Biomedical subjects

Graciela Vignolo

Publications and source records attributed to Graciela Vignolo.

7 recordsLinked to original sources

PubMed2006

Molecular view by fourier transform infrared spectroscopy of the relationship between lactocin 705 and membranes: speculations on antimicrobial mechanism.

Lactocin 705 is a bacteriocin whose activity depends upon the complementation of two peptides, termed Lac705alpha and Lac705beta. Neither Lac705alpha nor Lac705beta displayed bacteriocin activity by itself when the growth of sensitive cells was monitored. To obtain molecular insights into the lactocin 705 mechanism of action, Fourier transform infrared spectroscopy was used to investigate the interactions of each peptide (Lac705alpha and Lac705beta) with dipalmitoylphosphatidylcholine liposomal membranes. Both peptides show the ability to interact with the zwitterionic membrane but at different bilayer levels. While Lac705alpha interacts with the interfacial region inducing dehydration, Lac705beta peptide interacts with only the hydrophobic core. This paper presents the first experimental evidence that supports the hypothesis that Lac705alpha and Lac705beta peptides could form a transmembrane oligomer. From the obtained results, a mechanism of action of lactocin 705 on membrane systems is proposed. The component Lac705alpha could induce the dehydration of the bilayer interfacial region, and the Lac705beta peptide could insert in the hydrophobic region of the membrane where the peptide has adequate conditions to achieve the oligomerization.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2006

Direct molecular approach to monitoring bacterial colonization on vacuum-packaged beef.

Denaturing gradient gel electrophoresis allowed us to monitor total bacterial communities and to establish a pattern of succession between species in vacuum-packaged beef stored at 2 and 8 degrees C for 9 weeks and 14 days. Species-specific PCR was used to confirm the presence of Lactobacillus sakei and Lactobacillus curvatus. Multiplex PCRs using 16S rRNA-specific primers allowed differentiation between Leuconostoc species. These methods provided the desired information about microbial diversity by detecting the main microorganisms capable of colonizing this ecological niche.

Animals↗

PubMed2005

Monitoring the bacterial population dynamics during fermentation of artisanal Argentinean sausages.

The dynamics of the microbial community responsible for the artisanal fermentation of dry sausage produced in Argentina was investigated by using classical and molecular approaches. The combined use of RAPD analysis with primers M13, XD9, RAPD1 and RAPD2 and 16S rDNA sequencing were applied to the identification and intraspecific differentiation of 100 strains of lactobacilli and Micrococcaceae. DGGE analysis was used to monitor the dynamic changes in population after total microbial DNA was directly extracted from sausages and subjected to PCR using V3f (GC), Bact-0124f-GC and Univ-0515r primers. The sequence analysis of 16S rDNA of the dominant species was also carried out. Lactobacillus sakei and Lactobacillus plantarum were the dominant lactic acid organisms during the fermentation while Staphylococcus saprophyticus represented the dominant species of Micrococcaceae. It was demonstrated that the ripening process of Argentinean artisanal fermented sausage is driven by a limited number of Lactobacillus and Staphylococcus strains selected from environmental microbiota by the ability to best compete under the prevailing conditions of the ecological niche. The identification of dominant communities present in this artisanal fermented sausage can help in the selection of starter cultures consisting in well adapted strains to the particular production technology.

Animals↗

PubMed2005

PCR-DGGE analysis for the identification of microbial populations from Argentinean dry fermented sausages.

Different PCR-DGGE protocols were evaluated to monitor fermentation process and to investigate bacterial communities developed in two artisanal Argentinean fermented sausages. Bacterial universal primers frequently used in PCR-denaturing gradient gel electrophoresis (DGGE) were evaluated. Lactic acid bacteria (LAB) and staphylococci species isolated from Tucumán sausages were used to determine the experimental conditions for PCR amplification and DGGE differentiation. Total microbial DNA extracted directly from both fermented sausages was subjected to DGGE analysis. PCR-DGGE results were different for each set of primers used. Primers Bact-0124f(GC)-Uni-0515r and V1f(GC)-V1r showed to be efficient to differentiate LAB and Staphylococcus cultures while the set V3f(GC)-Uni-0515r allowed to demonstrate the succession of different Lactobacillus and Staphylococcus species during ripening process. An intense band corresponding to Lactobacillus sakei was observed to be present in both samples. Staphylococcus saprophyticus was only observed in Tucumán sausage while a band identified as Brochothrix thermophacta was detected in Córdoba sausage. PCR-DGGE analysis of different 16S rDNA amplicons was able to discriminate between LAB and Gram-positive, coagulase-negative cocci, resulting an effective tool to establish the microbiota developed in artisanal dry sausages.

Animals↗

PubMed2004

Meat-model system development for antibacterial activity determination.

Bacteriocins are antibacterial substances produced by many different bacterial species. Although the bacteriocins form a heterogenous group with respect to production of bacteria, antibacterial spectrum, mode of action, and chemical properties, they are by definition proteinaceous compounds that are bactericidal toward organisms taxonomically close to the producer. The ability of lactic acid bacteria (LAB) to compete and finally dominate in mixed fermentations has been attributed to the production of several antimicrobial metabolites such as organic acids, carbon dioxide, hydrogen peroxide, diacetyl, and bacteriocins. The antimicrobial activities of the LAB have long been known, but their bacteriocins have received limited attention until recently. Numerous strains of lactic acid bacteria associated with food systems are capable of producing bacteriocins, or antibacterial proteins with activity against foodborne pathogens and contaminants. Recently, considerable enphasis has been placed on the physicochemical, biochemical, and genetic characterization of these proteins. Many methods for the detection of bacteriocin production as well as the determination of the potency of bacteriocin preparations have been described. All the usual techniques are based on the fact that bacteriocins can diffuse in solid or semisolid culture media, which are subsequently inoculated with a suitable indicator strain. This method has long been shown to have a good perfomance in the characterization of new bacteriocins. Although results obtained from broth systems show that bacteriocins inhibit target organisms, applied studies must be performed to confirm their effectiveness in food. As many lactic acid bacteria associated with meat products were described to be important natural bacteriocin producers, it has been necessary to assay their inhibitory efficacy in meat or meat products. Because of the complexity of these kinds of foods a simplified meat-model system was developed to determine the inhibitory activity of a bacteriocin.

Animals↗

PubMed2004

Meat-model system development for proteolytic activity determination.

Many technological parameters that affect the nature and functional properties of proteins are involved in the preparation of meat products. The dry-curing process is quite complex because of the coexistence of enzymes from endogenous and bacterial origins. The protein breakdown that takes place during the ripening of dry fermented sausages leads to an increase in the concentration of peptides and free amino acids. The proteolytic events have been thoroughly investigated not only because of their physiological significance but also for their technological connotations in terms of texture and flavor development. Lactic acid bacteria and Staphylococcus or Kocuria are used as starter cultures in fermented meat products. In recent years, the proteolytic system of lactobacilli involved in meat fermentation became the focus of an increasing number of studies because of the technological roles of these organisms. Although results obtained from broth systems show proteolytic activity, studies involving food systems must be done to confirm their effectiveness. A simplified soluble muscle extract to determine the capacity to degrade meat proteins by lactic acid bacteria was developed using a spectrophotometric method based on the reaction of the alpha-amino groups released by hydrolysis with o-phtaldialdehyde and beta-mercaptoethanol to form an adduct (1-thioalkyl-2-alkylisoindoles) that is enhanced at basic pH and absorbs strongly at 360 nm.

Animals↗

PubMed2003

Mode of action of lactocin 705, a two-component bacteriocin from Lactobacillus casei CRL705.

Lactocin 705 is a bacteriocin whose activity depends on the complementary action of two peptides (705alpha and 705beta) of 33-amino-acid residues each and is produced by Lactobacillus casei CRL705. Biologically active, synthetic lactocin 705 was used to study the mode of action on sensitive cells of Lactobacillus plantarum CRL691. The addition of 90 nmol l(-1) of lactocin 705 to cells of L. plantarum dissipated both, the membrane potential (DeltaPsi) and the pH gradient (DeltapH). Energized membrane, obtained after the addition of glucose, were more susceptible to lactocin 705 action leading to the immediate release of intracellular K(+) and inorganic phosphate. When the role of various ions on sensitive cells were analyzed, only Ca(2+) ion exhibited a protective effect against lactocin 705. These data suggest that the presence of a proton motive force (PMF) promotes the interaction of the bacteriocin with the cytoplasmic membrane of energized cells, leading to pore formation which allows for the efflux of ions, thereby ensuring efficient killing of target bacteria.

Bacteriocins↗