Esteban Gerbino

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 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.

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.

Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Spray-Drying

In this work maltodextrins were added to commercial galacto-oligosaccharides (GOS) in a 1:1 ratio and their thermophysical characteristics were analyzed. GOS:MD solutions were then used as matrices during spray-drying of Lactobacillus plantarum CIDCA 83114. The obtained powders were equilibrated at different relative humidities (RH) and stored at 5 and 20°C for 12 weeks, or at 30°C for 6 weeks. The Tgs of GOS:MD matrices were about 20–30°C higher than those of GOS at RH within 11 and 52%. A linear relation between the spin-spin relaxation time (T2) and T-Tg parameter was observed for GOS:MD matrices equilibrated at 11, 22, 33, and 44% RH at 5, 20, and 30°C. Spray-drying of L. plantarum CIDCA 83114 in GOS:MD matrices allowed the recovery of 93% microorganisms. In contrast, only 64% microorganisms were recovered when no GOS were included in the dehydration medium. Survival of L. plantarum CIDCA 83114 during storage showed the best performance for bacteria stored at 5°C. In a further step, the slopes of the linear regressions provided information about the rate of microbial inactivation for each storage condition (k values). This information can be useful to calculate the shelf-life of spray-dried starters stored at different temperatures and RH. Using GOS:MD matrices as a dehydration medium enhanced the recovery of L. plantarum CIDCA 83114 after spray-drying. This strategy allowed for the first time the spray-drying stabilization of a potentially probiotic strain in the presence of GOS.

Use of Raman spectroscopy and chemometrics for the quantification of metal ions attached to Lactobacillus kefir

Aims:  To set‐up an experimental and analytical methodology to evaluate the feasibility of developing simple, accurate and quantitative models based on Raman spectroscopy and multivariate analysis for the quantification of metal ions adsorbed to the bacterial surface of Lactobacillus kefir.

Applications of Infrared and Raman Spectroscopies to Probiotic Investigation

In this review, we overview the most important contributions of vibrational spectroscopy based techniques in the study of probiotics and lactic acid bacteria. First, we briefly introduce the fundamentals of these techniques, together with the main multivariate analytical tools used for spectral interpretation. Then, four main groups of applications are reported: (a) bacterial taxonomy (Subsection 4.1); (b) bacterial preservation (Subsection 4.2); (c) monitoring processes involving lactic acid bacteria and probiotics (Subsection 4.3); (d) imaging-based applications (Subsection 4.4). A final conclusion, underlying the potentialities of these techniques, is presented.

Removal of cadmium by Lactobacillus kefir as a protective tool against toxicity.

The aim of this work was to evaluate the capacity of Lactobacillus kefir strains to remove cadmium cations and protect eukaryotic cells from cadmium toxicity. Lb. kefir CIDCA 8348 and JCM 5818 were grown in a 1/2 dilution of MRS broth supplemented with Cd(NO3)2 ranging 0 to 1 mM. Growth kinetics were followed during 76 h at 30 °C by registering optical density at 600 nm every 4-10 h. The accumulated concentration of cadmium was determined on cultures in the stationary phase by atomic absorption. The viability of a human hepatoma cell line (HepG2) upon exposure to (a) free cadmium and (b) cadmium previously incubated with Lb. kefir strains was evaluated by determining the mitochondrial dehydrogenase activity. Lb. kefir strains were able to grow and tolerate concentrations of cadmium cations up to 1 mM. The addition of cadmium to the culture medium increased the lag time in all the concentrations used. However, a decrease of the total biomass (maximum Absorbance) was observed only at concentrations above 0.0012 and 0.0011 mM for strains CIDCA 8348 and JCM 5818, respectively. Shorter and rounder lactobacilli were observed in both strains upon microscopic observations. Moreover, dark precipitates compatible with intracellular precipitation of cadmium were observed in the cytoplasm of both strains. The ability of Lb. kefir to protect eukaryotic cells cultures from cadmium toxicity was analysed using HepG2 cells lines. Concentrations of cadmium greater than 3×10(-3) mM strongly decreased the viability of HepG2 cells. However, when the eukaryotic cells were exposed to cadmium pre-incubated 1 h with Lb. kefir the toxicity of cadmium was considerably lower, Lb. kefir JCM 5818 being more efficient. The high tolerance and binding capacity of Lb. kefir strains to cadmium concentrations largely exceeding the tolerated weekly intake (TWI) of cadmium for food (2.5 μg per kg of body weight) and water (3 μg/l) addressed to human consumption, is an important added value when thinking in health-related applications.

Okara: A Nutritionally Valuable By-product Able to Stabilize Lactobacillus plantarum during Freeze-drying, Spray-drying, and Storage

Okara is a nutritionally valuable by-product produced in large quantities as result of soymilk elaboration. This work proposes its use as both culture and dehydration medium during freeze-drying, spray-drying, and storage of Lactobacillus plantarum CIDCA 83114. Whole and defatted okara were employed as culture media for L. plantarum CIDCA 83114. The growth kinetics were followed by plate counting and compared with those of bacteria grown in MRS broth (control). No significant differences in plate counting were observed in the three media. The fatty acid composition of bacteria grown in whole and defatted okara showed a noticeable increase in the unsaturated/saturated (U/S) fatty acid ratio, with regard to bacteria grown in MRS. This change was mainly due to the increase in polyunsaturated fatty acids, namely C18:2. For dehydration assays, cultures in the stationary phase were neutralized and freeze-dried (with or without the addition of 250 mM sucrose) or spray-dried. Bacteria were plate counted immediately after freeze-drying or spray-drying and during storage at 4°C for 90 days. Freeze-drying in whole okara conducted to the highest bacterial recovery. Regarding storage, spray-dried bacteria previously grown in whole and defatted okara showed higher plate counts than those grown in MRS. On the contrary, freeze-dried bacteria previously grown in all the three culture media were those with the lowest plate counts. The addition of sucrose to the dehydration media improved their recovery. The higher recovery of microorganisms grown in okara after freeze-drying and spray-drying processes and during storage was ascribed to both the presence of fiber and proteins in the dehydration media, and the increase in U/S fatty acids ratio in bacterial membranes. The obtained results support for the first time the use of okara as an innovative matrix to deliver L. plantarum. Considering that okara is an agro-waste obtained in large quantities, these results represent an innovative strategy to add it value, providing a symbiotic ingredient with promising industrial applications in the development of novel functional foods and feeds.

Development and characterization of iron-pectin beads as a novel system for iron delivery to intestinal cells

Iron deficiency is the most common nutritional deficit worldwide. The goal of this work was to obtain iron-pectin beads by ionic gelation and evaluate their physiological behavior to support their potential application in the food industry. The beads were firstly analyzed by scanning electronic microscopy, and then physical-chemically characterized by performing swelling, thermogravimetric, porosimetry, Mössbauer spectroscopy and X-ray fluorescence analyses, as well as by determining the particle size. Then, physiological assays were carried out by exposing the beads to simulated gastric and intestinal environments, and determining the iron absorption and transepithelial transport into Caco-2/TC7 cells. Iron-pectin beads were spherical (diameter 1-2 mm), with high density (1.29 g/mL) and porosity (93.28%) at low pressure, indicating their high permeability even when exposed to low pressure. Swelling in simulated intestinal medium (pH 8) was higher than in simulated gastric medium. The source of iron [FeSO4 (control) or iron-pectin beads] did not have any significant effect on the mineral absorption. Regarding transport, the iron added to the apical pole of Caco-2/TC7 monolayers was recovered in the basal compartment, and this was proportional with the exposure time. After 4 h of incubation, the transport of iron arising from the beads was significantly higher than that of the iron from the control (FeSO4). For this reason, iron-pectin beads appear as an interesting system to overcome the low efficiency of iron transport, being a potential strategy to enrich food products with iron, without altering the sensory properties.

Application of Principal Component Analysis to Elucidate Experimental and Theoretical Information

Principal Component Analysis has been widely used in different scientific areas and for different purposes. The versatility and potentialities of this unsupervised method for data analysis, allowed the scientific community to explore its applications in different fields. Even when the principles of PCA are the same in what algorithms and fundamentals concerns, the strategies employed to elucidate information from a specific data set (experimental and/or theoretical), mainly depend on the expertise and needs of each researcher.