I am still on planet earth

Dear all, it has been a while since my last post. As life sometimes has some different tasks and projects lined up for you, sometimes you simply cannot cope with everything in parallel. I would like to take the opportunity to talk about some news and the future of this blog.

As you may know, I share the dream of so many homebrewers to eventually make a living with brewing. And step by step, my brother and I created the brand Blackwell Brewery back in early 2012. And since late 2015, Blackwell is actually a legal company and we just need to wait now to get the approval for a liquor licence to make Blackwell an official Swiss brewery. In early 2014, we started brewing as gipsy brewers before investing in our first 400 L brewing kit. This step allowed us to refine our base recipes, playing around with some no-boils & mature beers in barrels for the first time. Without losing to much money (we had to dump a couple of batches though). Not to forget all the collaboration projects we got involved in that gave us more and more insight into the entire beer production work flow. And all our customers and good friends giving us the support and motivation to go the next steps. In less than a month from now, we will move to a new space where we have a good base for whatever might come next.

At Blackwell, we hand-craft all our products on a small quantity with focus on high quality – in effort to give our fans a different tasting product. We plan to brew a variety of styles with focus on Wild, Rustic, Sour, Dark, Light, Über-strong, Fermentation-driven beers. And everything else that floats our boat.

Carnivale Brettanomyces – 2016

I am very privileged and happy to announce that Blackwell got invited to the Carnivale Brettanomyces 2016 festival hosted in Amsterdam (NL) from the 23rd to 26th of June. Not only will there be beer on tap from Blackwell but I will lecture a yeast Master Class at the festival too. For more information about the festival and the lecture, please check-out the following links. Would be great to meet some of my readers in Amsterdam.

https://www.facebook.com/Brettfest/?fref=ts
http://www.wildegist.nl/

Discontinuation of the blog

Since I am still working my day-job and guiding Blackwell alongside, I came to the decision to discontinue publishing material on this blog. As I am a big believer in quality information, I simply don’t want to publish for the sake of publishing. However, as I am still doing my yeast projects, I will continue on publishing via the website of the brewery. I will put the new links on this blog as soon as I set-up the website’s blog part. The content of the blog will however stay where it is, I am still available for questions, yeast sharing etc but no new content will be uploaded.

Yeast Lab

What about the release of the EYL yeast strains? This is still a work-in progress project. At the moment, I have a Saccharomyces strain being tested by local brewers. Main reason for that is to check the packaging, propagation and shipping of the product on my side prior to selling the yeast products. The focus will be the supply of yeast products to homebrewers in Switzerland. Shipping outside of Switzerland might come in the future as well. Depending on the local demand. And the release & sell of the yeast will be supported by Blackwell. Meaning, all EYL strains are now owned and managed by Blackwell.

I think that’s enough information for today. The next post will very likely be the last one. I would like to take the opportunity to thank all my fellow blog readers and people who reached out to me by email or placing a comment. I am especially grateful to all the people who pointed out mistakes in the post. Or all the interactions that lead to great friendships. When I started the blog in January 2012, I would have never guessed that so many people would be interested in reading what I am writing. Especially as I am not an English native speaker (nor a very skilled writer). Thank you all.

Kind regards,

Samuel

Insight into the Dekkera anomala YV396 genome

As previously communicated in my last post, I am currently working on a Dekkera anomala proteome (strain YV396) to get a better understanding of the pathways associated with compounds found in beers like Lambics. I compiled my data and first results into a easier to read format (see below). In case there is something else of interest in the genome, I will likely publish these results on my blog as well.

View this document on Scribd

Insight into the Dekkera anomalus YV396 genome – part 1

It has been a while since I look at Brettanomyces genomes since there wasn’t that much data available to play around. And mainly B. bruxellensis data is available due to its importance in the wine industry. This all changed when I came across the deposited draft genome assembly of a Dekkera anomalus YV396 genome in June by Vervoort,Y et al. Since there was no annotation material available for this genome I quickly decided to give the annotation a shot myself. Simply because I am interested in certain pathways in Brettanomyces. Everything I needed was my Ubuntu notebook (which died during the annotation process), my new Ubuntu workstation (replacing the notebook) and some Python coding. No access to a cluster whatsoever. Is it possible to finish an entire annotation project at home? You will find out very shortly. As I am still compiling data for a post, I want to start sharing the material part as well as the first abstract. Just to give you a sneak-peek into the project. The remaining part of the genome & proteome project will get published very soon. Just give me some additional time to finish up the various pathway analysis and writing up the paper. Still a lot to be discovered in the new genome…

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I – Methods

Genome assembly

The draft genome assembly of Dekkera anomalus strain YV396 (isolated from a Belgian brewery) was retrieved from GenBank (accession number LCTY00000000.1; June 2015) deposited in May 2015 by KU Leuven [Vervoort et al.]. Illumina HiSeq data (100x coverage) was assembled into a genome using SOAPdenovo v.1.05. The statistics for the obtained assembly are summarized in Tab. 1.

Gene prediction

Gene prediction on contigs was performed using the AUGUSTUS web-service (AUGUSTUS parameter project identifier: pichia_stipitis, UTR prediction: false, report genes on both strands, alternative transcripts few, allowed gene structure: predict any number of (possibly partial) genes, ignore conflicts with other strand: false) [Stanke et al.2006, 2008]. The gene prediction statistics are summarized in Tab. 2.

Gene annotation

Gene annotation was performed by Blast2GO including remote blastx on NCBI and InterProScan for domain predictions [Conesa et al, 2005]. GO-term mapping and annotation performed by Blast2GO pipeline. Close to 3,000 out of the predicted 4,160 could be annotated by Blast2GO (Fig 3). Another subset of about 600 sequences could be mapped to a biological function without a GO term and about 460 sequences only resulted in BLAST hits which could not be further associated with a protein function.

Most abundant species associated with the best blastx hits were Dekkera bruxellensis, Ogataea polymorpha and Pichia kudriavzevi (not shown).

References

• Conesa, A., Götz, S., García-Gómez, J. M., Terol, J., Talón, M., and Robles, M. (2005). Blast2GO:a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 21(18):3674–3676.
• Stanke, M., Diekhans, M., Baertsch, R., and Haussler, D. (2008). Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics, 24(5):637–644.
• Stanke, M., Schoffmann, O., Morgenstern, B., and Waack, S. (2006). Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources. BMC Bioinformatics, 7(1):62.
• Vervoort,Y., Herrera-Malaver,B., Mertens,S., Guadalupe Medina,V., Duitama,J., Michiels,L., Derdelinckx,G., Voordeckers,K., Verstrepen,K.J. (2015) Purification and characterization of a novel Brettanomyces anomalus beta-glucosidase enzyme suitable for food bioflavoring – unpublished.

Hello, my name is Metschnikowia pulcherrima

Eureka, back to spoilage yeasts. Today, I would like to introduce another spoilage yeast called Metschnikowia pulcherrima (anamorph Candida pulcherrima): A killer yeast with applications in the wine industry, available at Lallemand’s (aka Flavia MP346) and results from one of my countless (beer) split batch experiments. I hope there is something in here for everyone. Put on your science hats, take out your pencils & notepads and start reading. Spoiler alert: you have to interpret the results yourself.

Where do I work?

M. pulcherrima can be isolated from grapes, cherries, Drosophila spp. (fruit fly), flowers and spoiled fruits [Kurtzman et al, 2011]. A yeast commonly found in nature then. If you want to know more about M. pulcherrima and its role in the wine production, go to http://wineserver.ucdavis.edu.

What about beer?

I could not find a source where M. pulcherrima is linked to beer or discussed as potential spoilage yeast thereof. This might not be that surprising since beer is, first of all, commonly not brewed with fruits and second, it’s not really the most alcohol resistant nor metabolically advanced yeast in the universe. Apparently, M. pulcherrima seems to have an alcohol tolerance of about 5% [wineserver.ucdavis.edu, 2015]. Not really high compared to brewer’s yeast with levels of around 12% (or even higher).

What is so special about me?

One very special character of M. pulcherrima makes it a very interesting yeast for the food industry: It’s a killer yeast! The killer activity is affecting blue mold (Penicillium sp.), Botrytis cinerea (gray mold) and couple of bacteria, yeasts [Kurtzman et al, 2011]. This inhibition/killer activity seems to be linked to pulcherriminic acid which is able to form insoluble compounds ultimately depleting the medium of iron ions (which are essential for the growth of other microorganisms) [Oro et al, 2014]. Scientists therefore studied efficacies of M. pulcherrima as a biocontrol agent against the molds mentioned above to eventually prolong the storage times of fruits. If you want to know more about the killer activity and the science behind it, go to PubMed and have a look at the publication from M. Sipiczki. I think this is a very nice example to show people not affiliated with science, that spending money to investigate very odd microorganisms may even result in discoveries that can have an industrial application. Or even have an impact on drug developments against pathological molds. Or put the yeast on the radar of a yeast hunter…

Where can you find me?

As mentioned in the introduction, Lallemand sells a M. pulcherrima product called Flavia MP346. One of the few advantages to brew in Switzerland is the fact, that everything around me is about wine. And getting wine yeasts is therefore rather easy. I therefore got myself a package of said yeast and tried to investigate the impact on beer.

According to Lallemand, Flavia MP346 is a strain isolated from Chile with the speciality of α-arabinofuranosidase secretion to increase terpenes and volatile thiols to enhance the aroma of a wine. Overall increasing the aroma complexity (mainly fruit components) in the finished product. The yeast is commonly added to the must first followed by a Saccharomyces pitch 24 h later on.

To test if there is any (detectable) effect on aroma complexity in a malt based beverage like beer, I performed a simple split batch experiment. I started with a straight forward Belgian inspired recipe (3 kg Vienna malt, 3 kg Abbey malt and 0.5 kg CaraMunich 2; 24 IBU with Saazer; OG about 1.066, brewed 12. December 2014), and split the wort in two parts. One part fermented with a Saccharomyces (US-05) only, the second part with a dose of M. pulcherrima for 24 h before pitching the same Saccharomyces strain. I was really curious to try this yeast because an effect seemed very unlikely to me since the yeast can mainly ferment glucose (see below) and is incapable of fermenting maltose. Not to forget the rather low alcohol tolerance. Bottled on 12th of January 2015 (TG_control: 1.023 (5.9 ABV), TG_Metschnikowia: 1.024 (5.9 ABV)).

First tasting performed in June, 2015 (beer approximately five month in the bottle):

Control:

Aroma: Lots of dark fruits like figs, prunes. Caramel notes as well as burnt sugar components. Very nice!

Appearance: Red-brown color, slightly cloudy, off-white head (see picture on the left). Lots of carbonation.

Flavor: Very similar to aroma. Caramel and malts. Not much else going on (yeast character or whatever).

Mouthfeel: Light body, average carbonation, malty/sweet-caramel/bitter finish. Very nicely balanced.

Overall Impression: Nice bitter:body balance. Very classical and easy to drink. Most of the character originates from choice of malts (caramalts). Not much flavor picked up from US-05 yeast (as planned).

Metschnikowia partial ferment:

Aroma: Different (compared to control): Besides the malt character (caramel, dark fruits, burnt sugar) notes of raspberries, pepper and wild funk (mostly phenolic acids). Very pleasant aroma profile.

Appearance: Red-brown color, slightly cloudy, off-white head (see picture on the left). Lots of carbonation. Similar to control.

Flavor: Similar to aroma without the fruity components (mainly caramel & malt). Finishes with a bitter overhang (balance toward bitter).

Mouthfeel: Very light body (lighter than control), higher carbonation than control (gusher),

Overall Impression: Typical Belgian dark ale with hints of fruits as well as phenolic funk in the nose. Body on the lower end resulting in an overhang of bitterness (not very well-balanced). Seems to have attenuated more than the control resulting in a low body and over-carbonated beer. In the end, I prefer the control. Although the aroma profile of the Metschnikowia beer is nice, it’s a less drinkable beer (my opinion).

So far for the plain results. Lets put them into perspective. According to Lallemand, the yeast is used in wine to promote the fruit components. And I have the feeling that I was able to pick up such an effect in my experiment as well. The beer dosed with M. pulcherrima had pronounced notes of fruits which were not present in the control. The beer had a higher attenuation level resulting in a lower body and a bitter balanced beer.

Great! I will leave the interpretation of the experiment as well as other applications of this yeast to you. The only thing that I don’t recommend is to ferment a beer with M. pulcherrima only. Simply because of its incapability to ferment the most abundant sugars in wort (just in case someone tries that, goes horribly wrong and wants to sue me for that).

Some biochemical stats about me for yeast ranchers

First some micrographs:

Yeast grows on Sabouraud agar like any other Saccharomyces yeast. I however noticed one difference: M. pulcherrima seems to be non-cycloheximide resistant (according to http://wineserver.ucdavis.edu, 2015). I plated the Lallemand product on Sabouraud agar supplemented with + 10 mg L-1 cycloheximide and I could not detect any colonies. Now some stats summarized from Kurtzman et al (2011).

Systematic name:	Metschnikowia pulcherrima (anamorph Candida pulcherrima)
Synonyms:	There are a lots of accepted synonyms for this yeasts. Just some examples: Torula pulcherrima, Saccharomyces pulcherrimus
Growth on malt agar:	Cell morphology:	Globose to ellipsoid, 2.5 µm x 4-10 µm. Pulcherrimin (reddish-brown) pigment present & diffuses into media
	Clustering:	Not described
	Pseudohyphae:	Not described
	Pellicle formation:	Not described
Fermentation:	Glucose:	Positive
	Galactose:	Weak
	Sucrose:	Negative
	Maltose:	Negative
	Lactose:	Negative
	Raffinose:	Negative
	Trehalose:	Negative

That’s all for today. Thanks for reading.

Bibliography

• Kurtzman CP, Fell JW, Boekhout T (2011) The Yeasts, a Taxonomic Study. Volume 1. Fifth edition. Elsevier (Link to sciencedirect)
• Oro L, Ciani M, Comitini F (2014) Antimicrobial activity of Metschnikowia pulcherrima on wine yeasts. J Appl Microbiol. 116(5):1209-17
• wineserver.ucdavis.edu (2015) http://wineserver.ucdavis.edu/industry/enology/winemicro/wineyeast/metschnikowia_pulcherrima.html

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)
   

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

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

   Media 3: Circular bacteria visible (very few LABs)

4. Media 4: LAB visible
   

   Media 4: LAB visible

5. Media 5: LAB visible
   

   Media 5: LAB visible

6. Media 6: LAB visible
   

   Media 6: LAB visible

7. Media 7: Lots of LAB
   

   Media 7: Lots of LAB

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

   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
   

   Media 6: Lots of LAB

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

   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.

Sugar composition of wort

Short one for today. I would like to share some information about the sugar composition of wort since I had to take this into consideration for an upcoming project I am preparing for publishing here soon (yes, its yeast related). Lets talk sugars today!

I am not sure how many homebrewers thought about the actual sugar composition of their wort before. And I am not speaking about fermentable and non-fermentable ones. The real composition like sucrose, maltose, glucose etc. The question now is why one might think about that problem in the first place. For example, if you are interested to know if a non-Saccharoymces yeast (capable of fermenting glucose only) can ferment something in a wort, you might need to know if glucose is even present in the first place (and this example is pretty close to the question I asked myself to eventually investigate the composition of sugars in wort).

The composition of sugars in wort has been addressed a couple of years ago and published in various papers. Like “Determination of the sugar composition of wort and beer by gas liquid chromatography” by Otter et al published in 1967 [get me to the paper]. I will not go into the scientific details as well as experimental setup of this paper but would like to discuss the results.

Fig 1: Paper header

Otter et al determined the concentrations of six sugars (fructose, glucose, sucrose, maltose, maltotriose and maltotetraose) in 15 different worts with various OGs (ranging from 1.027 up to 1.093). I averaged the sugar compositions of the 15 samples as amount of sugar X relative to the total amount of sugar present in wort. Just to give me a rough idea. So don’t read too much into the numbers here. It’s about the ratio or more like sugar X is highly abundant or not. And yes, I thought about effects of grain bill composition, mash schedules, mash pH, you name it on the sugar composition. Getting a rough idea here.

Fig 2:Sugar composition w[%] of total sugar as average of 15 different worts

About half of the sugars present in wort is maltose (Fig 2). Followed by maltotriose and glucose. And some smaller amounts of fructose, sucrose and maltotetraose. Maltotetraose by the way is a dextrin and can be counted as non-fermentable. Standard Saccharomyces cerevisiae strains are capable of fermenting all the present sugars except maltotetraose. Which might explain why S. cerevisiae is the working horse of brewers. In summary, maltose makes up about half of the total sugars followed by glucose and maltotriose. And some minor amounts of fructose, sucrose and maltotetraose. I am actually surprised about the amount of glucose present in wort. I did not expect that at all.

So there you go. I will address the initial problem about a specific sugar metabolism of a non-Saccharomyces yeast in a future post including some empirical data. Stay tuned!