**6.2 Precipitation assays: pros and contras**

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

**153**

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

astringency perception due to subjects' saliva characteristics [145].

Nephelometry is a method that allows a direct estimation of the amount of protein/tannin complexes by measuring the scattered light in the solution that results from the gradual formation of a cloudy precipitate corresponding to the soluble aggregate. Chapon [146] proposed this technique by studying the interactions between beer polyphenols and proteins involved in the colloidal instability of beer. Similarly, the haze formed between salivary proteins and polyphenols represents the first step in the development of astringency and can be measured with a turbidimeter [147, 148]. A continuous flow method was also used to study the interactions between grape extracts and wine with BSA at different concentrations [149]. Globular proteins and PRPs were used to measure a relative tannin specific activity of procyanidin oligomers from grape seeds [30], and PRPs showed the strongest affinity. Human salivary proteins have been considered as the most suitable model proteins. For this reason, in turbidity measurement, whole human saliva [148] and mucin, a high molecular weight salivary protein [150], were used as model proteins for astringency assessment. Based on polyphenol/mucin reactivity, a micro-plate assay was also developed [151]. Tannic acid [150], grape seed extracts [151], wine extracts [63], tannin fractions added to model solutions [152] were analysed by nephelometry. The turbidity of the solution, formed by the tannin-protein aggregates, linearly correlated with astringency. However, no direct analysis of wines was carried out. Lastly, instead, wine samples were analysed trough nanotechnology such as localised surface plasmon resonance (LSPR) combined with surface imprinted polymers, as a measure of the interactions of polyphenol with salivary protein and then astringency [153].

The sensory analysis represents the human response to wine tasting. A sensory

panel can provide information about the sensory properties of a product, but significant training is required before the panel becomes a reliable sensory instrument. Astringency is a difficult sensory attribute to evaluate, owing to particular

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

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

**6.3 Nephelometry: pros and contras**

**6.4 Sensory analysis: pros and contras**
