Ana C.A. Veloso

Separation and quantification of the major casein fractions by reverse-phase high-performance liquid chromatography and urea-polyacrylamide gel electrophoresis: Detection of milk adulterations

The separation and quantification of bovine kappa-, alpha- and beta-caseins by HPLC-UV using an RP column which contained polystyrene-divinilbenzene copolymer based packing was optimized and validated. Gradient elution was carried out at a flow-rate of 1 ml/min and a temperature of 46 degrees C, using a mixture of two solvents. Solvent A was 0.1% trifluoroacetic acid in water and solvent B was acetonitrile-water-trifluoroacetic acid (95:5:0.1). The effluent was monitored by a UV detector at 280 nm. The determinations were performed in the linear range of 0.038-0.377 mg/ml for kappa-casein, 0.188-1.883 mg/ml for alpha-casein and 0.151-1.506 mg/ml for beta-casein. The detection limits were 0.006, 0.019 and 0.015 mg/ml for kappa-casein, alpha-casein and beta-casein, respectively. The validity of the method was verified. The recoveries ranged from 91 to 100% for bovine milk. The precision of the method was also evaluated, the RSD being less than 3.67%. The same HPLC procedure was used for the separation of caprine and ovine caseins. Different chromatographic profiles were obtained for bovine, ovine and caprine milks, although it was only possible to detect and quantify additions of 5% or more of bovine milk to caprine milk. With respect to detection of milk adulterations, electrophoresis using urea-polyacrylamide gel electrophoresis (PAGE) analysis was more sensitive. The evolution of casein proteolysis in cheeses made from bovine milk and cheeses made from ovine milk, during 30 days of ripening was followed by HPLC-UV and urea-PAGE methodologies. The results obtained by these techniques were similar.

Single-cultivar extra virgin olive oil classification using a potentiometric electronic tongue

Label authentication of monovarietal extra virgin olive oils is of great importance. A novel approach based on a potentiometric electronic tongue is proposed to classify oils obtained from single olive cultivars (Portuguese cvs. Cobrançosa, Madural, Verdeal Transmontana; Spanish cvs. Arbequina, Hojiblanca, Picual). A meta-heuristic simulated annealing algorithm was applied to select the most informative sets of sensors to establish predictive linear discriminant models. Olive oils were correctly classified according to olive cultivar (sensitivities greater than 97%) and each Spanish olive oil was satisfactorily discriminated from the Portuguese ones with the exception of cv. Arbequina (sensitivities from 61% to 98%). Also, the discriminant ability was related to the polar compounds contents of olive oils and so, indirectly, with organoleptic properties like bitterness, astringency or pungency. Therefore the proposed E-tongue can be foreseen as a useful auxiliary tool for trained sensory panels for the classification of monovarietal extra virgin olive oils.

Monitoring of fed-batch E. coli fermentations with software sensors

Accurate monitoring and control of industrial bioprocess requires the knowledge of a great number of variables, being some of them not measurable with standard devices. To overcome this difficulty, software sensors can be used for on-line estimation of those variables and, therefore, its development is of paramount importance. An Asymptotic Observer was used for monitoring Escherichia coli fed-batch fermentations. Its performance was evaluated using simulated and experimental data. The results obtained showed that the observer was able to predict the biomass concentration profiles showing, however, less satisfactory results regarding the estimation of glucose and acetate concentrations. In comparison with the results obtained with an Extended Kalman Observer, the performance of the Asymptotic Observer in the fermentation monitoring was slightly better.

Sensory intensity assessment of olive oils using an electronic tongue

Olive oils may be commercialized as intense, medium or light, according to the intensity perception of fruitiness, bitterness and pungency attributes, assessed by a sensory panel. In this work, the capability of an electronic tongue to correctly classify olive oils according to the sensory intensity perception levels was evaluated. Cross-sensitivity and non-specific lipid polymeric membranes were used as sensors. The sensor device was firstly tested using quinine monohydrochloride standard solutions. Mean sensitivities of 14±2 to 25±6 mV/decade, depending on the type of plasticizer used in the lipid membranes, were obtained showing the device capability for evaluating bitterness. Then, linear discriminant models based on sub-sets of sensors, selected by a meta-heuristic simulated annealing algorithm, were established enabling to correctly classify 91% of olive oils according to their intensity sensory grade (leave-one-out cross-validation procedure). This capability was further evaluated using a repeated K-fold cross-validation procedure, showing that the electronic tongue allowed an average correct classification of 80% of the olive oils used for internal-validation. So, the electronic tongue can be seen as a taste sensor, allowing differentiating olive oils with different sensory intensities, and could be used as a preliminary, complementary and practical tool for panelists during olive oil sensory analysis.

Detecção de adulterações em produtos alimentares contendo leite e/ou proteínas lácteas

A critical review of the most relevant analytical methodologies for quality and authenticity control of dairy products and foods containing milk proteins is presented. Chromatographic, electrophoretic and immunological methods are used for: detection of cows milk in ewe and goat milks, detection of whey added to milk, detection of caseins and/or whey proteins in non-lactic foods and study compounds resulting from milk proteins degradation. Techniques based on polimerase chain reaction are also suitable for detection of cows milk on cheeses of ewe and goat milks.

Practical procedure for discriminating monofloral honey with a broad pollen profile variability using an electronic tongue

Colour and floral origin are key parameters that may influence the honey market. Monofloral light honey are more demanded by consumers, mainly due to their flavour, being more valuable for producers due to their higher price when compared to darker honey. The latter usually have a high anti-oxidant content that increases their healthy potential. This work showed that it is possible to correctly classify monofloral honey with a high variability in floral origin with a potentiometric electronic tongue after making a preliminary selection of honey according their colours: white, amber and dark honey. The results showed that the device had a very satisfactory sensitivity towards floral origin (Castanea sp., Echium sp., Erica sp., Lavandula sp., Prunus sp. and Rubus sp.), allowing a leave-one-out cross validation correct classification of 100%. Therefore, the E-tongue shows potential to be used at analytical laboratory level for honey samples classification according to market and quality parameters, as a practical tool for ensuring monofloral honey authenticity.

Evaluation of healthy and sensory indexes of sweetened beverages using an electronic tongue

Overconsumption of sugar-sweetened beverages may increase the risk of health problems and so, the evaluation of their glycemic load and fructose-intolerance level is essential since it may allow establishing possible relations between physiologic effects of sugar-rich beverages and health. In this work, an electronic tongue was used to accurately classify beverages according to glycemic load (low, medium or high load) as well to their adequacy for people suffering from fructose malabsorption syndrome (tolerable or not): 100% of correct classifications (leave-one-out cross-validation) using linear discriminant models based on potentiomentric signals selected by a meta-heuristic simulated annealing algorithm. These results may be partially explained by the electronic tongues capability to mimic the human sweetness perception and total acid flavor of beverages, which can be related with glycemic load and fructose-intolerance index. Finally, the E-tongue was also applied to quantify, accurately, healthy and sensory indexes using multiple linear regression models (leave-one-out cross-validation: Radj>0.99) in the following dynamic ranges: 4.7<glycemic load≤30; 0.4<fructose intolerance index≤1.5; 32<sweetness perception<155; 1.3<total acid flavor, gL(-1)<8.3; and, 5.8<well-balanced flavor≤74. So, the proposed electronic tongue could be used as a practical, fast, low-cost and green tool for beverages healthy and sensory evaluation.

Evolutionary algorithms for static and dynamic optimization of fed-batch fermentation processes

In this work, Evolutionary Algorithms (EAs) are used to control a recombinant bacterial fed-batch fermentation process, that aims to produce a bio-pharmaceutical product. Initially, a novel EA was used to optimize the process, prior to its run, by simultaneously adjusting the feeding trajectory, the duration of the fermentation and the initial conditions of the process. Finally, dynamic optimization is proposed, where the EA is running simultaneously with the fermentation process, receiving information regarding the process, updating its internal model and reaching new solutions that will be used for online control.

Tau Mislocation in Glucocorticoid-Triggered Hippocampal Pathology

The exposure to high glucocorticoids (GC) triggers neuronal atrophy and cognitive deficits, but the exact cellular mechanisms underlying the GC-associated dendritic remodeling and spine loss are still poorly understood. Previous studies have implicated sustained GC elevations in neurodegenerative mechanisms through GC-evoked hyperphosphorylation of the cytoskeletal protein Tau while Tau mislocation has recently been proposed as relevant in Alzheimer’s disease (AD) pathology. In light of the dual cytoplasmic and synaptic role of Tau, this study monitored the impact of prolonged GC treatment on Tau intracellular localization and its phosphorylation status in different cellular compartments. We demonstrate, both by biochemical and ultrastructural analysis, that GC administration led to cytosolic and dendritic Tau accumulation in rat hippocampus, and triggered Tau hyperphosphorylation in epitopes related to its malfunction (Ser396/404) and cytoskeletal pathology (e.g., Thr231 and Ser262). In addition, we show, for the first time, that chronic GC administration also increased Tau levels in synaptic compartment; however, at the synapse, there was an increase in phosphorylation of Ser396/404, but a decrease of Thr231. These GC-triggered Tau changes were paralleled by reduced levels of synaptic scaffolding proteins such as PSD-95 and Shank proteins as well as reduced dendritic branching and spine loss. These in vivo findings add to our limited knowledge about the underlying mechanisms of GC-evoked synaptic atrophy and neuronal disconnection implicating Tau missorting in mechanism(s) of synaptic damage, beyond AD pathology.

Assessment of physiological conditions in e. Coli fermentations by epifluorescent microscopy and image analysis

The development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Escherichia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both protein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fermentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes.