Lactobacillus delbrueckii questions

I've been fine-tuning a berliner weiss recipe that I plan to do in a couple of weeks, and I realized there were some things about lactobacillus delbrueckii that I don't know. I have a vial of the White Labs strain if it's relevant.

1) What exactly is it that the lactobacillus delbrueckii is eating in the wort? I know one subspecies eats only lactose, but I can't imagine that being the case when it is used in beer. What sort of sugars is it eating specifically? To my knowledge, lactobacillus delbrueckii does not produce alcohol of any type. If it is eating some of the same sugars that the saccharomyces (in my case, a Kolsch yeast) would be fermenting, then wouldn't an ABV estimate by, say, BeerSmith, actually be too high since not all of those sugars would be available for conversion to alcohol now? I'd like to take that into account if possible, so I know what my actual achieved ABV would be.

2) Does lactobacillus delbrueckii fall out of suspension like yeast would? Is it something particularly visible? I'm going to be making a lacto starter, and don't really want to pitch a liter of weak, bad beer into the batch if I don't have to. It would be nice if the lacto could be settled to the bottom like yeast, so just that is pitched into the fermenter.

shanecb said:

...Does lactobacillus delbrueckii fall out of suspension like yeast would?...

Click to expand...

I haven't actually used Lacto. D. (WL strain) in a real beer yet but did build it up in a starter to freeze some off.

It floccs out pretty well. Seems to do it's thing, clump up and drop out pretty quick. I would compare it to a medium-high flocculating Sacc strain.

I wouldn't know if it the combination of Lacto and Sacc in the same wort would change it's flocculation characteristics though.

denimglen said:

I haven't actually used Lacto. D. (WL strain) in a real beer yet but did build it up in a starter to freeze some off.

It floccs out pretty well. Seems to do it's thing, clump up and drop out pretty quick. I would compare it to a medium-high flocculating Sacc strain.

I wouldn't know if it the combination of Lacto and Sacc in the same wort would change it's flocculation characteristics though.

Click to expand...

Great, thank you! I was hoping that would be the case.

I think Lactobacillus delbrueckii consumes lactose in homofermentive metabolism producing lactic acid as a bi-product. The bacteria is quite small so unless it forms a bio-film or clumps together in suspension which seems to be common in beer-related microbes.

http://www.bd.com/ds/technicalCenter/inserts/Lactobacilli_MRS_Agar_&_Broth.pdf

onemanlan said:

I think Lactobacillus delbrueckii consumes lactose in homofermentive metabolism producing lactic acid as a bi-product. The bacteria is quite small so unless it forms a bio-film or clumps together in suspension which seems to be common in beer-related microbes.

http://www.bd.com/ds/technicalCenter/inserts/Lactobacilli_MRS_Agar_&_Broth.pdf

Click to expand...

Are those the same subspecies as the one that is available for brewing? I actually don't know which subspecies is used in brewing, and I haven't found it yet, I just know that *a* subspecies eats up only lactose. It could very well be the same one, though.

So, assuming that lactose is primarily what it is hitting, is there actually that much lactose in a typical wort? How much does the lacto really have to work with? I'd like to think that if it's limited, adding a little additional lacto could really bump up the sourness if wanted.

Just used pure WL lacto for the first time. It did seems to clump and flocc out, but I'm a little suspicious that it was actually a wild yeast strain that got in there (not much sourness in the starter or beer, and it took a couple days to get going).

Wild Brews suggests Lacto Del it is a homofermentive glucose eating lactic acid producing strain. Not sure how accurate that is though. The Wyeast strain seems to keep making lactic acid long after the yeast would have consumed all the glucose.

I’ve long suspected the same ABV lowering effect of acid production. That said it would depend on the relative amount of CO2 being produced, and anyway lactobacillus is only supposed to make a max of 1% lactic acid (so the effect is pretty minimal).

Might be a good post to send to the BBB, there are many more scientific minded sour brewers on there.

I know one subspecies eats only lactose

Click to expand...

If it eats lactose, it can eat galactose and glucose, the simple sugars that come from that disaccharide. Beyond that, bacteria are pretty good at scavenging carbon/energy compounds. Lactic acid itself is produced from pyruvic acid, a product of glycolysis (glucose breakdown).

JohnMc said:

If it eats lactose, it can eat galactose and glucose, the simple sugars that come from that disaccharide. Beyond that, bacteria are pretty good at scavenging carbon/energy compounds. Lactic acid itself is produced from pyruvic acid, a product of glycolysis (glucose breakdown).

Click to expand...

Good call on on it being a disaccharides though. I had completely forgot about it.

Lactic acid is a product of homofermative lactic acid metabolism where pyruvate is turned into lactic acid consuming 2 NADH. Glycolysis only takes 1Glucose -> 2 Pyruvate at the gain of +2 ATP and +2 NADH. The NAD+/NADH cycle is balanced in that way.

1Glucose -> 2Pyruvate -> 2Lactic acid - With major intermediates left out.

JohnMc said:

If it eats lactose, it can eat galactose and glucose, the simple sugars that come from that disaccharide. Beyond that, bacteria are pretty good at scavenging carbon/energy compounds. Lactic acid itself is produced from pyruvic acid, a product of glycolysis (glucose breakdown).

Click to expand...

Oldsock said:

Just used pure WL lacto for the first time. It did seems to clump and flocc out, but I'm a little suspicious that it was actually a wild yeast strain that got in there (not much sourness in the starter or beer, and it took a couple days to get going).

Wild Brews suggests Lacto Del it is a homofermentive glucose eating lactic acid producing strain. Not sure how accurate that is though. The Wyeast strain seems to keep making lactic acid long after the yeast would have consumed all the glucose.

I’ve long suspected the same ABV lowering effect of acid production. That said it would depend on the relative amount of CO2 being produced, and anyway lactobacillus is only supposed to make a max of 1% lactic acid (so the effect is pretty minimal).

Might be a good post to send to the BBB, there are many more scientific minded sour brewers on there.

Click to expand...

Thanks for the replies! So now it seems to be that it could be breaking down some glucose. I found it odd that it could have been a particular subspecies that only went after lactose, since I couldn't find any information leading me to believe there would be a significant amount of lactose in a typical wort. I'm now further wondering if adding some lactose would increase the possible sourness level without messing with the ABV, since only the lacto del would be able to do anything with it. I gave White Labs a call yesterday, and am just waiting to see if they get back to me.

Pardon, but what is the BBB?

onemanlan said:

Good call on on it being a disaccharides though. I had completely forgot about it.

Lactic acid is a product of homofermative lactic acid metabolism where pyruvate is turned into lactic acid consuming 2 NADH. Glycolysis only takes 1Glucose -> 2 Pyruvate at the gain of +2 ATP and +2 NADH. The NAD+/NADH cycle is balanced in that way.

1Glucose -> 2Pyruvate -> 2Lactic acid - With major intermediates left out.

Click to expand...

Biology I'd forgotten comes back to bite me in the ass again

I've based my original post on this wiki page: http://en.wikipedia.org/wiki/Lactobacillus_delbrueckii_subsp._bulgaricus

It's from there that I got wondering if the brewing subspecies is only a lactose eater or not. I'm thinking it's not at this point, but if it WAS that could be where it's getting the glucose to turn into lactic acid. As I said in a previous post, though, I haven't yet run across anything that leads me to believe there is any significant amount of lactose in a typical wort. I'm guessing the brewing subspecies of lacto del just starts at glucose?

shanecb said:

Pardon, but what is the BBB?

Click to expand...

Burgundian Babble Belt: http://www.babblebelt.com/newboard/forum.html?tid=1108752780&pg=1 lots of great technical talk about brewing sours.

http://infolib.hua.edu.vn/Fulltext/ChuyenDe2009/CD39/58.28.pdf is a good source of information, however its more oriented toward the industry side of things. Thats good because you'll be getting purified strains. In the article they grow Lb. delbruekii on molasses it seems. So I'm with you in the idea that they can get carbon sources from the components of the disaccharides or through other means.

3.2.2. Molasses
Molasses, a by-product of the sugar manufacturing
process, is used as an animal feed, and for ethanol and
yeast production [60]. In addition it could be used for LA
production [84 – 89]. The most abundant sugar is sucrose,
the high concentration of which makes the viscosity of
the liquid high [60]. Lb. delbrueckii has generally been used...

Starch
Another common substrate for LA production is starch
from crops or wastes [18,90 –92]. It has to be hydrolyzed to
glucose and maltose to be fermentable by LAB. Starch from
various origins has been used for LA production, including
wheat [20,93], corn [94,95], cassava [96], potato [97,98],
rice [93], rye [35], sorghum [99,100], and barley [95,101]....
Starch was often supplemented with
nutrients, mostly in the form of yeast extract [18] or peptone
[102]....

LAB
used for LA production from starch include: Lb. casei [92],
Lb. plantarum [109], Lb. delbrueckii [97,107], Lc. lactis
[18,108], and Lb. helveticus [28]...

Click to expand...

Note the use of yeast extract in this. When yeast die they lyse releasing their contents for other yeast or any other organism to scavenge for their own good. In the case of a large vat of wort and high yeast cell count the yeast contribute a good bit of nutritional value for the lactic acid bacteria to grow on.
It also contains a good diagram of the different types of fermentation. Yeast go through a method similar to heterofermative mixed acid fermentation, where as Lactobacilli go through homofermative lactic acid fermentation.

denimglen said:

It floccs out pretty well. Seems to do it's thing, clump up and drop out pretty quick. I would compare it to a medium-high flocculating Sacc strain.

Click to expand...

I did not have the same experience the past few times I used WL. Lacto.D. Lactobacillus is a bacterium, and is much smaller than yeast. I don't believe that it has surface lecithin and lecithin receptors like yeast use to facilitate flocculation. It will fall out of suspension but it takes a long time because of their small size.

Lactobacillus are generally nonmotile and can survive in both aerobic and anaerobic environments. L. delbrueckii, the type species of the genus, is 0.5 to 0.8 micrometre (μm; 1 μm = 10−6 metre) across by 2 to 9 μm long and occurs singly or in small chains. (http://www.britannica.com/EBchecked/topic/327373/Lactobacillus)

Click to expand...

I have also been working out how to do a no boil Breliner. so far I have tried two ways.

My first attempt was not vary sour after one month, but was starting to get sour.
I made up a large starter of lacto (2L) incubating it at 37ºC (98.6ºF) for two days. place in fridge for one week. Made a 1L ale yeast starter the day before brewing, then pitched the yeast at the same time , after decanting off the supernatant.

My second attempt was a bit more successful, with noticeable souring after 3 weeks. I think they will eventually get to the desired sour level after longer term aging.

for the second attempt, I did the same as the first. But I pitched the Lacto first into the wort that was cooled to ~110-120ºF, then allowed the wort to sit for 24hr and cool to 64ºF before pitching the yeast starter.

I think my next attempt will be to make a 4L lacto starter, pitch at 120ºF, wrap the carboy in insulation and wait 48hr before pitching the yeast.

Bsquared said:

I think my next attempt will be to make a 4L lacto starter, pitch at 120ºF, wrap the carboy in insulation and wait 48hr before pitching the yeast.

Click to expand...

Be careful, if the pH drops too low before the Sacch gets going you can end up with some weird flavors and poor attenuation.

I’ve had fine luck (plenty of sourness) pitching lacto/yeast at the same time. I also like to toss some other bugs in there after a few days to give it a bit of funk. Keeping the IBUs really low (not too hard with no-boil) and pitching lots of healthy lacto seem to be the two keys.

Oldsock said:

Be careful, if the pH drops too low before the Sacch gets going you can end up with some weird flavors and poor attenuation.

I’ve had fine luck (plenty of sourness) pitching lacto/yeast at the same time. I also like to toss some other bugs in there after a few days to give it a bit of funk. Keeping the IBUs really low (not too hard with no-boil) and pitching lots of healthy lacto seem to be the two keys.

Click to expand...

Thats a good point,I'll monitor my pH as I go next time.

I bought some 88% lactic acid from the HBS and did a bunch of dilutions into the first batch and found that 150µl of 88% lactic acid in to 50ml (that's a 1:333 dilution) of my berliner was a good level of tart that did not over power the funk from the bugs that got through the no-boil. That equates to about 0.25% lactic acid. I might do this dilution again and check where the pH is on that as a reference.

Bsquared said:

Thats a good point,I'll monitor my pH as I go next time.

I bought some 88% lactic acid from the HBS and did a bunch of dilutions into the first batch and found that 150µl of 88% lactic acid in to 50ml (that's a 1:333 dilution) of my berliner was a good level of tart that did not over power the funk from the bugs that got through the no-boil. That equates to about 0.25% lactic acid. I might do this dilution again and check where the pH is on that as a reference.

Click to expand...

I hadn't thought about pitching at a high temperature, and letting it cool down naturally. That's a good idea to make sure it is rather warm for the lacto to do its thing. How high do you think is too high? Also, what about the pH? I'm not familiar with how pH could lead to off-flavors in this regard. I'd always thought you wanted to cool down the wort as fast as possible to prevent off-flavors, so what measures need to be done to let the wort hot for awhile for the lacto?

Also, I finally got a call back from White Labs that definitely clarified some things for me. I'll post up the message they left me later tonight.

As for the max temp for Lacto, I don't know, but I'll take a SWAG (scientific wild ass guess) that based on the fact that Lacto's home in nature is in the gut of mammals that you should try to keep it in the range where mammals Live. That said though, for a typical sour mash, the procedure is to cool the wort to 120º and pitch in a hand full of grain in and hold it at 120º or close to that for 24hr. So I would say you would not want to go much over 120º, but I have not tried.

Cooling rapidly after the boil helps to reduce the amount of dissolved DMSO, (Dimethyl Sulphoxide)one of the precursors of DMS, and other compounds that are produced at high heat in the wort, but are off gassed during the boil. These compounds are formed above 170ºF but above 210ºF they are volatile enough to escape from the wort. By cooling rapidly they do not have as much of a chance to form when they can't get out.

So if you cool your wort from the boil down to 120 quickly you will minimize getting off flavors from these compounds.

Sacc. yeasts don't do so well when the pH gets to low(more acidic), where Brett likes the lower pH, so they can start to take over the fermentation and get the beer quite funky.

I sat through a good talk on "exploring Belgian sours" at NCH last week, I think they will be posting the talk on the AHA website soon. he had a good break down on what conditions the different bugs like to live in during the development to sour beer's.

I'll see if I can find it tonight.

Bsquared, that would be great if you would be able to find it.


Here is the info I got from White Labs. As said, the lactobacillus delbrueckii (in this case WLP677) is a homofermentative bacteria, with the end product being lactic acid. It will not utilize any pentoses, but will utilize a number of hexoses. These include glucose, fructose, maltose, maltotriose, raffinose, and all the way up to lactose. The guy who called me back named a couple it wouldn't use, and the only one I caught was xylo sugars.

So it seems that when lacto del is doing its thing, it could drop the ABV of your brew depending on the amount of sugars it eats up. The only way I can think of to take that into account is to sneak a gravity reading right before you pitch your yeast to see how much sugars you still have to work with, even though the lacto is still going to be eating some sugars after that. Does anyone have experience in how much sugars the lacto del might snatch up before the yeast?

I'm also now (relatively) firmly standing behind my idea that some added lactose could maybe increase the sourness, since it would only be available to the lacto del and not affect the FG/ABV (assuming it all gets used up).

The talks are not posted yet, but here is where they will be.

http://www.ahaconference.org/speakerspresentation.html

Great! Thank you.