More nerdy science stuff- This time, Lacto!

Reads 2916 • Replies 34 • Started Sunday, April 17, 2016 3:11:51 PM CT

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CLevar
places 23 º 15:11 Sun 4/17/2016

As some of you might know, I am currently working as the assistant brewer at a local craft brewery that has been doing a lot of kettle sours. Before I started full time in that role, they brewed their first kettle sour with a mixture of Lacto strains I provided them. This mixture worked pretty well, but we wanted to be able to have a single strain banked with a commercial lab so we could move towards something reproducible on a commercial scale.

In the end, we settled on a cool Lacto. species that I had isolated out of a beer aged in a red wine barrel. According to the brewer of this beer, no Lacto was pitched, so my current thought is that it was resident to the barrels from the malo-lactic fermentation that likely occurred. This strain has the ability to make lactic acid from trehalose (a carbon source released by lysing yeast cells), so it probably could exert its influence on finished beer even after most "good" sugars are consumed by yeast.

Some important characteristics:

*It produces only lactic acid (in my hands).
*It grows well on agar plates at room temp in atmospheric oxygen concentrations: No need for an incubator or the ability to produce low oxygen (candle jar) or anaerobic conditions (anaeropacks, etc)
*It sours well over a wide range of temperatures, from room temp (~65 F) to the temp we do our kettle souring at (107 F) with minimal gravity decrease.

We now have this strain banked, and are able to order pitchable quantities of it fairly regularly.

One of the things we’d like to be able to move towards is utilizing this strain in a "mixed fermentation" or "co-fermentation" capacity. There are a couple of reasons for this, but the most important one for us is that a co-fermentation sour does not tie up the kettle for an extra day and allows up to brew more beer. For this to happen, a few things are required of the strain:

*It must be able to produce lactic acid at a temp that other microbes (specifically, Sacch and Brett) are happy.
*It must be somewhat hop tolerant, as some of the beers we’d like to brew using this strain would have more than the 2-5 IBU that many say Lacto is sensitive to.
*It must be able to compete with other microbes (specifically, Sacch and Brett), such that a pH drop occurs even when other microbes are competing for carbon.

We know that we can check point one off, so it’s the second two that I’ve been working on lately. So the last few times that we’ve ordered this strain I’ve gotten a few small scale incubations going to see if the strain we already use could also be good for these mixed culture ferments.



A few caveats here: I’ve not normalized the pitch rates by anything other than volume. So, it’s possible that one of the "generation 1" (g1) pitches that we received had more or less viable cells in it than anot

 
CLevar
places 23 º 15:14 Sun 4/17/2016

A few caveats here: I’ve not normalized the pitch rates by anything other than volume. So, it’s possible that one of the "generation 1" (g1) pitches that we received had more or less viable cells in it than another pitch. All of the IBU values are calculated and achieved by using isomerized alpha-acid extract

So far, so good.

I’m planning on getting some more experiments going this week to test how well this strain does when Sacch. is added to the mix and it has to compete with something a bit more robust than Brett. I’m also really interested to see how the apparent hop tolerant nature of this strain compares to something like L. brevis, commonly thought of as being a hop tolerant Lacto species.

Any questions or suggestions, I’d love to hear them

 
CLevar
places 23 º 15:14 Sun 4/17/2016

Wow, I really borked that post. Stupid character limit!

 
Frank
beers 4464 º places 92 º 17:26 Sun 4/17/2016

If you are looking for a name for this strain, I recommend Frank.

 
CLevar
places 23 º 20:59 Sun 4/17/2016

Originally posted by Frank
If you are looking for a name for this strain, I recommend Frank.


Noted !

 
GarethYoung
beers 1111 º places 27 º 03:17 Mon 4/18/2016

Cool stuff!

Have you got a graph of the alcohol (or even gravity) change in the experiment with brett? Would be interesting to see how much of the acidification happened before the brettanomyces got to produce a substantial amount of alcohol.

Are you going to get a chance to repitch the strain much? You can train lactobacillus to increase its hop tolerance by reusing it in hoppy beers. I’d be interested to know how that worked out, if you’re planning to do it.

On kettle souring and tying up your kettle for an extra day: have you experimented with upping your lacto pitching rate? Most strains should be able to sour the beer in about 8 hours, if you pitch lots. That would let you do an evening mash and sour overnight, finishing up in the morning. Depends on how busy your brewery is, but that might help.

 
konstifik
beers 573 º places 37 º 03:40 Mon 4/18/2016

So, this strain looks like it has a stronger souring potential along with brett than on it’s own, at least in the presence of IAA, right? Or could it somehow be explained by the pitched lacto being of a second generation?

In the 2 Plato wort it seems like there’s a IBU-threshold somewhere between 0 and 5 IBU, above which the strain behaves more or less the same. A very simple (and uneducated) theory is that there is such a threshold for the 10 Plato wort as well, but that it’s higher than 30 IBU and thus doesn’t show in the experiments you have presented here. I would also guess that the strains behaviour will be more differentiated at lower levels of bitterness in the 2 Plato wort, but that the resolution in your data is to low to show that (e.g. if you would test it in a 2 Plato wort with IBU’s ranging from 0 to 5 in steps of, say, 0.5 the resulting chart would by my theory be similar to that of the 10 Plato wort).

If this guess where to hold true you could probably, with some more experimental data, work out a formula for how the strain behaves (approximatively) in different worts. Probably goes a bit deeper than your brewery have practical use of, but it would make for some nerdy fun.

 
HornyDevil
07:58 Mon 4/18/2016

Originally posted by CLevar
I’m planning on getting some more experiments going this week to test how well this strain does when Sacch. is added to the mix and it has to compete with something a bit more robust than Brett. I’m also really interested to see how the apparent hop tolerant nature of this strain compares to something like L. brevis, commonly thought of as being a hop tolerant Lacto species.


Looking forward to these. Looks like you’re getting some decent acidity in short periods of time with decently high IBUs. Anxious to see how sacch. affects this as brett seemed to have little to no effect. Probably because of its slower growth rate.

 
CLevar
places 23 º 08:19 Mon 4/18/2016

Originally posted by GarethYoung
Cool stuff!

Have you got a graph of the alcohol (or even gravity) change in the experiment with brett? Would be interesting to see how much of the acidification happened before the brettanomyces got to produce a substantial amount of alcohol.


These experiments were 50ml cultures- so not enough for measuring gravity change in any meaningful way.


Are you going to get a chance to repitch the strain much? You can train lactobacillus to increase its hop tolerance by reusing it in hoppy beers. I’d be interested to know how that worked out, if you’re planning to do it.


Not much reason to do this in the brewery, but it’s something I’m working on in the lab. Frankly, acidification down to 3.6 in a dry beer with 20-30 IBU in three weeks is enough as far as we are concerned. Anything more and things start to clash. So I’m fairly happy where things are at as far as IBU tolerance is.

On kettle souring and tying up your kettle for an extra day: have you experimented with upping your lacto pitching rate? Most strains should be able to sour the beer in about 8 hours, if you pitch lots. That would let you do an evening mash and sour overnight, finishing up in the morning. Depends on how busy your brewery is, but that might help.


We’ve played around a bit and target 3.2-3.5 in 16 hours. We actually just got a tank specifically for propping this strain and will now be able to pitch ~8% instead of 3%, but the same practical problem will exist. To explain- we have a 7.5 bbl system and brew into 15bbl FVs.

So what this means is:
Day one-mash batch one, lauter, boil 20 min, cool to 107, pitch LAB (2pm)
Day two-harvest LAB, boil batch one, mash batch two, lauter, boil 20 min, cool to 107, pitch LAB (2-3pm)
Day three- harvest LAB, boil batch two

We can get other things done of course, and have (when feeling especially saucy) knocked out a double brewday in addition on day three. But unless we can pick up a whole day in there...

We have, for re-pitched LAB on day two, gotten the pH down to 3.5ish by 8pm or so the day of pitching. But it’s not *quite* low enough, and firing up the kettle and mashing in another batch at that time would mean I’m brewing till 5am and thus worthless for getting anything done the next day.

 
CLevar
places 23 º 08:28 Mon 4/18/2016

Originally posted by konstifik
So, this strain looks like it has a stronger souring potential along with brett than on it’s own, at least in the presence of IAA, right? Or could it somehow be explained by the pitched lacto being of a second generation?


I think the small difference is due to a combination of factors.
First, gen two lacto.
Second, ion uptake by the yeast (pH drops from 5.2 to 3.9-4.5 depending on the brewers yeast used, I would expect a similar ion uptake by Brett though I’ve not tested this)
Third, possible acetic production by Brett. This was done in 50ml plastic conicals, so I’m sure there was a touch more oxygen than I would have liked.


In the 2 Plato wort it seems like there’s a IBU-threshold somewhere between 0 and 5 IBU, above which the strain behaves more or less the same. A very simple (and uneducated) theory is that there is such a threshold for the 10 Plato wort as well, but that it’s higher than 30 IBU and thus doesn’t show in the experiments you have presented here. I would also guess that the strains behaviour will be more differentiated at lower levels of bitterness in the 2 Plato wort, but that the resolution in your data is to low to show that (e.g. if you would test it in a 2 Plato wort with IBU’s ranging from 0 to 5 in steps of, say, 0.5 the resulting chart would by my theory be similar to that of the 10 Plato wort).

If this guess where to hold true you could probably, with some more experimental data, work out a formula for how the strain behaves (approximatively) in different worts. Probably goes a bit deeper than your brewery have practical use of, but it would make for some nerdy fun.


I actually think the data indicate the strain is *more* tolerant at the 2P concentration. If you look at the third panel on the figure I posted, you can see that the pH drops faster in the 2P wort than the 10P wort. Also, if you look at the different concentrations in the first and second panels, it seems like there is a pretty good "dose response" in the second (10P wort), whereas for the first (2P wort) the strain does about the same regardless of 5-30 IBU. Make sense?

I have a sorta explanation for this, but I want to do more reading first to see if I am on the right track.

 
CLevar
places 23 º 08:40 Mon 4/18/2016

OK, so this is very preliminary data from a while ago (and was what got me more interested in the house strain as a potential hop tolerant LAB). There are lots of problems with this (obviously), and I need to repeat this, lower temp, carry it out for longer, do it in 10 P wort, etc, etc, etc. BUT



One of the other big questions that I have is "What does it mean when we say ’Hop tolerant?’" Context matters. Pitch rates, wort composition, viability, etc. Depending on how we set up the experiment, a strain could look hop tolerant or not, or look like a good acid producer or not.

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