#61 Berliner Weisse the Second

Eureka, it’s time for another recipe blog entry. Another Berliner Weisse recipe. I finally found some spare time to write-up the following post. I did a Berliner Weisse before and let the mash turn sour by adding some grains (#44 Berliner Weisse). I added some Brettanomyces to one share of the batch and I am very happy how this beer turned out. You can find the tasting notes of my first batch here.

Because I am very inquisitive and got a package of Wyeast’s 3191 Berliner Weisse Blend, I could not resist to try the blend on pretty much the same Berliner Weisse recipe as mentioned above. The only difference was to leave the sour mashing aside because there should be some lactic acid bacteria in the blend to turn the beer sour. Lets go through the recipe and lets find out what Wyeast’s Berliner Weisse Blend can make out of the wort.

Recipe:	Berliner Weisse 2
Numbers:	Volume [L]	20 (5.3 gal)
	Original gravity	8.2°P (1.032)
	Terminal gravity	1°P (1.003)
	Color	Around 4 EBC
	IBU	N/A
	ABV	3.8% (v/v)
Grains:	Pilsner malt (4 EBC)	1.9 kg
	Wheat malt (4 EBC)	1.4 kg
Hops:	Hallertauer (4.2% AA)	26.6 g and added at mash in (mash hops)
Yeast:	Wyeast	#3191 Berliner Weisse Blend
Water:	Burgdorf	Mash: 8.5 L (2.2 gal), sparge: 18 L (4.8 gal) @78°C (172°F)
Rest:		Mash in @66°C (151°F), 60 min @66°C (151°F), 15 min @ 78°C (172°F)
Boil:		No boil
Fermentation:	Primary	Close to 6 months @20°C (68°F) in plastic fermenter
	Secondary	N/A
Maturation:	Carbonation (CO2 vol)	2 with sugar addition
	Maturation time	Weeks to months at 15°C (59°F)

08.01.2012: Brew day. Crushed malts, mashed in at 66°C (151°F) including adding the Hallertauer hops and left the mash rest for one hour. Then heated up to 78°C (172°F), sparged, cooled the wort down to roughly 20°C (68°F) and added a package of Wyeast’s 3191 Berliner Weisse Blend and left the fermenter untouched for nearly 6 months. I did not even rack the beer into a secondary fermenter.

10.20.2012: Bottled the beer with a sugar addition to a carbonation level of 2 vol of carbon dioxide and store the bottles at 15°C (59°F) since.

02.05.2013: Beer now 3.5 months in bottle. Its time for a first official tasting.

Aroma: Citrus character, some metallic notes as well and maybe some DMS (not sure)

Appearance: Straw yellow, clear, nice bubbles rise to the top and form a two finger white head

Flavor: Some citrus character, light Pilsner malt character (something between honey, corn and bread), no funk or even a hint of sourness…

Mouthfeel: Light body, average carbonation level (for the style), dry finish, hint of sourness in the aftertaste

Overall Impression: There are a lot of the typical Berliner Weisse aroma and taste characteristics present in this beer with one exception: the sourness. It is far from what I would expect from a Berliner Weisse. I don’t know if the sourness will increase by further maturating the beer. We will see.

04.14.2013: Six months in the bottle:

Aroma: Smells like a ripe apple. Very Champagne like aroma. No sourness, no funk, no hops or yeast character in the nose

Appearance: Straw yellow, light haze (got some of the sediment in the glass), white head and lots of bubbles

Flavor: Some cereal character, no hops or any yeast specific character such as esters. Hint of lactic acid sourness

Mouthfeel: Light body, average carbonation level, dry finish, some sourness in the finish

Overall Impression: Beside the cereal flavor, this beer is very much like a Champagne or a carbonated white wine. Compared to the previous tasting, the sourness level increased a bit. But still not at the level where I want it to be. I brought this beer to a party recently and described the beer as a Champagne-beer. And you know what? The tasters brave enough to taste it totally agreed and where really astonished that a beer can even taste like a Champagne.

Compared to the first version (#44 Berliner Weisse) where I used a spontaneous mashing technique, this beer’s taste is not even close to the first version. In my experience, the sour mashing step introduced a lot of the funk character (sourness, lactic acid smell, lemons) I would like to have in my version of a Berliner Weisse. In addition, the Brettanomyces I added back then did a very good job and added a very funky layer to the Weisse. We will see how this beer evolves. Cheers!

A glimpse into yeast flocculation

Eureka, science post! This is an entire review post about yeast flocculation. Flocculation describes the ability of yeast cells to aggregate into clumps/flocs and then drop out of suspension. This happens during the end of fermentation and the yeast cells form a sediment at the bottom of the fermenter. The flocculation treat is mainly genetically derived and thereby depends on the yeast strain. Flocculation characteristics can sometimes change and lead to early flocculation to occur or to loss of flocculation. Despite the genetics, there are a lot of ways a homebrewer can influence the yeast flocculation.

In this post I would like to cover the basic principles how flocculation functions on a genetic and biochemistry level, then speak about factors influencing the flocculation and end with some words about how a homebrewer can influence the flocculation of yeasts. Lets begin with a general overview about flocculation.

Yeast flocculation profiles can be distinguished into three groups:

• High flocculate strain (strongly sedimenting): Flocculation starts after 3-5 days (if kept at correct fermentation temperature). These strains tend to flocculate earlier during the fermentation and form a sediment at the bottom of the fermenter. Most of the English yeasts belong into this group. Such yeasts tend to lead to lower attenuation (higher terminal gravity) and sweet beers since the yeasts cells are not in suspension anymore and in contact with the sugars. In addition, a lot of fermentation byproducts stay in the beer such as diacetyl and esters for example.
• Medium flocculate strain (powdery): Flocculation starts after 6-15 days. Typical ale strains and lager strains. Such yeasts give you a clean and balanced beer. Such yeast stay in contact with the beer and can continue to ferment and metabolize fermentation byproducts such as diacetyl.
• Low flocculate strain (non-flocculate): Flocculation starts > 15 days. Most of the wild yeasts, Hefeweizen- and Belgian yeasts plus some lager strains belong into this category. Such yeasts tend to stay in suspension and lead to a cloudy, yeasty beer. In addition, such strains can make filtering of beer rather difficult.

Comparing the three different groups above, it is obvious that non-domesticated yeasts (named wild yeasts) are low flocculating. The flocculation character in domesticated yeast cells got improved by selective pressure. One easy way to do so it harvest the yeast from the bottom of the fermenter and therefore only harvest the highly flocculent yeasts. More about that later on.

How flocculation in yeast works

As already mentioned, the flocculation is mainly genetically driven. I would like to start with the phenotypes first and then get into the genetical setup since it might be easier to understand the different genes and what they do.

Cell biology of flocculation

One trait that influences flocculation is the charge surface charge of the yeast cell. The surface charge is mainly negatively charged but the charge depends on the strain, the phase of growth, the oxygen content in the wort, starvation of the cell and cell age. Most of these factors can be influenced by the brewer. Due to the negatively charged surface yeast cells repel each others. Such repulsions prevent yeasts from flocculating since flocculation involves yeast cells to get in contact first. Top-fermenting strains seem to have a less negatively charged cell surface than bottom-fermenting strains (Amory and Rouxhet, 1988).

The yeast cells have a cell membrane and a cell wall. The cell membrane’s function is mainly to regulate what gets in and out of the cell. The cell wall’s job is to stabilize the whole cell and is therefore responsible for the integrity of the yeast cell. One of the most important building blocks is mannan. We will come back to the cell membrane and mannan later on.

Non-flocculent yeast cells appear as smooth cells on a SEM (scanning electron microscope) micrograph and flocculent yeast cells appear to have some sort of hairs. Non-flocculent yeast strains collide but don’t form clumps. On the other hand, flocculent strains form clumps if they collide. As previously mentioned, yeast cells are in general negatively charged and therefore repel each other. What is the reason for the flocculent yeast strains to form clumps then?

Biochemistry of flocculation

Yeast cells like mammalian cells have a lot of surface proteins on/in their cell membranes. Such proteins are necessary for the yeast strains for signalling (interacting with the environment) and get molecules into and out of the cell. One could easily write books only about surface proteins and that’s why I will not get into further details here. One possible way to explain the interaction of flocculent yeasts is the lectin hypothesis.

Fig 1: Mannose and glucose structures

This hypothesis states that controlled interactions of specific surface proteins between different yeast cells are involved in the flocculation. One such protein is called zymolectin which is produced by the yeast cell and then incorporated into the cell wall. As one can tell zymolectin belongs to the family of lectins which is a family of proteins that bind sugars. Zymolectin can bind the sugar molecule mannose (Fig 1). In addition, it can also bind to mannan, the building block of the cell wall (Fig 2), which is made from mannose molecules. A bond between zymolectin and mannan (from different yeast cells) therefore links two cells together and initiates the formation of yeast flocs.

Fig 2: Mannan structure

The critical step for the flocculation to occur is the point where zymolectin gets active and establishes the connection to another yeast cell. Not much is yet known about the zymolectin expression. Zymolectin may become active at the end of exponential growth and might be triggered by depletion of nutrients such as sugars and an increase of fermentation byproducts such as ethanol. Lets have a closer look at the zymolectin family members.

• Flo1 (Flocculin-1): (http://www.uniprot.org/uniprot/P32768) Synonyms are FLO2 and FLO4. This protein selectively binds to mannan residues in the cell wall and is inhibited by mannose but not glucose, maltose, sucrose of galactose. The protein is 1,537 amino acids (aa) long and has a sugar recognition site between position 197- 240. Interestingly, there are 18 repeated domains (flocculin repeats) in this protein each with a length of 45 aa plus a PA14 domain which is responsible for binding sugars (Fig 3)
  

  Fig 3: FLO1 with 18 flocculin repeats (red) and a PA14 domain (blue) (Pfam)

• Flo5 (Flocculin-5): (http://www.uniprot.org/uniprot/P38894) 1,075 aa long. The protein consists of one P414 domain, 8 flocculin domains and 3 flocculin type 3 domains. Plus a sugar binding site
• Flo8 (Transcriptional activator FLO8): (http://www.uniprot.org/uniprot/P40068) 799 aa long. Putative transcription factor of FLO1, FLO9 and FLO11/MUC1
• Flo9 (Flocculin-9): (http://www.uniprot.org/uniprot/P39712) 1,322 aa long. The protein consists of one P414 domain, 13 flocculin domains and 3 flocculin type 3 domains. Plus a sugar binding site
• Flo10 (Flocculin-10): (http://www.uniprot.org/uniprot/P36170) 1,169 aa long. The protein consists of one P414 domain
• Flo11 (Flocculin-11): (http://www.uniprot.org/uniprot/P08640) 1,367 aa long. No conserved domains found. This protein is involved in filamentous growth (see next post)
• Lg-Flo1 (must be present in lager yeast)
• NewFlo: These proteins bind to mannose and glucose. Mannose, glucose, maltose and sucrose can inhibit zymolectin. There are two different proteins belonging into this group of zymolectins:Lg-Flo6p: (http://www.uniprot.org/uniprot/E9P9E1) 428 aa. Not much is known for this protein. However, there is a PA14 domain and 3 flocculin repeats presentLg-Flo10p: (http://www.uniprot.org/uniprot/E9P9E2) 492 aa. Not much is known for this protein either. However yet again, one PA14 domain and 5 flocculin domains

The longer the flocculin protein (the more flocculin repeats), the stronger the flocculation is (Vidgren et al 2011). Flo1 therefore shows a strong flocculation character. The NewFLo phenotype is very common in brewer’s yeast. Lets summarize, so far three groups of flocculation phenotype have been described:

• Flo1 type (is inhibited by mannose only). This phenotype occurs in Lager and ale yeast strains and is associated with FLO1 gene. Flocculation occurs independently on wort sugars (not suitable for brewing)
• NewFlo type (is inhibited by mannose, glucose, maltose, sucrose). Suited for brewing. Flocculation occurs if wort sugars are metabolized.
• Mannose insensitive. This phenotype occurs in ale but not in lager strains. Calcium ions are necessary. As it can be concluded from the name, this phenotype is not inhibited by mannose. Flocculation can be induced by low ethanol concentrations (Dengis et al, 1995). One possible mechanism for this phenotype might be by simply changing the cell surface charge. However, the evidence that small amounts of calcium are necessary and that FLO11 is involved points to an adhesion-mediated mechanism as well but not based on flocculin repeats.

In addition to the three groups, co-flocculation can occur as well if a non-flocculent and a flocculent strain get in contact. In this case the zymolectin from the flocculent strain binds to the mannose of the non-flocculent strain and pulls the non-flocculent strain down. Co-flocculation can occur with bacteria such as Acetobacter, Lactobacillus and Pediococcus as well (Vidgren et al 2011).

Genetical setup of flocculation

One to three genes are present in yeast strains which are inherited dominant. Flocculation therefore can be improved by crossing yeast strains: Cross a high flocculent strain with a low flocculent strain leads to a high flocculent yeast. Although the flocculent trait is dominantly inherited, flocculation can also decrease.

• FLO1 (Flocculin-1) Located on chr01 and encodes Flo1 protein https://www.ncbi.nlm.nih.gov/nuccore/NM_001178230.1 4,614 bp. No introns. This gene seems to be Saccharomyces specific since I could not find any other organism with similar genes.FLO2 and FLO4 are alleles of FLO1 and FLO5, FLO9 is a homologue of FLO1. Any expression of FLO1, FLO2, FLO4, FLO5 or FLO9 leads to the initiation of flocculation of the Flo1 phenotype.
• Lg-FLO1 can be found in Lager yeasts and is responsible for the NewFlo phenotype

The FLO genes are relatively unstable due to mutations and the highly repetitive pattern due to flocculin repeats. Highly repetitive sequences in the genome change more rapidly than regions with less repetitive motifs (Vidgren et al, 2011). A lot of mutations happen in the FLO genes and the most commons ones lead to deletions or any other alterations leading to a decrease of flocculation. In addition, FLO genes are near telomeres (ends of chromosomes) and can get transcriptionally silenced. Nevertheless, flocculation not solely relies on the FLO genes but implies physical interactions of yeast cells (collision of yeast cells).

Putting it all together. For flocculation to occur the following factors have to be true:

• Flocculins have to be expressed by the yeast and present in the cell wall (for Flo1 and NewFlo type)
• Physical interaction between yeast cells
• Absence of inhibitory sugars (in NewFlo type)
• Small amounts of calcium ions present. Calcium is necessary for the correct conformational shape of the zymolectin molecules
• Right environmental conditions

Environmental factors influencing flocculation

Now that we covered the biochemistry and genetics lecture part about flocculation, let’s have a look at some environmental factors affecting flocculation.

What environmental factors influence yeast flocculation?

• Fermentation temperature
  • Lower temperatures seem to initiate flocculation as well as higher temperatures above the recommended fermentation temperatures
• Wort pH. Top-fermenting yeast strains flocculate within a pH range of pH 3 – 4.5, bottom fermenting ones between pH 3.5 – 6
• Original gravity
• Oxygen content added
  • Poor wort aeration can result in an early flocculation. Oxygen content at pitching increases sterol and fatty acid content in cell membrane and increases the cell surface hydrophobicity
• Depletion of inhibitory sugars such as sucrose, glucose, maltose (all inhibit flocculation in NewFlo type only)
• Increase of fermentation byproducts such as ethanol can influence flocculation as well
• Factors increasing the chance that yeast cells collide
  • Pitching rate (higher pitching rate gives a higher yeast cell density)
  • turbulence by carbon dioxide production
  • Yeast age. Older yeast cells tend to have a rougher cell surface due to the undergone budding events and are therefore prone to stick to other cells
• Factors decreasing the cell surface charge (decrease of electrostatic repulsion)
  • Ethanol concentration
  • pH of wort
  • Changes in cell wall composition
  • Expression and incorporation of flocculins into the yeast cell wall
• Premature yeast flocculation-inducing factors (PYE) from the barley husks can lead to premature flocculation. Barley produces PYE as a response to microbial growth during the steeping process. Further investigations are necessary to fully understand the PYE influence on flocculation

This list might look very frightening to homebrewers. A closer look reveals some common factors which can be broken down into:

• Adequate oxygenation of the wort. Poor oxygenation not only leads to possible off-flavors but to incomplete fermentation due to delayed flocculation and reduced sterol content in the cell membranes
• Temperature. Flocculation is temperature dependent. In general a lower temperature favors yeast flocculation. However, this is very yeast strain dependent
• Pitching rates. Higher pitching rates increase amount of older cells and therefore favors flocculation. However, I do not recommend to overpitch to improve the flocculation character of a yeast strain

Beside oxygenation, temperature and pitching rates, how can a homebrewer lower the changes to encounter problems due to different flocculation behaviour?

• Choose the right yeast strain. If you plan on brewing a clear beer, better stick to a yeast strain with a high to very high flocculation potential. Flocculation behaviours can be looked up on the yeast suppliers webpages
• Decrease temperature to 0°C (32°F) after the fermentation reached terminal gravity. Lowering the temperature results in higher flocculation rates and leads to clearer beers. Don’t chill the beer too early
• Get yeast out of beer by filtration or centrifugation (if you can’t wait for the yeast to drop out itself)
• Add collagens (positively charged) and pull down the yeast cells. This is commonly used in real ales by adding Isinglass. By doing this, one can use the positive character a low flocculate yeast strain might contribute to a beer without having a cloudy pint of beer in the end
• Collect yeast from the bottom of the fermenter or from kräusen and thereby select for the highly flocculate yeast cells. If you collect yeast from the yeast cake, the most flocculate yeasts will be in the middle part of the yeast sediment. The non-flocculate or poorly flocculate yeasts will be in the top layer and older cells, dead cells in the bottom layer
• Yeast storage. Use a method without excessive stressing the yeast cells such as low/high osmolarity of the storage media. Storing yeast at lower temperatures (4°C) can result in reduced flocculation. However, these effects are strain dependent
• Keep acid washing steps at a minimum. Washing cells with acid can change the surface protein composition and therefore might have an impact on the surface charge and surface hydrophobicity
• Avoid excessive re-pitching of the same yeast over and over again. Don’t re-pitch your yeast for more than 5 – 10 times.

What to do if your yeast does not flocculate as before?

• A change of flocculation behaviour can have several causes such as mutations, mixed cultures (infections), different environmental factors. Finding the cause for the different flocculation behaviour might be hard. Therefore:
• Don’t use the same strain for another batch of beer. Start with a fresh yeast
• If a high flocculent strain is used, get the yeast back into suspension by either swirling or venting some carbon dioxide into the fermenter

To keep in mind:

• Flocculation character of a yeast directly impacts the flavor and fermentation performance of a beer. Therefore choosing the right flocculate yeast strain is very important in the first place
• Keep as much of the fermentation factors as consistent as possible. This includes fermentation temperatures, pitching rates, oxygenation etc.
• Keep record to be able to observe changes in flocculation
• Flocculation itself depends on yeast strain and its FLO genes, environmental factors and the physical interaction between yeast cells

Flocculation seems to be Saccharomyces yeast specific and a lot of research is still done to further understand how flocculation works. I hope I could give you a small glimpse into the topic and got you an idea what flocculation is all about. Including some advice what influences flocculation and what a (home)brewer can do about it to keep flocculation behaviours as constant as possible. The next post concerning flocculation will cover the biological function of FLO genes and therefore the biological function of flocculation for the yeasts cells and further insights into other flocculins and their biological role in Saccharomyces. Cheers!

References:

• Amory DE, Rouxhet PG (1998) Surface properties of Saccharomyces cerevisiae and Saccharomyces carlsbergensis: chemical composition, electrostatic charge and hydrophobicity. Biochim. Biophys. Acta, 938: 61 – 70
• Dengis PB, Nélissen LR, Rouxhet PG (1995) Mechanism of Yeast Flocculation: Comparison of Top- and Bottom-Fermenting Strains. Appl Environ Microbiol, 61(2): 718 – 728
• Fix G (1999) Principles of brewing science: a study of serious brewing issues. Brewers Publication, 2nd edition
• Narziss L (2005) Abriss der Bierbrauerei. WILEY-VCH, Weinheim, 7th edition
• Verstrepen K, Industrial Microbiology Part II – Fermentation, Katholieke Universiteit Leuven, http://www.biw.kuleuven.be/dtp/cmpg/G%26G1/assets/internal/IM-class2-Fermentation-v4.pdf
• Verstrepen KJ, Derdelinckx G, Verachtert H, Delvaux FR (2003) Yeast flocculation: what brewers should know. Appl. Microbiol. Biotechnol, 61: 197 – 205
• Vidgren V, Londesborough J (2011) 125th Anniversary Review: Yeast FLocculation and Sedimentation in Brewing. J. Inst. Brew. 117(4): 475 -487
• White C (2012) Flocculation Basics. www.whitelabs.com/beer/Flocculation_help.pdf
• White C, Zainasheff J (2010) Yeast: The Practical Guide to Beer Fermentation. Brewers Publication, 1st edition
• Wikipedia (2012) Flocculation, http://en.wikipedia.org/wiki/Flocculation
• Wyeast (2012) Flocculation/Clarification http://www.wyeastlab.com/hb_clarification.cfm