Andrea Gómez-Zavaglia

Low-temperature solid-state FTIR study of glycine, sarcosine and N,N-dimethylglycine: observation of neutral forms of simple α-amino acids in the solid state

Neutral forms of glycine and their N-methylated derivatives, sarcosine (N-methylglycine) and N,N-dimethylglycine were, for the first time, observed in the solid state pure compounds. The substances were sublimated under high vacuum, quickly deposited onto a cold CsI substrate at 9 K and examined using FTIR spectroscopy within the temperature range 9–300 K. For all the compounds studied, the spectra obtained at 9 K after deposition revealed the presence of both the neutral and zwitterionic amino acid forms. The identification of the neutral forms and their vibrational signatures could be unequivocally established by taking into consideration the available data for the matrix-isolated neutral monomeric and aggregated amino acids as well as solid state data for acid and alkaline salts of glycine. Upon temperature increase, neutral forms convert non-reversibly to the zwitterionic forms, as noticed by the systematic increase in the relative intensities of the infrared bands ascribable to the latter species. Observation of aggregates of neutral amino acids in the solid phase, together with the fact that the amount of the neutral forms observed reduces with increasing both the temperatures of the cold substrate and of the vapour prior to deposition, enabled to conclude that the observed (neutral form) → zwitterion conversion occurs in the solid state, to the therein most stable zwitterionic species, and that intermolecular interactions in this phase should play an important role in the mechanism of proton transfer.

Galacto-oligosaccharides as protective molecules in the preservation of Lactobacillus delbrueckii subsp. bulgaricus.

In this work, the protective capacity of galacto-oligosaccharides in the preservation of Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 was evaluated. Lactobacillus bulgaricus was freeze-dried or dried over silica gel in the presence of three commercial products containing galacto-oligosaccharides. The freeze-dried samples were stored at 5 and 25°C for different periods of time. After desiccation, freeze-drying or storage, samples were rehydrated and bacterial plate counts were determined. According to the results obtained, all galacto-oligosaccharides assays demonstrated to be highly efficient in the preservation of L. bulgaricus. The higher content of galacto-oligosaccharides in the commercial products was correlated with their higher protective capacity. Galacto-oligosaccharides are widely known by their prebiotic properties. However, their role as protective molecules have not been reported nor properly explored up to now. In this work the protective capacity of galacto-oligosaccharides in the preservation of L. bulgaricus, a strain particularly sensitive to any preservation process, was demonstrated. The novel role of galacto-oligosaccharides as protective molecules opens up several perspectives in regard to their applications. The supplementation of probiotics with galacto-oligosaccharides allows the production of self-protected synbiotic products, galacto-oligosaccharides exerting both a prebiotic and protecting effect.

Effect of sugars and growth media on the dehydration of Lactobacillus delbrueckii ssp. bulgaricus

Aims:  The efficiency of trehalose, sucrose and maltose to protect Lactobacillus bulgaricus during drying has been evaluated in bacteria grown at low water activity.

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.

DNA fingerprinting of thermophilic lactic acid bacteria using repetitive sequence-based polymerase chain reaction.

DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR). The method made it possible to identify 37 isolates from raw milk. industrial starters and yogurt. Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp bulgaricus and Str. thermophilus. BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.

Effect of physical properties on the stability of Lactobacillus bulgaricus in a freeze-dried galacto-oligosaccharides matrix

The ability of galacto-oligosaccharides (GOS) to protect Lactobacillus delbrueckii subsp. bulgaricus upon freeze drying was analyzed on the basis of their capacity to form glassy structures. Glass transition temperatures (T(g)) of a GOS matrix at various relative humidities (RH) were determined by DSC. Survival of L. bulgaricus in a glassy GOS matrix was investigated after freezing, freeze drying, equilibration at different RHs and storage at different temperatures. At 32 °C, a drastic viability loss was observed. At 20 °C, the survival was affected by the water content, having the samples stored at lower RHs, the highest survival percentages. At 4°C, no decay in the cells count was observed after 45 days of storage. The correlation between molecular mobility [as measured by Proton nuclear magnetic resonance (¹H NMR)] and loss of viability explained the efficiency of GOS as cryoprotectants. The preservation of microorganisms was improved at low molecular mobility and this condition was obtained at low water contents and low storage temperatures. These results are important in the developing of new functional foods containing pre and probiotics.

Volume recovery, surface properties and membrane integrity of Lactobacillus delbrueckii subsp. bulgaricus dehydrated in the presence of trehalose or sucrose

Aims:  Although the practical importance of adding sugars before drying is well known, the mechanism of protection of bacteria by sugars is not clear. The response of the dehydrated micro‐organisms to rehydration is analysed in terms of structural and functional changes, and correlated with their potentiality to grow in rich media. These aspects are related with the membrane integrity and the metabolic state of the rehydrated bacteria, measured by means of surface properties and permeability. To attain this objective, Lactobacillus delbrueckii subsp. bulgaricus was dehydrated in the presence and in the absence of sucrose and trehalose. The bacterial response upon rehydration was investigated by determining: (i) the lag time of the bacterial growing in rich media, (ii) the restoration of the surface properties and the cellular volume and (iii) the membrane integrity.

Role of S-layer proteins in bacteria

S-layers are paracrystalline bidimensional arrays of proteins or glycoproteins that overlay the cell surface of several genus and species of bacteria and archaea. As the outermost layer of several genus and species of microorganisms, S-layer proteins (SLP) are in direct contact with bacterial environment and thus may be involved in many of their surface properties, including adherence to various substrates, mucins and eukaryotic cells, aggregation and coaggregation with yeasts and other bacteria. In addition, SLP have been reported to be responsible for the bacterial protection against detrimental environmental conditions and to play an important role in surface recognition or as carriers of virulence factors. In this mini-review, we bring together the latest evidences about functional and mechanical properties of bacterial SLP from two different perspectives: (A) their role on bacterial adherence to different substrates and surfaces, and (B) their role as mechanical barriers in bacterial harmful environments.

Use of whey permeate containing in situ synthesised galacto-oligosaccharides for the growth and preservation of Lactobacillus plantarum

Galacto-oligosaccharides (GOS) are prebiotics that have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. GOS are commonly produced from lactose in an enzymatic reaction catalysed by β-galactosidase, named transglycosylation. Lactose is the main constituent of whey permeate (WP), normally wasted output from the cheese industry. Therefore, the main goal of this work was to optimise the synthesis of GOS in WP using β-galatosidase from Aspergillus oryzaea. WP and whey permeate enzymatically treated (WP-GOS) were used as culture media of Lactobacillus plantarum 299v. Lb. plantarum 299v attained the stationary phase in approximately 16 h, reaching 3·6 and 4·1×108 CFU/ml in WP and WP-GOS, respectively. The in situ synthesised GOS were not consumed during growth. No significant differences were observed in the growth kinetics of microorganisms in both media. After fermentation, microorganisms were dehydrated by freeze-drying and spray-drying and stored. The recovery of microorganisms after fermentation, dehydration and storage at 4 °C for at least 120 d was above 108 CFU/g. These studies demonstrated that WP is an appropriate substrate for the synthesis of GOS and the obtained product is also adequate as culture medium of Lb. plantarum 299v. The coexistence of GOS and dehydrated viable probiotic microorganisms, prepared using an effluent as raw material, represents the main achievement of this work, with potential impact in the development of functional foods.

Rotational isomers of lactic acid: first experimental observation of higher energy forms

Lactic acid {2-hydroxypropionic acid [CH(CH3)OHCOOH]} monomer was studied by matrix isolation FT-IR spectroscopy and molecular orbital calculations undertaken at both DFT(B3LYP)/6-311++G(d,p) and MP2/6-31G(d,p) levels of approximation. The theoretical calculations predicted the conformer (SsC) with the carboxylic group and both the CCOH(alcohol) and OCCO moieties in a cis configuration as the most stable form. In this conformer, the α-hydroxy hydrogen atom is involved in an intramolecular hydrogen bond with the carbonyl oxygen atom. The second most stable conformer (GskC) also shows a cis carboxylic group and differs from SsC in the CCOH(alcohol) and OCCO dihedral angles, which are equal to 43.3° and 156.8° respectively. These angles are equal to −51.5° and −149.9° in the third most stable conformer (G′sk′C). These forms are characterized by showing a relatively weak intramolecular H(alcohol)⋯O(acid) hydrogen bond. In the AaT conformer, the carboxylic group is trans and the CCOH(alcohol) and OCCO dihedral angles are 162.2° and 174.1°, respectively. This conformer shows a relatively strong H(acid)⋯O(alcohol) hydrogen bond. In consonance with the theoretical results, the matrix isolation experiments confirmed the predominance of conformer SsC in argon and xenon matrices, and provide the first experimental evidence of conformers GskC and AaT. Since the barrier for interconversion G′sk′C ↔ GskC is only ∼2 kJ mol−1, these two conformers are in equilibrium in the matrices and, at low temperature, the population of the less stable G′sk′C form is too small to enable its observation. Full assignment of the observed spectra was undertaken on the basis of comparison with the theoretical data and temperature variation studies. Water doping of matrices enabled the identification of spectral features due to weakly bound complexes of lactic acid with water.