Flocculation

Flocculation is the ability of yeast cells to agglomerate or adhere to one another in the form of clumps. When lager yeasts flocculate, the clumps have a density greater than that of the beer and settle to the bottom. Ale yeasts, in contrast, form clumps or flocs that entrap CO2 bubbles and have a lower density, and, therefore, rise to the surface.

The ability of yeast cells to clump or flocculate, and the time at which flocculation occurs, are very important properties in beer manufacture. In most cases, flocculation should occur at the end of the fermentation, when all of the monosaccharides (glucose and fructose), disaccharides (sucrose and maltose), and trisaccharides (maltotriose) have been fermented. The yeast will have done its job, fermentable sugars are depleted, and the beer is considered to be fully attenuated.

Importantly, the beer will be clear or "bright," even in the absence of additional clarification steps. If, however, flocculation occurs prematurely, before the end of the fermentation, fermentable sugars will remain in the beer (a situation brewers refer to as a "hanging fermentation").These residual sugars can affect maturation and flavor. In particular, these unat-tenuated beers have a lower than normal ethanol concentration and are relatively sweet which, depending on the intent of the brewer, may or may not be desirable.

Of course, the presence of sugars in the beer also provides growth substrates for other organisms. In contrast, yeast cells that fail to flocculate, and instead remain in the beer, are difficult to remove, causing cloudiness problems. These yeasts may later autolyze in the beer, releasing enzymes that contribute to yeasty flavor defects.The onset of flocculation appears to be triggered by several factors, including entry into stationery phase, low pH, low temperature, low sugar concentration, and high ethanol concentrations. Hops and calcium (see below) also promote flocculation activity.

Despite the importance of flocculation in the brewing process, the physical-chemical basis of this property is not well understood. Ale yeasts, in general, have been reported to be more hy-drophobic than lager yeasts, due to differences in cell surface charges, but it is not clear that this property affects flocculation. Rather, it now appears that flocculation occurs as a result of lectin-like domains, contained within cell surface proteins, that bind in the presence of ionic calcium to mannans (mannose-containing chains) located on the surface of adjacent cells. Floccu-lation is also a heritable property, meaning it has a genetic basis. Several genes have been identified in S. cerevisiae that code for proteins involved in flocculation, and efforts are now under way to manipulate floc gene expression during beer making (Box 9-7).

As noted earlier, beer manufacture, at first glance, would appear to be a rather simple exercise: barley is converted to malt, which is converted to wort, which is then fermented by yeast.The problem is that at this point the beer still contains yeasts, other microorganisms, and other insoluble and non-dissolved materials that give it an undesirable cloudy and hazy appearance. In addition, this so-called "green" or immature beer may contain chemical constituents that impart off-flavors. Finally, little or none of the carbon dioxide produced during the primary fermentation is retained in the beer, leaving the product without one of its key components.Thus,the challenge for the brewer is to promote acceptable flavor development and maturation, to remove the undesirable cloud- and haze-forming materials, and to introduce carbona-tion into the beer. Indeed, it can be argued that the post-fermentation steps are as important as any of the preceding activities.

Flocculation should occur as the fermentation ends, or more precisely, when the fermentable sugars are depleted and the beer is fully attenuated. Once the yeasts have flocculated, they are usually collected promptly and removed from the beer. It is important to recognize that, even though most of the yeast cells are removed by the sedimentation or skimming step, the beer still contains yeast cells. In addition, this green beer typically contains undesirable flavor compounds, including sulfur dioxide and diacetyl, as well as proteins and tannins that can potentially form haze complexes. Therefore, it is essential that the beer be "conditioned" to enhance sedimentation of the remaining yeasts and haze-forming proteins, promote dissipation of off-flavors, and produce beer with a mature or finished flavor. Since yeast growth also occurs during the conditioning period, a secondary fermentation may take place, resulting in formation of CO2. Depending on the specific conditions, enough CO2 may be produced to naturally carbonate the beer.

Box 9—7. Flocculation—A Case of Beer Yeasts Sticking Together

The ability to flocculate, consistently and at the right time during the fermentation, is one of the most important traits of a good brewing yeast.If flocculation occurs too early or too late, beer flavor, clarity, and overall quality are bound to suffer.Yeast floccuation is now thought to be mediated via a lectin-like mechanism in which the N-terminal region of a cell wall-associated protein recognizes and binds, in a calcium-dependent manner, to mannose or trimannoside oligosaccharides located on the surface of other cells (Figure 1; Kobayashi et al., 1998; Javadekar et al., 2000; Verstrepen et al., 2003). Although both flocculating and non-flocculating cells contain this man-nan receptor, only flocculating cells produce the binding protein. Importantly, flocculation is influenced by several physiological and environmental factors, including growth phase, ethanol and sugar concentrations, and calcium ion availability (Table 1).These observations suggest that flocculation depends not only on physical interactions, but is also subject to genetic regulation.

Five genes (FLO1, FLO5, FLO9, FLO10,and FLO11) currently have been identified in Saccha-romyces cerevisiae that encode for proteins involved in adhesion (Halme et al., 2004). At least two of these genes, FLO5 and FLO9, have high sequence homology to FLO1. However, several

Flocculation Cells

Figure 1. Model for yeast flocculation. Flocculating cells produce cell wall-attached lectin-like proteins that recognize and bind to mannan regions on the surface of receptor cells. The man-nan receptors are present on most cells; thus, it is the expression of the lectin-like proteins that is responsible for the flocculation phenotype. Adapted from Verstrepen et al., 2003.

Table 1. Factors affecting flocculation.

Figure 1. Model for yeast flocculation. Flocculating cells produce cell wall-attached lectin-like proteins that recognize and bind to mannan regions on the surface of receptor cells. The man-nan receptors are present on most cells; thus, it is the expression of the lectin-like proteins that is responsible for the flocculation phenotype. Adapted from Verstrepen et al., 2003.

Table 1. Factors affecting flocculation.

Factor

Effect

Fermentable sugars Nitrogen, other nutrients Temperature pH

Oxygen Ethanol Cell age

Inoculum handling

Inhibitory Little effect

Strain-dependent, with broad range Optimal flocculation at pH 3.5—5.8 No direct effect

Strain-dependent, affects cell surface Old cells flocculate more than young cells Ambient temperature increases flocculation

Box 9—7. Flocculation—A Case of Beer Yeasts Sticking Together (Continued)

different adhesion phenotpyes exist, including cell-to-cell adhesion (i.e. flocculation), as well as adherence to other surfaces.

The expression of these dominant genes is complex and unstable, and appears to be controlled by both genetic and epigenetic mechanisms (Halme et al., 2004).Whereas FLO11 is ordinarily expressed (in a lab strain), the other four genes are silent. Expression of FLO11 gives a non-flocculating (but adhesion positive) phenotype, whereas expression of the silent FLO genes (via a heterologous promoter) results in flocculating adherence. Epigenetic silencing of FLO11 also causes a morphological change in the appearance of the cells, from yeast cells in a filamentous form to yeasts that grow as discreet cells.

Depending on growth and environmental conditions, four different flocculation phenotypes have been described for brewing strains of S. cerevisiae (Soares et al., 2004). In cells with a FLO1 phenotype, flocculation is inhibited by mannose.The NewFlo phenotype is inhibited by mannose and glucose.The MI phenotype is mannose insensitive. Finally, flocculation can be ethanol-dependent. While flocculation by most FLO1 yeasts is constitutive, flocculation by NewFlo cells occurs during stationary phase.Thus, most lab strains have the FLO1 phenotype and most brewing strains have the NewFlo phenotype (Javadekar et al., 2000).

Brewing yeasts ordinarily do not flocculate in the presence of glucose and other fermentable sugars. Furthermore, when fermentable sugars are added to flocculant cells, the flocs will disperse and subsequent flocculation is lost (Soares et al., 2004). It also appears that, under experimental conditions, the rate of fermentation-dependent loss of flocculation correlates with growth rate and sugar use.That is, the faster the cells grow, the faster the flocs come apart. Despite these findings, however, it is not clear at a molecular level how the cells sense sugar availability and then respond by inducing or repressing flocculation. Recently, it was suggested that the onset of flocculation occurs as a result of the combined effects of nutrient shortage (of either fermentable sugars or nitrogen) and the presence of ethanol (Sampermans et al., 2005).

Efforts to modify or improve flocculation properties of brewing yeast are being pursued actively as part of current strain improvement programs. Originally, plasmid vectors were used to deliver flocculation genes (e.g., FLO1), but because expression was constitutive, these transformed strains had limited application for brewing. In one recent study, however, researchers sought to manipulate the onset of flocculation by using an inducible promoter (Verstrepen et al., 2001).A non-flocculating wild-type strain, harboring a genomic copy of the FLO1 gene (encoding

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