Optimization of a microplate-based assay to assess esterase activity in the alga Pseudokirchneriella subcapitata

The present work describes the optimization of a short-term assay, based on the inhibition of the esterase activity of the alga Pseudokirchneriella subcapitata, in a microplate format. The optimization of the staining procedure showed that the incubation of the algal cells with 20 μmolL−1 fluorescein diacetate (FDA) for 40 min allowed discrimination between metabolic active and inactive cells. The shortterm assay was tested using Cu as toxicant. For this purpose, algal cells, in the exponential or stationary phase of growth, were exposed to the heavy metal in growing conditions. After 3 or 6 h, cells were subsequently stained with FDA, using the optimized procedure. For Cu, the 3- and 6-h EC50 values, based on the inhibition of the esterase activity of algal cells in the exponential phase of growth, were 209 and 130 μg L−1, respectively. P. subcapitata cells, in the stationary phase of growth, displayed higher effective concentration values than those observed in the exponential phase. The 3- and 6-h EC50 values for Cu, for cells in the stationary phase, were 443 and 268 μgL−1, respectively. This short-term microplate assay showed to be a rapid endpoint for testing toxicity using the alga P. subcapitata. The small volume required, the simplicity of the assay (no washing steps), and the automatic reading of the fluorescence make the assay particularly well suited for the evaluation of the toxicity of a high number of environmental samples.

Protein–polyphenol interaction on silica beads for astringency tests based on eye, photography or reflectance detection modes

Astringency is an organoleptic property of beverages and food products resulting mainly from the interaction of salivary proteins with dietary polyphenols. It is of great importance to consumers, but the only effective way of measuring it involves trained sensorial panellists, providing subjective and expensive responses. Concurrent chemical evaluations try to screen food astringency, by means of polyphenol and protein precipitation procedures, but these are far from the real human astringency sensation where not all polyphenol–protein interactions lead to the occurrence of precipitate. Here, a novel chemical approach that tries to mimic protein–polyphenol interactions in the mouth is presented to evaluate astringency. A protein, acting as a salivary protein, is attached to a solid support to which the polyphenol binds (just as happens when drinking wine), with subsequent colour alteration that is fully independent from the occurrence of precipitate. Employing this simple concept, Bovine Serum Albumin (BSA) was selected as the model salivary protein and used to cover the surface of silica beads. Tannic Acid (TA), employed as the model polyphenol, was allowed to interact with the BSA on the silica support and its adsorption to the protein was detected by reaction with Fe(III) and subsequent colour development. Quantitative data of TA in the samples were extracted by colorimetric or reflectance studies over the solid materials. The analysis was done by taking a regular picture with a digital camera, opening the image file in common software and extracting the colour coordinates from HSL (Hue, Saturation, Lightness) and RGB (Red, Green, Blue) colour model systems; linear ranges were observed from 10.6 to 106.0 μmol L−1. The latter was based on the Kubelka–Munk response, showing a linear gain with concentrations from 0.3 to 10.5 μmol L−1. In either of these two approaches, semi-quantitative estimation of TA was enabled by direct eye comparison. The correlation between the levels of adsorbed TA and the astringency of beverages was tested by using the assay to check the astringency of wines and comparing these to the response of sensorial panellists. Results of the two methods correlated well. The proposed sensor has significant potential as a robust tool for the quantitative/semi-quantitative evaluation of astringency in wine.