Yeast flocculation

Yeast flocculation typically refers to the clumping together of brewing yeast once the sugar in a beer brew has been converted into alcohol. In the case of ale yeast Saccharomyces cerevisiae the yeast floats to the top of an open tank, whereas with lager yeast Saccharomyces pastorianus the yeast will sink to the bottom of the tank.

Cell aggregation occurs throughout microbiology, in bacteria, filamentous algae, fungi and yeast (Lewin, 1984; Stratford, 1992). Yeast are capable of forming three aggregates; mating aggregates, for DNA exchange; chain formation, for development and differentiation; and flocs as a survival strategy in adverse conditions (Calleja, 1987). Brewing strains are polyploid so mating aggregates do not occur. Therefore only chain formation and flocculation are of relevance to the brewing industry.

Flocculation is distinct from agglomeration (‘grit’ formation), which is irreversible and most commonly in bakers yeast strains of fail to separate when resuspended (Guinard and Lewis, 1993). Agglomeration only occurs following the pressing and rehydration of yeast cakes and both flocculent and non-flocculent yeast strains have been shown to demonstrate agglomeration (Guinard and Lewis, 1993). It is also distinct from the formation of biofilms, which occur on a solid substrate.

Pasteur first described flocculation of brewer’s yeast in 1876 (Pasteur, 1876) which has since been the subject of many reviews (Stewart et al, 1975; Stewart and Russell, 1986; Calleja, 1987; Speers et al, 1992; Stratford, 1992; Jin and Speers, 1999, Smart, 2001). Flocculation has been defined as the reversible, non-sexual aggregation of yeast cells that may be dispersed by specific sugars (Burns, 1937; Lindquist, 1953, Eddy, 1955; Masy et al, 1992) or EDTA (Burns, 1937; Lindquist, 1953). The addition of nutrients other than sugars has been demonstrated not to reverse flocculation (Soares et al, 2004). This is as opposed to mating aggregates formed as a prelude to sexual fusion between complimentary yeast cells (Calleja, 1987; Stratford, 1992).

Flocculation is a bimodal process in which a non-flocculent population develops into one comprising of flocculent and non-flocculent cells (Miki et al, 1982). The efficiency of flocculation is determined by the timing of flocculation onset and the rate of flocculation in conjunction with the ratio of flocculent to non-flocculent cells (Stratford and Keenan, 1987; 1988; van Hamersveld et al, 1996). The rate-limiting step is doublet formation, requiring the presence of active surface proteins (Stan and Despa, 2000). The mechanism by which this occurs is thought to be the lectin interaction theory.

Lectin Interaction Theory The accepted mechanism of flocculation involves a protein-carbohydrate model (Miki et al, 1982) (figure 1.3). Fully flocculent yeast cells exhibit carbohydrate α-mannan receptors and protein lectins (section 1.5.4). It has been suggested that lectin like interactions between the two results in the flocculation phenotype (section 4.1) with Ca2+ ions required for the correct conformation of the flocculation lectins. Coflocculation between Kluyveromyces and Schizosaccharomyces has been shown to be by a “lectinic” mechanism (El-Behhari et al, 2000). This theory explains the essential role of calcium and how deproteinisation affects flocculation. Flocculation Lectins and Phenotypes Three flocculation phenotypes have been elucidated based on the lectins they produce: Flo1 (Stratford and Assinder, 1991) NewFlo (Stratford and Assinder, 1991) and Mannose Insensitive (MI) (Masy et al, 1992; Dengis and Rouxhet, 1997). These flocculation phenotypes differ in the time of the onset of flocculation and the sugar inhibition of flocculation. Flocculation has also been classified according to time of onset and floc morphology.

Gilliland Class	Flocculation Characteristics I	Completely Dispersed II	Flocculating into small, loose lumps late in fermentation III	Flocculating into dense masses late in fermentation IV	Flocculating very early in fermentation owing to non-separation of daughter cells

The genetic control of yeast flocculation has not been extensively studied. Recent reports suggest genes encoding lectin-like proteins exhibit close sequence homology (Jin and Speers, 1991, 1999; Smart, 2001). Furthermore it seems that FLO genes have interchangeable functions that can compensate for one another (Guo et al, 2000).

Flocculation Phenotypes The Flo1 phenotype is inhibited by mannose (Burns, 1937; Miki et al, 1982; Nishihara and Toraya, 1987; Kihn et al, 1988; Stratford, 1989; Stratford and Assinder, 1991) occurs in both ale and lager strains (Miki, 1982; Stratford and Assinder, 1991; Masy et al, 1992; Smit et al, 1982; Stratford, 1993; Stratford and Carter, 1993; Teunissen et al, 1993; Teunissen et al, 1995a, b; Bony et al, 1997; Braley and Chaffin, 1999; Fleming and Pennings, 2001; He et al, 2002; Verstrepen et al, 2003) and is associated with the FLO1 gene (Watari, 1991 Masy et al, 1992; Stratford, 1993; Stratford and Carter, 1993; Teunissen et al, 1993; Teunissen et al, 1995a, b; Bony et al, 1997; Braley and Chaffin, 1999).

The NewFlo phenotype differs from that of FLO1 in several ways. Firstly NewFlo flocculation is inhibited by mannose, glucose and maltose (Stratford and Assinder, 1991; Masy, 1992; Rhymes, 1999). Secondly the NewFlo lectin is putatively encoded by the FLO10 gene (Guo et al, 2000; Smart, 2001) and is not expressed until stationary phase onset (Stratford, 1989; Stratford and Assinder, 1991; D’Hautcourt and Smart, 1999). Thirdly lectin maturation occurs some fourteen hours after the cessation of cell division (Stratford, 1989; Stratford and Assinder, 1991; Masy 1992; D’Hautcourt and Smart, 1999) and is therefore not concomitant with entry into stationary phase, although this is strain dependent (D’Hautcourt and Smart, 1999; Verstrepen et al, 2003).

The MI phenotype appears to occur in ale (Saccharomyces cerevisiae, but not lager (saccharomyces pastorianus) strains (Masy et al, 1992; Dengis and Rouxhet, 1997; Jin and Speers, 1999) and is considered to be a rare phenotype. The FLO11 gene has however been identified as being essential for flocculation in S. bayanus (Ishigami et al, 2004) and characteristics such as invasive growth and pseudohyphal formation in Saccharomyces cerevisiae (Lo and Dranginis, 1998; Gagiano et al, 1999; Gancedo, 2001; Gagiano et al, 2002; Gagiano et al, 2003; Verduzco-Luque et al, 2003; Vivier et al, 2003; Guldener et al, 2004). Although this flocculation phenotype has not been fully characterised, it is differentiated from other flocculation phenotypes by a lack of inhibition of the lectin like reaction in the presence of mannose (Dengis and Rouxhet, 1997; Guo et al, 2000; Smart, 2001).