Yeast flocculation

Yeast flocculation typically refers to the clumping together (flocculation) of brewing yeast once the sugar in a beer brew has been fermented into ethyl alcohol. In the case of "top-fermenting" ale yeast ("Saccharomyces cerevisiae"), the yeast sinks to the bottom of an open tank; as it does with ["bottom-fermenting" [lager] yeast ("Saccharomyces pastorianus").

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.

Yeast flocculation is distinct from agglomeration (‘grit’ formation), which is irreversible and occurs most commonly in bakers yeast when strains 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.

Louis 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). 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 complementary yeast cells (Calleja, 1987; Stratford, 1992).

For flocculation to occur the yeast must be flocculent and the environmental conditions (such as low amount of agitation, absence of sugars, a microamount of Ca2+, ethanol, etc.; Jin and Speers 1999). Several factors are important in cell-to-cell binding such as surface charge, hydrophobic effects and zymolectin interactions (see following). The importance of these forces in brewing yeast flocculation is debated but recent work by Speers et al. (2006) [Speers, R.A., Wan, Y-Q., Jin, Y-L., and R. J. Stewart, R.J. 2006. Effects of fermentation parameters and cell wall properties on yeast flocculation. J. Inst. Brew. 112:246-254.] have indicated the importance of zymolectin and hydrophobic interactions. As the cells are too large to be moved by Brownian motion, for binding of two or more cells to occur the cells must subjected to low level of agitation.

Zymolectin 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 zymolectins (section 1.5.4). Zymolectins are so termed as they may not be true bivalent lectins (Speers, Smart, Stewart and Jin,1998) [Speers, R.A., Smart, K., Stewart, R., Jin, Y-L., 1998. Zymolectins in Saccharomyces cerevisiae. Letter J. Inst. Brew., 104:298.] It has been suggested that zymolectin interactions between the protein and mannan moities 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 Zymolectins and Phenotypes

Three flocculation phenotypes have been elucidated based on the zymolectins 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.

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 may 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 picture is complicated by changes in cell surface hydrophobicity which confounds flocculation measurements often erroneously solely attributed to zymolectin interactions.

The "MI" phenotype appears to occur in ale but not lager 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 zymolectin-like reaction in the presence of mannose (Dengis and Rouxhet, 1997; Guo et al, 2000; Smart, 2001).

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