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

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

enzymes spectrophotometrically [123].

**6.2 Precipitation assays: pros and contras**

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

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].

Protein precipitation assays are of particular interest because the interaction of proteins with tannins can be used to model astringency perception [130]. The ability of gelatin to precipitate phenols, including tannins, has been observed since 1934 [131]. The same phenomenon was observed when hide powder or polyvinylpyrrolidone were used in high concentrations [132]. Bate-Smith [130] noted that protein of skin differed from proteins of saliva, which caused the "puckery" sensation induced by tannin. For measuring the relative astringency of tannins, a spectrophotometric technique based on the precipitation of the haemoglobin with tannin was then introduced [130]. Similarly, another spectrophotometric technique measured the inhibition of β-glucosidase after the precipitation with tannic acid and condensed tannins [133]. Alternatively, Hagerman and Butler [134] used bovine serum albumin (BSA) as a precipitant agent, which was successively taken by Harbertson et al. [135] for wine analysis. Glories [136] proposed the gelatin index, in which tannins were precipitated by gelatin protein. This procedure required the measure of proanthocyanidin concentration before and after precipitation with an excess of gelatin. Besides, gelatin is a heterogeneous mixture of proteins, and its composition may change amongst the different commercial products, leading to a source of variability and imprecision of data. For this, some researchers replaced gelatin with ovalbumin [137]. Another tannin assay used the methylcellulose to precipitate tannin (MCP) [138, 139]. The MCP tannin assay is based on the formation of an insoluble polymer-tannin complex, which can be separated by centrifugation. The total phenolic content (absorbance at 280 nm) is measured in control and treated samples. However, if the assays utilise synthetic agent or protein different from saliva, the binding reaction seems not to reproduce the physiological conditions during the wine tasting, because the binding affinity of the protein is not comparable to that of salivary protein. In the case of bovine serum albumin, it has been shown that the salivary protein has a higher affinity for tannin than BSA. In fact, in the presence of an excess of BSA, the tannin preferentially bound the salivary protein. Other proteins, including dietary proteins, may not complex any tannin in the presence of the salivary tannin-binding protein [8]. The use of salivary proteins has been proposed to represent the model system for astringency better. In precipitation

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assays, fractionated [8, 140] or whole [141, 142] human saliva has been used. Mixing whole saliva and grape polyphenols give rise to a "soft cloudy" precipitate, which gathered after centrifugation on the bottom of the tube so that the supernatant was easily recovered without disturbing this pellet. The binding reaction was performed at 25°C, and the complex formed was successively precipitated by centrifugation at 4°C in order to stop further reactions. The induced precipitation allowed to separate the proteins bound to polyphenols from whose remained in the solution that not reacted with them. Both the nature of condensed tannin [141] and salivary proteins [142] involved in the precipitation were analysed. In both works, the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of human saliva was carried out, Sarni-Manchado et al. [141], analysed the tannins in the supernatant and pellet. In contrast, Gambuti et al. [142], analysing the supernatant, revealed the proteins mainly reactive with polyphenols by comparison with the control saliva. Evidence of the qualitative and quantitative changes in salivary protein profile after tasting tannin solutions and wines was also made by HPLC [143]. Interactions and precipitation of low molecular weight salivary proteins with procyanidins confirmed the involvement of different families of salivary proteins in the development of astringency [144]. The use of salivary proteins involves the collection of human saliva from different healthy volunteers according to a specific protocol, and it must take into account the salivary flow to limit the effect of individual differences in astringency perception due to subjects' saliva characteristics [145].
