements in presence of AOT reverse micelles suggest that the secondary structure of the peptide upon interaction is an α-helix. In this membrane model, the hydrophobic side of the α-helix is inserted into the micelles, leaving the lysines exposed to the solvent and interacting with the polar moieties of AOT. The fluorescence data point out that the N-terminal tyrosine residue is close to the micellar interface. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

Bacteriocins produced by lactic acid bacteria (LAB) are proteins or peptides with inhibitory activity against related and non related species, associated with food spoilage and food borne illnesses. The aim of this study was to purify and characterize bacteriocins produced by a strain of Lactobacillus plantarum[1].