100% Lactobacillus fermentations cannot achieve complete attenuation (test results included)

Discussion in 'Homebrewing' started by lshaner, Jul 21, 2015.

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  1. lshaner

    lshaner Initiate (0) Mar 16, 2014 Illinois

    People may have seen me post in other threads that fully attenuated (defined as >70% apparent attenuation) 100% Lactobacillus fermentations are not possible and those reported as such were in reality a result of unintentionally introducing yeast. To prove that point, I performed an experiment at Omega Yeast Labs to address the question. Here is what I did: I obtained the two Lacto strains from White Labs (delbrueckii and brevis) and Wyeast (buchneri and brevis), and the Omega L. plantarum strain. I grew them on AOAC plates (a Lactobacillus medium) supplemented with cycloheximide. Propagated the lacto in liquid AOAC at ~90F (no stirring) to maximum cell density (achieved in about 3 days). Pitched 200 mL of these cultures into 2 liters of autoclave-sterilized unhopped wort with a starting gravity of 1.037. Incubated for 2 weeks at ~90F, periodically checking gravity and pH.

    Final Results:
    WL brevis: pH 3.14, 1.035
    WL delbrueckii: pH 3.72, 1.033
    WY brevis: pH 3.72, 1.035
    WY buchneri: pH 3.61, 1.034
    OYL plantarum: pH 3.17, 1.036

    None of the cultures, whether heterofermentative or homofermentative, produced a significant gravity drop. None produced a krausen or obvious CO2 evolution.

    We're going to send the samples off to be tested for alcohol levels so we can get an idea of how much alcohol the heterofermentative strains produce. I'll update this post when we have those results.

    Here is the take home message: If you see significant krausen development or gravity drops in your Lacto starters or kettle sours, you have a yeast infection. It's not a result of the Lactobacillus.
     
    walows, DarrenE, IPeteA91 and 5 others like this.
  2. Brew_Betty

    Brew_Betty Disciple (394) Jan 5, 2015 Wisconsin

    I thought Omega lacto contained two lactos? Brevis and plantarum.

    I also thought delbrueckii wasn't supposed to produce any booze, yet it attenuated the most. Yeast infection!
     
  3. lshaner

    lshaner Initiate (0) Mar 16, 2014 Illinois

    delbrueckii isn't supposed to produce alcohol. That's correct. WL, however, has said that their delbrueckii is heterofermentative. We'll find out when we have alcohol levels tested. There's no yeast. We checked.

    And yes, Omega Lacto contains plantarum and brevis. Only the plantarum was included in this test.
     
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  4. psnydez86

    psnydez86 Initiate (0) Jan 4, 2012 Pennsylvania

    Awesome info. Much appreciated. Can't wait to see more.
     
  5. lshaner

    lshaner Initiate (0) Mar 16, 2014 Illinois

    Here are the updated numbers from running the samples through an Anton Paar Alcolyzer:

    WL brevis: 0.10% ABV, 1.03386 (apparent specific gravity)
    WL delbrueckii: 0.29%, 1.03255
    WY brevis: 0.18%, 1.03386
    WY buchneri: 0.24%, 1.03279
    OYL plantarum: 0.10%, 1.03394

    Everything except delbrueckii was known to be heterofermentative. delbrueckii has classically been defined as homofermentative but White Labs has told people over the years that it was heterofermentative. So it's either an unusual delbrueckii or it's misidentified.

    I think an important point to come out of this study is that the amount of alcohol produced by known heterofermentative Lactobacilli such as brevis is so low that the choice of a homofermentative vs. heterofermentative Lactobacillus for kettle souring is inconsequential. This is a good thing too because it looks like home brewers can't get a homofermentative Lactobacillus from an easily available yeast lab source.
     
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  6. Brew_Betty

    Brew_Betty Disciple (394) Jan 5, 2015 Wisconsin

    Since the Brett Trois flap, I have very little confidence in White Labs' ability to correctly identify the microbes they sell. It wouldn't surprise me if their Delbrueckii strain isn't Delbrueckii.
     
  7. Tebuken

    Tebuken Aspirant (289) Jun 6, 2009 Argentina

    So , I would like to ask you a simple question : My lambic beer is still fermenting , I brewed it on may 1th and a week ago after a month of no airlock activity it started to have new krausen(1 inch) and airlock works again. I took 3 lts of this beer ,have pasteurized,bottled and sampled. There is almost no sourness-tartness in the flavor, it is barely noticeable. Does this second started fermentation what is going to sour this beer?


    Edit : I fermented it with wild yeast captured from fruits
     
  8. Brew_Betty

    Brew_Betty Disciple (394) Jan 5, 2015 Wisconsin

    If lacto is pitched in ample quantity at the right temperature, sourness happens in less than a month. Pedio takes several months to produce sourness. If you have a smaller than optimal pitch of lacto, it could take a few months to sour.

    What yeast/bacteria did you pitch?
     
  9. Tebuken

    Tebuken Aspirant (289) Jun 6, 2009 Argentina

    Sorry it is not my intention to hijack the OP post.

    It was fermented with captured yeast from fruits
     
  10. Brew_Betty

    Brew_Betty Disciple (394) Jan 5, 2015 Wisconsin

    In that case, you won't know what you pitched unless you use a microscope. Truly wild fermentations like yours can contain more than 20 different types of microbes. If you get a good result, the beer God smiled upon you.
     
  11. Brew_Betty

    Brew_Betty Disciple (394) Jan 5, 2015 Wisconsin

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  12. Brew_Betty

    Brew_Betty Disciple (394) Jan 5, 2015 Wisconsin

  13. MrOH

    MrOH Savant (986) Jul 5, 2010 Maryland

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  14. OldSock

    OldSock Zealot (576) Apr 3, 2005 District of Columbia
    Industry

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  15. koopa

    koopa Poo-Bah (1,763) Apr 20, 2008 New Jersey

    I've had a beer attenuate down to 1.012 using only wlp677 within 3 days once in the fairly recent past, but I didn't look at it under a microscope to determine whether or not some yeast was in the mix.
     
  16. lshaner

    lshaner Initiate (0) Mar 16, 2014 Illinois

    The results of our experiment say that without a doubt, you had yeast in the mix.
     
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  17. koopa

    koopa Poo-Bah (1,763) Apr 20, 2008 New Jersey

    FWIW: White Labs advertises wlp677 as having 75-82% apparent attenuation range (funny when I looked it up about a month ago, I thought the site said 65-72% at that time). I believe I read somewhere that the wlp677 strain is capable of producing an enzyme which allows it to ferment maltose, maltotriose, and raffinose, ensuring a dry finished beer without the aid of Saccharomyces. I think what was more in question about wlp677 wasn't its ability to attenuate wort, but its ability to produce ethanol as the pyruvate it forms gets converted to lactic acid. I can't find the references for this subject at this time though, sorry!
     
    #17 koopa, Jul 25, 2015
    Last edited: Jul 25, 2015
  18. Brew_Betty

    Brew_Betty Disciple (394) Jan 5, 2015 Wisconsin

    He pitched 200ml of lacto into 2L. If WLP 677 attenuates 75-82%, then they clearly didn't ship him the lacto strain they claim to have the magical ability to ferment wort like an ale yeast. He verified there was no sacc in the 677 he received.

    Perhaps they used to add some sacc to their "magic lacto" and now they don't or perhaps there are random quality control issues. Either way, White Labs has some 'splaining to do...again.
     
  19. jbakajust1

    jbakajust1 Meyvn (1,286) Aug 25, 2009 Oregon
    Moderator Society

    He grew it on a medium designed to target Lacto growth and inhibit Sacc.
     
  20. Tebuken

    Tebuken Aspirant (289) Jun 6, 2009 Argentina

    I think the first 36 gravity points from the 51 attenuated in my lambic beer were fermented by sacc. thus the samples I took haven´t got much sourness, now there must be other microbes working on the last 15 points(I really have no idea wich are they). Do you think these last points will be enough to produce a noticeable sourness?
     
  21. lshaner

    lshaner Initiate (0) Mar 16, 2014 Illinois

    The only step where I included cycloheximide (inhibits yeast growth) was the initial plating of the samples from the vials/packs (because the 677 and 672 had yeast in them). That allowed me to inoculate the first 200 mL Lacto cultures with confidence that I wasn't inadvertently introducing yeast. There was no cycloheximide in the wort used to do the attenuation experiment. I just used good old fashioned aseptic technique.
     
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  22. lshaner

    lshaner Initiate (0) Mar 16, 2014 Illinois

    Perhaps it's easier to say it gets >70% apparent attenuation than it is to keep yeast out of the prop so they roll with it. In any event, the bacteria in the 677 vials -- purified away from the yeast -- cannot achieve +70% apparent attenuation.
     
  23. ryane

    ryane Initiate (0) Nov 21, 2007 Washington

    I know I know, Ive let it slide quite a bit.......Now there's so many things Ive done in the past year I dunno even where to start
     
  24. MrOH

    MrOH Savant (986) Jul 5, 2010 Maryland

    Seriously man, you were one of three homebrew bloggers that I regularly followed.
     
    jbakajust1 likes this.
  25. koopa

    koopa Poo-Bah (1,763) Apr 20, 2008 New Jersey

    No clue as to whether it would allow for >70% aa or not without yeast, but here is some more info on the enzyme expression within the subspecies of delbrueckii that I remember reading about...

    "Significant to the four L. delbrueckii subspecies are the number and types of substrates it can metabolize. As noted, such properties are restricted to enzyme expression within its genome.

    L. delbrueckii
    subsp. bulgaricus and subsp. indicus can metabolize lactose, glucose, fructose, and mannose.

    In addition to these, L. delbrueckii subsp. lactis catabolizes galactose, sucrose, maltose, trehalose and other modified carbohydrates.(4)"

    4) [Kitazawa, H., Yasuyuki, I., Uemura, J., Kawai, Y., Saito, T., Kaneko, T., Noda, K., and Itoh, T. “Augmentation of macrophage functions by an extracellular phophopolysaccharide from Lactobacillus delbrueckii ssp. bulgaricus”. Food Microbiology. 2000. Volume 17. p.109-118. ]
     
  26. Brew_Betty

    Brew_Betty Disciple (394) Jan 5, 2015 Wisconsin

    Ahh ha! So both White Labs lacto strains had yeast in the vial. Now all I need to ponder is whether or not the yeast is put there intentionally or accidentally. Either way, White Labs has some 'splainin' to do...again. The choice between admitting a deceptive business practice or a production negligence seems like a no win situation. It could be better to remain silent.
     
  27. ryane

    ryane Initiate (0) Nov 21, 2007 Washington

    Not saying anyone is wrong about yeast inclusion in the lacto packs, but in my beer I DID NOT use White labs, I used the Wyeast PC 5223 Lacto Brevis
     
  28. Brew_Betty

    Brew_Betty Disciple (394) Jan 5, 2015 Wisconsin

    The interesting anomaly with your beer is the very low FG of 1.004. No doubt there was something more than brevis in your batch. Perhaps the pitch contained some saison sacc or brett. Perhaps the batch was contaminated with yeast during the brewing process.
    :grimacing:

    I've used Wyeast Brevis a few times and didn't experience high attenuation in the starter or the batch with one week of fermentation for each at 80F. The batches were finished with sacc and made good sour beers. I have never tried to completely ferment a beer with any type of lacto.
     
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