BBA/EBY Brett Experiment Update 2

Hello fellow BBA/EBY experiment collaborators. This is the second update concerning the BBA/EBY Brett Experiment. The first update can be found here. Once again thanks to all the participants and all the people offering to send me some unique dregs and yeasts as well. I would like to proceed with some numbers:

  • 36 collaborators are officially in for the experiment (me not included)
  • 347 samples will be sent out for the experiment
  • 9 collaborators will test the entire 20 strains (awesome!)
  • From the 36 collaborators, 1 is not from the US (and it isn’t me). Don’t worry, I only looked up the cities and not the entire addresses 🙂

collaboratormapCan I still sign up for the experiment?

I am sorry to announce that I don’t accept any further collaborators. If you are interested in the strains, please subscribe to https://groups.google.com/forum/#!forum/eureka-brewing-yeast to get email alerts of future strain releases

Yeast shipping

As previously mentioned, the yeasts will be sent out on Monday, the 26th of August. Please read the first update post what has to be done after you received the yeasts.

First results about the strains

I would like to publish first results about some of the strains

  • EBY001 B. girardin I: The strain which will be released is different from the one I originally isolated. The original strain looked and behaved like normal brewers yeasts and I therefore replaced it with another Girardin Brettanomyces strain I have
  • EBY005 B. cantillon I, EBY008 B. cantillon II, EBY009 B. cantillon III, EBY011 B. cantillon V, EBY013 B. cantillon VII and EBY016 B. lembeek I are really slow growers. Expect to wait longer to get signs of fermentation
  • All strains can form colonies on agar plates. Therefore all strains are viable, even the slow-growing ones

IMG_20130814_201831That’s it from me already. I hope that everyone can revive the yeasts without problem and gets some nice beers out of them. Over and out!

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Review on Rehydrating Dry Yeasts and Viability Stains

Eureka and welcome to a new science post. I would like to discuss the results from a paper about rehydrating yeasts and viability staining. Since dry yeasts are widely used in the homebrewing scene and even on industrial scale, lots of discussions are about the effects of rehydrating the dry yeasts before use. Some rehydrate the active dry yeast (ADY) in some water and others just sprinkle the yeast in the beer. Some prefer to use warm water and others prefer colder temperatures.

During the drying process, the water flows out of the yeast cells rather rapidly and leads to a collapse of the cytoskeleton (Rodriguez-Porrata et al (2008)). During the first minutes of the rehydration process, the cell’s membrane are not functionally active yet and lead to membrane leakage. In this process, molecules from within the cell flow out of the cell. During the rehydration process, the cellular membranes get repaired and thus stop the membrane leaking. If the cells cannot stop the leakage, it is going go die.JenkinspaperJenkins et al studied the effects of rehydration conditions on yeast viability and came to some remarkable results. The authors studied three different yeast strains (LAL1 a lager strain, LAL2 Nottingham strain and LAL4 a Munich strain), rehydrated the cells at different temperatures and for a different amount of time. In addition, they measured the viabilities at certain time points during the rehydration process using four different techniques: Slide cultures, methylene blue, MgANS (8-anilino-1-naphthalene-sulfonic acid hemi-magnesium salt hydrate) and Oxonol staining. For the slide cultures, a small volume of yeast suspension was added to a small amount of agar on a slide and the arising microcolonies from the yeast cells within the agar were counted after 18 h. Thus a staining independent method to asses viability.

For this test, Jenkins et al used 1 g of ADY and added 10 times the weight of water (water temperatures 25°C and 30°C). Time point A was taken immediately after adding the ADY to the water. Time point B was taken after leaving the ADY rehydrate for 15 min. The yeast-water mixture was then mixed and samples were taken after additional 15 min each (time points C1 to C4).

Lets look what they found out. In case of the Lager strain, the viabilities at the different time points are shown in Fig 1. I would like to leave the different temperatures aside since its effect is strain dependent and not important for my main message here. What they could observe is a lower viability at time point A compared to the other time points. This effect seems to be independent of the water temperature (not shown). Further on to notice are the different viability values one obtained using the four techniques. For the Lager strain, measuring the viability using methylene blue lead to lower values compared to the other two staining techniques.

Test

Fig 1: Viabilities of Lager strain at different time points at 25°C by Jenkins et al (2011)

The lower viabilities at time point A could also be observed for the Nottingham (Fig 2) and the Munich yeast (Fig 3). Again, the lower viabilities seem not to depend on the water temperature of the water used for the rehydration process (not shown).

JenkinsCharts2

Fig 2: Viabilities of Nottingham strain at different time points at 25°C by Jenkins et al (2011)

JenkinsCharts3

Fig 3: Viabilities of Munich strain at different time points at 25°C by Jenkins et al (2011)

Putting this observations together. Measuring the yeast viability using staining methods such as methylene blue within the first minutes of rehydration seems to lead to significant lower values compared to values obtained from later time points. What is the reason for this you may ask? This is where it gets interesting.

The first question one has to address is how yeast viabilities can increase in the first place (as observed in the figures from time point A to B and C1 in case of the staining). Please note that the viability measured with slide cultures in case of the Munich strain are highly similar to later time points (Fig 3). Viability, in a biological sense, can only increase by the formation of new, viable cells. One other way would be for dead yeasts to get alive again which is not very likely in my opinion (though I don’t have any proof for my statement). Due to the chosen time points, it is very unlikely for the yeasts to undergo divisions and thus increasing the viabilities again from time point A to B and C1. There has to be a different effect.

The answer to the question lies within the methods. As previously mentioned, dried yeasts don’t have active membranes. During the rehydration process, the membranes get repaired again (Rodriguez-Porrata et al). Since most of the viability stainings, such as methylene blue rely on active membranes (the dye should only be taken up by dead cells with inactive membranes), dry yeasts behave much like dead cells within the first time points and thus leading to lower viability values. With time, the membranes get active again, and the dye is probably exported from the cells and thus increasing the viability. This is a very nice example of a method’s limitations.

However, there seems to be an effect in the slide culture method as well. This might be due to osmotic pressure issues when the dry yeasts get in contact with a highly osmotic agar which enhances the leaking of the cells.

Assessing viability measurements using methylene blue on dry yeasts is a valid method but has to be done with caution. Especially if one wants to assess whether dry yeasts have a higher viability if added to water first or pitched to wort directly. Consider the right time points for such experiments.

I hope this was interesting to read and might give you a better insight into viability stains and its limitations.

Bibliography

Jenkins DM, Powell CD, Fischborn T, Smart A (2011) Rehydration of Active Dry Brewing Yeast and its Effect on Cell Viability, J. Inst. Brew. 117(3), 377-382 (http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.2011.tb00482.x/abstract)

Rodriguez-Porrata B, Novo M, Guillamon J, Rozès N, Mas R, Cordero Otero R (2008) Vitality enhancement of the rehydrated active dry wine yeast. International Journal of Food Microbiology. 126, 116-122
(http://www.sciencedirect.com/science/article/pii/S0168160508002560)

BBA/EBY Brett Experiment Update 1

Hello fellow BBA/EBY experiment collaborators. This is the first update concerning the BBA/EBY Brett Experiment. I would like to begin by thanking Jeff for the good collaboration so far and all the other people who are willing to take the risk testing some of my strains. Additional thanks to Luke and Ryan for their contributions about the evaluation sheets. Cheers to all that. And thanks for all the people offering to send me some unique dregs and yeasts as well. I would like to proceed with some numbers:

  • 32 collaborators are officially in for the experiment (One subscriber, George Peterson did not write me any email yet)
  • 308 samples will be sent out for the experiment so far (might further increase)
  • From the 32 collaborators, 1 is not from the US (and it isn’t me)
  • 7 collaborators will test the entire 20 strains (awesome!)

I would like to give you further information about the experiment today and cover some other questions I got asked so far.

Can I still sign up for the experiment?

You signed up but would like to test more/less strains?

  • Write me an email with your request. We will find a solution

Updates concerning the recipe/process:

  • Fermentation temperatures and fermentation times. We left this one open so people can pick what they feel most comfortable with. Please feel free to play with fermentation temperatures and times if you like. One request though. Please remember the numbers such as fermentation temperatures and times for the evaluation later on
  • Please try to measure the terminal gravities before bottling. This is necessary to get the attenuation levels for the different strains
  • Split batch sizes. Take whatever fermentation volume you feel most comfortable with. Jeff and I will both brew a 10 gal (40 L) batch and split the batch into 0.5 gal shares to test all the 20 strains. If someone would like to brew more than that, please adjust the pitching rate
  • Pitching rates. Expect to get about 1.2 million yeast cells per tube (see picture below) and go from there.2013-07-23-19-55-27Pitching the 1 mL liquid culture into a 200 mL DME unstirred starter should give you roughly 25 billion (+/- 3 billion) yeast cells after 10 to 14 days. Corresponding to roughly 12 mL of yeast slurry. This should be enough yeast to pitch 0.5 gal directly. If you need bigger cell counts, use the http://yeastcalc.com/ and use 25 billion cells as initial cell count if you have done a 200 mL starter first. As far as I can tell from my Brett starters, the starter volumes are quite comparable to normal yeast but Brett need more time. So don’t expect the Bretts to eat through a starter within 24 h. To evaluate how much yeast you got after each starter, try to estimate the yeast sediment volume (in mL) and multiply it by two to get to the cells in billion. For example, 12 mL of yeast slurry are equal to 24 billion yeast cells
  • When should I bottle the beers? Bottle the beers as soon as you think they are ready. It is hard to make any predictions here since I have no idea how the strains perform. Some strains might be done fermenting after seven days, others might need more time. I for my part will give the primary fermentation about two weeks and then evaluate which beers to bottle

Yeast shipping

  • I am currently stepping up all the 20 strains to have enough viable yeast for the shipping. This will take another few days for sure before I can prepare the first tubes. I plan to send out the yeasts on Monday, the 26th of August. According to my post office, you should get the yeasts after three to seven days
  • The yeasts will be sent out without any cooling pads as Bretts should be fine with ambient temperatures. Further on, I will supply the yeasts with fresh media to give them something to do during the shipping. As long as the parcels are not exposed to sun light for several hours, they should be fine. If someone expects problems with this approach, please let me know
    2013-08-02-17-37-00

I got the yeasts, what’s next to do?

  • I would advise you to prepare small starters (200 mL at max) some days after the 26th of August to be prepared for the yeasts. For a 200 mL starter, add 20 g of dry malt extract to 200 mL of water and sterilize it using a pressure cooker if possible. Mason jars could be well suited for the starters. Any smaller volumes works as well. Just don’t go beyond a starter volume of 10 mL.
  1. Wash your hands thoroughly with soap first
  2. Flick at the bottom of the tubes to get the yeast pellet on the bottom of the tube back into suspension. Prevent any vivid shaking of the tubes
  3. Sanitize the tubes if possible with alcohol (Vodka etc) or a sanitizer
  4. Remove the Parafilm wrap from the top of the tube. There might be some pressure forcing some gas out of the tubes or even lift the lid. So be prepared. I would press down the lid with my thumb (or any other finger you like) while removing the parafilm and then gently open the lid
  5. Open the tube, avoid touching any inner parts of the tube lid and pour the entire content of the tube into your yeast starter
  6. Shake your starter a bit and leave it as it is
  • If you planned to do the batch for the experiment later on, please prepare the starters as well. The Bretts can be stored in the starters for weeks to months and it would be better for the yeasts to let them recover from the shipping procedure
  • If this in no option, store the tubes at a cool place. Don’t freeze them! They might not survive that
  • Expect to wait at least 10 days before you see signs of activity in the starters. I know this might be hard for some of you but please don’t write to me complaining about dead yeasts before the 10 days mark
  • If you encounter any problems during the starter steps (no signs of fermentation visible after 10 days, contaminations etc) please let me know what problem you encountered (contacteurekabrewing@gmail.com). I will send you some fresh yeasts immediately

What about the evaluation process?

If there is anything left unanswered, please let me know. That’s it for the first update. Thanks for your help and cheers, Sam