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Thread: Refermentation in Packaged beer due to Diastaticus

  1. #16
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    Quote Originally Posted by mobcraftbeer View Post
    Has anyone who has been reading this thread had any experience with starch based agar for isolating/detecting Diastaticus?

    Even with Membrane filtration on LWYM and LCSM i have not been able to find any traces of growth (on some of our beers anyway) even when PCR screening says its there.

    I'm going to experiment around with a few variations of starch agars and see what I come up with, will post results here, if no one has any other experience they'd like to share.

    The idea behind using starch agar is that the only source of carbohydrates in the media would be those that require glucoamylase to break them down, and consequently see growth (since culture yeast typically can't handle any sugars bigger that di or tri saccharides). If this was coupled to a membrane filtration of say, between 100-200mls of finished beer in the bbt (clearest beer you could possible get so as not to clog the filter) Then i wouldnt see any reason why you wouldnt be able to detect it, at least in theory.
    My simple first thought was try PDA without the D. Just potato water agar. No dextrose. Thats where I would start.

    In fungi the mushroom growers have also tried starch agars as a way to get selective media for higher fingi. Varying levels of success. Its hard to get media that grows mushroom forming fungi but not all the other molds.
    Last edited by Yeast; 10-24-2017 at 01:09 PM.
    I hope I encouraged you!

  2. #17
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    Mar 2013
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    Found this little Gem after combing through my Bact 103 Lab notebooks

    Starch Agar: organisms streaked onto this media and incubated for a few days, after incubation, the media is flooded with Iodine solution and poured off, clear zones around growth indiciate amylitic capability, and anything that turns blue, black or dark purple means the starch has not been hydrolyzed, I will be testing a variation on this media along with another media that uses maltodextrin instead starch.

    I will be starting this project today and will post up results here.


    https://catalog.hardydiagnostics.com...tarchAgar.html

  3. #18
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    Oct 2017
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    South Salt Lake, UT - USA
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    Unhappy Same issue

    Hello Guys,

    My name is Marcio Buffolo. I'm the Head Brewer and the Scientist from Shades of Pale Brewing at Salt Lake City, UT.

    We have the same problems and we found very similar results.

    So far, we got two distinct problems due to diastaticus.
    1- Bottles explode after 3 months in self. All beers were rest for diacetyl rest, and the final Plato was 3. constant. period. After a month in bottles, same gravity. After three to four months, gravity close to 1 in shelf condition, not in the cold room.

    2- We have a yeast propagator, so, we never collect yeast after fermentation. All used yeast is disposed and a new yeast from a yeast prop tank was introduced to the beer. We got a new pitch from Wyeast and after two days in the yeast prop tank, the slurry was added to 4 beers. All, this time, finish around 1.4. Two first ones didn't smell spice at all. The other two, are saison like in the tank.

    We feed the yeast with a new wort, which is tested and so far (2 weeks later) didn't show any sign of contamination.

    PS: We never use Saison yeast in the brewhouse. Only in barrels in the back of the brewery. That yeast never touches any fermenter or bottling line.

    I got the prove of contamination by plating the yeast tank in WL medium with chloraphenicol (to inhibit bacteria growth). But, this took me 3 days to seen a sigh of contamination. I'm try to develop a PCR technique to identify faster this problem.

    But, at this point, I lost 120bbl of beer. My only thinking is the Wyeast pitch was contaminated.

    Any advace in primer seuqence that I should be usign and any easy protocol for DNA extration?

    Thanks

  4. #19
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    Mar 2013
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    Milwaukee WI
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    PCR Assay

    We've had two different labs using commercial Primer Sequences for Diastaticus give back two different results, positive and negative, which tells me that even the "Pros" developing these assays don't even have a full handle on the genetics (or expression) of this organism yet.

    From the limited research I have done, all the assays target the STA1 region of the genome.

    STA1 is supposed to contain the genes for production AND excretion of glucoamylase,

    It is to be noted that not every yeast that has the STA1 region contains both sequences for production AND secretion of glucoamylase. Secretion is required for Diastaticus to cleave complex dextrins outside of the cell since it cant transport them inside the cell.

    Not every organism that contains these genes is capable of full expression, there are several strains that contain these genes but so far have NOT been shown to be able to express them (as is evidenced by this post from White Labs Website https://www.whitelabs.com/yeast-bank...ison-ale-yeast), but certainly at a broad level, if you don't want known Var. Diastaticus in your beer, PCR is a great tool to help detect potential problems.

    From my understanding, the precise mechanism of regulation and expression of these genes is not completely understood yet but seems to be a reaction to stress (ie low sugar content, high alcohol conditions of fully fermented beer)

    I am right now in the middle of experimenting with different media compositions containing starch and maltodextrins against a known Var. Diastaticus and a known NON Var. DIastaticus yeast.

    The idea being that you will be able to detect glucoamylase through its presence and action as opposed to whether or not the yeast contains the genes for expression of glucoamylase.

    I just started trials and will be sharing my results on this thread as i get them.

  5. #20
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    Dec 2003
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    Strongsville, Ohio
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    So... Has anyone else heard anything in regards to this issue recently...say in the last week and a half?


    ...and yes I am being vague.

  6. #21
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    I don't know if you are referring to something in particular, but earlier this week my hometown paper (Denver Post) published an article on Left Hand suing White Labs over yeast contaminated with S. diastaticus....

    http://www.denverpost.com/2017/11/21...k-stout-yeast/

    Could be a problem with other suppliers as well.

  7. #22
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    Quote Originally Posted by UnFermentable View Post
    I don't know if you are referring to something in particular, but earlier this week my hometown paper (Denver Post) published an article on Left Hand suing White Labs over yeast contaminated with S. diastaticus....

    http://www.denverpost.com/2017/11/21...k-stout-yeast/

    Could be a problem with other suppliers as well.
    yep....that's it. Wondered if anyone saw that too.

  8. #23
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    Oct 2013
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    Cincinnati, OH
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    I will definitely be watching this closely as I know first hand that WL response is always just to ship a fresh pitch, and not necessarily address issues from their customers. I personally doubt Left Hand will win the suit, but I firmly believe they are correct in their deductions. This probably should have been settled quietly outside of the public spectrum, but I am sure one mans ego got in the way of that.

    With an employee owned business this is a super big deal. Especially at the scale they both operate at. Know handful of people from both companies personally and it is an unfortunate incident to say the least.

  9. #24
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    Toronto, Canada
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    any idea what strains of yeast this issue has affected? dates? lot numbers? production facilities?

    I can accept that a supplier could have problems from time to time, but it is crucial that they share the information with their customers rather than burying it and hoping nobody notices.

  10. #25
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    Oct 2017
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    South Salt Lake, UT - USA
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    Hey @mobcraftbeer

    Yes, indeed the STA1 gene is the main marker for diastaticus. Although, STA2 also could be express in Saison yeast. Thus, I'm trying to develop some primers to use in regular PCR (PCR/gel) to identify diastaticus, Brett and LAB's. The idea is developing primers with different sizes where i can include all in one reaction and and run a gel. I have more than 8 years of lab experience and 5 years of brewing (2 as head brewer). Medium plates are great, but takes more than 4 days to get good results. PCR take me 5 hours total.

    And we have problem with Wyeast. 1272 pitch was contaminated. They denied and there isn't much what we can do.

  11. #26
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    Sep 2005
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    Louisville, KY
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    I believe they spoke specifically of San Diego Super being one of the contaminated yeasts. My next question is... I wonder if this is coming from WL Ashvillle location as well? I switched to Ashville when it opened. I use lots of San Super. J
    Joel Halbleib
    COO / Zymurgist
    Goodwood Brewing Co
    636 East Main St
    Louisville, KY
    goodwood.beer

  12. #27
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    Mar 2013
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    Milwaukee WI
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    Media Vs PCR

    @Buffalo, i am well aware of the time restrictions of using media, i was only looking at it out of desperation, as the one lab i would trust to run PCR and give a correct result had thier thermocycler break down over the last two months, however they just got it back up and running so just today I sent out samples for analaysis. (I will share these results since we have switched yeasts over a few months now once they come in)

    There are limitiations to Gel Electrophoresis as well, but as was mentioned it will work great for giving a yes/no answer as to whether or not the genes you are looking for are present, qPCR is nice because it does introduce some level of quantitative analysis into the picture, which can help you more in tracking down a specific point of infection in your process than a simple yes/no result.

    As far as my experiments using media were concerned, after initial round of experimentation revealed inconclusive resullts (my diastaticus strain was not able to demonstrate hydrolyzation of starch) I have abandoned it in favor of just sending samples out for testing and looking at getting my own PCR thermocycler in house (whether i will be using gel electrophoresis or qPCR remains to be seen, im still doing my homework on that front)

    Also: PCR is much more expensive than simple media plating, so if i could find a simple assay that other small brewers could perform without specialized lab experience operating thermocyclers, all the better

    I may have time to return to it one day, but for now i have too much on my plate to devote too much more time to this.

    As far as the Lawsuit with left hand and white labs, the outcome (regardless of whose favor it is ruled in) will have important implications, and i will be following it to see how it plays out.

  13. #28
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    Mar 2014
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    Elisa

    @mobcraftbeer

    I'm not sure if there is a way to make a canned assay that any brewer could use just because setting up a PCR reaction is straightforward but you have to be really careful about contamination by stray genetic material. Doubly so in qPCR. I wonder if there's a way to design an ELISA assay to detect diastaticus.

  14. #29
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    Mar 2013
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    Milwaukee WI
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    ELISA assay

    @TheRonch

    I had thought about an ELISA assay, as it would be far superior not just because of genetic contamination issues, but also because some strains of yeast contain the genes for glucoamylase (Notably Saison strains) but are not able to express them. An ELISA would ensure that the actual presence of glucoamylase is detected, however i was not able to find any companies currently making ELISA for Glucoamylase, especially the type of Glucoamylase produced by Yeast (unsure the differences in structure of plant derived versus fungal derived Glucoamylase enzymes)

    I'm sure there is a way to design an ELISA assay, but you would have to invlove your local University and my locals Food Science program has shown no interest in using lab resources to develop an assay like this unfortunately.

    I used ELISA all the time when i worked at the ethanol plant, we were required to test for Aflatoxin and Vomitoxin because all of our corn products were certified for livestock feed use. It would be a better, simpler assay to perform, with less margin for error, but would be impossible to develop without the resources of a Large biochem/microbiology lab.

    For those of you out there looking for a silver bullet, the best advice i can offer is to closely monitor your warm retain beers for drop in gravity. We also incubate our samples post packaging for a forced aging test, and measure after one month in the incubator for drop in gravity, its not ideal, but better to recall product off the market PRIOR to customers discovering a flaw than after.

  15. #30
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    Sep 2013
    Location
    Cape May, NJ, USA
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    Cleaning & Sanitizing?

    There is so much good information in this thread! Thank you to all of those who posted here sharing their experiences.
    My brewery just got a positive hit for diastaticus yesterday and are in the process of limiting its spread and containing it. Luckily, we caught it while the beer was still in the FV, so it's currently quarantined to that vessel. It's a Belgian ale that used Wyeast 3711 French Saison strain, which I have heard others say is a good candidate for being a var. diastaticus strain (it was pointed out earlier in this thread that the White Labs French Saison strain is indeed a var. diastaticus). We have been testing for diastaticus with the Invisible Sentinel PCR machine since November and this is our first positive result.
    I would like to ask those on this thread that have dealt with it before about how they went about cleaning any equipment that came into contact with the contaminated beer. I read about the Ecolab Vortex, and will be asking my supplier if they have anything similar, we currently use PAA as a sanitizer. However, I am wonder if anything special was done when it came to equipment such as centrifuges, filters, and bottling or canning lines.
    Our running theory here is that we have had diastaticus in the brewery (we've been using this yeast strain for almost 6 years now) and that our CIP and sanitizing procedures have been adequate, we've never had a complaint for an over-foaming can/bottle or an over-carbonated one (except for the occasional bottle conditioned Brett or sour ale) in the time that I've been here, which would support this theory. But ignorance is bliss and now that we do know it is present I want to make sure we are doing everything we can to prevent it from contaminating other beers. Any information would be helpful.
    Thank you all again and happy brewing!

    CHEERS

    Jimmy

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