I’m still growing the fellow, though I’ve pitched some into a saison just for fun, but it will be a while before I’m drinking that. It has various brett strains and lacto, so it won’t be easy to pick out the specific contribution of the debaryomyces, I don’t think.
There could be saccharomyces in there, but they ferment for at least 9 months at the brewery and it had been in the bottle for a while, so I’m guessing it should be mostly debaryomyces.
Just in case, though, once I’ve got enough to spare, I’m going to put some in a 10% salt solution of wort. That should be high enough to inhibit everything except the debaryomyces, and I’ll see if the resulting yeast seems different from the rest of the stuff I’ve got. |
I think there’s definitely a fair whack of saccharomyces in this. In the initial starter from the dregs a pellicle, and some funky aromas, arrived fairly quickly. I stepped it up to 1 litre and had a pretty healthy fermentation, but with no signs of a pellicle, and a pleasant but non-wild aroma. Once fermentation had died down, a pellicle arrived and some more interesting aromas (nothing sour about it, so hopefully it’s the strain I want).
I’ve stepped it up to 2.5 litres of 1.025 wort with 10% salt to see if that helps increase the quantity of debaryomyces. I guess we’ll find out if the strain really is debaryomyces, since if it’s not (or it’s some non-salt-resistant kind), it’s not going to be a very exciting fermentation. |
Just to bump this, Gareth was kind enough to send me a sample of this, which I received yesterday. I am currently working on isolating it, and hope to be able to send it out to other people if and when that happens (With Gareth’s permission of course!). |
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Great stuff. Send it everywhere! |
Just a quick update-
I’ve got stuff up on plates and in tubes.
What I did-
I made 1:10 serial dilutions of what Gareth sent me in a medium made of Yeast Extract, Malt Extract, Peptone, and Glucose (YPGM). From the 10^-3 dilution, I streaked onto agar plates made of the same medium+1.5g/L agar powder. Tubes were incubated at 25, and plates were incubated at 25, 30, and 37 degrees C.
All of the tubes show growth, with the 10^-1, 10^-2, and 10^-3 showing pelicle formation. There is only slight turbidity in the 10^-4 tube, so I’d expect once the cell number get high enough we will also see a pelicle there.
I also got two distinct colony morphologies on the plates incubated at 30 degrees. Only one type of colony was present on the 25 degree plates, and no growth was observed on the 37 degree plates. The colonies on the 25 degree plates are identical to one of the morphologies on the 30 degree plates, so we are dealing with a mixed culture of at least two organisms. For now, I’m calling them "Deb 1" and "Deb 2". Deb 2 looks like run of the mill Sacch to me, whereas Deb 1 looks like some of the environmental isolates I’ve cultured before; Flat, spreading, white-grey in color.
Because Debaromyces is supposed to be very resistant to chlorine dioxide, I used both isolates, the sample Gareth sent me, and one of the dilution tubes to set up a series of incubations with or without chlorine dioxide at an initial concentration of 0.3mg/L. If one of the isolates grows, that would be great. Otherwise, the chlorine dioxide will serve to enrich the mixed population for whatever (very) small number of Debaromyces are there, and then I will attempt again to isolate on a plate.
Cheers!
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Little update.
I ran PCR on the two isolates I have using a technique nearly identical to the one described in this blog post by another person keen on beer microbiology: http://beer.suregork.com/?p=3598
Turns out this technique has been used a number of times for the rapid identification from brewery "contaminants"- See Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants and Sequence-based identification of species belonging to the genus Debaryomyces for examples.
Here are the results of that PCR (I’ve inverted the colors, as I prefer to see the bands in black...plus, it saves on ink if/when I print the picture for my notes!)
Lanes 1 and 6 are 1KB ladder from NEB.
Lane 2 is a Brett isolate from WLP.
Lane 3 and 4 are the two isolates from the sample I was sent.
Lane 5 is an isolate from Black Label Gueuze that I wanted to identify (A Brett, for reasons that you should be able to work out by the end of this)
Lane 2 shows me that the primers and my PCR protocol work (on Brett at least).
Lane 3 and 4 indicate that the two isolates are distinct (pretty easy to tell from morphology too), and that neither of the isolates are likely Brett, due to the different size bands.
Ideally, I would have had a Sacch isolate to run as well, but I didn’t have any grown up, but I think that it’s a fairly safe bet that Lane 4 is a Sacch.
Lane 3 I’m not so sure on. BUT, suffice it to say that it’s NOT the size that Debaromyces has been reported to be using these primers.
So, I’m currently working on doing some other stuff to try to pull out what Debaromyces might be present in the samples.
Stay tuned!
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Oh, I should mention that I didn’t do the restriction enzyme digestion due to not having the specific enzyme needed. So only the PCR was done. |
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So is this stuff tasting good or what?
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