9D Influence of Phenolics on Wine Organoleptic Properties 553
the hydroxyl groups of phenolic compounds and carbonyl and amide groups of pro-
teins. Hydrophobic interaction can occur between the benzenic ring of phenolic
compounds and the apolar side chains of amino acids such as leucine, lysine or
proline in proteins (Hatano and Hemingway 1996; Jobstl et al. 2006; Oh et al. 1980;
Wroblewski et al. 2001).
The proline residue is generally considered to be a good binding site as it pro-
vides a flat, rigid, hydrophobic surface that is favourable to interactions with other
planar hydrophobic surfaces such as benzenicrings. Effectively, NMR studies per-
formed by the Haslam research team in the University of Sheffield have shown the
existence of hydrophobic stacking between the benzenic ring of several phenolic
compounds and the apolar face of the proline ring of peptides rich in this amino
acid (Baxter et al. 1997; Charlton et al. 2002a; Murray et al. 1994). On the other
hand, a carbonyl group of tertiary amides, as it occurs in a protein with proline
residues, is a hydrogen acceptor more efficient than a carbonyl in a secondary or
primary amide. Hence, hydrogen bonding involving the carbonyl group of proline
is predictable, concomitantly with hydrophobic interactions. Although studies per-
formed by other authors suggest that the driving force of protein-tannin interactions
are mainly hydrogen bonding (Frazier et al. 2006; Simon et al. 2003), it seems
more prudent to accept that both type of interactions are present, strengthening
each other, depending on the protein and tannin structure and medium conditions
(Artz et al. 1987; Hagerman et al. 1998; Oh et al. 1980).
Protein-tannin interactions are importantly affected by pH (Calderon et al. 1968;
Naczk et al. 1996). Apparently there is higher protein precipitation at pH values
close to the protein isoelectric point (pI) where protein-protein electrostatic repul-
sions are minimized (Hagerman and Butler 1981): proteins with acidic pI have
higher affinities to complex with tannins at lower pH, whereas basic proteins aggre-
gate preferentially at higher pH (Yan and Bennick 1995). An interesting study by
NMR performed by Charlton et al. (2002b) with EGCG and a basic PRP fragment
of 19 amino acids, showed that pH greatly affects precipitation of aggregates but
does not change the protein-tannin binding affinities.These authors suggested that
protein-tannin particles are in a colloidal state, stabilized by repulsions of proteins
of the same charge. When this charge is minimized (at pH=pI), there is no repul-
sion and particles aggregate together and precipitate. Therefore, although the initial
binding is due to specific protein-tannin interactions, the aggregation process seems
to be due mainly to surface charge effects.
The affinity of tannins to bind proteins is favored by their ability to work as
multidentate ligands (cross-linking) in which one tannin is able to bind to more
than one protein at one time (Fig. 9D.11b) or to bind to more than one point in
the same protein (Charlton et al. 2002a; Siebert et al. 1996). These associations
between tannin and protein could result in aggregates that precipitate depending on
the ratio of protein/tannin and also the concentration of protein (Frazier et al. 2003;
Poncet-Legrand et al. 2006).
Besides the relative concentrations, those interactions are affected by the struc-
tures of both tannin and protein.