**5. Polyphenol-protein interactions**

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 binding involved in the astringent sensation.

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.

**151**

*Salivary Protein-Tannin Interaction: The Binding behind Astringency*

The stability of these complexes depends on the tannin dimension and number of free phenolic groups, as well as the nature of the protein involved [81, 109].

The driving factors that determine the binding between tannins and salivary proteins were identified to be the critical micelle concentration value (CMC), tannin structure preferences, and tannin colloidal state [113]. Below the values observed in wine (from 1.5 to 2.9 mM), procyanidins specifically interacted with peptide through hydrophilic recognition. A network of interactions can be formed depending on tannin conformation, and precipitation of the complex can occur, or if an intramolecular staking Π-Π of phenolic groups is preferred, the precipitation is not observed. Above these values, tannins spontaneously tend to form aggregates that, at first through specific interactions bind proteins, and then surrounded by the hydrophobic residues, stabilise the complex by hydrophobic bonding. To summarise, both hydrophilic and hydrophobic interactions contribute to form a complex network, which determines the precipitation of salivary proteins with tannins.

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

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

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],

*DOI: http://dx.doi.org/10.5772/intechopen.93611*

**6. Assessments of astringency**

the real phenomenon of wine tasting.

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

relatively simple method.

### *Salivary Protein-Tannin Interaction: The Binding behind Astringency DOI: http://dx.doi.org/10.5772/intechopen.93611*

The stability of these complexes depends on the tannin dimension and number of free phenolic groups, as well as the nature of the protein involved [81, 109].

The driving factors that determine the binding between tannins and salivary proteins were identified to be the critical micelle concentration value (CMC), tannin structure preferences, and tannin colloidal state [113]. Below the values observed in wine (from 1.5 to 2.9 mM), procyanidins specifically interacted with peptide through hydrophilic recognition. A network of interactions can be formed depending on tannin conformation, and precipitation of the complex can occur, or if an intramolecular staking Π-Π of phenolic groups is preferred, the precipitation is not observed. Above these values, tannins spontaneously tend to form aggregates that, at first through specific interactions bind proteins, and then surrounded by the hydrophobic residues, stabilise the complex by hydrophobic bonding. To summarise, both hydrophilic and hydrophobic interactions contribute to form a complex network, which determines the precipitation of salivary proteins with tannins.
