Yeast flocculation
Encyclopedia
Yeast flocculation typically refers to the clumping together (flocculation
) of brewing yeast once the sugar in a beer
has been fermented into ethyl
alcohol
. In the case of "top-fermenting" ale
yeast (Saccharomyces cerevisiae
), the yeast creates a "crust" on the top of the liquid, unlike with "bottom-fermenting" lager
yeast (Saccharomyces uvarum) where the yeast falls to the bottom of the brewing vessel.
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
is erroneously credited with first describing flocculation of brewer’s yeast. Brewers yeast flocculation has been the subject of many reviews (Stewart et al., 1975; Stewart and Russell, 1986; Calleja, 1987; Speers et al., 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).
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 was unrecognized but recent work by Speers et al. (2006) 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.
receptors and protein zymolectins (section 1.5.4). Zymolectins are so termed as they may not be true bivalent lectin
s (Speers, Smart, Stewart and Jin,1998) 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.
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).
(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). It is argued that 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). However the molecular proof of this maturation is near non-existent. The picture is complicated by changes in cell surface hydrophobicity and CO2 driven shear which confounds flocculation measurements often erroneously solely attributed to zymolectin interactions.While flocculation (clumping and settling) occurs at this time this flocculation occurs as a result of changes in hydrophobicity and a decline in shear due to which is in turn to low CO2 evolution.
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).
Flocculation
Flocculation, in the field of chemistry, is a process wherein colloids come out of suspension in the form of floc or flakes by the addition of a clarifying agent. The action differs from precipitation in that, prior to flocculation, colloids are merely suspended in a liquid and not actually...
) of brewing yeast once the sugar in a beer
Beer
Beer is the world's most widely consumed andprobably oldest alcoholic beverage; it is the third most popular drink overall, after water and tea. It is produced by the brewing and fermentation of sugars, mainly derived from malted cereal grains, most commonly malted barley and malted wheat...
has been fermented into ethyl
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
alcohol
Alcohol
In chemistry, an alcohol is an organic compound in which the hydroxy functional group is bound to a carbon atom. In particular, this carbon center should be saturated, having single bonds to three other atoms....
. In the case of "top-fermenting" ale
Ale
Ale is a type of beer brewed from malted barley using a warm fermentation with a strain of brewers' yeast. The yeast will ferment the beer quickly, giving it a sweet, full bodied and fruity taste...
yeast (Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
), the yeast creates a "crust" on the top of the liquid, unlike with "bottom-fermenting" lager
Lager
Lager is a type of beer made from malted barley that is brewed and stored at low temperatures. There are many types of lager; pale lager is the most widely-consumed and commercially available style of beer in the world; Pilsner, Bock, Dortmunder Export and Märzen are all styles of lager...
yeast (Saccharomyces uvarum) where the yeast falls to the bottom of the brewing vessel.
Cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
aggregation occurs throughout microbiology, in bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
, filamentous algae, fungi and yeast
Yeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
(Lewin, 1984; Stratford, 1992). Yeast are capable of forming three aggregates; mating aggregates, for DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
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
Agglomeration
In the study of human settlements, an urban agglomeration is an extended city or town area comprising the built-up area of a central place and any suburbs linked by continuous urban area. In France, INSEE the French Statistical Institute, translate it as "Unité urbaine" which means continuous...
(‘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
Louis Pasteur
Louis Pasteur was a French chemist and microbiologist born in Dole. He is remembered for his remarkable breakthroughs in the causes and preventions of diseases. His discoveries reduced mortality from puerperal fever, and he created the first vaccine for rabies and anthrax. His experiments...
is erroneously credited with first describing flocculation of brewer’s yeast. Brewers yeast flocculation has been the subject of many reviews (Stewart et al., 1975; Stewart and Russell, 1986; Calleja, 1987; Speers et al., 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
EDTA
Ethylenediaminetetraacetic acid, widely abbreviated as EDTA , is a polyamino carboxylic acid and a colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely used to dissolve limescale. Its usefulness arises because of its role as a hexadentate ligand...
(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).
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+
Calcium
Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...
, ethanol, etc.; Jin and Speers 1999). Several factors are important in cell-to-cell binding such as surface charge, hydrophobic
Hydrophobe
In chemistry, hydrophobicity is the physical property of a molecule that is repelled from a mass of water....
effects and zymolectin interactions (see following). The importance of these forces in brewing yeast flocculation was unrecognized but recent work by Speers et al. (2006) have indicated the importance of zymolectin and hydrophobic interactions. As the cells are too large to be moved by Brownian motion
Brownian motion
Brownian motion or pedesis is the presumably random drifting of particles suspended in a fluid or the mathematical model used to describe such random movements, which is often called a particle theory.The mathematical model of Brownian motion has several real-world applications...
, 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-mannanMannan
Mannan is a plant polysaccharide that is a polymer of the sugar mannose.Detection of mannan leads to lysis in the mannan-binding lectin pathway.It is generally found in yeast, bacteria and plants. It shows α linkage. It is a form of storage polysaccharide.-See Also:Mannan Oligosaccharides...
receptors and protein zymolectins (section 1.5.4). Zymolectins are so termed as they may not be true bivalent lectin
Lectin
Lectins are sugar-binding proteins that are highly specific for their sugar moieties. They play a role in biological recognition phenomena involving cells and proteins. For example, some viruses use lectins to attach themselves to the cells of the host organism during infection...
s (Speers, Smart, Stewart and Jin,1998) 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
Kluyveromyces
Kluyveromyces is a genus of ascomycetous yeasts in the family Saccharomycetaceae. Some of the species, such as K. marxianus, are the teleomorphs of Candida species....
and Schizosaccharomyces
Schizosaccharomyces
Schizosaccharomyces is a genus of fission yeasts. The most well-studied species is S. pombe. At present four Schizosaccharomyces species have been described . Like the distantly related Saccharomyces cerevisiae, S. pombe is a significant model organism in the study of eukaryotic cell biology...
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.| 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 mannoseMannose
Mannose is a sugar monomer of the aldohexose series of carbohydrates. Mannose is a C-2 epimer of glucose. It is not part of human metabolism, but is a component of microbial cell walls, and is therefore a target of the immune system and also of antibiotics....
(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
Glucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
and maltose
Maltose
Maltose , or malt sugar, is a disaccharide formed from two units of glucose joined with an αbond, formed from a condensation reaction. The isomer "isomaltose" has two glucose molecules linked through an α bond. Maltose is the second member of an important biochemical series of glucose chains....
(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). It is argued that lectin maturation occurs some fourteen hours after the cessation of cell division
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
(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). However the molecular proof of this maturation is near non-existent. The picture is complicated by changes in cell surface hydrophobicity and CO2 driven shear which confounds flocculation measurements often erroneously solely attributed to zymolectin interactions.While flocculation (clumping and settling) occurs at this time this flocculation occurs as a result of changes in hydrophobicity and a decline in shear due to which is in turn to low CO2 evolution.
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).