Evaluate starter media to propagate Lactobacillus sp.

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

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

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

#83 Grätzer/Grodziskie

Eureka, its time for another recipe publication. Not only am I fascinated about the new arising beer styles in the world but really like to look back as well. One of the rediscovered beer styles is a Polish wheat beer called Grätzer or Grodziskie named after the place where it was originally brewed. This beer style is made with oak smoked wheat malt and with a healthy dose of hops. The result is a clear, smoky and very refreshing beer. Because this beer style heavily depends on the malt and no such malt was easily available up to know, only the ones willing to smoke their own malt had the ingredients to make a real Grätzer. This all changed as soon as the German maltster Weyermann revived the oak smoked wheat malt which is now available again. With this, the most important ingredient for this kind of beer is yet again commercially available.

I came across this beer style a while ago and got really excited as I encountered Eichenrauchmalz (= oak smoked wheat malt) at my local homebrew store. I instantly ordered a small quantity of this particular malt to give this beer style a go. And below is my recipe.

Recipe: Grätzer
Numbers: Volume [L] 20 (5.3 gal)
Original gravity 7.8°P (1.030)
Terminal gravity 2.6°P (1.010)
Color Around 5 EBC
IBU 20 IBU
ABV 2.7% (v/v)
Grains: Smoked wheat malt (4 EBC) 3 kg (5.5 lbs)
Hops: Hallertauer (4.2% AA) 25 g and boiled for 60 min
Hallertauer (4.2% AA) 10 g and boiled for 30 min
Yeast: Wyeast 1 part WY1338 European Ale and 2 parts WY1007 German Ale
Water: Mash: 8 L (2.1 gal), sparge: 12 L (3.2 gal) @78°C (172°F)
Rest: Mash in @38°C (100°F), 30 min @38°C (100°F), 30 min @52°C (126°F), 10 min @66°C (151°F), 30 min @70°C (158°F), sparge at 78°C (172°F)
Boil: Boil for 60 min
Fermentation: Primary 8 days at 20°C (68°F)
Secondary 10 days at 20°C (68°F)
Maturation: Carbonation (CO2 vol) 3.6
Maturation time 2 weeks, cold

06/29/13: Brew day as normal. Mashed in as stated in the recipe, rested at the given temperatures and sparged as usual. I did not add any kind of rice hulls or barley to facilitate the lautering. The lautering went as smooth as ever. I am aware that a lot of people say that it is not possible to sparge a 100% wheat malt mash. Well, I kind of experienced different things in the past and don’t care about any other experiences. I use a perforated metal plate as a filter (see picture below) and this is maybe on of the best investments in my homebrewing career. Since then, the lautering is not a pain any more. However, I encounter difficulties with this plate when I sparge the soured Berliner Weisse mash. Then the mash gets very easily stuck because the entire mash is very mushy.

IMAG07992

Fig 1:Mash kettle with perforated metal plate at bottom

After sparging and collecting the wort in my boiling kettle, I boiled the wort with the additions of the Hallertauer hops, cooled down the wort and pitched a yeast blend of two commercial yeasts (WY1008 German Ale and WY1338 European Ale). Before I cooled down the wort, I filled a 2 L growler with hot, boiled wort for the carbonation step later on. The reason for using two yeast strains is to get the WY1008 out of suspension (low flocculent) with a highly flocculent yeast strain (WY1338 in this example). This is very important because the beer has to be very clear in the end. Primary fermentation at 20°C (68°F).

07/07/13: Racked beer after 8 days to a secondary fermenter.

07/17/13: Bottled beer with addition of wort to a carbonation level of 3.6 vol of carbon dioxide. Added 2 L of unfermented wort to 18 L of green beer.

Now the most interesting part, what about the taste?

83_gratzerAroma: Smoke is dominant aroma with character of smoked bacon, bonfire. Thanks to Anna who describes this aroma in “one” word: “dried green beans”. Spot on in my opinion

Appearance: Yellow, clear, fluffy, white head with lots of bubbles rising to the top (see picture)

Flavor: Pretty much the same as in the aroma: bonfire, ash, some wood flavors as well. Nice level of bitterness and well integrated

Mouthfeel: Light body, average carbonation level, rather dry, light smoky finish. Very easy drinkable

Overall Impression: Very refreshing and surprisingly not too thin for the 2.7 vol alcohol. I am quite intrigued how this batch turned out. Even after three months in the bottles, the beer still has a very prominent smoky, ashy character. This experiment is yet another successful one.

BBA/EBY Brett Experiment Update 3

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

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!

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)