Introduction

Unlike many other food products, wine is consumed primarily for its sensory properties, highlighting the importance of visual, olfactory and gustatory aspects in quality appreciation. Colour and mouthfeel properties are two main criteria for assessing red wine quality. Native anthocyanins, or evolved anthocyanins (e.g., pyranoanthocyanins and oligomeric and polymeric pigments formed as a result of reactions between anthocyanins and tannins (Cheynier et al., 2006)) are responsible for red wine colour. Ideal mouthfeel properties are considered to be soft and unaggressive astringency, balanced with sweetness and acidity. Astringency is a sensory stimulus that is experienced as a dryness or roughness in the mouth often characterised as graininess (De Freitas and Mateus, 2002). It is mostly ascribed to the polymerised forms of flavan-3-ol monomers (catechin, epicatechin and epigallocatechin), known as proanthocyanidins or condensed tannins. Anthocyanins, phenolic acids, and flavonols, also contribute to astringency, and they sometimes amplify this perception, as has been observed for phenolic acids (Ferrer-Gallego et al., 2015b; Ferrer-Gallego et al., 2014; Ferrer-Gallego et al., 2016). The astringency mechanism involves complexation between tannins and salivary prolin-rich proteins (PRPs), which precipitate on the mucosal pellicle and thereby alter its lubrication (Ployon et al., 2018; Soares et al., 2020). Complexes between mucins (other salivary proteins) and anthocyanins can also be involved (Torres-Rochera et al., 2023). PRPs can also prevent tannins from interacting with the mucosal pellicle, epithelium cells and chemoreceptors (Canon et al., 2021). In any case, binding with PRPs - with or without precipitation - has been found to contribute to most astringent compounds (Soares et al., 2020). Tannin composition influences the perception of astringency. Differences have been observed between skin and seed tannins: when tasted as purified fractions, skin tannins containing (epi)gallocatechin (tri-hydroxylated monomeric units) were preferred to seed tannins containing only (epi)catechin (di-hydroxylated monomeric units) (Ferrer-Gallego et al., 2015a). Moreover, a higher galloylation rate of seed tannins increases the perception of their astringency (Vidal et al., 2003a; Soares et al., 2020). However, the composition of tannin is highly modified during aging. The acidic conditions in wine lead to tannin cleavage and therefore a drop in their mean degree of polymerisation (Vidal et al., 2002). Simultaneously, tannins are involved in reactions with different compounds (e.g., other tannins, anthocyanins, flavonols and phenolic acids), resulting in molecules with higher molecular weight and giving the wine better sensorial properties, as observed for flavanol units (Ferrer-Gallego et al., 2015a). After transformation due to chemical reactions, seed tannins lose their astringency, as do skin tannins; as a consequence, aged wines are less astringent (McRae and Kennedy, 2011). We cannot rule out the hypothesis that the contribution of seed tannins to mouthfeel is equivalent to that of skin tannins; extraction of seed and skin tannins are both considered desirable in this review.

Several methods have been developed to address the issue of the substantial losses of anthocyanins and tannins occurring during red wine making (Sacchi et al., 2005). For example, only 25 % of berry tannins have been recovered in wine (Bindon et al., 2010). Such losses can be explained by oxidation and binding to the cell walls. Oxidised polyphenols are highly reactive; they polymerise and can be lost in precipitates when the molecular weight of the molecules is too high. Tannin quantity can be underestimated due to artifacts forming, such as phloroglucinolysis on evolved tannins, when depolymerisation methods are used for quantification. Newly formed bonds between polyphenols are resistant to acidic catalysed cleavage (Poncet-Legrand et al., 2010), thus leading to the underestimation of tannin quantity (Vernhet et al., 2020). However, methods based on size exclusion chromatography (SEC) result in general but more accurate quantifications. On the other hand, wine polysaccharides can play a positive role, as the complexes they form with polyphenols contribute to a softer, less aggressive astringency; for example, pectins have been shown to affect the binding of PRPs salivary proteins with polyphenols (Gonzalez-Munoz et al., 2021). Therefore, more attention should be paid to polysaccharides and their interactions with polyphenols.

Based on a literature review, the aim of this paper is to propose a simple explanation of the relationships between polyphenols and polysaccharides, from mature red grape berries to wine tasting, in the context of polyphenol extraction and stabilisation during or after wine making.

For simplification, the term ‘polyphenols’ will hereafter be used to refer to anthocyanins and tannins, on which there is extensive literature. More precisely, ‘tannins’ will refer to proanthocyanidins and their adducts with, for example, other proanthocyanidins, anthocyanins, flavonols and phenolic acids.

An exploration of anthocyanins, tannins and polysaccharides

Before introducing our model, in this section we draw on relevant literature to describe the location of anthocyanins and tannins, and how they bind to cell walls.

1. Location of anthocyanins, tannins and polysaccharides in the berries

Berries can be divided in three parts: skins, pulps and seeds. At harvest, most anthocyanins and tannins are located in either the skins (anthocyanins and tannins) or the seeds (tannins). At the cell scale, tannin concentrations in the pulp and skin cell walls are equivalent. However, pulp cell walls represent roughly 10 % of skin cell walls (Ortega-Regules et al., 2008); therefore, the quantity of wine tannins that come from pulp tannins is negligible, and this paper will focus on the extraction of polyphenols from skins and seeds.

1.1. Location in the skins

In a study by Fougere-Rifot et al. (1996), polysaccharides were stained red by PAS (Periodic Acid Schiff) to reveal the skin cell walls (Figure 1). Anthocyanins, naturally red in colour, were also visible in spheric inclusions. Epidermic cells were homogeneous and had thick walls containing anthocyanins. No tannins are visible in this image, but other works have shown tannins to be mainly located in the vacuole (Amrani-Joutei et al., 1994), as were anthocyanins, but in separate inclusions. Such homogeneity has not been observed for the hypoderm: some hypodermic cells were the epidermic-cell type, while others had thick cell walls, similar to epidermic cells but no anthocyanins, similar to pulp cells. The hypoderm is a transition zone, but due to its thick cell walls, it is collected along with the skin and not the pulp when the berries are peeled. The epiderm is protected by a layer of wax, known as the pruine; therefore, it is likely that polyphenols and polysaccharides located in the epidermic cells have to cross the hypodermic zone prior to dissolving in the must (Gao et al., 2019).

Figure 1. Polysaccharides and anthocyanins in the cell walls (Fougere-Rifot et al., 1996).

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CD = cell type A, CB = cell type B, Ep = external epiderm, Hyp = hypoderm, Lm = medium lamella, M = Meatus, Pd = plasmodesma, PI = plaste, PU = pulp, V = Vacuole, VT = tannic vacuole, and N = nucleus.

1.2. Location in the seeds

Seeds do not contain anthocyanins, but they nonetheless represent a large part of total tannins in the berry depending on the variety: around 60–70 % in Cabernet-Sauvignon (Hanlin et al., 2010) and Pinot noir (Mane et al., 2007; Terrier et al., 2009), and only 30 % in Maccabeo (Terrier et al., 2009). Seed tannins are composed of the three monomeric units catechin, epicatechin and epicatechin-3-gallate (Prieur et al., 1994) and not epigallocatechin units, which are only found in berry skin (Souquet et al., 1996; Llaudy et al., 2008). Seeds are made up of several layers (from outer inner): the cuticle, epidermis and tegument, then the endosperm and embryo. Most seed tannins are located in the tegument (Cadot et al., 2006), which is in the external part of the seed and is therefore favourable for tannin extraction. However, other factors can affect tannin extractibility, namely lignification (mostly occuring at maturation), epidermis thickness which considerably varies from one side of the seed to the other, and polyphenol oxidation (Cadot et al., 2006).

Proposed model

The proposed model will be described first, with an explanation of the behaviour of polyphenols and polysaccharides from grape berries to wine. The terms adducts, aggregates and complexes refer to covalent bonds between polyphenols, weak associations between polyphenols (same family) and weak associations between polyphenols and polysaccharides (different families) respectively. Polyphenols, especially anthocyanins and tannins, are very reactive molecules, prone to forming adducts with or without oxidative context (Cheynier et al., 2006). Precipitation, which causes losses, is triggered by high molecular weights and other parameters (e.g., absence of glycosylation), as well as the chemical environment; for example, polysaccharides are protective colloids in wines and they therefore limit precipitations (Lubbers et al., 1993). Polyphenol-polysaccharide complexes have long been observed (Glories, 1978). Red wines contain three main families of polysaccharides (Vidal et al., 2003b): i) cell wall polysaccharides, such as polysaccharides rich in arabinose and galactose (PRAGs), ii) type II (RG-II) rhamnogalacturonan in monomer or dimer form, and iii) polysaccharides released by yeast during winemaking, such as mannoproteins (MPs). PRAGs mainly comprise arabinogalactan-proteins (AGPs), but they also comprise arabino-galactans (AGs) and arabinans. Concentrations have been observed in the ranges of 18–616 mg/L for PRAGs, 67–291 mg/L for RG-II, and 76–216 mg/L for MPs (Boulet et al., 2016). The stabilisation properties of these three families (i.e., their ability to prevent enlarged aggregation) have been studied using tannin fractions prone to forming aggregates and precipitating in model wine: the MPs clearly stabilised the polyphenols (Riou et al., 2002), preventing enlarged aggregation and precipitation, and low and medium molecular weight MPs (51 and 62 kD) were more efficient for the stabilisation of tannins than higher molecular weight MPs (337 kD) (Poncet-Legrand et al., 2007). For AGPs and RG-II, conclusions cannot be drawn so easily. The observed molecular weights of the AGPs were between 48 and 192 kDa (Vidal et al., 2003b), which is within the 30 to 400 range - the molecular weight range of MPs that form complexes with polyphenols (Poncet-Legrand et al., 2007). AGPs and MPs both comprise neutral chains of polysaccharides linked to a protein which represents 5–10 % of the AGP or MP weight. Therefore, like MPs, AGPs should stabilise polyphenols. Fractions of AGPs have been purified according to their uronic acid content, from neutral AGP0 to acidic AGP4 (Pellerin et al., 1995; Vidal et al., 2003b). AGP4 has been found to inhibit the formation of large polyphenol aggregates, while AGP0 has not (Riou et al., 2002). These results align with the literature: acidic polysaccharides have been shown to be more effective than neutral polysaccharides at stabilising polyphenols (Le Bourvellec et al., 2004). Therefore, the efficiency of AGPs should depend on the proportions of AGP0 and AGP4 respectively, which seem to dramatically vary. AGP4 and AGP3 are similar in terms of their composition: they both contain galacturonic acid, and a higher concentration of rhamnose and a lower concentration of proteins than AGP0, AGP1 and AGP2 (Vidal et al., 2003b). AGP3 plus AGP4 have been found to represent 19 % (Pellerin et al., 1995) and 78 % (Vidal et al., 2003b) of total AGPs, thus showing high variability. Moreover, AGPs can provide colloidal stabilisation at very low concentrations. The aggregation of polyphenols has been shown to be dramatically limited by the addition of 50 mg/L of AGP4 (Riou et al., 2002) and even 12 mg/L of an AGP extract from Acacia senegal gum (Nigen et al., 2019). RG-II is present in the form of a monomer or a dimer. The RG-II monomer has been found to slightly limit tannin aggregation and the RG-II dimer to increase it. The explanation for this lies in the hydrophobic region of RG-II, which is able to bind polyphenols. Having two hydrophobic regions, the RG-II dimer was able to link the polyphenol aggregates, while RG-II monomer was not. The efficiency of the RG-II monomer increased as it increased in concentration, but these were too low to have a real effect (Riou et al., 2002).

To conclude, monomeric RG-II, MPs and acidic AGPs have been found to contribute to polyphenol stabilisation. Some explanations for this have been proposed. Wine polyphenols are encapsulated by a layer of polysaccharides, which may provide them steric protection (Luck et al., 1994; Saucier, 1997; Le Bourvellec et al., 2004; Mateus et al., 2004; Poncet-Legrand et al., 2007; Brandao et al., 2017). As regards large branched polysaccharides (e.g., from MP or AGP families), polyphenols may be nested between the branches (Carn et al., 2012) (Figure 2). A synergic effect likely occurs between RG-II, AGPs and MPs, which each surround a part of the polyphenol pool by encapsulating or nesting, depending on the polysaccharide family, size and conformation.

Figure 2. Polysaccharides (blue and orange) surrounding polyphenols (red) via encapsulation (left) and nesting (right).

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Observed under non-wine conditions, the decrease in density of polyphenol-polysaccharide complexes when compared to polyphenol aggregates enhanced stabilisation (Carn et al., 2012). Polyphenol-polysaccharide complexes have been observed in wine in an AF4 analysis (Marassi et al., 2021). Proteins have been evidenced in polyphenol aggregates (Maury et al., 2016; Marassi et al., 2021), which may or may not belong to AGPs or MPs (not discussed here, since proteins not belonging to AGPs or MPs are not within the scope of this article, and they do not alter the general picture). To summarise, the previously described model (e.g., polyphenol aggregates surrounded by polysaccharides), is largely supported by the literature. However, it does not address the involvement of oligosaccharides in encapsulating tannins, nor the presence or not of free tannins, i.e. not surrounded by polysaccharides, in wines. Unpublished studies by our team show that there are interactions between oligosaccharides and polyphenols, which supports the theory that oligosaccharides contribute to encapsulating tannins, as some polysaccharides do. Furthermore, if significant quantities of free tannins were present in the wines, then the oligosaccharide contribution to astringency would be negative (as was the polysaccharide contribution) and not positive as has been observed (Boulet et al., 2016). It should be noted that an alternative model was considered that relied on tannin-oligosaccharide complexes that had higher astringency than free tannins - themselves more astringent than tannin-polysaccharide complexes; however, this model was dismissed as explanations could not be provided. In the end, the most probable model from our point of view is as follows: to remain stable in musts or wines, anthocyanins or tannins need to be entirely surrounded by polysaccharides or oligosaccharides. The time scale for stability is measured in years. The wine-making process, from berry crushing to wine tasting, will hereafter be reviewed in the light of the proposed model.

1. Crushing of berries

Maturation involves the degradation of the middle lamella located between the walls of two adjacent cells, which increases the porosity of the cell walls and is associated with a wavy appearance (Bindon et al., 2014), enabling better access to the binding sites within the cell walls (Hanlin et al., 2010). As soon as the cell walls, and thus the vacuole membrane, are disrupted, anthocyanins and tannins are released, of which one part bind to the cell walls and the other will dissolve in the must.

1.1. Polyphenols bound within the cell walls

Cell walls have a very high capacity for binding polyphenols (Renard et al., 2001; Hanlin et al., 2010), those of skins more so than those of pulp (Abi-Habib et al., 2023). It has been hypothesised that hydrophobic cavities in the cell walls are able to store polyphenols (Le Bourvellec et al., 2004). The stacking of polyphenols within these cavities very likely occurs, thus explaining such high binding capacity (Renard et al., 2001; Le Bourvellec and Renard, 2005; Hanlin et al., 2010). Of the cell wall polysaccharides, pectins have the highest affinity for polyphenols (Le Bourvellec et al., 2005; Ruiz-Garcia et al., 2014). Pectins can be categorised as linear homogalacturonan (HG) or hairy rhamnogalacturonans, namely rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II).

1.1.1. Skin cell wall homogalacturonan

HGs are linear chains of galacturonic acids and the methylation rate of their acidic functions affects binding capacity. Highly methylated HGs have been found to have a high affinity for tannins, especially those with high mean degrees of polymerisation (mDP) (Watrelot et al., 2013), as well as a high affinity for para-coumaroylated anthocyanins (Goncalves et al., 2012). On the other hand, low methylated HGs have been found to have a higher affinity for non acylated anthocyanins in studies on cyanidin-3-o-glucoside and blueberry anthocyanins (Fernandes et al., 2020a; Koh et al., 2020). The capacity of HGs for binding anthocyanin increases during ripening (Campbell et al., 2021) due to a decrease in methylation rate. The methylation rate in green berries is around 70 %, dropping to around 20 % on average in ripe berries (Barnavon et al., 2001). Because this methylation rate is low at maturation, anthocyanins are expected to bind more easily than tannins. We thus propose the following scenario, which is illustrated in Figure 3. A first layer of polyphenols is formed when they bind to the HGs: non-acylated anthocyanins bind to slightly methylated HGs, tannins to highly methylated HGs, and para-coumaroylated anthocyanins bind between non-acylated anthocyanins and tannins, since they are more hydrophobic than non-acylated anthocyanins but not as hydrophobic as tannins. Thus a first layer is formed, as already suggested by Campbell et al. (2021), which contains more anthocyanins than tannins. Then, a second layer is formed by stacking (Renard et al., 2001), and so on, until the cavity has been filled. This scenario provides an explanation for why tannin recovery in wines increases with anthocyanin concentration (Singleton & Trousdale, 1992; Kilmister et al., 2014; Campbell et al., 2021). The contribution of anthocyanins to binding capacity will be discussed in detail hereafter.

Figure 3. Binding of anthocyanins and tannins onto homogalacturonans within the cavities of skin cell walls. Left: filling of the first layer. Right: filling by stacking. The number of layers is illustrative.

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1.1.2. Skin cell wall rhamnogalacturonan-I

RG-Is are linear chains of rhamnose and galacturonic acid, which support lateral branches of arabinans, galactans and arabinogalactans (Watrelot et al., 2014). Limited interactions have been observed between tannins of mDP 9 and rhamnogalacturonans when compared to HGs. However, arabinans, which are side chains of RG-Is, are of interest in the binding mechanism. Due to their higher binding capacity than that of RG-Is (Watrelot et al., 2014), arabinans are another significant contributors to binding after HGs (Fernandes et al., 2020b), non-branched arabinans showing high efficiency. Another role of arabinans within the cell walls may be to faciliate the access of polyphenols, since they increase cell wall porosity (Verhertbruggen et al., 2009).

1.2. Polyphenols released in the must

After the berries have been crushed, the conditions in the must are not favorable for anthocyanins and tannins. Oxidative enzymes associated with dissolved oxygen lead to the formation of adducts of higher molecular weight that are more unstable. This situation is worsened by the presence of proteins, and by laccase in the case of harvest with Botrytis cinerea (Ribereau-Gayon et al., 2000). Polysaccharides contribute to either the precipitation or the stabilisation of polyphenols. An example can be given with HGs and arabinans based on the results presented in Table 1 and our model. Soluble HGs result from the degradation of the pulp and around 100 mg/L is present in fresh must (Vidal et al., 2000). They have a high capacity for binding polyphenols (Watrelot et al., 2013), but not for surrounding them due to their linear form and their lower flexibility. Therefore, homogalacturonan-polyphenol complexes precipitate: this is supported by no HGs being found in wines (Pellerin and Cabanis, 1998). In contrast, soluble arabinans, however, surround polyphenols and therefore lead to stable complexes due to their high flexibility (Verhertbruggen et al., 2009). However, the concentration of arabinans is limited in musts, with values of 26 mg/L having been found in a fresh Ugni blanc must (Vidal et al., 2001). Other stabilising polysaccharides present in musts are RG-II (30–50 mg/L) and AGPs (113 mg/L) (Vidal et al., 2001), but their concentrations are also low. Thus a minor part of polyphenols released into the must before alcoholic fermentation starts can form stable complexes with polysaccharides, but on the whole, they end up being lost and never recovered in wine.

Discussion

In order to provide support for our model, the following five points are discussed in this section drawing on the literature: 1) oenological practices, 2) seed tannins, 3) anthocyanins, 4) enzymes, 5) cell wall material.

Conclusion

The aim of this paper is to describe the interactions between polyphenols and polysaccharides from grape to wine. Inspired by unexplained observations, the proposed model corresponds with most of the literature on the subject. Its novelty lies in the potential positive contribution of polysaccharides/oligosacharides to the recovery and stabilisation of anthocyanins and tannins in wines. The main idea is that all stable tannins and anthocyanins are surrounded by a layer of polysaccharides or oligosaccharides, namely RG-II, PRAGs and MPs, which are already known as protective colloids. While homogalacturonans do not have this stabilisation property, they can bind polyphenols. Such binding capacity enhances polyphenol recovery in wine when it occurs within the cavities of the skin cell walls. The contribution of anthocyanins to the recovery of tannins can be explained by their ability to bind to lowly methylated homogalacturonans; tannins can then stack on this anthocyanin layer. However, polyphenol binding on homogalacturonans in the must, whether in soluble form or in pulp tissues, will early precipitation.

We are aware that further research is required to reinforce and develop this initial study. Nevertheless, it seems clear that oligo and polysaccharides play a central role in the control of anthocyanins and tannins; therefore, a characterisation of both oligosaccharides and polysaccharides would be required to increase our understanding of anthocyanin and tannin behaviour. Such an approach would be particularly valuable for carrying out quick but accurate evaluations of the oenological potential of new varieties.

Acknowledgements

This review was triggered by the results of the Interface project funded by the French National Research Agency (reference: ANR-10-LABX-001-01), and from the PF4CEPIA AIC funded by the INRAE Transform division.

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