**6. Assessments of astringency**

*Chemistry and Biochemistry of Winemaking, Wine Stabilization and Aging*

or even flavanols structures, similarly to procyanidins [98].

34 mg/L, depending on the cultivar [101].

**5. Polyphenol-protein interactions**

binding involved in the astringent sensation.

proteins forming soluble aggregates [96], and even precipitates, being the cinnamoylated the most reactive fraction (precipitation between 6.5 and 17.5%), also influencing the astringency perception [97]. Pyranoanthocyanins, anthocyaninderived pigments that can form during red wine ageing, seems to be involved in astringency, since they are able to interact with salivary proteins by phenol, catechol

Flavonols (kaempferol, quercetin, and myricetin) are present in grapes and wine as glycosides (sugar attached). In the plant, they act as a natural sunscreen in the skin of grape berries. In wine, they can be hydrolysed and act as cofactors for colour enhancement. Flavonol glycosides, such as 3-O-glucosides and 3-O-galactosides of quercetin, syringetin, and isorhamnetin, have been reported to be astringent at low detection threshold levels and characterised by a velvety astringency [99]. The addition of quercetin 3-O-glucoside (2 g/L) to wine increased astringency, leading to the formation of complexes with saliva at 200 μM [100]. However, such concentrations are not naturally present in red wine, in which quercetin 3-O-glucoside can range from 2 up to

Many sensory active non-volatile compounds comprising hydroxybenzoic acids, hydroxycinnamic acids, flavon-3-ol glycosides, and dihydroflavon-3-ol rhamnosides were identified as the key inducers of the astringent mouthfeel of red wines using a molecular sensory approach [99]. The phenolic acids in wines, especially hydroxycinnamic and benzoic acid derivatives, have been reported to be more puckering astringent. These compounds have also been correlated with astringency in free-run and pressed wine [102]. The trans-*p*-coumaric, cis-aconitic, and transcaftaric acids seem to participate in the astringency of Spanish wines [103].

Given that the carbonyl function of salivary proteins is a very effective hydrogen bond acceptor [104], it would appear that it would play a significant role in bonding to polyphenols hydroxyls [10, 105]. Nowadays, the interaction between proteins and proanthocyanidins is widely recognised to be a combination of hydrogen bonding and hydrophobic effects in the acidic wine matrix. However, covalent bonding is also possible between proteins and polyphenols during oxidation [106] and nucleophilic addition processes [107]. In this chapter, we focused on the non-covalent

Physico-chemical quantities (binding constants, stoichiometry, and atomic structure of complexes, driving forces for the association) have been utilised to understand the multifaceted sensation of astringency. Many techniques including circular dichroism (CD) [108], isothermal titration microcalorimetry (ITC) [109], fluorescence spectroscopy [50], dynamic light scattering (DLS) [110], and nuclear magnetic resonance (NMR) [111] have been employed to understand the formation mechanism of protein/polyphenol aggregates in solution. Generally, these studies focused on interactions between protein segment from human saliva PRPs proteins family and selected procyanidins, because it represents the easiest way to simulate such a complex phenomenon. They can reveal the hydrophobic interactions formed between the phenolic rings of the procyanidins and proline residues, and the hydrogen bonding between the hydroxyl groups on the phenolic B-ring and hydrogen acceptor sites of the peptide bond [52, 112]. The aggregation of procyanidin with peptide seems to be firstly mediated by hydrophobic forces, and then hydrogen bonding has been postulated to provide directional and robust bonding that stabilises the complex. The peptide is coated by polyphenols, which provides a crosslink between two or more peptides up to a critical point, after which precipitation begins.

**150**

A method for measuring astringency remains one of the great analytical challenges in wine chemistry and oenology. The interest in investigating the mechanisms and interactions between polyphenols and proteins can allow us to find the optimal way to simulate and evaluate what happens during the red wine tasting. Quite often, sophisticated techniques rely on the purification of both tannin and protein fractions, the extrusion from the wine content, and the omission of matrix components during reactions, and all contribute to send away astringency from the reality that is: wine polyphenols interacting with salivary proteins in mouth, causing drying sensations.

Several procedures have been carried out during the last decades for measuring tannins. Additionally, analyses of soluble (turbidimetric analysis) and insoluble (precipitation protein assays) protein-polyphenols complex have been developed for assessing astringency. The sensory analysis represents the human response as an analytical tool to evaluate wine perception. Many training and tasting sections are necessary over a long period involving a high number of tasters to form a reliable panel. In the case of astringency, it is complicated to discern amongst tastes and brings on fatigue. A method capable of estimating tannin palatability has to be the most objective as possible and must correlate with sensory data in order to reflect the real phenomenon of wine tasting.
