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Flocculation, the non-sexual aggregation of cells to form clumps, is a key determinant of the suitability of brewing strains for use in brewing. The ability to flocculate in late fermentation assists with separating the yeast crop from green beer. Strains that do not show this property to the appropriate degree lead to high cell counts in processes down-stream of fermentation, with concomitant high loss rates and inefficiencies in primary filtration. Conversely, highly flocculant strains may cause premature separation and thereby increase the risk of stuck fermentation and poor VDK removal.

There is a relationship between sugar availability and the onset of flocculation. This makes perfect sense from the standpoint of yeast. When sugar is plentiful in early fermentation, flocculation is inhibited and this ensures good cell dispersion and equal access to carbon. When sugar becomes exhausted, flocculation can occur and from the standpoint of survival it is advantageous for cells to form flocs, which provide a sheltered environment for those in the interior.

Flocculation is a cell-surface phenomenon and therefore its expression is linked to cell wall structure. It has been established that flocculation involves interactions between lectin-like proteins (flocculins) and cell surface mannans. The former are present on all cells, whereas the flocculins only occur in those strains possessing the appropriate genotype. Calcium ions are required for flocculation to occur and it is thought that these mediate binding by causing an essential conformation change on the lectin structure. Abundant wort sugars such as glucose and maltose preferentially bind to lectins and thereby inhibit the process. In addition to mannan-lectin interactions, floc stabilization may occur via hydrophobic interactions and hydrogen bonding (Soares, 2011). Two main phenotypes are recognized based on the patterns of inhibition by sugars. NewFlo types, which include many brewing strains, do not flocculate in the presence of mannose, glucose, maltose, or sucrose. In Flo1 types, flocculation is inhibited by mannose. A rarer third type, M1, occurs in some strongly top-cropping ale strains and appears to operate via direct protein-protein interactions and does not occur in the absence of ethanol.

Flocculation is conferred by a number of FLO genes: FLO1 and its alleles, FLO2 and FLO4, together with FLOS, FLO9, and FLO10, which show high homology to FLO1. Collectively, these are responsible for the Flo1 phenotype. Lager strains alone possess another gene, lg-FLO1, which confers the NewFlo phenotype and codes for a flocculin that binds a broader range of sugars than Flo1 types (Verstrepen et al., 2003). Other genes are involved in the regulation of flocculation. The product of FLO8 is a transcriptional activator of FLO1 and another gene FLO11, which is implicated in a stress response in which in some strains' growth becomes pseudohyphal (Bayly et al., 2005). Regulation of FLO11 is particularly complex and both transcriptional and post-translational mechanisms are recognized. Several signalling pathways are involved in its expression. These include a mitogen-activated protein kinase pathway, cAMP protein kinase A pathway, and those involved in quorum sensing and nutritional status (Verstrepen and Klis, 2005). In NewFlo strains, flocculation is triggered when exponential growth ceases. Aside from exhaustion of sugars and the consequent availability of Flo-mediated lectins, regulation involves metabolic activities in which nutrient-sensing pathways via intracellular kinases control regulation of FLO genes.

Flocculation requires cell-to-cell contact to occur and it follows that mechanical agitation is important. Other factors, apart from the presence of sugars, are the concentrations and range of cations, pH, temperature, oxygen, and ethanol. Some of these effects may be purely physical; for example, lower fermentation temperature will decrease natural agitation rates and therefore lessen the probability of cell-to-cell collision. Several cations (Ba2+, Sr2+ and Pb2+) inhibit flocculation, possibly by competing with calcium ions. Others, especially Mn2+ and Mg2+, promote flocculation. Optimum pH for flocculation is in the range pH 3.0-5.0 and is inhibited at wider ranging values. This explains the observation that acid washing of pitching yeast is accompanied by slurries adopting a more fluid, less viscous form. The presence of ethanol promotes flocculation by an unknown mechanism but possibly by its influence on cell hydrophobicity (Soares, 2011).

Flocculation impinges in two ways on the practice of serial repitching. It has been observed that older cells are more flocculent compared with those of a younger generational age, presumably a consequence of increased size of the latter and possibly linked to age-related changes in the cell surface, maybe the presence of greater concentrations of chitin (Powell, 2003). Changes in flocculation have also been linked to genetic instability. Some strains appear very stable in this regard, in others an abrupt shift from flocculant to non-flocculant has been observed in the course of serial repitching. Thus, Powell and Diacetis (2007), working with two brewing strains, observed no changes in flocculence behaviour over several generations. Conversely, Sato et al. (2001) reported that, for a bottom-fermenting strain, loss of flocculence was the most commonly observed change and this was related to loss of the lg-FLO1 gene.

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