**3. Salivary proteins**

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

seasonally demanding input of nutrients to the soil [1].

constantly increased in different research areas.

**2. Perception of astringency**

In higher plants, tannins are primarily reserved as a chemical defence against pathogens. The complex with macromolecules such as cellulose and pectin, send out the exo-enzymes capable of utilising cellulose or pectin, either as a carbon source or for branching cell wall barriers to more nutrient-cytoplasm, depriving of the substrate or binding sites to these substrates. Another important function of tannin complexes is to impede the decomposition of plant litter, also when the leaf is fallen. This provides the delay in decomposition, which allows a constant input or

In the other processes, proteins of animal or human saliva interact with tannins of the unripe fruit, forages, or vegetable-derivates such as red wine, tea, and chocolate. Tannin molecules can bind proteins or enzymes at the level of specific amino acids, and modify the folding, the molecular weight, and the core binding site, to form soluble complexes or precipitates, which can alter protein function or inhibit enzyme activity [8]. This binding is at the basis of the astringent sensation experienced when tannins precipitate salivary proteins, and as a result, they lose their ability to lubricate the epithelial membranes of the mouth [6]. This sensation in mouth discourages the animal from feeding the unripe fruit or high-tannin forages and determines the unpleasantness of consumers for some tannin-rich products. These are the reason why, in the last decades, the interest in astringency has been

The term astringency derives from the Latin verb, *ad-stringere* that means tightly bind, strongly join. It refers to the propensity of vegetable tannins to complex with macromolecules, such as proteins and polysaccharides, and alkaloids. Bate-Smith [9] first speculated that astringent sensations were caused by the increase in friction between the mucosal surfaces, which resulted from a reduction in lubrication in the oral cavity as astringent compounds bound salivary proteins. The binding between polyphenols/salivary proteins forms soluble complexes and/or precipitates that can cause the rupture of the salivary pellicle [10], interact with oral cells [11], and stimulate and activate mechanoreceptors (MRs) hold in the mouth [12]. MRs are nerve endings that function like those of the skin, except that they have smaller receptive fields and lower activation thresholds [13]. They are selectively sensitive to different stimulus properties, such as particle size and/or mouth movements, and project such information to the central nervous system [14]. Besides, the activation of G-coupled proteins also seems to be involved in the perception of astringency, activating signal transduction pattern as that of taste recognition [15]. Some brain regions (hippocampus and anterior cingulate cortex) that have been shown to respond to basic tastes were activated by the intensity and pleasantness of astringency [16]. In particular, the right ventral anterior insula that responded to astringent stimuli contributed to the ability to recognise the qualitative features of astringency. The activation of the trigeminal nerve, chorda tympani, and brain regions involved in memory and emotions could explain astringency as a multi

Whilst the chemical definition of astringency is related to the ability of tannins to bind proteins, in sensory terms, it is described as different and concomitant feelings of drying, puckering, and roughing [17, 18]. Astringency can be defined as a tactile sensation, because: (i) it is perceived on non-gustatory surfaces such as on the soft palate, gingiva, lips [12], (ii) does not show adaptation but also (iii) increases upon repeated ingestion [19], leading to carry-over effects during the tasting. However, side tastes as bitterness, sourness, and sweetness can highly modulate the overall

**146**

perceptual phenomenon.

Saliva is a biological fluid primarily produced by the three pairs of "major" salivary glands (parotid, submandibular, and sublingual glands) in mouth and by the minor ones by 10% [39]. In the whole, saliva are presently more than 2000 different proteins and peptides [40, 41], which are the result of protein post-translational modifications before being secreted in the mouth [42]. Although saliva is predominantly a watery fluid (99.5%) with a complex mixture of proteins (0.3%; 1–2 mg/mL), ions and other organic compounds (0.2%) are also present. The whole saliva continuously baths the oral cavity and having a pH ranging from 6.2 to 7.4 acts as a buffering system. The saliva is continuously secreted (0.3–7 mL/min) and plays a role in protecting the tooth and mucosal integrity, in antibacterial and antiviral activity, digestion of food, speech, lubrication, taste, and represents a biomarker tool for some diseases [41, 43]. The main families of proteins include enzymes (amylase, carbohydrase, lipase), lactoferrin, high (M1), and low (M2) molecularweight glycoproteins (mucins), peptides as agglutinins, immunoglobulins, prolinerich proteins (PRPs), cystatins, histatins and statherins [44].

There is evidence that saliva may affect the way we perceive the taste and mouthfeel of foods in various ways [45–47]. During the wine tasting, saliva transports and dissolves the stimuli substances [48]. Saliva constituents are of great importance for establishing protein-tannin interactions. In particular, the PRPs, histatins, mucin, amylase are the main salivary proteins involved in the binding with polyphenols eliciting astringency [49]. The differences between the binding of the same polyphenol to different proteins result from differences in the amino acid sequences [50].

The PRPs account for approximately 70% of the total secretory protein and are subdivided into acidic, basic, and glycosylated PRPs. They are characterised by an abundance of proline, glutamic acid/glutamine, and glycine [51]. The presence of these four amino acids, especially proline, which are the so-called alpha-helix breakers, enables the protein to form secondary structures, which assumes a random coils conformation in solution [10, 52]. This feature may allow PRPs to universally bind various types of polyphenols, mainly tannins with different sizes and structures. Some species, such as humans, rats, and mice, produce PRPs containing about 40% proline [53, 54]. However, some species produce salivary proteins, which are rich in proline but do not show a high affinity to tannins due to extensive glycosylation [54].

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 basic residues and proline [59].

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 sequence that enable the binding with polyphenols [50].
