*7.1.1 Enological practices*

In winemaking, the clarification process is fundamental to stabilise and clarify the wine by adding exogenous proteins into wine [169]. Proteins used for fining interact with wine tannins by a mechanism similar to that occurring during the tasting. The interaction protein-tannin, binding, and precipitation determine a decrease in polyphenolic compounds responsible for the sensation of astringency [170]. The SPI was used to evaluate the efficacy of the fining of different proteins at different concentrations in Aglianico [171], and Sangiovese wines [172]. In Aglianico, the gelatin (animal protein) and patatin (plant protein) showed similar efficacy in diminishing wine polyphenols reactive towards salivary proteins, and then astringency, whilst in Sangiovese it depended on the polyphenolic content of the wine. The information provided by SPI was useful to understand that each wine, with peculiar polyphenolic composition, should be treated maintaining the ratio anthocyanins and tannins such as to assure a modulation of astringency and at the same time a correct evolution of the colour during ageing.

A common practice is the utilisation of enological tannins as a substitute for oak barrels to improve colour stability and taste and is authorised by the International Organisation of the Vine and Wine (OIV) for musts and wines clarification [173]. Commercial preparations of tannins of different origins showed different abilities in precipitating salivary proteins: condensed tannins resulted in higher SPI and astringency than hydrolysable tannins. The addition of tannins in wines modify the astringency or not depending on the wine phenolic content. The SPI was useful to understand the effect of tannins addition on wine astringency in order not to compromise overall wine quality [83]. Similarly, after a moderate oxidation (21 mg/L of oxygen equivalent), the addition of 2 g/L of enological tannins did not result in an increase in the reactivity of wine tannins towards salivary proteins after 30 days of treatment. This effect was also shown in the oxidation process in the presence of acetaldehyde [174]. The SPI seems to be sensitive to reaction-products such as polymers of flavanols and anthocyanins formed directly or via a molecular bridge (e.g., acetaldehyde) [31, 175], and new-formed proanthocyanidins [93, 176]. This may explain why during the oxidation of red wines, the SPI followed a different trend from BSA reactive tannins [174, 177, 178].

#### *7.1.2 Ageing*

The decrease of astringency with time has been shown to depend on the reduced concentration of tannins due to precipitation [31, 68], but the trend is not strictly related to the age of wine [179]. The astringency of red wine decreases during ageing because of the changes in the structure of tannins due to cleavage reactions generating low molecular weight species [31], polymerisation without the participation of anthocyanins and subsequent precipitation [95], direct or indirect condensation with anthocyanins [180], and the formation of flavan-3-ol sulfonates by SO2 [181]. Wine becomes soft and mellow for the decline of tannin mean degree of polymerisation [182], velvet and mouth-coating for the formation of the polymeric pigments [24], or satin for lower content of flavans and astringent tannins (measured by SPI), and higher formation of polymers [183] after ageing. Studies on Sangiovese wine revealed that the astringency profile changed from an unripe, dry astringency towards rich, full-body, and mouth-coating sensations after about 2 years of ageing [184]. However, pucker sensations can appear if the oxidation is excessive [24, 25]. Astringency subqualities have been able to discriminate wines of different

**157**

**8. Conclusions**

improve wine quality.

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

conferring positive subqualities of astringency to red wines [162].

Astringency is still a complex phenomenon, and despite the many efforts from researchers, it is not fully understood. However, the different *in vitro* assessments have been shown to be useful in evaluating the wine astringency. They could replace the sensory evaluation when there is no possibility of tasting wines: for low sample availability, when tasting is not permitted (as in the pandemic period due to Covid-19) or unsafe, or when too many samples must be tasted. An analytical method for astringency may be potentially useful not only in research purposes but also in the optimisation of the winemaking process and may help wine producers to

denominations with a chemical age of 3–5 years, more than other wine parameters [25]. Red wine benefits of a moderate oxygenation during ageing favouring changes in tannin structures that, affecting their reactivity towards proteins, can modulate wine astringency. The SPI was utilised to objectively evaluate changes in astringency as a function of oxygen uptake before and after bottling [185]. Although conflicting results were reported for astringency after micro-oxygenation of wines, a significant variation of wine reactivity towards salivary proteins and, then, in wine astringency was observed after 42 months of ageing in bottle only in low pH wines. Moreover, oxygen permeating towards closures determined changes in wine phenolics detectable only using SPI. It was significantly lower when the bottles were sealed with closures at high oxygen transfer rate (OTR). Such differences were not perceived by sensory analysis, demonstrating that SPI can be more sensitive in revealing slight differences in the reactivity of tannins. Lastly, the effect of ageing on the precipitation of salivary proteins is a function of ageing time, wine pH and phenolic composition, and oxygen level in red wine. The decisive role of pH on wine astringency has been confirmed in a recent work of Forino et al. [92], in which the SPI was used to measure wines with different pH levels (3.7–3.2) obtained by adding strong acids or bases, which made the wine unsafe to taste. The binding and precipitation of wine tannins with saliva proteins was favoured at low pH values, and this effect was dominant with respect to the tannins content. Previously, the tartaric acid addition in wine, modifying the pH, resulted in high SPI [186], due to the increase of tannins in the phenolate form, and therefore to an increase of hydrogen bonding with salivary proteins. It is also likely that at low pH increases the accessibility of the binding sites leading to enhanced Van der Waal interactions and hydrogen bonding between proteins and polyphenols [187]. However, other parameters, such as ethanol, fructose, and mannoproteins have been shown to influence astringency and SPI [186]. The effect of mannoproteins on the inhibition of salivary protein precipitation was also showed in Aglianico and Sangiovese wines after 12 months of ageing. The sensory analysis confirmed a reduction in wine astringency. Some mannoproteins interact with tannins forming higher molecular weight structures that prevent the binding with salivary proteins, and thus are not able to elicit astringency [94]. Mannoproteins can also act as steric stabilisers limiting the binding with tannins [112]. Wine polysaccharides inhibit tannin-salivary proteins interaction by a mechanism that involves the formation of protein-tannin complex firstly, probably ruled by hydrophobic interactions and stabilised by hydrogen bonds, and then the polysaccharides can act by a ternary mechanism through the encapsulation of this complex, increasing its solubility. However, the efficiency depends on the polarity of both salivary proteins and tannins [188]. Beyond the molecular mechanism, mannoproteins can highly influence the qualitative sensory perception of astringency,

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

## *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*

the same time a correct evolution of the colour during ageing.

trend from BSA reactive tannins [174, 177, 178].

In winemaking, the clarification process is fundamental to stabilise and clarify the wine by adding exogenous proteins into wine [169]. Proteins used for fining interact with wine tannins by a mechanism similar to that occurring during the tasting. The interaction protein-tannin, binding, and precipitation determine a decrease in polyphenolic compounds responsible for the sensation of astringency [170]. The SPI was used to evaluate the efficacy of the fining of different proteins at different concentrations in Aglianico [171], and Sangiovese wines [172]. In Aglianico, the gelatin (animal protein) and patatin (plant protein) showed similar efficacy in diminishing wine polyphenols reactive towards salivary proteins, and then astringency, whilst in Sangiovese it depended on the polyphenolic content of the wine. The information provided by SPI was useful to understand that each wine, with peculiar polyphenolic composition, should be treated maintaining the ratio anthocyanins and tannins such as to assure a modulation of astringency and at

A common practice is the utilisation of enological tannins as a substitute for oak barrels to improve colour stability and taste and is authorised by the International Organisation of the Vine and Wine (OIV) for musts and wines clarification [173]. Commercial preparations of tannins of different origins showed different abilities in precipitating salivary proteins: condensed tannins resulted in higher SPI and astringency than hydrolysable tannins. The addition of tannins in wines modify the astringency or not depending on the wine phenolic content. The SPI was useful to understand the effect of tannins addition on wine astringency in order not to compromise overall wine quality [83]. Similarly, after a moderate oxidation (21 mg/L of oxygen equivalent), the addition of 2 g/L of enological tannins did not result in an increase in the reactivity of wine tannins towards salivary proteins after 30 days of treatment. This effect was also shown in the oxidation process in the presence of acetaldehyde [174]. The SPI seems to be sensitive to reaction-products such as polymers of flavanols and anthocyanins formed directly or via a molecular bridge (e.g., acetaldehyde) [31, 175], and new-formed proanthocyanidins [93, 176]. This may explain why during the oxidation of red wines, the SPI followed a different

The decrease of astringency with time has been shown to depend on the reduced concentration of tannins due to precipitation [31, 68], but the trend is not strictly related to the age of wine [179]. The astringency of red wine decreases during ageing because of the changes in the structure of tannins due to cleavage reactions generating low molecular weight species [31], polymerisation without the participation of anthocyanins and subsequent precipitation [95], direct or indirect condensation with anthocyanins [180], and the formation of flavan-3-ol sulfonates by SO2 [181]. Wine becomes soft and mellow for the decline of tannin mean degree of polymerisation [182], velvet and mouth-coating for the formation of the polymeric pigments [24], or satin for lower content of flavans and astringent tannins (measured by SPI), and higher formation of polymers [183] after ageing. Studies on Sangiovese wine revealed that the astringency profile changed from an unripe, dry astringency towards rich, full-body, and mouth-coating sensations after about 2 years of ageing

[184]. However, pucker sensations can appear if the oxidation is excessive

[24, 25]. Astringency subqualities have been able to discriminate wines of different

**7.1 Applications of SPI in winemaking**

*7.1.1 Enological practices*

**156**

*7.1.2 Ageing*

denominations with a chemical age of 3–5 years, more than other wine parameters [25]. Red wine benefits of a moderate oxygenation during ageing favouring changes in tannin structures that, affecting their reactivity towards proteins, can modulate wine astringency. The SPI was utilised to objectively evaluate changes in astringency as a function of oxygen uptake before and after bottling [185]. Although conflicting results were reported for astringency after micro-oxygenation of wines, a significant variation of wine reactivity towards salivary proteins and, then, in wine astringency was observed after 42 months of ageing in bottle only in low pH wines. Moreover, oxygen permeating towards closures determined changes in wine phenolics detectable only using SPI. It was significantly lower when the bottles were sealed with closures at high oxygen transfer rate (OTR). Such differences were not perceived by sensory analysis, demonstrating that SPI can be more sensitive in revealing slight differences in the reactivity of tannins. Lastly, the effect of ageing on the precipitation of salivary proteins is a function of ageing time, wine pH and phenolic composition, and oxygen level in red wine. The decisive role of pH on wine astringency has been confirmed in a recent work of Forino et al. [92], in which the SPI was used to measure wines with different pH levels (3.7–3.2) obtained by adding strong acids or bases, which made the wine unsafe to taste. The binding and precipitation of wine tannins with saliva proteins was favoured at low pH values, and this effect was dominant with respect to the tannins content. Previously, the tartaric acid addition in wine, modifying the pH, resulted in high SPI [186], due to the increase of tannins in the phenolate form, and therefore to an increase of hydrogen bonding with salivary proteins. It is also likely that at low pH increases the accessibility of the binding sites leading to enhanced Van der Waal interactions and hydrogen bonding between proteins and polyphenols [187]. However, other parameters, such as ethanol, fructose, and mannoproteins have been shown to influence astringency and SPI [186]. The effect of mannoproteins on the inhibition of salivary protein precipitation was also showed in Aglianico and Sangiovese wines after 12 months of ageing. The sensory analysis confirmed a reduction in wine astringency. Some mannoproteins interact with tannins forming higher molecular weight structures that prevent the binding with salivary proteins, and thus are not able to elicit astringency [94]. Mannoproteins can also act as steric stabilisers limiting the binding with tannins [112]. Wine polysaccharides inhibit tannin-salivary proteins interaction by a mechanism that involves the formation of protein-tannin complex firstly, probably ruled by hydrophobic interactions and stabilised by hydrogen bonds, and then the polysaccharides can act by a ternary mechanism through the encapsulation of this complex, increasing its solubility. However, the efficiency depends on the polarity of both salivary proteins and tannins [188]. Beyond the molecular mechanism, mannoproteins can highly influence the qualitative sensory perception of astringency, conferring positive subqualities of astringency to red wines [162].
