Category Archives: Experimental

Evaluate starter media to propagate Lactobacillus sp.

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Welcome back everyone. Yes, I am still alive. Although my job in science absorbs lots of my spare time lately, I still find time now and then to brew on a semi-professional scale. Which unfortunately leaves yeast science and this blog at lower priorities. I still do yeast work at home but it all shifted to more practical applications like establishing and testing blends, evaluating yeast isolates and playing around with some full size wine barrels.

What I want to share today are the results of an evaluation experiment I performed a couple of months ago to look for MRS media alternatives to propagate lactobacillus at home. MRS media is kind of the golden standard used to propagate lactobacillus. It works very well but with the disadvantage of being a quite expensive media. I therefore tested a couple of cheaper alternative media and compared the growth/propagation efficiency with MRS. Please notice that the experiment and results are added in a rather short kind of way. That’s all from me now. Take out your pencils & notepads and start reading. Over&Out.

Goal of project

  1. Set up starter conditions and protocols to propagate Lactobacillus sp. to pitchable amounts
  2. Compare growth properties to MRS broth as reference
  3. Determine most efficient media to propagate Lactobacillus sp.

Material & Methods

The following media were tested:

  1. Lactobacillus media 1: 100% apple juice
  2. Lactobacillus media 2: 100% apple juice + CaCO3 (20 g L-1)
  3. Lactobacillus media 3: 100% apple juice + CaCO3 (20 g L-1) + yeast nutrients
  4. Lactobacillus media 4: 10°P DME
  5. Lactobacillus media 5: 10°P DME, 10% (v/v) apple juice
  6. Lactobacillus media 6: 10°P DME, 10% apple juice + CaCO3 (20 g L-1)
  7. Lactobacillus media 7: 10°P DME, 10% apple juice + CaCO3 (20 g L-1) + yeast nutrients
  8. MRS-Bouillon (as reference, CarlRoth prepared according to manual)

10°P DME starter and MRS media was autoclaved (15 min at 121°C) and mixed with additional components at room temperature. Pasteurized apple juice was used. Each media (50 mL in total) was inoculated with 1 mL of bacteria culture (Wyeast 5335 L. delbruecki/L. buchneri; Wyeast 5223 L. brevis). Propagation performed at room temperature (no shaking, no aeration).

To address the efficiency of the media, the culture densities were estimated based on microscope observations after 7 days of propagation.

Results

WY 5335 L. delbrueckii/L. buchneri:

  1. Media 1: None-few LAB cells (rod-shaped) visible
  2. Media 2: Yeast & circular bacteria cells visible (contamination)

    Ldelbrueckii_Med2_01

    Media 2: Yeast & circular bacteria cells visible (contamination)

  3. Media 3: Circular bacteria visible (very few LABs)

    Ldelbrueckii_Med3_01

    Media 3: Circular bacteria visible (very few LABs)

  4. Media 4: LAB visible

    Ldelbrueckii_Med4_01

    Media 4: LAB visible

  5. Media 5: LAB visible

    Ldelbrueckii_Med5_01

    Media 5: LAB visible

  6. Media 6: LAB visible

    Ldelbrueckii_Med6_01

    Media 6: LAB visible

  7. Media 7: Lots of LAB

    Ldelbrueckii_Med7_01

    Media 7: Lots of LAB

  8. MRS control: Lots of LAB maybe more than media 7

    Ldelbrueckii_MRS

    MRS control: Lots of LAB maybe more than media 7

WY 5223 L. brevis:

  1. Media 1: None-few LAB
  2. Media 2: Circular cells, few rod-shaped bacteria
  3. Media 3: Circular cells, few rod-shaped bacteria
  4. Media 4: LAB visible
  5. Media 5: LAB visible, more or less the same as for media 4
  6. Media 6: Lots of LAB

    Lbrevis_Med6_01

    Media 6: Lots of LAB

  7. Media 7: Lots of LAB, maybe same as media 6

    Lbrevis_Med7_01

    Media 7: Lots of LAB, maybe same as media 6

  8. MRS control: Lots of LAB, same as media 7

pH-measurements after propagation

Unfortunately, I was not able to measure the pH of the media prior to the propagation. Just received my fancy pH-meter a bit to late for that. Below the pH measurements of the media after propagation.

Media // L. delbrueckii // L. brevis
1 // 3.21 // 3.23
2 // 5.80 // 5.87
3 // 6.54 // 5.92
4 // 4.08 // 3.32
5 // 3.10 // 3.22
6 // 5.68 // 4.57
7 // 5.42 // 4.82
MRS // 4.18 // 4.44

Summary & Conclusions

  • LAB grow very well in MRS media (room temperature, no aeration)
  • Both LAB samples tested grew in various of the tested media. Apple juice, even in presence of other components, does not lead to optimal growth efficiencies compared with the MRS controls
  • Propagation in Lactobacillus Media 7 (10°P DME + 10% (v/v) apple juice + 2% (w/v) CaCO3 and yeast nutrients leads to growth efficiencies close to MRS media

In conclusion, growing/propagating LAB in Lactobacillus Media 7 seems to be the most efficient media tested in this series with results similar to MRS media.

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BBA/EBY Brett Experiment Update 5

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Eureka, we are back to Brett experiment updates. I would like to take the opportunity here to remind people to fill in evaluation data they have so far. At this point, only 6 people (out of 38 collaborators) sent in data either via the provided google doc form or by posting their results on blogs. If you haven’t sent in any data so far, may I kindly ask you to do so? Sending me your evaluation sheets as PDF works as well.

That’s it already. More yeast posts to come soon.

BBA/EBY Brett Experiment Update 4

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Hello everyone. This is the fourth update concerning the BBA/EBY Brett Experiment and should give you some preliminary results for some of the strains and be a reminder concerning the evaluation process. I hope the experiment is working fine so far. As mentioned in the last update (BBA/EBY Brett Experiment Update 3) the planned time points for the evaluation are

  • Month 1 (uncarbonated at bottling), Month 2, Month 6, 1 Year

and the results should be put into the following list

Whenever possible, please transfer all your data from whatever form you used to the Google Form mentioned above. If you include any other (commercial) Brett strains in the experiment, please use the same form and just mention the strain in the appropriate field (yeast code).

Preliminary results

As far as I know, two bloggers published first results so far.

If someone else published evaluation data on their blog (or wherever) and is not mentioned here, please let me know. For the Google Form above, only one person filled in information so far. A bit too early to make any conclusions so far

Small lab update

I would like to end with a short update about the current status of the EBY lab. I am still isolating new yeasts from various new beers (mostly from the US now) and am now especially interested to get some Lactobacillus strains as well. And I finally detected some living bacteria in a dreg sample (see picture below) which might be Lactobacillus (will not reveal the brewery/beer at this point). On the other hand, I got some non-Saccharomyces yeasts from various dregs as well. One might be especially interesting as it is from a brewery from the UK isolated from a barrel aged beer. And I am quite sure they did not add Brettanomyces artificially. In addition, I finally got my hands on a Berliner Weisse brewed with an old Brettanomyces strain isolated from a very old, traditional Berliner Weisse. Will see if I can manage to get some living yeast from the bottle though. Further on, I am playing around blending various strains and dregs to get some unique and aggressive blends. That’s it so far.

dreg001On yet another unrelated notice, I would be interested using the legendary Conan yeast for a future batch. However, it is impossible to get the beer in Europe to isolate the yeast myself nor any yeast shipped to Europe. If anyone out there willing to send me some Conan, please write an email. Thanks for reading and stay tuned

BBA/EBY Brett Experiment Update 3

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Hello everyone. This is the third update concerning the BBA/EBY Brett Experiment and should give you further information about the evaluation of the beers and yeast strains. After some issues with leaking tubes, it all seems to be fixed now. At least I did not receive any feedback so far about problems with any of the yeast strains. I learned one lesson already which is to use a different kind of tube to send out yeasts in the future.

If you are interested in any upcoming strain releases and general news about my yeast lab, please go to https://groups.google.com/forum/#%21forum/eureka-brewing-yeast

I would like to thank all the people for sending me the money for the shipping and am especially thankful for all the ones who sent me some extra as well. This money will flow back into the lab and any yeast related projects. Thank you for that. Unfortunately, there are still three people who did not send me the money for their shipping yet. I might have to cover these shipping costs myself as it seems.

And I got my first yeast package as well. My thanks to Barrett for sending me some dregs of Jester King’s Das Wunderkind and Green Flash’s Rayon Vert. Already excited to have a closer look at these dregs. And since I am already talking about dregs, I recently put aside the dregs of Lost Abbey’s Amorosa de Framboise and Mo Betta Bretta. Again, really excited to see what I can get out of these dregs. I already have enough material for a second round of Brett Experiment…

Sensory evaluation and the Brett score sheet

Jeff put together a pretty nice Brett score sheet in the beginning which was transformed into a Google Form by Luke later on. There is nothing wrong in using Jeff’s Brett sheet for the evaluation process as I prefer to do my tastings with a pencil and paper. The Google Form includes the same structure as Jeff’s form but makes it a bit easier for us to do the evaluation a bit easier afterwards. Whenever possible, please transfer all your data from the Brett sheet to the Google Form. We ask some background information in the Google Form to have additional information available for the evaluation process later on. Here are the links to the mentioned score sheets:

If you include any other (commercial) Brett strains in the experiment, please use the same form and just mention the strain in the appropriate field (yeast code).

When do I sample the beers?

The planned tasting time points are as following:

  • Month 1 (uncarbonated at bottling), Month 2, Month 6, 1 Year

Just mention the closest time point if you for example taste the beers after seven months instead (like 6 months for this example).

How/where to sample the beers?

As Ryan pointed out, to get more evaluation data, it is a good idea to share the beers with other homebrewers, professional brewers, beer judges, beer geeks, friends etc. Either by sitting together to do the actual tasting or sharing bottles with others. How you do it is up to you and it is not mandatory to trade nor share the beers with others. It’s just an idea. Jeff for example is thinking of offering some spots for a tasting panel in the San Diego area (http://jeffreycrane.blogspot.com). On my side (although I haven’t actually brewed the batch yet) will share the beers with a recently set up Wild Ale/extreme beer geek group in Switzerland at one of our upcoming meetings. If you are from/living in Switzerland and are interested in either our newly formed group or be present at the tasting of the beers, please write me an email (contacteurekabrewing@gmail.com) and we will figure out things.
The question here is how to do the organization. As I do not want to give away any collaborator’s email address without even asking, we could use the HBT forum thread about this experiment to organize swaps and meetings. If you have better ideas how to do the organization, please let me know.

I would like to finish with a few links to other blogs writing about this experiment. Please let me know if I forgot someone.

BBA/EBY Brett Experiment Update 2

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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!

BBA/EBY Brett Experiment Update 1

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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