Encapsulation of lactic acid bacteria in W1/O/W2 emulsions stabilized by mucilage:pectin complexes.

Opuntia silvestri mucilage obtained from dried stems was explored as an emulsifier to prepare double emulsions aiming to encapsulate Lactiplantibacillus plantarum CIDCA 83114. W1/O/W2 emulsions were prepared using a two-step emulsification method. The aqueous phase (W1) consisted of L. plantarum CIDCA 83114, and the oil phase (O) of sunflower oil. The second emulsion was prepared by mixing the internal W1/O emulsion with the W2 phase, consisting of 4 % polysaccharides, formulated with different mucilage:(citric)pectin ratios. Their stability was assessed after preparation (day 0) and during storage at 4 °C (28 days). Determinations included creaming index, color, particle size, viscosity, turbidity, and bacterial viability, along with exposure to simulated gastrointestinal conditions. Significant differences were evaluated by analysis of variance (ANOVA) and Duncan's test (P < 0.05). After 28 days storage, bacterial viability in the W1/O/W2 emulsions was above 6 log CFU/mL for all the pectin:mucilage ratios. Emulsions containing mucilage and pectins showed lower creaming indices after 15 days, remaining stable until the end of the storage period. Formulations including 1:1 pectin:mucilage ratio exhibited the highest bacterial viability under simulated gastrointestinal conditions and were more homogeneous in terms of droplet size distributions at day 0, hinting at a synergistic effect between mucilage components (e.g., proteins, Ca2+) and pectin in stabilizing the emulsions. These results showed that Opuntia silvestri mucilage enhanced the stability of emulsions during refrigerated storage, highlighting its potential for encapsulating lactic acid bacteria. This presents an economical and natural alternative to traditional encapsulating materials.

Quantification of the environmental impact arising from the utilization of whole and defatted Okara in fermentative and dehydration processes.

Okara is the insoluble pulp that remains after the grinding and filtration of soybeans during the production of soymilk and tofu. As it retains a noteworthy quantity of nutrients, there has been an increasing emphasis in the utilization of this residue for the development of sustainable processes. This study focused on assessing the environmental impact of employing okara as a medium for fermenting and dehydrating probiotic bacteria at laboratory scale. The evaluation was carried out using the Life Cycle Assessment (LCA) methodology, considering the entire process lifecycle. Whole okara and defatted okara were used as culture media for Lactiplantibacillus plantarum CIDCA 83114, followed by dehydration (either freeze-drying or spray-drying) and subsequent storage. For the purpose of comparison, both scenarios (whole and defatted okara) were evaluated using 1 kg of dehydrated final product for storage, as functional unit. Based on experimental results, the conservation of the received okara and the dehydration-storage (e.g., freezing and freeze-drying) phases were identified as the most significant environmental hotspots responsible for the most substantial impacts of the processes. The use of LCA facilitated the measurement of the environmental effects linked to the reutilization of okara as an agro-industrial residue, thus providing quantitative support when engineering its sustainable valorization.

Microfluidics potential for developing food-grade microstructures through emulsification processes and their application.

The food sector continues to face challenges in developing techniques to increase the bioavailability of bioactive chemicals. Utilising microstructures capable of encapsulating diverse compounds has been proposed as a technological solution for their transport both in food and into the gastrointestinal tract. The present review discusses the primary elements that influence the emulsification process in microfluidic systems to form different microstructures for food applications. In microfluidic systems, reactions occur within small reaction channels (1-1000 µm), using small amounts of samples and reactants, ca. 102-103 times less than conventional assays. This geometry provides several advantages for emulsion and encapsulating structure production, like less waste generation, lower cost and gentle assays. Also, from a food application perspective, it allows the decrease in particle dispersion, resulting in a highly repeatable and efficient synthesis method that also improves the palatability of the food products into which the encapsulates are incorporated. However, it also entails some particular requirements. It is important to obtain a low Reynolds number (Re < approx. 250) for greater precision in droplet formation. Also, microfluidics requires fluid viscosity typically between 0.3 and 1400 mPa s at 20 °C. So, it is a challenge to find food-grade fluids that can operate at the micro-scale of these systems. Microfluidic systems can be used to synthesise different food-grade microstructures: microemulsions, solid lipid microparticles, microgels, or self-assembled structures like liposomes, niosomes, or polymersomes. Besides, microfluidics is particularly useful for accurately encapsulating bacterial cells to control their delivery and release on the action site. However, despite the significant advancement in these systems' development over the past several years, developing and implementing these systems on an industrial scale remains challenging for the food industry.

Dataset on the Life Cycle Assessment of fructo- and galacto-oligosaccharides (FOS and GOS) produced by synthesis or hydrolysis.

The prebiotics like FOS and GOS are receiving special attention in the food industry due to their potential health benefits. They can be produced by enzymatic synthesis by using disaccharides or other substrates as raw materials or by extraction and hydrolysis from different natural sources (roots, legumes). However the environmental footprints of these different production schemes are lacking. This dataset presents Life Cycle Assessment (LCA) of the production of FOS and GOS by enzymatic synthesis from glucose (to get FOS) or lactose (to get GOS) and hydrolytic production from extraction of yacon potato (to get FOS) or chickpea (to get GOS). A cradle-to-gate approach was considered in the two scenarios under assessment (the phases of use and/or final disposal of FOS/GOS were not considered). The functional unit was defined as 100 g of FOS/GOS produced. LCAs were performed using data collected at the laboratory scale during experiments, supplemented with data from technical and scientific literature. Ecoinvent database provided background data. SimaPro was used for the LCA modeling with the midpoint impact EF2.0 characterization method to calculate environmental impacts. For each scenario (FOS produced by synthesis, FOS produced by hydrolysis, GOS produced by synthesis, GOS produced by hydrolysis), the Life Cycle Inventory (LCI) and the Life Cycle Impact Assessment (LCIA) are provided. These data can be used (i) to identify the main environmental hotspots of the production process, (ii) to compare the different process alternatives between them and (ii) to suggest eco-design options to upscale these processes. They could also be re-used in other LCA studies which would include FOS and/or GOS in the production system.

Green synthesis, characterization and applications of iron and zinc nanoparticles by probiotics.

The synthesis of nanoparticles (NPs) by microorganisms is one of the most promising areas of research in modern nanotechnology since microorganisms can easily act as real nanofactories of industrially relevant compounds. Recent studies suggest that probiotic bacteria have an intrinsic potential to synthesize metal NPs when grown in the presence of metal ions. In such conditions, they can reduce metal ions through different biochemical mechanisms occurring both intra and extracellularly, and leading to the production of NPs. Different approaches have proposed the synthesis of silver, gold, titanium or selenium NPs from probiotics, with promising health related effects. However, their use for the production of iron and zinc NPs has been scarcely reported. Considering the nutritional relevance of iron and zinc, a thorough approach about the synthesis of iron and zinc NPs by probiotics was addressed, including the factors affecting the synthesis processes, the mechanisms of synthesis, and the physical and chemical characterization of NPs. The impact of products containing probiotics and minerals has applications in many different fields going beyond the food industry and representing a powerful strategy as economic engine for very diverse industries and countries.

Microencapsulation of Lactobacillus plantarum in W/O emulsions of okara oil and block-copolymers of poly(acrylic acid) and pluronic using microfluidic devices.

Okara oil is a by-product remaining from defatting okara, the solid residue generated after extracting the aqueous fraction of grounded soybeans in the elaboration of soy beverages. The goal of this work was to encapsulate the probiotic Lactobacillus plantarum CIDCA 83114 into W/O emulsions composed of a block-copolymer constituted of pluronic® and acrylic acid (PPP12) and okara oil, prepared in microfluidic devices. For comparative purposes, alginate was also included as a second dispersed phase. Lactobacillus plantarum CIDCA 83114 was suspended in PPP12 or alginate giving rise to dispersed phases with different compositions, named I, II, III and IV. Controls were prepared by suspending microorganisms in water as dispersed phase. 6-carboxyfluorescein was added as bacterial marker in all the emulsions. The presence of green dyed bacteria in the dispersed phases, inside the droplets of the emulsions and the absence of fluorescence outside them, confirmed the complete encapsulation of bacteria in the dispersed phases. After being prepared, emulsions were freeze-dried. The exposure to gastric conditions did not lead to significant differences among the emulsions containing polymers. However, in all cases bacterial counts were significantly lower than those of the control. After exposing emulsions to the simulated intestinal environment, bacterial counts in assays I, II and III (emulsions composed of only one dispersed phase or of two dispersed phases with bacteria resuspended in the PPP12 one) were significantly greater than those of the control (p < 0.05) and no detectable microorganisms were observed for assay IV (emulsions composed of two dispersed phases with bacteria resuspended in the alginate one). In particular, bacterial cultivability in emulsions corresponding to assay I (only PPP12 as dispersed phase) exposed to the intestinal environment was 8.22 ± 0.02 log CFU/mL (2 log CFU higher than the values obtained after gastric digestion). These results support the role of PPP12 as an adequate co-polymer to protect probiotics from the gastric environment, enabling their release in the gut, with great potential for food or nutraceutical applications.

Release of health-related compounds during in vitro gastro-intestinal digestion of okara and okara fermented with Lactobacillus plantarum.

Okara is a highly perishable by-product remaining after filtration of the smashed soybeans seeds in the production of soymilk. Due to its nutritional value, different approaches have been developed to use it as functional ingredient. Fermentation of okara appears as an interesting strategy to preclude spoilage, providing a more stable matrix to be incorporated in the formulation of functional foods. Okara has antioxidant compounds but the effect of fermentation, and their bioaccessibility still need to be investigated. To achieve this aim, the phenolic compounds (as determined by TPC and TFC assays) and the antioxidant properties (as determined by ABTS ·+, DPPH · , O2 ·- assays) of okara and okara fermented with Lactobacillus plantarum CIDCA 83114 were assessed both before and after exposure to simulated gastro-intestinal conditions. Before digestion, okara showed higher values of TPC and TFC than the fermented counterpart. Although a decrease of TPC and TFC was observed after exposing okara to gastric conditions, no significant differences between okara and fermented okara were detected. No further decrease of TPC were observed in intestinal conditions. Okara showed higher antioxidant activity than fermented okara. There was a considerable decrease in the antioxidant activity for both samples when exposed to gastric and intestinal conditions. A good correlation between TFC and antioxidant activities was detected, suggesting that flavonoids play an important role as antioxidants. As a whole, this work provides a solid support for the stability of phytochemicals along the digestive process of both okara and fermented okara.

Infrared spectroscopy with multivariate analysis to interrogate the interaction of whole cells and secreted soluble exopolimeric substances of Pseudomonas veronii 2E with Cd(II), Cu(II) and Zn(II).

Extracellular polymeric substances (EPS) are bacterial products associated to cell wall or secreted to the liquid media that form the framework of microbial mats. These EPS contain functional groups as carboxyl, amino, hydroxyl, phosphate and sulfhydryl, able to interact with cations. Thus, EPS may be considered natural detoxifying compounds of metal polluted waters and wastewaters. In this work Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) in combination with multivariate analysis (Principal Component Analysis-PCA-) were used to study the interaction of Cd(II), Cu(II) and Zn(II) and Pseudomonas veronii 2E cells, including bound EPS and cell wall, and its different soluble EPS fractions, previously characterized as Cd(II) ligands of moderate strength. Amino groups present in exopolysaccharide fraction were responsible for Zn(II) and Cu(II) complexation, while carboxylates chelated Cd(II). In lipopolysaccharide fraction, phosphoryl and carboxyl sites were involved in Cd(II) and Cu(II) binding, while Zn(II) interacted with amino groups. Similar results were obtained from cells. These studies confirmed that FTIR-PCA is a rapid analytical tool to provide valuable information regarding the functional groups in biomolecules related to metal interaction. Moreover, a discrimination and identification of functional groups present in both EPS and cells that interacted with Cd(II), Zn(II) and Cu(II) was demonstrated.

Pectin Hydrolysates from Different Cultivars of Pink/Red and White Grapefruits (Citrus Paradisi [macf.]) as Culture and Encapsulating Media for Lactobacillus Plantarum.

Citrus pectin hydrolysates (Citrus paradisi [Mafc.]) from "Foster," "Red Shambar," "Tangelo Orlando," and "Citrumelo Swingle" cultivars were obtained by partial chemical hydrolysis and their properties as culture media (sole carbon/nutrient source) and encapsulating agents of Lactobacillus plantarum CIDCA 83114 were evaluated. The concentration of neutral sugars was maximal after 2-hour hydrolysis. All hydrolysates were rich in glucose >xylose >galactose >galacturonic acid >mannose >arabinose. "Citrumelo Swingle" cultivar was the one with the highest concentration of xylose. After 24 hr of fermentation with L. plantarum CIDCA 83114, bacterial viability increased from 6.76 ± 0.14 to almost 9 log CFU/mL, and lactic acid concentration, from 2.63 ± 0.41 to 7.82 ± 0.15 mmol/L in all hydrolysates. Afterwards, bacteria were entrapped in pectate-calcium beads by ionotropic gelation. Bacterial viability did not significantly decrease after freeze-drying and storage the beads at 4 °C for 45 days. PRACTICAL APPLICATION: Pectin hydrolysates were adequate culture media for microorganisms, as determined by the viabililty and lactic acid production. Considering that citrus peels are agro-wastes obtained in large quantities, their use as encapsulating materials provides a solution to overcome the environmental problem they entail.

Pectin-decorated magnetite nanoparticles as both iron delivery systems and protective matrices for probiotic bacteria.

The goal of this work was to investigate biophysical stability of iron-pectin nanoparticles and analyze the feasibility of using them as delivery systems for the probiotic strain Lactobacillus plantarum CIDCA 83114. Iron oxide (Fe3O4) nanoparticles were synthesized from 0.25M FeCl2/0.5 M FeCl3.6H2O, and coated with citrus pectins. Their physico-chemical properties [FTIR, X-ray diffraction (XRD), ζ-potential, particle size, SEM, TEM] and their effect on bacterial stabilization (viability after freeze-drying/storage, stability when exposed to simulated gastro-intestinal conditions) were assessed. XRD indicated the almost exclusive presence of magnetite crystalline phases. FTIR spectra confirmed the adsorption of pectin on magnetite nanoparticles surface. SEM and TEM images evidenced agglomerated nanoparticles, and a morphological surface change after adsorption of pectin. DLS and ζ-potential results proved the solvation of the ionizable groups in the hydrophilic network which induced chain expansion and agglomeration. Iron from nanoparticles demonstrated to be non-toxic for microorganisms up to 1.00 mg/mL. Simulated saliva and gastric solutions prevented nanoparticles from dissolution. The higher pH of the intestinal conditions (solvated -COO- and Fe-O- groups) facilitated the dispersion and partial dissolution of nanoparticles. Pectins adsorption on magnetite nanoparticles significantly enhanced electrostatic repulsion, which aided the solvation of ionized iron forms. The soluble species diffused out from the aggregates, being detected in the simulated intestinal fluid. Regarding bacterial viability, no decays were observed neither when pectin-decorated nanoparticles were exposed to simulated fluids nor when stored at 4 °C for 60 days. The composites engineered in this work appear as adequate delivery systems for probiotic bacteria, whose target is the gut.

Characterization of Pectins Extracted from Different Varieties of Pink/Red and White Grapefruits [Citrus Paradisi (Macf.)] by Thermal Treatment and Thermosonication.

The physical and chemical properties of pectin extracts obtained from different white and pink/red varieties of grapefruit [Citrus paradisi (Macf.)], using both conventional heating (CHE) and thermosonication (TS), were investigated. The content of galacturonic acid (GalA), degree of esterification (%DM), color and antioxidant capacity were analyzed. Fourier-Transform Infrared Spectroscopy (FTIR) associated with multivariate analysis enabled a structural comparison among the pectin extracts, and differential scanning calorimetry (DSC) completed a full landscape of the investigated extracts. Pectin extracts obtained by CHE showed mostly higher GalA than those obtained by TS. All the extracts had a high antioxidant capacity, as determined by 2,2 diphenyl 1-picrylhydrazyl (DPPH* ) and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS* +) assays, and a high correlation with the GalA content. The main differences observed in the FTIR spectra occurred in the 1200 to 900 cm-1 region (differences in GalA). The glass transition temperatures (Tgs) of all extracts were above 85 °C, making them interesting as stabilizing agents for the food industry. PRACTICAL APPLICATION: A wide database for the characterization of pectin extracts from grapefruits was obtained. The relationship between the extraction method and the source of pectins, with the physicochemical and antioxidant properties provided great support for their application in the food industry.

A Combined Approach of Infrared Spectroscopy and Multivariate Analysis for the Simultaneous Determination of Sugars and Fructans in Strawberry Juices During Storage.

In this work, a Fourier transform mid-infrared spectroscopy (FTIR)-based method was developed for simultaneously quantifying simple sugars and exogenously added fructooligosaccharides (FOS) in strawberry juices preserved for up to 14 d using nonthermal techniques (geraniol and vanillin+ultrasound). The main spectral differences were observed in the 1200 to 900 cm-1 region. The presence of FOS was identified by the typical bands at 1134, 1034, and 935 cm-1 . During storage, a significant decrease of sucrose was concomitant to an increase of glucose and fructose in juices stored without any previous preservation treatment, as determined by high-performance liquid chromatography (HPLC). A principal component analysis was performed on the FTIR spectra corresponding to the different treatments. The groups observed explained more than 94% of the variance and were related to changes in the carbohydrate composition during storage. Then, different partial least square models (PLS) were defined to determine the concentrations of glucose, sucrose, fructose, and those of exogenously added FOS with degrees of polymerization within 3 and 5. The carbohydrates' concentrations determined by HPLC were used as reference method. The models were validated with independent sets of data. The mean of predicted values fitted nicely those obtained by HPLC (correlation and R2  > 0.97), thus supporting the use of the PLS models to monitor the quality of strawberry juices in unknown samples. In conclusion, FTIR spectroscopy appears as an adequate analytical tool to quick assess whether juice formulations meet specifications in terms of authenticity, contamination and/or deterioration. PRACTICAL APPLICATION: FTIR spectroscopy provided a method potentially transferable to the food industry when associated with the multivariate analysis. The robust 21 PLS models defined in this work provided reliable tools for the rapid monitoring of juices' authenticity and/or deterioration. In this regard, FTIR associated to multivariate analysis enabled the determination of different sugars in a single measurement without the need of pure sugars as standards. This experimental simplicity supports the use of FTIR at the production line, and also contributes to save time in determining carbohydrates' composition and stability, in an environmentally friendly way.

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.

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.

Effect of protective agents and previous acclimation on ethanol resistance of frozen and freeze-dried Lactobacillus plantarum strains.

The aim of this work was to study the protective effect of sucrose, trehalose and glutamate during freezing and freeze-drying of three oenological Lactobacillus plantarum strains previously acclimated in the presence of ethanol. The efficiency of protective agents was assessed by analyses of membrane integrity and bacterial cultivability in a synthetic wine after the preservation processes. No significant differences in the cultivability, with respect to the controls cells, were observed after freezing at -80 °C and -20 °C, and pre-acclimated cells were more resistant to freeze-drying than non-acclimated ones. The results of multiparametric flow cytometry showed a significant level of membrane damage after freeze-drying in two of the three strains. The cultivability was determined after incubation in wine-like medium containing 13 or 14% v/v ethanol at 21 °C for 24 h and the results were interpreted using principal component analysis (PCA). Acclimation was the most important factor for preservation, increasing the bacterial resistance to ethanol after freezing and freeze-drying. Freeze-drying was the most drastic method of preservation, followed by freezing at -20 °C. The increase of ethanol concentration from 6 to 10% v/v in the acclimation medium improved the recovery of two of the three strains. In turn, the increase of ethanol content in the synthetic wine led to a dramatic decrease of viable cells in the three strains investigated. The results of this study indicate that a successful inoculation of dehydrated L. plantarum in wine depends not only on the use of protective agents, but also on the cell acclimation process prior to preservation, and on the ethanol content of wine.

Role of S-layer proteins in the biosorption capacity of lead by Lactobacillus kefir.

The role of S-layer proteins (SLP) on the Pb(2+) sequestrant capacity by Lactobacillus kefir CIDCA 8348 and JCM 5818 was investigated. Cultures in the stationary phase were treated with proteinase K. A dot blot assay was carried out to assess the removal of SLP. Strains with and without SLP were exposed to 0-0.5 mM Pb(NO3)2. The maximum binding capacity (q max ) and the affinity coefficient (b) were calculated using the Langmuir equation. The structural effect of Pb(2+) on microorganisms with and without SLP was determined using Raman spectroscopy. The bacterial interaction with Pb(2+) led to a broadening in the phosphate bands (1,300-1,200 cm(-1) region) and strong alterations on amide and carboxylate-related bands (νCOO(-) as and νCOO(-) s). Microorganisms without SLP removed higher percentages of Pb(2+) and had higher q max than those bearing SLP. Isolated SLP had much lower q max and also removed lower percentages of Pb(2+) than the corresponding whole microorganisms. The hydrofobicity of both strains dramatically dropped when removing SLP. When bearing SLP, strains do not expose a large amount of charged groups on their surfaces, thus making less efficient the Pb(2+) removal. On the contrary, the extremely low hydrofobicity of microorganisms without SLP (and consequently, their higher capacity to remove Pb(2+)) can be explained on the basis of a greater exposure of charged chemical groups for the interaction with Pb(2+). The viability of bacteria without SLP was not significantly lower than that of bacteria bearing SLP. However, microorganisms without SLP were more prone to the detrimental effect of Pb(2+), thus suggesting that SLP acts as a protective rather than as a sequestrant layer.

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.

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.

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.