**4.1 Other stimuli**

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

basic residues and proline [59].

Parotid and submandibular secretions also contain several low molecular-weight

histidine-rich peptides [55, 56]. Amongst 12 forms, the histatin 1, 3, and 5 are predominant and vary in size from 7 to 38 residues. These peptides show a high content of basic residues, such as lysine, arginine, and histidine [57]. They tightly bind tannins, even if some peptides are devoid of proline [58]. Conversely, others observed high tannin precipitation by histatins thanks to the interactions formed by

Amongst the low molecular weight salivary proteins, there is a selectivity in binding polyphenols (as PGG, procyanidin trimer, epicatechin, malvidin-3-glucoside): the acidic PRPs considerably form soluble and insoluble complexes with PGG and trimer but not with epicatechin; basic PRPs and glycosylated PRPs seem to not interact with trimer, whilst basic PRPs show a high affinity for epicatechin, malvidin-3-glucoside, and a mixture of both; the statherin shows no selectivity [60, 61]. Mucins are the major constituents of the viscous layer coating hard and soft tissues in the oral cavity. Mucins are generally composed of a peptide core (apomucin) enriched in serine, threonine, and proline residues and carbohydrate side chains (oligosaccharides) that are linked O-glycosidically to threonine or serine. M1 is a polymeric mucin due to the formation of disulfide linkages between cysteine residues in non-glycosylated domains, whilst M2 is a monomer [62]. Average proline content of 10% seems to be responsible for protein-phenol interactions [63].

Amylase is secreted mainly by the parotid gland in both glycosylated and nonglycosylated isoforms [64]. It is an enzyme capable of hydrolysing bonds within amylose and amylopectin and is composed mainly of amino acids like aspartic acid > glutamic acid > arginine [65]. However, amino acids as tyrosine and tryptophan seem to be crucial for interaction with polyphenols [66]. The non-glycosylated form of amylase contains 22 proline and 16 tryptophan amino acid residues in its

Astringent wines are commonly defined as "tannic" because tannins are the main polyphenolic compounds involved in the sensation of astringency. Swain and Bate-Smith [67] provided the first useful phytochemical definition of tannin, being "water-soluble phenolic compounds, having molecular weights lying between 500 and 3000, which have the ability to precipitate alkaloids, gelatin, and other proteins". Tannins can be classified in condensed tannins, phlorotannins, and hydrolysable tannins. Condensed tannins are large macromolecules that consist of two or more monomeric (+)-catechin or (−)-epicatechin units called procyanidins, whilst prodelphinidins consist of (+)-gallocatechin or (−)-epigallocatechin units. In plants, condensed tannins are found as oligomers (2–10 monomer units) or polymers (>10 monomer units). The number of monomer units in a polymer may be as high as 83 units [68]. The subunit composition varies amongst tannins from grape skins, seeds, and stems [69–71]. The phlorotannins are present in marine brown algae as polymers of phloroglucinol (1,3,5 trihydroxy-benzene) in different ranges of molecular sizes (126 Da–650 kDa). They are analogous to the terrestrial condensed tannins since they do not contain a carbohydrate core [72]. Hydrolysable tannins, structurally perhaps the most complex tannins, comprise three subclasses such as simple gallic acid, poly-galloyl esters of glucose (gallotannins), and esters of ellagic acid (ellagitannins). Derivatives of gallic acid contain one to five galloyl groups that can be esterified to either glucose (e.g., pentagalloyl glucose) or quinic acid (e.g., monogalloyl quinic acid). Gallotannis can contain six or more galloyl

groups and can be characterised by having one or more digalloyl groups

sequence that enable the binding with polyphenols [50].

**148**

**4. Tannins**

Compounds able to elicit sensations as tastes and mouth feelings are called *stimuli*. Chemically diverse astringents such as complex salts such as aluminium sulfate (alum), acids, and other phenolics, have also been shown to evoke astringency [17, 86]. Five organic acids and one inorganic elicited astringency and astringent subqualities [87], and dryness has also been reported [86, 88]. The addition of malic and lactic acid in red wine at the same pH did not differ significantly in astringency despite the difference in titratable acidity [89]. However, these acids were defined astringent in addition to their sour taste [90]. Wines more abundant in malic acid showed higher reactivity towards saliva proteins and then higher potential astringency than tartaric acid-rich wines at the same pH, probably due to different buffer capacities [91]. The astringency of acids is attributed either to the direct contribution of H+ ions or to the hydrogen bonding capabilities of the hydroxyl groups on the anion or un-dissociated acid [17]. Denaturation of proteins in the saliva could also affect the binding and dissociation of phenolic compounds and their precipitation. The intensity of astringency linearly increases as a function of pH reduction [19], implying significant precipitation of salivary proteins [92].

Anthocyanins, composed of a sugar bound to the anthocyanidin moiety (cyanidin, peonidin, delphinidin, petunidin, and malvidin), impart colour to the grapes and red wine and can be modified by different enological practices [93]. Controversial is the studies of the influence of anthocyanins on astringency. An anthocyanin fraction added in model wine solution was felt as "rough and chalk," and slightly contributed to the overall astringency probably for contamination of the fraction with unknown phenolic compounds [94]. Successively, the isolated fractions of anthocyanidin–glucosides and anthocyanin coumarates did not influence astringency of wine solutions either the "coarse," "chalk," or "dry" astringent subqualities [95]. However, anthocyanins were able to interact with human salivary proteins forming soluble aggregates [96], and even precipitates, being the cinnamoylated the most reactive fraction (precipitation between 6.5 and 17.5%), also influencing the astringency perception [97]. Pyranoanthocyanins, anthocyaninderived pigments that can form during red wine ageing, seems to be involved in astringency, since they are able to interact with salivary proteins by phenol, catechol or even flavanols structures, similarly to procyanidins [98].

Flavonols (kaempferol, quercetin, and myricetin) are present in grapes and wine as glycosides (sugar attached). In the plant, they act as a natural sunscreen in the skin of grape berries. In wine, they can be hydrolysed and act as cofactors for colour enhancement. Flavonol glycosides, such as 3-O-glucosides and 3-O-galactosides of quercetin, syringetin, and isorhamnetin, have been reported to be astringent at low detection threshold levels and characterised by a velvety astringency [99]. The addition of quercetin 3-O-glucoside (2 g/L) to wine increased astringency, leading to the formation of complexes with saliva at 200 μM [100]. However, such concentrations are not naturally present in red wine, in which quercetin 3-O-glucoside can range from 2 up to 34 mg/L, depending on the cultivar [101].

Many sensory active non-volatile compounds comprising hydroxybenzoic acids, hydroxycinnamic acids, flavon-3-ol glycosides, and dihydroflavon-3-ol rhamnosides were identified as the key inducers of the astringent mouthfeel of red wines using a molecular sensory approach [99]. The phenolic acids in wines, especially hydroxycinnamic and benzoic acid derivatives, have been reported to be more puckering astringent. These compounds have also been correlated with astringency in free-run and pressed wine [102]. The trans-*p*-coumaric, cis-aconitic, and transcaftaric acids seem to participate in the astringency of Spanish wines [103].
