14 2  The Brewing Yeast

Fig. 2.1  Schematic view of the NewFlo yeast phenotype under different situations of beer
­fermentation, where a flocculation is established because free sugars (e.g., glucose) have been
exhausted, calcium ions are present and associated with the N-terminals of flocculins, and
­mannan residues in cell wall are phosphorylated; b flocculation cannot occur because there
are neither calcium ions nor phosphorylated mannans; and c flocculation is prone to occur, but
the sugar-binding domains of flocculins are occupied with free sugars of the unfinished beer
­fermentation

Ogata et al. (2008) further confirmed that Lg-FLO1 was a S. pastorianus-specific

gene located on S. cerevisiae-type chromosome VIII. However, Lg-FLO1 was also
found in some S. cerevisiae (ale) strains proving the flocculation gene variability in
industrial brewing yeast strains (Van Mulders et al. 2010). More recently, Sim et al.
(2013) demonstrated that Lg-Flo1 flocculins would bind to phosphorylated mannans
rather than non-phosphorylated mannans in the yeast’s cell wall.
Both environmental (e.g., pH, metal ions, and nutrients) and genetic factors
affect flocculation. However, these factors should never be considered separately
as the environment may influence the expression of FLO genes (Verstrepen and
Klis 2006). Because flocculation is mainly a defense mechanism, nutrient starva-
tion and stress conditions will trigger the expression of flocculins (Stratford 1992).
Nothing represents this better than the competitive attachment of simple sugars to
the flocculin binding sites, working as a signaling mechanism of nutrient availabil-
ity. Indeed, Ogata (2012) has suggested that yeast expresses Lg-FLO1 in response
to nutritional starvation, and it is regulated by a nitrogen catabolite repression-like
mechanism. In fact, FLO genes are under tight transcriptional control of several
interacting regulatory pathways such as Ras/cAMP/PKA, MAPK, and main glu-
cose repression (Verstrepen and Klis 2006; Gagiano et al. 2002).
Ethanol has a positive effect on flocculation as it reduces the negative electrostatic
repulsion between cells (Dengis et al. 1995) and increases cell-surface hydrophobic-
ity (Jin et al. 2001). Moreover, it has also been suggested that ethanol acts directly on
the expression of FLO genes (Soares et al. 2004; Soares and Vroman 2003).
Hydrodynamic conditions may also have an impact on flocculation as liquid
agitation increases the chance of cell collision; however, vigorous movement may
also break up cell clusters (Klein et al. 2005). Additionally, concentration of yeast
cells in suspension must be sufficient to cause the number of collisions necessary