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Triple Freak
09/09/07 09:00 AM  
Brett Starters
How would one go about making a Brettanomyces bruxellensis starter? I know how to make a regular starter. I also have a stir plate & several 4 L flasks.

Would I just use regular DME or what?

Thanks for any help.

Brendan
09/09/07 09:15 AM  
Re: Brett Starters
Seeing how brett is yeast? why do anything different from a "regular starter"?

DME? Water? nutrient? Sugar? hops? maybe a combo of all 5?

Are you making an all brett beer? or trying to build up cells to innoculate in secondary. In my experiences, Brett doesn't need a whole bunch of cells. I lean on time, and let the slow growers do their thing.

ewanzel
09/10/07 09:48 AM  
Re: Brett Starters
I'm in the process of stepping up WLP Brett C & L. I used typical starter methods (wort pulled post boil from a mild with a SG 1.038) and they are chugging along. From my experience (which is limitted) they take a little longer to establish themselves then sacch strains when provided with the same conditions. What I did was to initially start them in 1L of wort let them ferment out and then I stepped them up to 3L (which is what I plan to pitch). Hope that helps.
Triple Freak
09/11/07 07:22 AM  
Re: Brett Starters
I was under the impression that Brettanomyces bruxellensis needed something special to make a starter with.

I know it feeds more on glucose than maltose. I was thinking a simple sugar solution would provide better results than using DME.

Maybe I'll experiment with it. Maybe make a 2 L starter with pure glucose (corn sugar) & make another starter with maltose (DME) & see which one grows quicker.

Baums
09/11/07 10:17 AM  
Re: Brett Starters
"I know it feeds more on glucose than maltose."

I am not sure exactly what you mean by this? I do agree that just about any yeast will get a bit more from an equal weight of glucose vs. maltose, just because some energy is necessary to transport and break maltose into two glucoses. So a starter with pure glucose (+ nutrients) will therefore probably "do better" in some sense than a starter with pure maltose (+ nutrients), which of course DME would roughly provide. But, I'm not sure why this would apply more to b. brux than any other yeast, say WY1056.

On the other hand, the original b. anomalous strain was so-named because it apparently did not ferment maltose well (or maybe at all). I don't think this is true of the common brux, claussenii, or lambicus strains though--and in fact it's not even true of some other "b. anomalous" strains.

But, if you just want to speed up the starter process (with any yeast) then I think a higher-glucose starter makes sense. I would go with Al's suggestion from a while back, to just add some glucose to the DME, which seems like the easiest way of making sure there's a decent base of nutrients as well.

SteveG
09/11/07 10:40 AM  
Re: Brett Starters
The best guys to comment here are N8 or Al, in general though it is important to NOT look at brett as just another yeast. When Al has worked them up for me he's handed of a few hundred mils of pure slurry. I think providing lots of O2 in the growing process is important.

Brendan, the reason to treat it differently is - as ewanzel mentioned - it does take a lot more time and help to really get going. When it is going it ferments like a school of pharana fish, but until that time it can be suceptable to unwanted outside influences as can any yeast. And mostly the containers we all use are not completely inpermeable to air exchange.

There is really good info in the "Brettanomyces Brewing E-Symposium Transcript", the link can be found in the blue box up top the main homeBBBrewBoard page.

Al B
09/11/07 11:23 AM  
Re: Brett Starters
Brett starters can be handled like other yeast starters - which are certainly necessary for all-breet brews. One can add 1% CaCO3 as a buffer, but its not crucial. As long as you handle living cells properly - you'll be fine. Nutrients (amino acids), sugar, and O2 is all that you need for propagation.

For example, my next batch of lambicus - I've decided to grow the cells on basic agar plates containing (40g Dextrose,10g amino acids, 15g agar - pH 5.6). Growth was fast, healthy, and abundant in 24-48hrs at room temp.

And yes, generally glucose is consumed faster than disaccharides and trisaccharides, and so forth.

Al B

Baums
09/11/07 03:03 PM  
Re: Brett Starters
The only sizeable brett starters I've made have been Wyeast b. lambicus, and for me it behaved in a starter just like your average saccharomyces would (except for aroma/taste of course). So, take that for what it's worth.

It does seem like other strains can be slower (claussenii for instance), and I think each brett strain needs to be considered very individually. But I also think Al's advice to "handle living cells properly" is a good way to think of it. O2, nutrients, carbohydrate(s), temperature, sanitation, are important for growing any sacc or brett. Two potential differences that have been talked about here (for brett versus saccharomyces) are

1. Speed of fermenting various carbohydrates

2. Potential for making harmful levels of acid in the presence of lots of O2

Whether or not these are serious issues for particular strains is something that I guess we're in the process of finding out. I can say that *for me* neither of these was a problem in a stirred starter with WY lambicus in Munton's DME (at 1.040) + Wyeast nutrient added at recommended propagation levels. For my part I am still not sure about WLP b. claussenii.

Brendan
09/11/07 06:43 PM  
Re: Brett Starters
Steve,

<<Brett starters can be handled like other yeast starters.>>

And all yeast need O2 to build up glycogen for reproduction.

As I posted before, time is an issue. However, I believe in my sanitation procedures, and am not afraid of my starters being infected whether it's 1056 or Brett.

Do you worry about O2 permiablity with starters? Did I read that right?

Al B
09/11/07 07:34 PM  
Re: Brett Starters
Baums -

I haven't had an issue either with propagating Brett (except for WL Clausenii - that I attributed to an old culture). Once clausenni was up + running, it fermented as fast as any yeast culture....but overall growth kinetics depending on the strain can vary slightly. I do think storage temperature/storage medium/and age are factors.

Baums
09/12/07 11:30 AM  
Re: Brett Starters
Al, I feel like I've heard several similar comments about the WL claussenii, to add to my own experience (with a very fresh culture) where getting it "up and running" from colonies on agar was troublesome. Getting it going on the agar was easy, but it didn't transfer to growth in liquid as easily as a sacc or WY b. lambicus.

I only tried twice, before my claussenii experiments were sidetracked by some other odd things I noticed and don't fully understand yet. I would be interested to hear about anyone's experience with growing WL claussenii on plates.

There's an interesting description in one of those very old papers available on PubMed, of how there's a significant lag when the brett they were using transitions from aerobic to anaerobic growth. There is an anaerobic step in my process when I propagate from agar--all the liquid starters are stirred except for the initial 10 mL in a test tube. Perhaps eliminating that step, or somehow making it aerobic, would help get the claussenii going faster.

Al B
09/12/07 02:17 PM  
Re: Brett Starters
The phenomenon you are experiencing with clausenii has happened to me as a microbiologist using bacterial isolates as well. Certain isolates when subcultured from one agar type to another lose viability, and its necessary to go back to the original culture to regain growth. Why?

Colonies of growth on agar (or broth) are many generations in population. Lots of oxygen available for sterol production for healthy membranes, etc etc. But an environmental shock can throw healthy cells out of whack.

(pH differences, water availability, too much nutrients can cause osmolarity shock, alcohol, and so forth). This isn't an issue most of the time, cells can recover usually.

A note on stir plates: they can get freakin' warm transferring the heat to the culture. This speeds up things, but not necessarily O2 diffusion to the culture. Its really amazing how fast a small culture can go from respiration to fermentation......and possibly to cell death. I've had a culture spinning on a stir plate once, and I stopped it - bubbles of fermentation were in full swing! Closed the cap tight, waited, then reopened - yep CO2. Because of this, I've been using an air pump + stone for liquid starters.

Baums
09/12/07 02:40 PM  
Re: Brett Starters
Those are interesting points. Maybe using more dilute media makes sense for at least the first propagation. Instead of 10 mL at 1.040, maybe 50 mL at 1.010, which might allow a stir plate to be used as well, so that the ferment (or possibly respiration?) can stay aerobic.

Thankfully my homemade stir plate doesn't get hot at all. There's a route for the muffin fan to suck in fresh cool air and blow it back out a different way.

As for respiration vs. fermentation... wouldn't you see fermentation with the air pump + stone as well? My understanding is that for reasonably high sugar levels (certainly above 1.020), saccharomyces will ferment instead of respire even in the presence of lots of O2 (but at higher biomass yield when more O2 is available). Or are you incrementally feeding to stay below the Crabtree limit?

Does Crabtree apply to brett? The ethanol-to-acetic metabolism is a bit of a monkey wrench there.

Al B
09/12/07 03:07 PM  
Re: Brett Starters
Yeah, incrementally feeding, if not, refrigerate promptly.

Seems to be a bit crazy w/ Brett in this regard, seems each Brett is bit different in its kinetics. That is, lambicus from WY is much slower in resp/ferm. vs clausenii (so far from I've seen). It took alot of effort. Maybe I should just buy alot of pouches, but the growth on agar for lambicus is awsome .....and I have plenty of plates....

I have clausenii on a slant, I'll streak it out and see how its doin.

Corky Stewart
10/18/07 03:10 PM  
Re: Brett Starters
I made a starter using DME and Orval dregs(2 bottles). Within 2 days I had thick krausen in the flask and it smelled like Orval. I pitched it into a saison I had in secondary(See my post "Bubbles"). It still smells heavenly.
Ryan
10/22/07 10:23 AM  
Re: Brett Starters
SO Al and Baums

when you guys make starters with Brett do you do more than oxygenate the starter vessel at first pitching? How many days of constant feeding do you use before you're confident that you have a good colony?

finally, does that starter matter only in the case of a 100% brett ferment or do you do this for secondary as well?

ryan

Al B
10/22/07 10:40 AM  
Re: Brett Starters
You should treat it like normal yeast (see above) and for a 100% brett brew, aim for a pitch rate of a lager (higher amount) - depending on the size of the batch of brew.

It is not necessary to dose a high amount for secondary fermentation.

Baums
10/22/07 12:29 PM  
Re: Brett Starters
WY b. lambicus seems to act just like a saccharomyces for me. I've gone from agar colonies to 10 mL of wort in a test tube, with basically no aeration, and it shows obvious signs of fermentation in a day or two. Then it grows well on a stir plate after that.

I don't have much experience/suggestions with 100% brett brews--just a couple small (1 G) experiments that fermented much like sacc would.

I agree with Al about not needing much brett for a secondary ferment. 1 mL of starter (not even slurry) added to a 375 mL bottle for bottle conditioning, adds an obvious effect after a couple months. That works out to a 50 mL starter in 5 gallons of beer, and I'm certain that's not the minimum.

Ryan
10/22/07 02:16 PM  
Re: Brett Starters
Baums

<<1 mL of starter (not even slurry) added to a 375 mL bottle >>

<<That works out to a 50 mL starter in 5 gallons of beer>>

when you measure X ml of starter (that isn't slurry), is there a qualitative way for you to describe the density of cells in suspension? I don't have a way to count cells, and I'm always curious what people mean by "slurry" versus "liquid"

Baums
10/22/07 04:04 PM  
Re: Brett Starters
I'm not sure what's a good way to define how dense a "slurry" is, other than diluting and counting cells.

I think it's easier with a "starter" though. Since people like to talk about cell counts, I guess the question is how many cells do you grow in a liter of starter. Answers vary, but at least people have taken a shot at this (for instance, Designing Great Beers has a table). 100B cells/L is a number I've seen from a few different experiment reports, for saccharomyces in a 10P starter on a stir plate with loose cover. When I say "50 ml starter" I'm talking about a 10P starter, with Wyeast nutrient at recommended levels, on a stir plate with loose cover. Absolute cell counts might be different with brett but everything should still scale the same.

So, if you were doing the same process then I think we'd be close. Otherwise we'd have to consider what difference there might be our cell yields.

Ryan
10/22/07 04:10 PM  
Re: Brett Starters
Well, for one, I usually have an airlock on my flask. Its on a stir plate, and I've always wondered if the airlock made the stirplate moot, since it can't get any more air than is already in the flask. But I hit it with pure 02 and add yeast nutrient. I usually innoculate a 1L 10P starter with a WYeast smack pack.

Is a loose cover the way to go (like aluminum foil)? I've always worried about infecting the starter if using sacch.

shane
10/22/07 07:42 PM  
Re: Brett Starters
foil is fine, just boil your starter with it on and no worries. just make sure it overlaps well and is snug
Baums
10/23/07 10:41 AM  
Re: Brett Starters
An airlock doesn't make the stirring entirely moot (since the stirring expels excess CO2 from solution, and makes nutrients more available) but the big point of stirring is continuous aeration and yeah, an airlock negates that. With an airlock you drop to ~50B cells/L.

Large amount of uncertainty on all of these numbers of course. Maybe +/- 30% or more. But you have to start somewhere.

Anyway as Shane says many people use loose foil covers without infection trouble. I suspect that it's very possible to crimp the foil cover so tight that when it's really going, the outflowing CO2 doesn't allow any air to get in. That would defeat the purpose, so I try to keep the cover very loose.

I think in labs (Al?) they use reusable stainless caps that have a gap to let air in (or porous foam closures, or roller bottles, or other tricks) for aerobic culturing. A loose foil cover is pretty close to that kind of cap.

Al B
10/23/07 11:03 AM  
Re: Brett Starters
Yes Baums, SS caps are sometimes used. I have been using screw cap loosely on my pyrex bottles. Although the optimum method would be to use an air pump with the stir plate. Oxygenation and agitation together. After a couple of days, amounts of sugars/nutrients will be depleted. At this point, it is important to decant off a large portion of the slurry for storage in the refrigerator if you continue to grow more cells OR step up to larger bottles/starter volumes. Otherwise glycogen reserves in the cells will be converted and they will not be ready for quick fermentation.
Brin
12/03/07 04:21 AM  
Hello
Hello, nice site :)
DrunkenPanther
01/11/08 03:49 PM  
Re: Brett Starters
Quick question on bumping brett starters....decant off just like yeast and it will form a new pellicle and such if ya let it sit?
 
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