**6.1** *Stimuli* **analysis: pros and contras**

Amongst *stimuli* able to elicit astringency, tannins are the main compounds responsible for this sensation. Tannins are intrinsically amphiphilic molecules with high reactivity, have a diverse range of structures, and are often found in matrices with other phenolic molecules containing similar functional groups. Besides using sophisticated equipment and analytical techniques, there is also a great interest in a relatively simple method.

In the past, many colourimetric techniques were developed to analyse phenolics compounds spectrophotometrically. The first one used the Folin-Denis reagent [114], which was successively modified [115, 116], and lastly into the Folin-Ciocalteau assay [117]. However, they were not specific for tannins but detected any phenolic compound. More specific colour reactions were used to measure condensed tannins and their precursors. Depolymerisation in HCl and n-butanol of proanthocyanidins yield anthocyanidins that can be quantified spectrophotometrically [118, 119]. Others used vanillin reagent for flavanols [6, 120],

or *p*-dimethylaminocinnamaldehyde for a more specificity and colour stability [121, 122]. Only the flavonoid-based condensed tannins can be detected with these reagents. As tannins can inhibit the catalytic activity of enzymes [6], many methods used the interaction with proteins in solution to measure the inhibition of different enzymes spectrophotometrically [123].

Other methods, based on the acid-catalysed condensation reactions with benzyl mercaptan (thiolysis) and phloroglucinol (phloroglucinolysis), can determine both the chain length (mDP) and composition by HPLC [124, 125]. Most of our current knowledge about the general composition and structure of grape and wine tannins have been obtained by depolymerisation [126]. Poor yields due to reaction product instability, reactions with non-proanthocyanidin compounds, and side reactions also contribute negatively to the utility of thiolytic methods [124]. The problem with phloroglucinolysis, on the other hand, is that it produces low yields, and only a fraction of the tannin is converted to known flavan-3-ol products [127]. Normalphase HPLC (NP-HPLC) method has also been developed to quantify the proanthocyanidins into low and high molecular-weight polymers [128]. A simple method based on Fourier transform mid-infrared (FT-MIR) spectroscopy combined with multivariate data analysis, was successfully used to measure the tannin concentration of 86 red wines, previously purified by solid-phase extraction (SPE) [129].
