E. Anibal Disalvo

Critical water activity for the preservation of Lactobacillus bulgaricus by vacuum drying.

Lactobacillus delbrueckii subsp. bulgaricus was dried under vacuum at different temperatures and its preservation evaluated analyzing the evolution of three parameters throughout the process: lag time, percentage of membrane damage and zeta potential. Microorganisms were dehydrated at 30, 45 and 70 degrees C in a vacuum centrifuge for different times. The aw achieved for each time of drying was correlated with the cell recovery at all the temperatures assayed. The recovery of microorganisms was evaluated by means of: a) kinetics of growth in milk after drying, as a measure of the global damage; b) quantification of the membrane damage using the fluorescent dyes SYTO 9 and PI; c) determination of changes in the superficial charges (zeta potential) as measured of the increase in the hydrophobic residues exposed in the bacterial surface after dehydration. These changes correlate well with the bacterial damage occurred during the dehydration process. The Pages equation allowed fitting of aw and time of drying, thus making possible the determination of the appropriate dehydration conditions (time-temperature ratios) for which no cell damage occurs. The evaluation of three parameters (lag time, percentage of membrane damage and zeta potential) allowed us to conclude that at the lowest temperature of dehydration, the first target of damage is the cell membrane. However, this damage is not decisive for the bacterial recovery after rehydration, as are the increase in the lag time and the changes in the zeta potential, as was observed for L. bulgaricus dehydrated at 45 and 70 degrees C for larger times.

Action of trehalose on the preservation of Lactobacillus delbrueckii ssp. bulgaricus by heat and osmotic dehydration

Aim: This work determines the efficiency of trehalose on the preservation by heat or osmotic drying of a strain of Lactobacillus delbrueckii ssp. bulgaricus. Cell recovery at different trehalose concentrations during drying correlated with the surface properties and osmotic response of cells after rehydration.

Surface characterization and adhesive properties of bifidobacteria.

Publisher Summary This chapter discusses the surface characterization and adhesive properties of Bifidobacteria. The colonization of different portions of the intestinal tract by beneficial microorganisms constitutes the first defensive barrier against the invasion of pathogenic microorganisms or toxic substances. The ability to adhere to the intestinal epithelia and to compete with other microorganisms seems to be crucial for a probiotic strain to colonize the gastrointestinal tract. Bacteria of the genus Bifidobacterium are normal inhabitants of the gut of humans and animals, where they play an important role in the prevention of gastrointestinal disorders. Bifidobacteria have been successfully employed to prevent antibioticrelated diarrhea and acute infant diarrhea. One approach to obtaining information regarding adherent behavior in the intestinal ecosystem is to study the surface properties of the selected strains. The adhesion of microorganisms to natural environments can be inferred by studying the surface properties of bacteria, employing a combination of methodologies. This procedure includes several determinations such as partitioning of bacteria in hydrocarbon-aqueous interfaces, ζ potential, binding to solid surfaces, capacity to aggregate particles, and adhesion to monolayers of cell cultures. These methodologies are applied to a collection of bifidobacteria isolated at CIDCA (Centro de Investigacion y Desarrollo en Criotecnologia de Alimentos, La Plata, Argentina) and to reference strains.

Role of guanidinium group in the insertion of l-arginine in DMPE and DMPC lipid interphases

l-Arginine (Arg) is a positively charged amino acid constituent of peptides and proteins, participating in diverse mechanisms of protein-membrane interaction. The effect of Arg on phosphatidylcholine (PC) membranes has been previously related to water structure changes and to the presence of water defects in the hydrocarbon region. However, no information is available with regard to phosphatidylethanolamine (PE), another important component of lipid membranes. For this reason, the aim of this study is to determine the effect of Arg on DMPE membranes and partially methylated PEs in comparison to DMPC. The adsorption of the amino acid onto the lipid membranes was followed by determining the changes in the surface potential as a function of the bulk amino acid concentrations. The effects of Arg on the surface properties were also measured by changes in the surface pressure and the dipole potential. The onset of the transition temperature was measured with a fluorophore anchored at the membrane interphase. The results provide a new insight on amino acid-PE interactions, which can be ascribed to specific perturbations in the head group region induced by the guanidinium residue.

Molecular Dynamics Study of the Interaction of Arginine with Phosphatidylcholine and Phosphatidylethanolamine Bilayers

In this work, the differential interaction of zwitterionic arginines with fully hydrated dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers was analyzed by molecular dynamics simulations. In both systems, arginine binds to lipids with the carboxylate moiety oriented toward the aqueous phase, in agreement with previous experimental determinations of ζ potential of DMPC and DMPE liposomes. The guanidinium groups are found at different depths within the bilayers indicating that some arginines are buried, especially in DMPE. We observe, in the DMPE system, that the strongest interaction occurs between the guanidinium group and the carbonyl oxygen of the lipid. In the case of DMPC membranes, the strongest interaction is found between the guanidinium groups of the arginines and the phosphate groups of the lipids. Unexpectedly, arginine zwitterions are stabilized through the creation of hydrogen bonds (HB), either with water or with polar groups of the lipids. The mechanisms of interaction seem to be different in both membranes. In DMPE bilayers, arginines insert by breaking the inner HB network of the polar head groups, consequently increasing the occupied area per lipid molecule. In the DMPC bilayers the arginines insert by replacing the already present water molecules within the membrane, without significant effects on the area per lipid.

Configuration of Carbonyl Groups at the Lipid Interphases of Different Topological Arrangements of Lipid Dispersions

The purpose of this work is to analyze the conformation of the carbonyl groups of acyl phospholipids at the hydrocarbon-water interphase in different topological ensembles and phase states, such as micelles and bilayers. The separation of the band components in lipids dispersed in D(2)O is compared with that of PCs in a low hydrated state. When hydrated, the differences in the frequencies of the band components corresponding to the carbonyl groups identified as low hydrated and hydrated populations increase when dimyristoylphosphatidylcholine (DMPC) bilayers go from the lamellar gel to the ripple corrugated phase at the pretransition temperature. Below the pretransition, at which the membrane in the gel state is planar, the two components overlap making the deconvolution unreliable. A further analysis shows that the frequency of the highly hydrated population increases more noticeable than that corresponding to the low hydrated one following the sequence: micelles, fluid phase, ripple gel phase, and lamellar gel phase. This is confirmed by the increase in the separation of the band components when the liposomes are subjected to an osmotic dehydration suggesting that the hydrated population loses water and the dehydrated one partially hydrates. It is concluded that this behavior is a feature conferred by hydration of the different topological arrangements. The relevance of these results on the interphase properties of lipid membranes is discussed.

Aggregation induced by concanavalin A of lipid vesicles containing neoglycolipids

Abstract Concanavalin A (Con A) adsorbs on dipalmitoylphosphatidylcholine (DPPC):cholesterol bilayers but aggregation is only produced if neoglycolipids such as mannose–mannose–mannose-distearylglycerol (3M-diC18), glucose–mannose–glucose-distearylglycerol (GMG-diC18) or glucose–mannose–glucose-cholesterol (GMG-cho) are present. The phase state of the lipid, modified by the inclusion of cholesterol in the mixture, does not affect the aggregation of GMG-diC18:DPPC vesicles induced by lectin. With GMG-diC18 lipids, aggregation appears to be produced by the formation of a strong complex between the neoglycolipids and the protein. This interaction affects the hydrocarbon core of DPPC bilayers containing GMG-diC18. The greatest changes are observed when GMG-diC18 is present in the vesicle composition. The aggregation induced by Con A is maximal with 3M-diC18. In this case, the changes in surface properties and in the protein are less pronounced, evidencing an alternative mechanism of aggregation. The type of sugar unit and the lipid moiety by which neoglycolipids are anchored to the bilayer, determine the magnitude of the above mentioned processes.

Coordination forces between lipid bilayers produced by ferricyanide and Ca2

Attractive forces usually invoked to take place in membrane-membrane contact in aggregation are hydrogen bonding cross-linkings and hydrophobic interactions between opposing surfaces. However, little is known in relation to the presence of coordination forces in the membrane-membrane interaction. These are understood as those that may be favoured by the formation or the participation of coordination complexes between surface specific groups. In this work, we have analyzed the formation of this type of aggregates between phosphatidylcholine vesicles mediated by a coadsorption of ferricyanide and Ca(2+) ions to the interface. The results obtained by surface potential measures, optical and electronic microscopy, FTIR and (1)H NMR spectroscopies indicate that ferricyanide [Fe(CN)(6)](3-) but not of ferrocyanide [Fe(CN)(6)](4-) can form the complex when Ca(2+) has been adsorbed previously to the membrane surface. In this condition, the anion is likely to act as a bridge between two opposing membranes causing a tight aggregation in which geometry and the polarizability of the ligands to Fe(3+) play a role.