Clarification & Flocculation

CLARIFICATION:

Clarification of beer involves many factors from biochemical to mechanical. A bright beer is the result of good brewing techniques, an understanding of the fundamentals of clarification, a good filtration system, or a combination of all three.

During the brewing process is possible to face three different kind of haze:

1. Permanent haze caused by bacterial contamination or by the starch conversion through the mashing process not being completed.
2. Chill haze in some cases is created by a protein polyphenol bond or by changes in temperature, appearing at cold temperatures and dissolving at warmer temperatures.
3. Yeast haze will depend on yeast strain flocculation characteristics, which will vanish during the cooling process. In wheat beer style is a favorable attribute.

A hazy beer is commonly blamed on yeast that will not settle, but that is one of several possibilities. Yeast does not stay in suspension without help.

FLOCCULATION:

Flocculation refers to the ability of yeast to aggregate and form large flocs and then drop out of suspension. The definition of flocculation is, “reversible, asexual and calcium dependent process by which cells adhere to form flocs.”

It is very important to understand the basics of flocculation and what affects it because the flocculation and sedimentation process is the easiest and least expensive way to get bright beer. Flocculation also effects fermentation performance and beer flavor. Ideally, yeast will stay non-flocculent and in suspension until the desired final gravity is reached and then become flocculent and drop out of solution. As any brewer knows, yeast do not always cooperate with this concept.

Yeast strains have different levels of flocculation characteristics from non-flocculent (1007 German Ale) to highly flocculent (1968 London ESB).

NON-FLOCCULENT CELLS:

Non-flocculent yeast have cells that appear smooth under a scanning electron microscope and that have a negative surface charge. When these cells are close and moving slowly, they repel each other. If these cells are moving toward each other fast enough, they will overcome the repulsion and collide but will not stick together.

FLOCCULENT CELLS:

Flocculent cells are cells that appear to be covered in hairs or spines under a scanning electron microscope. These cells also have a negative surface charge that causes repulsion between two cells. However, when these cells collide they overcome the repulsion and stick together.

LECTIN HYPOTHESIS:

The Lectin hypothesis is the current hypothesis that describes how yeast flocculate. This hypothesis explains flocculation as controlled by cell-wall to cell-wall interactions, specifically the binding of zymolectins to mannose residues of mannan in the cell wall of yeast cells.

Zymolectins are proteins produced in the cell and then secreted into the cell wall. Zymolectins bind to sugar molecules and require calcium ions to maintain proper configuration for binding these sugars. Zymolectins also bind to cell wall mannose residues of mannan.

Mannan consists of long and branched mannose sugar chains that are present in the cell wall. Mannan is present in the cell walls of all yeast cells. Mannan is attached to long peptide chains anchored in the cell wall.

The zymolectins and cell wall mannans basically work like Velcro. What causes the production and activation of zymolectins is not well understood. It is believed that zymolectins become active at the end of exponential growth and during the stationary phase. Most likely depletion of nutrients and increase of fermentation byproducts (ethanol and pH changes) trigger production and activation of zymolectins.

There are two phenotypes in brewing strains that are defined by the type of zymolectin they produce.

FLO1 PHENOTYPE:

In the Flo1 phenotype, the zymolectins produced bind to only mannose residues and the zymolectins are inhibited only by mannose. In this yeast type, flocculation is not affected by the growth stage of the yeast. Many ale strains fall into this category.

NEWFLO PHENOTYPE:

In the NewFlo phenotype, the zymolectins produced bind to mannose and glucose residues and are inhibited by mannose, glucose, maltose, and sucrose. Flocculation is typically expressed late in the exponential phase and into the early stationary phase. This group contains most lager strains and some ale strains.

CO-FLOCCULATION:

Co-flocculation can occur when a flocculent and a non-flocculent strain are used together. The combination of the two flocculation types can cause both strains to flocculate because the zymolectins of the flocculent strain bind to mannans of the non-flocculent strain. It is difficult to predict whether two strains will exhibit co-flocculation so it is always important to run small scale fermentation trials before using two strains together.

FACTORS THAT PROMOTE CELL AGGREGATION AND ATTRACTION (FLOCCULATION):

The Lectin Hypothesis describes the mechanism that makes yeast cells stick together, but what factors promote this mechanism?

• Genetic background of the strain:
  • For flocculation to occur, the strain must carry the FLO genes responsible for encoding and regulating the production of FLO proteins
  • FLO genes are very unstable and have extremely high frequencies of mutation
  • Inherent instability leads to deletion of FLO genes and loss of flocculation characteristics
• Zymolectin concentration:
  • An increase of zymolectin concentration in the cell wall causes flocculation to occur
  • Factors that increase in zymolectin include:
    • Depletion of nutrients
    • Increase in fermentation by-products
    • Temperature increase
• Mechanical factors that increase collisions between cells and cell aggregation:
  • Turbulence caused by CO₂ production
  • Temperature gradients
  • Higher cell concentration
• Factors that decrease repulsive electrostatic charge:
  • Ethanol concentration
  • pH
  • Changes in the cell wall composition
• Factors that increase Cell Surface Hydrophobicity or CSH:
  • Increases in surface protein concentration
  • Increases in zymolectin density due to the hydrophobic regions of this protein
  • Change in the ratio of phosphorus-rich to nitrogen-rich polypeptides in the cell wall
  • An increase in the production and accumulation of oxylipids, sterols, and fatty acids in the cell wall
• Reduction of zymolectin inhibiting sugars. Over the course of fermentation, sugars that competitively bind to zymolectins will be consumed by yeast; this will make these sites available to cell wall mannans.
• Cell Age:
  • Older yeast cells tend to have rougher and more wrinkled cell walls than virgin cells, which tends to increase their binding ability
  • Older cells tend to have a more filamentous growth and may have a higher density of zymolectins in the cell wall

WHAT DOES ALL OF THIS MEAN TO THE HOMEBREWER?

Flocculation and clarification are complex issues and are affected by many factors. Some of these factors are beyond the homebrewer’s control while others are well within reach. Manipulating factors that affect flocculation will have a direct impact on flavor and aroma of the finished product.

Oxygenation:

• Poor or low wort aeration can result in early and incomplete flocculation
• Adequate aeration can result in delayed and more intense flocculation
• Affects sterol and fatty acid synthesis and presumably cell surface hydrophobicity (CSH)

Temperature:

• Optimum flocculation temperature can vary between strains
• Research trials of lager strains has shown flocculation to be optimal at 50°F (10 °C) and significantly decreased below 41 °F (5 °C)
• Flocculation for one lager strain increased when temperature was raised from 41 °F (5 °C) to 77 °F (25 °C)
• In other research trials, flocculation was repressed at 77 °F (25 °C) and optimal at 41 °F (5 °C)
• By lowering the fermentation temperature, the CO₂ production by the yeast is diminished, causing less turbulence and will promote sedimentation
• Good record keeping will help to determine the optimum temperature range

pH:

• Flocculation is influenced by the wort pH
• Flocculation can occur in a broad range of 2.5 to 9.0
• Optimum range is 3.5 to 4.8 and will vary by strain
• Brewing strains of the NewFlo phenotype occur at a pH of 3.9 to 5.5, with a very strain-specific optimal range

Ethanol Concentration:

• Research trials show that both increases and decreases in ethanol levels can enhance flocculation
• Very strain dependent
• Too high of concentration (10%) becomes toxic to the yeast

Pitch Rate:

• Research has shown a NewFlo strain increase flocculation with gradual pitch rate increase
• Pitch rate increased from 1 million cells/mL to 15 million cells/mL
• Flocculation increased from 58% to 71%
• Higher pitch rates can yield populations with higher percentages of older cells

Trub:

• Influence of trub levels on flocculence varies greatly across yeast strains
• At pH levels below 4.0, electrostatic interactions take place among trub and yeast cells leading to sticky yeast beds in the production of low carbohydrate beers

YEAST HANDLING AND FLOCCULATION/CLARIFICATION:

Pitch Rates:

Standardize pitch rates

• This will remove one factor from the equation to determine sources for changes in flocculation

Yeast Cropping/Harvesting:

• Cropping of yeast for subsequent repitching is very important for maintaining proper flocculation characteristics.
• Cropping from different layers can be used to adjust and maintain flocculation characteristics. Cropping from the middle layer of yeast in the yeast bed will select for the highest flocculation. However, in homebrewing the different layers will be difficult to determine since the carboy is flat at the bottom. Harvesting will result in a combination of yeast from all layers that will vary in viability, vitality, and flocculation characteristics.

Generation:

• The flocculence of a yeast strain will change with serial repitching. This is due to changes in the cell wall composition and genetic variation.
• Very strain dependent; some strains are much more stable than others.

CONCLUSION:

Flocculation is one of the most complex and least understood mechanisms that yeast have. It is very difficult to determine exactly why a yeast strain has had a change in flocculation characteristics. Good and consistent record keeping combined with good and consistent brewing techniques will minimize the number of unknown factors affecting yeast.