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12/27/08 07:45 AM  
Brett research and general knowledge
Hi everyone hope it was a good Christmas and everyone is looking forward to the new year.

I'm a active reader here but not a poster as I my only experience with Brett so far is a secondary bottle ferment/conditioning with Orval dreg bottles to my 'turbid mashed, american pale ale, cascade dry hopped, with 3.0 oz of tamarind fruit from Colombia added during the boil'. Although I've got a big stout that could under go some brett funk and souring, like with the old english stock ales...

I'm a masters student doing my masters in Brewing and Distilling and am currently writing my Lit review for what may lead to my dissertation on Brettanomyces.. as with most of you I have an obsession with Brett. So I currently spend my holidays and free time reading all the scientific articles I can find on brett ferments, brett physiology, aeration results...so on and so forth...wildBREWS great read, along with the transcript 'horse of a different color'.

Now the thing is there is a big difference from how things work in a lab and how they work in a fermenter...

So my question are..

What types of brett has every one used. Brett anomalus? brett L,B,C. C is only claussenii or people have used custersianus also? What about Brett intermedius?

If anyone is using oxygen after pitching Brett at what level are you achieving for dissolved oxyen in the wort? even timed bursts and the rate of the pump? Are you oxygenating to culture up before pitching into an oxygen free enviro?

How are you keeping your brett cultures for re pitching, or are you buying new each time from WLP,WY..

And finally if you had three topics you think are most important to Brett and would be interested to read or learn more about with regards to use in the Brewery what would those be.

Hope this wasn't to long of a run on thread. I would be interested in swapping emails with a few of the microbiologists and more experienced brett users for some detailed conversations and some knowledge AlB, Mike T, Steve G, N8, any interested?


Al B
12/28/08 11:03 AM  
Re: Brett research and general knowledge
Lots of good questions, I've used all of the commercially available bretts and several cultured ones from various bottles. B. intermedius and B. custerianus are not available unless one is willing to shelve out $200 for a

lyophilized stock culture. I store all of them on YM agar slants in my refrige. I propagate them using a stir-plate + air pump/pure O2 canister. Continous aeration with the air pump + stirring, Pure O2 bursts in the first hour of propagation.

01/05/09 11:25 AM  
Re: Brett research and general knowledge
Hi Chad,

Taking hints from Al and converting to my non-lab apartment setup, I store bretts on slants and petri dishes (using DME and glucose together as carbon sources, plus a bit of Wyeast nutrient) and build up from these by looping a colony or two into wort made from 5% DME and 5% glucose and Wyeast nutrient. B. claussenii I continuously aerate from the start, the others go into a once-shaken test tube of wort and from there propagate to continuously aerated starter. Works for me.

As you probably know, the distinctions "brux", "lambicus", or "custersiansis" are not sufficient to specify a strain. For instance, there are over 20 strains classified as brux but which have wildly different properties (this shown in a recent wine paper on 4EP production). So, I think that for brewing purposes each strain has to be judged and considered on its own, and the classifications are not very useful.

Here are 2 questions I think are very interesting and possibly open. I've read a lot of the lit, but not Verachtert's lambic papers so some of this may be in those. Anyway:

1. does any brett produce significant lactic acid under any conditions

2. does any brett produce significant citric acid under any conditions--you can find the basis for this question by google-patent-ing 3733253 and 3801455, in which huge production is claimed. That would seem to be a definitive "yes", however in the 37 years since these patents very little mention has been made of citric acid in relation to brett. BTW I think the brett used in those papers is the type strain of claussenii (which may be the same as, or totally unrelated to, WLP claussenii).

01/22/09 03:38 PM  
Re: Brett research and general knowledge
Sorry for the hiatus I had to finish writing up the Bretta paper over the holidays.. And attended the big beers fest in Vail, CO. a plethora of wild ales and brett funk beers to be found...

Baums I think you make two good points in the Brett IPA thread..and then expanding on them here too.

I did a search of the two patents although I didn't read the study that was done or the patent write up. I was able to find the species which were studied as they are held by NCYC depository, but it was Dekkera bruxellensis which was found to produce citric acid. Baring a discussion on the differences between the two, this was what I found. On the 3801455 patent I'm guessing it was maybe the same? Couldn't find data about the species used. Although in these patents many other species of Candida, Pichia were also all found to produce citric acid atleast in the 3733253 patent, so I'm not sure what was going on, was it a biochemical process with a medium similar to wort with these yeast in which they were able to then produce citric acid? (I'll expand on my thoughts later..)

As for lactic acid I often read about it here on these boards as being produced from Bretta, but the 25 papers or so I have, which from what I can tell is nearly every possible paper published scientifically in English or translated anyways spanning from Clausen 1904 paper up to a paper or two released in 2008, there has never been lactic acid observed to be produced by Bretta. The lactic acid is always created by Lactic Acid Bacterias? Right? Thats what I gathered.. Hence why Peter Bouckaert takes the first running of wort inoculates with lactobacillus at about 100ppm and then re-pitches into the kettle during boil. (By the way Peter went up about a week later and Brewed with Elysian Brewing in Seatle and they made an all Brett beer in this way as he always recommends) Back on track with the Lactic acid. As it has not been observed, this may be due to I also never remember any of the studies actually monitoring for lactic acid production by bretta only acetic acid production. So maybe it has not been observed since it has not been looked for. Anyone remember differently?

As for citic acid production.. this is a interesting idea. Many people are noticing the citrus flavors coming through in brett beers. Even Peter Bouckaerts hinted at it. That would would be a very skilled palate as New Belgium has daily taste panels and with his experience he would be one of the most skilled tasters trained to look for such flavors.

Also on this note, while at Avery's in Boulder, CO. last week having at look at Brabant before all the barrels are blended together and readied for bottling Andy the barrel aging guy wheeled out "Bad Sali" They have a barrel that is producing amazing aromas/flavors. They are taking their Salvation golden strng and barrel aging it. On first taste I was amazed at the Orange Bomb flavors as i instantly blurted out. I have never had a sour beer taste this amazing before. Days earlier I had just had Santification, Consecration, Temptation from Russian River, along with Cerise Casee from Cambridge brewing, New Belgium has produced a speacial La Folie for bottlewoks with Lambicus this time around, and none of those while some having some lemon zest and others stronger horsy barnyard had this amazing orange citrus flavor. So is citric acid being produced by Bretta? I think that will become part of a parameter in my upcoming masters project which is taking shape.

On that note I would like to present what I have come up with as my proposed masters research project. These projects are only supposed to encompass about 2-3 months of research and lab work (The usual UK masters doesn't do the large thesis like in the US it is more geared to getting you skilled to enter the work place so they do "Masters Dissertations"or projects instead to finish out the Masters degree) so I may be getting in over my head here and this could be cut back but here is my hopes..

First, I plan to look into is propagation of Brettanomyces for primary fermentation.

Starting possibly from single colonies streaked or kept on slants, then grown up into vial tubes, then propagated into 500mL Erlenmeyer flasks always with a standard stock solution of sugars and nutrients similar to wort.

The variables I will be looking into are:

-Optimal dissolved oxygen levels/aeration rates for cell biomass production with monitoring acetic acid production.

-Also looking at with or without the use of a stir plate/agitation on cell biomass and acetic acid production.

-Optimal pH and Temperature range when propagating.

-Looking at cell viability while propagating and the time necessary till a second step is needed in order to get cell counts high enough for pitching into a primary fermentation.

-Basically the optimal parameters for propagating up Brettanomyces.

Second, I plan to look at pitching rates of Brettanomyces when used for primary fermentation.

Multiple strains of Brettanomyces being pitched at multiple cell counts per mL or wort, grown up using the optimal conditions found previously in the study. The fermentation would be from wort brewed up on the 2hL pilot plant we have at the University. A completely anaerobic fermentation conducted in 20L batches.

The variables I will be looking into are:

-Days till fermentation is complete.

-Attenuation rate as a primary fermenter.

-Optimal fermentation temperature.

-Looking at any lag phases due to possible observance of Cutsers effect and how the phenomenon is possibly over come due to pitching rates or glucose levels...during a primary fermentation.

-Viability of Brettanomyces once fermentation commences.

-Glucose consumption and ethanol production in comparison with acetic acid production.

-I will be looking for an optimal Brettanomyces fermentation, the reaction of each yeast strain as a primary fermenter, possibly playing around with the amount of fermentable sugars available for an optimal clean fermenation, keeping acetic acid production low as possible and attaining the highest level of ethanol production.

Third, I plan to look at the aroma/flavor compounds being produced being during a Brettanomyces primary fermentation.

It is already well known about the production of ethyl acetate, ethyl lactate, lowered levels of isoamyl acetate, ethyl caproate, ethyl caprylate, ethyl caprate, 4-ethyl phenol, 4-ethyl guiaicol...

I will conduct multiple mini ferments (20L) under the optimal conditions found in the earlier experiments I conducted and then see what kind of compounds each strain is producing by running Headspace GC on each mini ferment. Along with testing bottles donated from Vinnie Cilurzo (Supplication) and Avery (Avery 15). I will be looking for possible esters which have net yet been reported due to a Brettanomyces fermentation or the possible lack of production when used as a primary fermenter.

So now any one see any ways I can tweak this?

As you can see in the end I'm really interested in trying to also discover compounds which have not been observed in Bretta spp. fermentations yet. When talking with Vinnie of Russian River we both felt there were un-known compounds/esters being produced during the fermentation with Bretta spp. Hence his giving me a bottle for research purposes. I also got a Avery 15 for research too. None of this work is underway yet as I'm re shaping and re- writing. I will work the citirc acid in as said earlier too.

So if not all of this something from this will be conducted.. Oh yeah and White Labs 3 varieties will be used plus I'm asking Chris White if they have more as they mention it at the bottom of the page that they will be testing more. Wyeast has teh brux and the lambicus which i will also try to include in and that would make 5 so I may acquire more but at least two of each (brux, lambicus, anomalus(claussenii) would be nice. If anyone has a unique culture I would be interested in working out a way of shipping it to Edinburgh, Scotland and I don't mind covering the cost obviously.


01/22/09 06:20 PM  
Re: Brett research and general knowledge
Your project sounds very cool (and also pretty ambitious for 2-3 months time). I have some thoughts but they might wander pretty far off topic for the board--you mentioned swapping emails?

Anyway in general I think a fair bit is known about what brett can, might, or sometimes will do, but a lot less is known about how to control it. Personally I'd be most interested to see research that fills that gap.

Bad Sally is awesome.

01/23/09 12:46 PM  
Re: Brett research and general knowledge
you can reach me at chadyakobson at gmail dot com

After I saw the size of my post I was sure no one would take the time to read through it...

I know it is over ambitious for the 2-3 months which is why I'm trying to get started now as I have until about August to rap up the lab work and fermentations and then finish writing by Sept but the sooner the better..

The objective of the overall research project would be how to optimally control brett in order to obtain more consistent brett fermentations and certain characteristic aroma/flavors more consistently. If that is possible?

Al B
01/23/09 02:05 PM  
Re: Brett research and general knowledge
What pitch rates are you considering CFU/ml wort?

My thoughts on my experiment were a log difference:

~4.0 x 10^6 High

~4.0 x 10^5 Low

For WL B. clausenii

Al B
01/23/09 02:10 PM  
Re: Brett research and general knowledge

You may want to look at the Homebrewers Conf 09 presentations(beertown.org). There is a Brett study performed by WY that may offer some insight or info.


01/23/09 02:55 PM  
Re: Brett research and general knowledge
Just took a look at it...Yes that is along the lines of what I'm looking to do. Didn't know anything like that had been done yet. Good, I can use some of that to build upon initial ideas. Thanks for that.

Hopefully I can build on what they presented by using both WY strains and at least three from WL. Its possible they have some unreleased strains according to their website...

I'm surprised to see they reported Orval only uses 5000 cells/mL. What are other people using for secondary in bottle?

~4x10^6 with what degreesP ?

I think log differences would be best to play around with. I know Vinnie under pitches as he likes the results and Avery was saying they liked to pitch to standard, but this implies there is already an industry standard so I was planning to email them back to get the exact figures. But otherwise I hadn't formulated and opinion yet. What have your person results been like with those rates so far? Any reason for using 4x10^6 instead of 10^5cells/mL or 10^6cells/mL per degreeP or something along those lines?

Al B
01/23/09 10:00 PM  
Re: Brett research and general knowledge
<<~4x10^6 with what degreesP ?>> Oh, I was thinkin of 15 Plato

<<Any reason for using 4x10^6 instead of 10^5cells/mL or 10^6cells/mL per degreeP or something along those lines>>

Not really, perhaps off of "normal" pitch rates for Normal yeast.

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