Astringency and Colour of Wine: Role, Significance, Mechanism and Methods of Evaluation

M. Teresa Escribano-Bailon[1], Alba M. Ramos-Pineda and Ignacio Garria-Estevez

Polyphenols Research Group, Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Pharmacy, University of Salamanca, Campus Miguel de Unamuno, E-37007, Salamanca, Spain

1. Introduction

Alcoholic beverages including wines and brandies have been consumed and enjoyed by the mankind for centuries (Joshi et ah, 2011). Wine is the fermented juice of the grape. Fruit wines, derived from fruits other than grapes, include cider from apple, репу from pears, plum wine, cheny wine and others from various benies (Joshi et ah, 1999; Kosseva et al., 2017). With the advances in the technology, horticultur al practices or newer sources of wine, brandy or other exotic drinks are developed, testing of their acceptance by consumers has become very important (Narasimhan and Stephen, 2011). Consumer acceptance is defined as the reaction of the user to a product determined by personal factors as age, like, dislike, familiarity, economics etc. This is an “affective” response and the major focus is on a population, its behaviour/response. Sensory' analysis on the other hand, is testing of product under bias free conditions, using human beings as tools.

Sensory evaluation is a scientific discipline used to evoke, measure, analyze and interpret the reactions to characteristics of food/beverage or materials as perceived by senses of sight, smell, taste, touch and hearing (Narasimhan and Rajalakshmi, 1999). The measurements are not simple physical quantities but involved interpretations of the sensations evoked and perceived at a psychological level, in response to various physical and chemical stimuli. It is certainly different from casual examination of products.

In evaluation of wine, colour and astringency are very important characteristics. The acceptance of a quality wine depends greatly on how its colour- is perceived or liked while the astringency influences the acceptance of the taste of a wine. Regarding astringency, recent findings on the molecular mechanisms of astringency and the instrumental analysis performed to assess the astringency from a molecular point of view are discussed. As for colour, the available oenological tools to enhance colour extraction and to improve wine colour- stability are also addressed. The consequences of global climate change have several environmental impacts including the warming of the Northern Hemisphere and the alteration of the rainfall patterns. These changes are expected to have high impact in the viticulture and oenology of the Southern European wine-producing regions. Indeed, the effects have already been observed especially in warm vintages: an important gap between the technological maturity (coming earlier) and the phenolic and aromatic maturity (which come later), resulting in wines with unbalanced astringency or with irregular, poor or unstable colour. This chapter is focused on the astringency and colour of red wine with the aim to understand these two sensory properties.

2. Astringency

Astringency has been defined as “the complex of sensations due to shrinking, drawing or puckering of the epithelium as a result of exposure to substances such as alums or tannins” by the America Society for Testing Materials (ASTM, 2004). Although the bases of the astringency mechanism are not well understood yet, it is known to be engendered by different classes of astringent compounds, including salt of multivalent metallic cations (particularly aluminum salts), dehydrating agents (ethanol and acetone), mineral and organic acids and polyphenols (Bajec and Pickering, 2008; Joslyn and Goldstein, 1964).

2.1. Mechanisms for Astringency

The word astringency is derived from the Latin word, ad stringent, meaning ‘to bind’, showing the basis of this primary chemical process. The first mechanism for astringency was proposed by Bate-Smith (1954) indicating the main reaction whereby astringency develops is via the precipitation of proteins and mucopolysaccharides in the mucous secretions (Bate-Smith, 1973). This view is still broadly accepted, although widely different opinions exist around the astringency process.

Lee and Lawless (1991) proposed that astringency can be broken down into multiple sub-qualities. Their data suggested that the tactile attributes of diying and roughing were the most closely associated with astringency, implying changes in the texture of the oral mucosa. Green (1993) also pointed to a tactile origin of the astringency sensation, mainly caused by the precipitation of salivary proteins and possibly cross-linking of proteins in the mucosa. Since some wine polyphenols are able to bind salivary proteins, they can form insoluble tannin-protein precipitates in the mouth, causing a loss of lubrication and increased friction in the oral cavity, which would explain its astringency (Baxter et al, 1997). The most accepted mechanisms to explain these facts was proposed by Siebert et al. (1996). Regarding this mechanism (Fig. 1), a protein has a fixed number of sites to which tannin can bind while each polyphenol also has fixed number of binding sites. When the number of binding sites in the polyphenol equals the number of binding sites in the protein, the largest network and the maximum protein precipitation will be produced. Depending on the ratio of protein or tannin used, different protein-polyphenol complexes will be formed (Soares et al., 2012).

Moreover, the interaction process between polyphenols and peptides has been divided into three stages (Charlton et al, 2002). Initially, reversible associations between the hydrophobic face of the aromatic rings of the polyphenol and the pyrrolidine ring of the proline residues of the protein give a soluble complex. In general, several molecules can bind to the same peptide. In the second stage, two peptides are cross-linked by the addition of more polyphenols, which can bind to the peptide acting as a linker between two peptides, by cooperative weak intennolecular binding interactions, leading to a bigger and insoluble complex which starts to precipitate. Finally, the complex aggregates into larger or smaller

Model for protein/polyphenol interaction (Adapted from Siebert el al., 1996) particles, seen as a phase separation process. The 3-stage model was later confirmed and expanded by Jobstl et al. (2004)

Figure 1. Model for protein/polyphenol interaction (Adapted from Siebert el al., 1996) particles, seen as a phase separation process. The 3-stage model was later confirmed and expanded by Jobstl et al. (2004).

Protein-polyphenol aggregates have been described as both soluble and insoluble, and its stability depends on several variables like protein: polyphenol ratios, pH, temperature and ionic strength of solution, but also on the types of polyphenol and protein used (Bajec and Pickering, 2008).Furthermore, the existence of synergistic effect between phenolic compounds has also recently been suggested. It has been proposed that it could be due to a cooperative behaviour between phenolic compounds when binding proteins, and it could explain why astringency of wine is more influenced by the qualitative phenolic composition than by the total concentration (Ferrer-Gallego et al., 2014; Ramos-Pineda et al., 2017). Even more, the existence of a synergistic effect of the coexistence of different salivary protein families on the interaction with wine flavanols has been recently proposed (Ramos-Pineda et al., 2019).

Although precipitation of salivary proteins, namely PRPs (proline-rich proteins), is one of the most accepted mechanisms, not all astringents cause salivary protein precipitation, evidencing there must be other mechanisms implicated in astringency development. For instance, Lee and Vickers (2012) studied the influence of loss of salivary lubricity on the development of the astringency sensation. They determined both sensory and instmmentally if different astringent compounds could induce changes in saliva lubricity, evincing that precipitation of PRPs or mucins is not a requirement to the development of astringency. Considering these results, they proposed that changes in friction or lubricity are not necessary conditions for astringency and they stipulated that direct tissue effects are related with tannin astringency while acid astringency could be related with coating disruption (Lee et al., 2012).

Other approaches have suggested that astringency could be engendered by activation of specific taste receptors (Tachibana et al., 2004) or even by direct interactions between tannins and oral epithelial cells (Payne et al., 2009). In this study, a cell-based assay was developed with which demonstrated that interactions between grape seed tannins occur (Payne et al., 2009). Gibbins and Carpenter (2013) proposed that the complex sensation of astringency could involve multiple mechanisms occurring simultaneously (Fig. 2): aggregation of salivary proteins, salivary film disruption, decrease in salivary lubrication, receptors exposure and mechanoreceptors stimulation in the oral mucosal epithelium.

Proposed astringency mechanisms

Figure 2. Proposed astringency mechanisms: (A) Protein aggregation and complex formation by the interaction between polyphenols and salivary proteins; (B) Astringency development: (I) Free polyphenols and soluble polyphenol-protein aggregates could disrupt the salivary film and reach the pellicle or even activate specific taste receptors. (II) Insoluble aggregates are rejected against salivary film, causing a loss of lubrication and increased friction in the oral cavity. (Ill) Direct interaction between polyphenols an oral epithelium (Adapted from Ma et at., 2014)

Nevertheless, since there are no conclusive results, the scientific community is still discussing the different proposed mechanisms that explain this complex phenomenon. Although the literature suggests several theories related to astringency, we could differentiate two main hypotheses: astringency as a tactile or taste sensation.

2.2. The Theory of Astringency as a Tactile Sensation

According to this theory, astringency is a feeling not a taste, attributing its tactile nature to the increased friction between oral surfaces after the loss of lubrication. Many authors have furthered the tactile theory of astringency, suggesting that protein precipitation could lead to oral tissue constriction due to a loss of oral lubrication, perceived as an increase in oral friction (Clifford, 1997). Other authors have suggested that astringent compounds could cause a sensation of roughness when changing the oral epithelium (Jellinek, 1985). Breslin et al. (1993) contribute with this theory presenting additional evidence that astringency is a tactile sensation resulting from the stimulation of mechanoreceptors during movement of the oral mucosa, consistent with Bate-Smith’s (1954) previous speculation. Moreover, Green (1993) explained not only how dehydration can lead to sensations of diyness, but also how the cross-linking of proteins may contribute to the development of astringency. The main reason probably lies in the effects that astringents have on the lubricating capacity of saliva. Cross-linking mucoproteins could induce astringents to precipitate, leaving a losing of viscosity and lubrication in the oral fluid. Additionally, free precipitated proteins are able to adhere to the mucosa and dentition, where they can form a sticky residue. Both effects may increase the coefficient of friction between the surfaces of the oral cavity, changing the tactile sensation perceived (Green, 1993).

Although most studies have assumed that the oral perception of astringency is related to salivary protein precipitation, mainly PRPs, other authors have also proposed the possibility of free astringent stimulus that interact directly with oral tissue through receptors (Schwarz and Hofmann 2008, Lee et al., 2012). Moreover, several studies have shown that the precipitation of PRPs is not required for astringency development, but they could play a protective role. PRP-binding reaction could prer ent astringent compounds to interact with the oral mucosa (Home et al., 2002).

As discussed above, the interaction between salivary proteins and wine polyphenols can lead to the formation of insoluble aggregates, decreasing lubricity in the oral cavity. The increased friction stimulates mechanoreceptors in the oral mucosa, which are responsible to pressure, touch, vibration, tension and stretch responses. These mechanoreceptors are both superficial slow-adapting (SA) and rapidly adapting (RA) receptor units (Van Aken, 2010) and could be the most susceptible gustatory receptors to stimulation by this mechanism (Breslin et al., 1993). However, Breslin et al. (1993) and Lim and Lawless (2005) revealed direct physiological data to explain astringency as a tactile sensation that is mediated by non-gustatory mechanisms. In these studies, they tested some astringent compounds in an area of the mouth exempt of taste receptors, finding that they could elicit the sensation of astringency. Based on these results, these authors considered the possibility that taste receptors were not responsible for astringency, so the presence of these receptors was not an essential factor. On the contrary, Green (1993) suggested that “the mechanoreceptors responsible for astringency may be RA receptor units that have been identified in the chorda tympani and lingual nerve” In order to prove the tactile origin of astringency, Breslin et al. (1993) confirmed by an experimental study that astringency was evoked on non-gustatory surface between the upper lip and gums.

Another explanation against the taste hypothesis has been proposed, since it seems that perceived astringency intensifies with repeated sampling. Guinard et al. (1986) and Lyman and Green (1990) demonstrated that the astringency of wine and beer increased over three and five exposures, respectively, and that the rate of ingestion affected the rate of increase, a typical feature of trigeminal, but not taste, sensations.

Finally, many astringent compounds have shown its ability to precipitate mucins, large salivary glycoproteins produced by epithelial tissues. However, published research studies focused on the role of mucins in the development of astringency are scarce, although it is broadly assumed its importance in oral lubrication. It has even been proposed that mucosa pellicle could be more important than the salivary film in the perception of astringency (Nayak and Carpenter 2008, Lee et al., 2012). In relation with the tactile theory of astringency, it has been suggested that polyphenols compounds could disrupt the salivary mucosal pellicle, causing an increased friction in the oral surface, which could stimulate mechanoreceptors as it has been previously explained (Gibbins and Carpenter, 2013). More recently, some authors have explored sensoiy astringency using “oral tribology” approaches. Tribology is the science of adhesion, friction, and lubrication of interaction surfaces that are in relative motion (Upadhyay et al.,2016). The latest studies using this approach have found a relationship between sensoiy and friction, but no conclusive results have been obtained clarifying astringency perception, despite being a potential tool (Brossard et al., 2016, Laguna et al., 2017).

2.3. The Theory of Astringency as a Taste Sensation

Taste is firstly recognised on the tongue, where epithelial receptors cells are located, organised into taste buds in papillae. Gustatory papillae can be divided into three types: fungiform papillae, foliate papillae and circumvallate papillae (see Fig. 3). Mammalian taste receptors cells transmit action potentials to neurons of the gustatory fibers that innervate taste buds. Fungiform papillae are placed in the anterior two-thirds of the tongue, and receive innervation from the chorda tympani branch of the facial nerve (cranial nerve VII). The facial nerve connects gustatoiy papillae of this area of the tongue, ending on the lingual branch of the trigeminal nerve (CN V). Foliate papillae are found on the lateral edges whereas circumvallate papillae are found in the posterior one-third of the tongue. These papillae are innervated by the glossopharyngeal nerve (CN IX) (Scott 2005, Bajec and Pickering 2008). Those three nerves (facial nerve, glossopharyngeal nerve and trigeminal nerve) provide innervation to the oral cavity (Matthews 2001), receiving taste information that is transmitted to the thalamus and finally to the gustatory areas of the cortex (Scott 2005).

Gustatoiy papillae location

Figure 3. Gustatoiy papillae location

Nowadays, astringency is not considered one of the five basic taste modalities, including salty, sour, umami, sweet and bitter. These taste modalities are sensed by taste buds on the tongue and the information is transmitted to the brain through taste nerves, as explained above. It is not clear if any receptor exists that recognise astringents, or the signaling cascades downstream (Jiang 2014).However, several authors have supported a possible interpretation of astringency as a taste sensation. Kawamura et al. (1969) demonstrated that tannic acids do not interact directly with mechanoreceptors. Subsequently, Schiffman et al. (1992) attribute the tactile, thermal, and pain response to the lingual nerve, although it is also known that this nerve is responsible to chemical stimulation (Wang et al., 1993). Moreover, they established that the chorda tympani branch of the facial nerve (which innervates taste cells) was sensitive to astringent compounds, whereas the lingual branch of the trigeminal nerve was not. They conclude that mechanoreceptors could not be responsible for the perception of astringency due to the lack of stimulation of the lingual nerve by the astringent compounds. However, these studies did not provide a definitive proof of the gustatory basis of astringency. The studies by Simon et al. (1992) also supported this theory. These authors published electrophysiological investigations showing that some astringent compounds affect ion transport through Na~ channels in the lingual epithelium. In the same way, the ability of some astringent compounds to change the membrane potential of a lipid taste sensor has been presented as additional evidences supporting the theory of astringency as a taste sensation (Iiyama et al., 1995). More recently, several studies have shown that polyphenols could directly activate functional transient receptor potential channels in oral epithelial cells, producing intracellular calcium concentration changes when they are opened (Kurogi et al., 2012, Wang et al, 2011). Nevertheless, Carpenter (2013) foimd that most human oral epithelial cells did not respond to either the transient receptor potential agonist or a black tea solution containing polyphenols.

On the other hand, the cellular effects of astringent substances in cortical signaling have been studied. Critchley and Rolls (1996) investigated the cortical representation of the taste of tannic acid, which produces the taste of astringency. They suggested a sub-population of neuron specific for tannic acid and proposed that it’s astringent taste should be considered as a distinct taste quality apart from the five basic taste modalities (Critchley and Rolls, 1996).

Moreover, in the last few years, some research has focused on the study of receptors that could be involved in oral sensing of astringent compounds. Studies in cancer cell lines have identified a receptor for epigallocatechin-gallate as the mammalian 67 kDa laminin receptor (Tachibana et al, 2004), a protein also identified as an extracellular matrix receptor in the oral mucosa (Hakkinen et al, 2000). Other studies have suggested that several astringent wine phenols activate bitter taste receptors (Soares et al, 2013).

Deepening in the neural basis of this complex sensation, Shovel et al (2014) proposed that human astringency perception was mediated by trigeminal nerves through the activation of a G-protein and adenylyl cyclase, and analyze the downstream signaling holding this response. They proved in human subj ects that only when trigeminal and taste nerves were both blocked, they lost their astringency sensation, while this sensation was not affected when only blocking taste nerves, indicating that astringency is more likely a trigeminal sensation for humans. Moreover, they tested whether astringents could directly activate trigeminal neurons in vitro, suggesting the existence of a trigeminal G protein-coupled receptor for galloylated astringent phenols (Schobel et al, 2014).

It has been demonstrated that the mechanism of wine astringency goes much beyond the traditional view focused on mechanosensation. The theories discussed here are not exclusive, so a possible explanation of the mechanism for astringency lies in the sum of several mechanisms working together. In this way, caution should be taken when interpreting results and further investigation will be needed to better understand this enigmatic sensation.

2.4. Methodologies for Astringency Determination

Because of the complexity of astringency sensation, it is not easy to find an objective methodology for measuring and characterizing it. That is way, nowadays, sensory analysis is still the method regularly used for the determination of wine astringency. However, new approaches using different instrumental techniques have been developed for solving the several dr awbacks related to sensory analysis. These new methodologies aim to unravel the astringency mechanisms and/or to predict the astringency sensations elicited by different compounds, which may allow providing an objective explanation for the astringency of different food and beverages, such as wines.

2.4.1. Sensory Analysis

Sensory analysis requires a group of testers (comprised of 8-20 people) that have been previously trained using a set of reference compounds and descriptors, to familiarise them with the astringency sensation and terminology and to standardise the criteria used for evaluating (quantitatively and qualitatively) astringency. Different scales have been employed for quantifying astringency in wines, from the simplest linear magnitude estimations to more complex alternatives involving non-linear spaces, the later usually providing better results in astringency quantification (Green et al, 1996). Among these alternatives, the Labeled Magnitude Scale (LMS) was introduced by Green and co-workers for rating perceptual magnitudes related to taste or aroma (Green et al, 1993). This scale is a quasilogarithmic scale with verbal label descriptors ranging between “barely detectable” to “strongest imaginable” that is not affected by the ceiling effect, thus improving other scales (Pickering et al., 2004).

From a qualitative point of view, to divide astringency into different sub-qualities is helpful for characterizing it. The terminology traditionally employed for describing wine astringency (astringent, puckering, roughing, diying...) can turn out to be quite general and insufficient and, that is why, Gawel and co-workers proposed a structured vocabulary for assisting tasters in the interpretation of the mouth- feel sensations elicited by red wines (Gawel et al., 2000). These authors suggested the terms paticulate, diying, harsh and unripe for grouping the negative sub-qualities of astringency whereas the terms surface smoothness, complex and dynamic would group the positive ones (Gawel et al., 2000). Thus, it seems that sensoiy analysis allowed a comprehensive description of astringency. In fact, it has been possible to develop predictive models for wine astringency from sensory results (Cliff et al., 2002). However, sensoiy analysis shows important drawbacks: it is time-consuming, expensive and it usually leads to important standard deviations in the determination even when trained panelist are involved, due to the fact that astringency perception is highly subjective (Simoes Costa et al., 2015). The use of instrumental techniques for determining astringency mainly tries to solve the latter issue (Garcia-Estevez et al., 2018).

2.4.2. Instrumental Analysis

Most of the instrumental analyses employed for assessing astringency in wines are based on the premise that the key mechanism for astringency development is the interaction between salivary proteins (namely PRPs) and different compounds (mainly tannins and other phenolic compounds). For this reason, the simplest and most used approaches for assessing the astringency properties of phenolic compounds studied its ability for interacting with proteins. This is the basis of the gelatin index, which measures the extent of precipitation of phenolic compounds by means of a gelatin solution. The main limitation of this method is related to the high variability of the results obtained, which is mainly due to the fact that gelatin is a complex and non-standardised mixture of proteins (Llaudy et al., 2004). Moreover, it has been established that this method does not provide good results for high levels of tannins (Goldner and Zamora, 2010). For this reason, other commercial proteins showing more similarities to salivary proteins are used for assessing astringency. Among them, the most utilised are bovine serum albumin (BSA) and a-amylase obtained from porcine pancreas, the latter showing a high degree of homology to salivary a-amylase (Soares et al., 2009). The use of these proteins for assessing the astringency of different wine compounds has provided successful results from a quantitative point of view (Hofmann et al., 2006, Llaudy et al., 2004). However, to be close to the real conditions in the astringency development, recent studies have used purified salivary proteins (SP) for assessing astringency (Silva et al., 2017, Soares et al., 2018).

Moreover, the precipitation of salivary proteins has been studied by means of different techniques, such as SDS-PAGE (Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis), which has been used for assessing the changes in the salivary protein profile after the interaction with different phenolic compounds. As a result, the reactivity of the different salivary proteins (SPs) and the ability of different wine phenolic compounds for interacting with SPs can be determined (Gambuti et al., 2006, Rinaldi et al., 2010, Sami-Manchado et al., 1999). A similar approach but using liquid chromatography (LC) has been used, allowing the determination of not only the most reactive families of SPs but also the formation of soluble aggregates (Kallithraka et al., 1998; Quijada-Morin et al., 2016). LC coupled to mass spectrometry has been used for proteomics studies about the effect on salivary profile of different astringent stimuli, thus providing qualitative and quantitative information about the interaction (Delius et al., 2017). Moreover, mass spectrometry, namely MALDI-TOF (Matrix Assisted Laser Desorption/' Ionisation-Time of Flight), has also been employed for assessing the identity of wine phenolic compound- salivary proteins soluble aggregates (Ferrer-Gallego et al, 2015a; Garcia-Estevez et al, 2017; Perez- Gregorio et al., 2014).

Other recent approaches have studied the interaction process using techniques further than precipitation of proteins, such as infrared spectroscopy, electronic tongues, fluorescence, nephelometry, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), circular dichroism (CD), nuclear magnetic resonance (NMR) or isothermal titration calorimetry (ITC). Several of these techniques allow studying the interaction even if it does not lead to protein precipitation. For instance, middle infrared spectroscopy (MIR) has been used as a predictive tool for wine astringency. The studies using MIR assess the most important wavelengths for estimating astringency and they build predictive models by means of chemometric tools, such as Partial Least Square regression (PLS) (Simoes Costa et ah, 2015; Vera et ah, 2010). PLS was also used to build calibration models from near infrared (NIR) spectroscopy that results to predict the sensory attributes related to astringency of grape skin and seed, pointing out the potential of infrared spectroscopy to predict different astringency parameters (Fener-Gallego et ah, 2013). Similarly, electronic tongues based on spectroscopic, potentiometric and/or electrochemical sensors can be used to estimate astringency. Those allows making measurements that, when calibrated, have been used trying to simulate sensory analysis (Diako et al., 2016). The results obtained by using electronic tongues seems promising, allowing the discrimination and classification of wines (Gay et ah, 2010). However, although these methodologies usually show good correlations to sensory analysis, they do not provide any information about the astringency process.

Fluorescence quenching measurements study the reduction in the intrinsic fluorescence of proteins (mainly due to the tryptophan residues (Soares et ah, 2009)), as a result of the interaction between proteins and phenolic compounds. The study of the fluorescence quenching allows measuring the extent of the interaction. Results usually showed that the higher concentration of wine phenolic compounds is assayed, the higher quenching effect is observed (Jauregi et ah, 2016; Yao et ah, 2010). However, Fener- Gallego et ah (2012) have pointed out that higher phenolic contents do not involve larger affinity towards proteins since the structural featur es of the wine phenolic compounds (molecular size or galloylation in the structure) could modify the affinity, thus affecting quenching results.

Compound astringency can also be assessed by means of nephelometry, which studies the formation of phenolic compound-protein aggregates by measuring the scattered light when a beam of light is passed through a solution containing suspended particles. Studies carried out by using this technique showed a direct relationship between the level of phenolic compounds and the nephelometric values (Fener- Gallego et ah, 2012; Monteleone et ah, 2004), which, in turn, are also related to the ability of wine phenolic compounds to induce astringency in sensorial analysis (Monteleone et ah, 2004). Nephelometry has also been used to assess the astringency mechanism when an agent for modulating astringency of wines is involved (Brandao et ah, 2017). However, nephelometry measurements are affected by several factors, mainly by the size of aggregates, since all the particles should be small and of identical size (Monteleone et ah, 2004). Thus, to avoid the formation of larger aggregates, nephelometry measurements should be done after a short reaction time. Dynamic light scattering (DLS) could help for solving this problem since it measures the relaxation rate of particles in a solution that scatter light and, thereby, permits an estimation of their diameter (Charlton et ah, 2002) providing size distribution of aggregates. This technique provides good results, showing a direct relationship between the size of aggregates and the concentration of wine phenolic compound assayed, suggesting the formation of complexes or metastable aggregates (Fener-Gallego et ah, 2016; Jauregi et ah, 2016; Ramos-Pineda et ah, 2018). The size distribution of the aggr egates for a large range of particle sizes can also be studied by SAXS. This technique measured the scattered radiation (X-rays in this case) by a solution containing the aggregates when it is irradiated with an X-ray collimated beam. Measurements are done very close to the primary beam (“small angles”) and, depending on the angle, different ranges of aggregate sizes can be studied (Petoukliov and Svergun, 2013). Moreover, this technique allows obtaining quantitative information about the strength of the interaction (Pascal et ah, 2008).

Nuclear magnetic resonance (NMR) allows a deeper study of the interaction, since it could provide not only quantitative information, measuring the strength of the interaction, but also qualitative, by providing information about the number and the nature of binding sites (Cala et ah, 2010a, b). Experiments usually involves a phenolic compound titration, maintaining the protein concentration constant throughout the entire titration. The chemical shifts of protein protons obtained from this experiments are used for obtaining information about the binding sites and the type of aggregates (soluble or insoluble) (Faurie et ah, 2016). The association constants can be obtained both from the chemical shifts (Cala etah, 2010b) and from saturation transference difference (STD) experiments (Fener-Gallego et ah, 2015b). In the latter, the subtraction of the on-resonance spectnun (in which protein was selectively saturated by irradiating at a region of the spectnun in which protein protons appear) from the off-resonance spectnun (that recorded without protein saturation) is done. In the difference spectrum, only those protons of the wine phenolic compounds that are close to protein via binding will appear, since they could receive saturation transfer from the protein (Viegas et al., 2011). This technique also allows the determination of the binding epitope of the phenolic compound, i.e. the protons of the astringent compound that are closer to the protein upon binding (Yiegas et al., 2011). For instance, recent studies support that some procyanidins could be multidentate ligands and that the first epitopes involved in the interaction between these wine tannins and salivary proteins depends on the structure of the procyanidins, being rings В and E in the case of non-galloylated procyanidins dimers and galloyl ring for galloylated dimers (Soares et al., 2018). Moreover, NMR diffusion experiments can be useful for detecting the formation of small aggregates between phenolic compounds and proteins, allowing the determination of the number of binding sites and the association constants by following the changes in the diffusion of the protein throughout the titration (Cala et al., 2010a). Structural information about the aggregates (size, binding epitopes in protein and phenolic compound, etc.) can be obtained from two-dimensional NMR experiments, such as TOCSY (Total Correlation Spectroscopy), NOESY (Nuclear Overhauser Spectroscopy). ROESY (Rotating-frame Overhauser Spectroscopy), HSQC (Heteronuclear Single Quantum Correlation) and DOSY (Diffusion Ordered Spectroscopy) (Faurie et al., 2016; Pascal et al., 2009; Simon et al., 2003). Qualitative information aboirt protein-phenolic compounds aggregates can also be obtained from circular dichroism (CD) spectroscopy. CD studies the difference in the absorption of the left and right circularly polarised light of the solution containing the protein and/or aggregates, providing information aboirt the bonds and structures responsible for this chirality (Rodger et al., 2005). As for studies aboirt wine astringency, it is usually employed the far-ultraviolet (190-370 nm) spectra for obtaining information about the conformational changes in the protein structure because of interaction (Pascal et al., 2009, Simon et al., 2003).

As for the mechanism of aggregation, ITC (Isothermal Titration Calorimetry) could be a helpful tool for establishing the main forces driving the interaction. In fact, from the titration curves the change in energies (enthalpy (ДН), Gibbs free energy (AG) and entropy (AS)) can be determined. Titration at different temperatures helps for distinguishing among the different forces driven the interaction (Kilmister et al., 2016, McRae et al., 2010). From the values of energy changes, it could be ascertained the type of forces involved in the interaction: important AH negative values; i.e. enthalpy drives the interaction, are related to exothermic hydrogen bonding between protein and phenolic compounds. On the contrary, hydrophobic interactions can be considered as the main force of the interaction when the process is entropy-driven, i.e. when positive values for AH and for AS are obtained (Kilmister et al., 2016, McRae et al., 2010, Ramos-Pineda et al., 2017). Moreover, from ITC result, the stoichiometry and the binding constants of association can also be obtained, thus helping to assess the whole interaction process (Garcia-Estevez et al., 2018). As for wine tannins, it has been pointed out that the situation is not so well dichotomised and that both hydrophobic and hydrophilic interactions can play complementary roles in the network formation depending on the protein and procyanidin structures (Soares et al., 2018). However, this study also reports that some proteins such as aPRPs, which show in their structure an N-tenninal with acidic residues, seem to preferentially establish H-bonds in the interaction with wine procyanidins, mainly when these compounds present a galloyl group in the structure.

In addition to these experimental techniques, molecular dynamics (MD) simulations have been employed to establish theories about the strength, the mechanisms and the number of molecules involved in the interaction. Calculations are done by using model peptides (Cala et al., 2012; Ferrer-Gallego et al., 2017) or salivary peptides (Soares et al., 2018) to simulate the interaction with one or several molecules of ligand.

Moreover, it has been stated that quantifying only the extend of protein binding could not be enough for explaining sensory perception and that it is necessary to also consider other free astringent stimulus in the saliva liquid (Schwarz and Hofmann, 2008). For this reason, new methodologies, based on salivary rheology and oral tribology, are being used with the aim to explain astringency not only based on protein complexation but also in a wider sense. These approaches, which study the modifications in friction and lubrication of saliva when mixture with astringent compounds for explaining astringency (Brossard et al., 2016) have shown a great potential for establishing relationships to the perceived texture and the mouthfeel attributes of different foods, including wine (Upadhyay et al., 2016).

3. Colour

Colour is an important sensory property for evaluating the quality of red wine and it is one of the most influential factors when consumers make their choice. The colour of a red wine is determined mainly by the composition and concentration of anthocyanins and anthocyanin derived pigments. Furthermore, colourless or poor coloured compounds like catechins, proanthocyanidins or flavonols, have been found to play an important role in the protection of wine colour since they can act as anthocyanin co-pigments and also contribute to the development of anthocyanin derived pigments during wine aging.

3.1. Chemical Stabilisation of Coloured Pigments

The chemical stabilisation of pigments in wine is one of the main research areas of enology. The stability and quality of wine colour is related to its phenolic composition. The anthocyanin stabilisation by copigmentation and polymerisation mechanisms is highly dependent on the concentration and nature of other colourless phenols also extracted from grape skins and seeds during maceration. Several strategies have been tried to enhance red wine colour and stability. One of them is the incorporation of extra copigments in the wine. This can be achieved by:

  • • the external addition of phenolic extracts or oenological tannins, that may consist of condensed or hydrolysable tannins or a mixture of them,
  • • co-vinification of different grape varieties, each contributing with additional cofactors, and
  • • addition of winery by-products like seeds or skins from white grapes, in order to provide supplementary sources for the extraction of phenolics.
  • 3.1.1. External Addition of Oenological Tannins to Improve Wine Colour

The objective of this practice is mainly to compensate wine quality deficiencies. Oenological tannins could supply compounds to the wine that can take part directly or indirectly in reactions with anthocyanins favouring the synthesis of derivative pigments or can provide compounds that participate in copigmentation interactions or that protect them from oxidation. The usefulness of the addition of enological tannin, containing condensed and hydrolysable tannins (ellagitannins), in chemical stabilisation of the colouring matter of the wines has been demonstrated (Alcalde-Eon, 2014a). This chemical stabilisation implies the increase of more stable anthocyanin-derived pigments in relation to control wines (Alcalde- Eon, 2014b), which can be formed by condensation reactions favoured by the presence of ellagitannins and other compounds that can promote the formation of reactive oxygen species (Garcia-Estevez et ah, 2015). Furthermore, greater and faster extraction of the anthocyanins in wines treated with oenological tannin has been observed (Boulton, 2001; Darias-Martin et ah, 2001; Gonzalez-Manzano et ah, 2009). Although the effect of this oenological practice depends on the characteristics of the grapes at harvest, which in turn may change from one vintage to another (Bautista-Ortm et al., 2007). Also, it has been observed that the addition after alcoholic fermentation has better effect on the phenolic composition than the addition before alcoholic fermentation (Neves et ah, 2010).

3.1.2. Co-vinification of Different Grape Varieties, Each Contributing with Additional Cofactors

The extraction and retention of colour in red wines is mainly influenced by the content of cofactors in it. Since not all grape varieties have the same quantities and percentages of individual anthocyanins and other polyphenols, that could act as colour cofactors, co-maceration of different grape varieties could compensate cofactor deficiencies favouring and increasing the content of anthocyanins and contribute to an increase in the co-pigmentation process (Garcia-Marino et ah, 2010). It has been observed that blends from different wines could lead to wines with a more balanced anthocyanin/flavanol ratio (Monagas et ah, 2006; Garcia-Marino et ah, 2013). Pigment extraction and retention in Tempranillo wines seems to be increased by the incorporation of the Graciano variety during the pre-fennentative maceration step and may be linked to the fact that the flavanols from grape skins of the Graciano variety are better copigments than those of Tempranillo (Garcia-Marino et ah, 2010, 2013). When Monastrell grapes were co-fennented in the presence of Cabernet Sauvignon and Merlot grapes, an increase in the phenolic extraction was observed which influences the colour of the finished wine (Lorenzo et al., 2005).

3.1.3. Addition of Winery By-products like Seeds or Skins from White Grapes

Grape-skins and seeds from white grapes are considered a good source of phenolic compounds (catechin, procyanidins, quercetin glycosides, etc.), which have enological interest to be used in co-fermentations with red grape musts, especially when red grapes do not present a good balance between the concentrations of anthocyanins and co-pigmentation cofactors (Gordillo et al., 2014). It has been observed that the addition of white-grape pomace during wine fermentation increases the extraction of phenolic compounds from grapes. Therefore, it has a positive effect on co-pigmentation and on the colour, although the effect of the addition depends on the doses applied (Gordillo et al., 2014). Recently, Rivero and coworkers observed that the addition of overripe seeds from white grapes during the fermentative stage of red winemaking leads to wines with higher pigment extraction, darker colours and with bluish tones (Rivero et al., 2017). This underlines the potential of the use of by-products for enhancing the stability of red wine colour.

3.2. Addition of Mannoproteins to Stabilise Red Wine Colour

Mannoproteins are glycoproteins of Saccharomyces cerevisiae cell wall that can be excreted to the wine during alcoholic fermentation or be released to the wine during yeast autolysis (Ribereau-Gayon, 2000). They are considered as protective colloids that protect wine from protein haze (Dupin et al., 2000) and that can prevent tartrate precipitation (Moine-Ledoux and Dubourdieu, 2002). It has also been reported that mannoproteins can improve colour stability (Escot et al., 2001), probably due to stabilisation effect of the colloidal colouring matter (Alcalde-Eon et al, 2014) and that could increase the monomeric anthocyanin content (Ghanem, 2017). Nevertheless, some other studies have shown that mannoproteins do not affect wine colour (Guadalupe et al., 2010) and even could provoke losses of colour (Guadalupe et ah, 2008). These discrepancy shows that it maybe due to the yeast strain used to obtain the mannoprotein or the technique used for obtaining it (acidic hydrolysis, enzymatic hydrolysis, type of enzyme utilised) that could lead to differences in mannoprotein composition. In fact, Fernandez and coworkers observed that wines treated with mannoproteins obtained by acidic and enzymatic treatment showed higher intensity of colour than those treated with mannoproteins obtained only by acidic hydrolysis (Fernandez et ah, 2011).

4. Summary and Conclusion

As a consequence of the global wanning, the viticulture and the oenology have different challenges to address. The gap between technological and phenolic maturity that leads to wines with low astringency quality and problems of colour stability during the wine ageing is one of the concerns of oenologists in the last years. This chapter is focused on astringency and on colour of red wines with the aim to understand these two sensoiy properties. Regarding astringency, despite its importance on the quality of red wines, and therefore, its economic importance in the winemaking industry, the mechanisms of astringency are not well understood, neither the possibility of an objective determination. Several aspects that have been outlined in this chapter such as the utilisation of new methodologies as molecular dynamics simulations or tribology and the application of instrumental techniques to unravel the astringency mechanisms and/or to predict the astringency sensations elicited by different compounds, may allow providing an objective explanation for the astringency of wines. This knowledge is essential, for example, to successfully deal, in a non-empirical way, with processes of modulation of astringency and stabilisation of colour in wineries, for example using biopolymers. The addition of oenological products based on cell wall material, obtained from winemaking by-products, could be a suitable and sustainable strategy that worth to be further investigated to modulate wines with unbalanced astringency and to enhance their colour stability.

Acknowledgments

The authors thankFEDER-Interreg Espaiia-Portugal Programme (Project ref. 0377_IBERPHENOL_6_E) and the Spanish MINECO (Project ref. AGL2017-84793-C2-1-R co-ftmded by FEDER) for the financial support. IGE thanks University of Salamanca for the postdoctoral contract and AMRP thanks MINECO for the FPI scholarship.

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