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Thread: Refermentation in Packaged beer due to Diastaticus

  1. #31
    Join Date
    Mar 2013
    Location
    Milwaukee WI
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    28

    Updates, Information, and Protocols

    Providing an update on this thread and hopefully some useful information:

    We ended up caving and getting the qPCR unit from Chai Biosciences, the thermocycler is designed to work with the PCR kits produce by PIKA Wienhenstephan in Germany. The unit has been working well, and has helped us immensely in IDing potential sources of infection in our brewery and truly taking a proactive approach to dealing with this organism.

    They do recommend that you run an enrichment specifically when looking for diastaticus as it can be hard to detect at low concentrations, we are running side by side analysis right now on finished beers of enriched and unenriched samples to determine if it is actually necessary for us.

    In the meantime, with the help of a laboratory intern, we have set out to isolate the specific strain of diastaticus growing in our brewery with the hopes of developing a low cost method/assay for detecting Diastaticus, and we beleive we have stumbled onto something that might work well, we are still collecting data, but basically we have figured out how to achieve a nearly 100% Maltodextrin media broth for enrichment of Diastaticus for Durham tube fermentation tests (if anything ferments in pure maltodextrin, then you know you have a problem), we will be validating the protocol with PCR and are also experimenting with it as an enrichment media for our beers for Diastaticus detection. As soon as the final results are in, i will post the method and protocol up here along with the data for feedback and potential validation by other interested folks.

    This is also coinciding with our attempts to better characterize our particular Diastaticus strain, now that we have isolated it (our isolated were validated as positive by our PCR machine), we will be testing its growth efficacy on various conventional and unconventional medias in an attempt to be able to better isolate this organism using traditional micro methods in addition to PCR screening.

  2. #32
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    Mar 2013
    Location
    Milwaukee WI
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    Preliminary Data Analysis and Low Cost Assay for Diastaticus Detection

    We are still running testing on our isolated strains of diastaticus, but one of the first, and more terrifying things we have found from our preliminary analysis is that this strain of Diastaticus appears to be Copper Sulfate Sensitive, meaning that it is inhibited by Copper Sulfate (at least partially, we havent had the time to experiment with different concentrations yet), this would explain why we were not seeing any growth on our LCSM Plates.

    It also explains why we had so much trouble trying to isolate it in the first place, as others have said, and my testing seems to confirm this, some strains of diastaticus behave EXACTLY like your house yeast in most conventional microbiological medias.

    Which brings me to one of the methods we have started to develop to help us isolate it in the first place, followed by PCR testing to confirm that it does infact contain Diastaicus Genes. Below i have detailed the premise and baseline protocol, and would encourage others to contribute thoughts, ideas or practical experience in order to improve the method as a low cost alternative to PCR to detect Diastaticus.


    GOC MaltoDextrin Fermentation Test


    Premise: Diastaticus breaks down long chain sugars that most "traditional" brewers strains cannot through the use of a secreted glucoamylase enzyme to break down long chain sugars into simpler sugars, which are then consumed by the yeast.

    Solution: Produce a selective nutirent broth composed of complex carbohydrates capable of being broken down by Diastaticus but completely deviod of simple sugars that can be fermented by "traditional" brewers yeast. The idea being that, if you see fermentation in a tube (via a Durham tube test) in a broth made entirely up of Complex carbohydrates, you have something in there that can metabolize complex carbohydrates (not necessarily yeast, but any bug that can do that is going to a problem).

    Background and Issues: Commercially produced MaltoDextrins are not pure, and traditional purification is an expensive proposition. The reason being is that commercial maltodextrins are produced in a very similar fashion to how beer is mashed in the brewing process. You have a bioreactor that you feed pure gelatinized cornstarch. Glucoamylase enzymes are then added to the bioreactor, just like mashing, you can, to a certain degree using temperature, control the chain length of the maltodextrins so that they are extremely long or extremely short, the shorter sugar chain, the closer you get to something like corn syrup (glucose and fructose). Thge longer the chain, the closer you get to something like an actual amylose or amylopectin chain (starch). We aquired maltodextrin with a very high chain length, the problem is, as a result of this reaction, you will ALWAYS get simple fermentable sugars produced during this reaction, just like mashing, even the longest chain maltodextrins available are still something like 6-12% Glucose or GLucose equivalents (mono, di and tri saccharides) by composition.

    So we have an impure maltodextrin that if we were to add to a nutrient broth, we would get fermentation no matter what because there is always just a little bit of Glucose in there, and a durham tube test ISNT Quantitatve, its Qualitative. SO how do we obtain a pure or relatively pure maltodextrin solution?

    First we make up our regular broth solution (right now it just DI Water, M040 Maltrin from Grain Processing Corporation, yeast nutrient), autoclave and then add our house yeast which is a confirmed negative for Diastaticus. This yeast can only metabolize simple sugars, so we use this yeast to ferment out all the simple fermentable sugars, leaving behind an almost pure maltodextrin solution (there is a way to figure out how pure, all you would have to do is centrifuge and dry pre and post fermentation samples in an oven and compare the leftover solids content of each sample to figure out precisely how much mass was lost during fermentation). Once this intial fermentation is completed, we re-autoclave and then add our target samples (this is all done as a Durham tube test btw). The yeast we had already added to ferment is autolysed during the 2nd autclaving and provides further nutrients to organisms in target samples and, as long as you are not using a Diastaticus strain for your initial fermentation, you can even use this as an enirchment for samples you plan to run using PCR (enrichments are always recommended for Diastaticus since they are effective at such low concetrations)

    If we see fermentation/gas production, we know we have a high likliehood of glucoamylase acitivity in the target sample. You could also add a pH indicator dye such as Phenol Red to look for acid production as well, and be able to differentiate between acid production (LAB) or simply fermentation (Diastaticus)

    The data we have collected so far on known yeast strains confirms this as an effective method (all known Diastaticus strains are able to ferment while non diastaticus strains we not able to ferment the 2nd time around), but next week we will be running side by side validations using PCR in order to confirm the assay is Valid.

    Any thoughts on this method? Any criticisms? ANy ways to potentially increase its selectivity and effectiveness?

  3. #33
    Join Date
    Mar 2018
    Location
    Chicago, IL
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    10
    I have been pretty intrigued by this issue as well. Currently helping out a local Chicago brewery that may have a contamination, and have been to a few presentations on the subject (I'm QA).

    Are you saying that you think there are multiple different strains of diastaticus? Just curious because I was always aware of it being its own thing. I need to go back through the rest of this thread and read it a bit more closely too lol.

    One of the clues that led me to believing it was diastaticus in their brewery was that I had WY colonies growing on YM + CuSO4, and had learned at a recent MBAA meeting that diastaticus will grow in the presence of a Sacch. inhibitor. So I wonder if it is concentration dependent in your case? We are currently using 3.75ml of 16,000ppm CuSO4 /100ml YM media, and that was giving me colony growth.

    They were having this continued fermentation, but it wasn't till cans were sitting on shelves for months that the damage was being done. Here we're some of the tests we did to try to verify...

    - ABV went up 2% from initial canning
    - CO2 was also elevated
    - Higher than normal cell count in the finished can, which is indicative of diastaticus as it is pretty immune to cold crashing (Studies have shown that a diastaticus cell count of 170.00 x10^6 cells/ml cold crashed for 7 days only dropped out to a count of 90.00 x10^6 cells/ml, which would be similar to a peak fermentation cell count for us). Throw that can on a room temp shelf and that dextrin is going to break down again.
    - Membrane filtered finished cans after 6 months on YM + CuSO4 and saw WY colonies, which should've inhibited Sacch. but allowed this to grow.

    Unfortunately we only have kits and primers to run beer spoiling bacteria on our RT-PCR, but I believe we are working on getting Pall GeneDisk plates to read yeast strains as well.

  4. #34
    Join Date
    Mar 2013
    Location
    Milwaukee WI
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    28

    Diastaticus Strains

    Based on my research, here are the conclusions that i have come to about the precise mechanics of Diastaticus Infection:

    There are multiple different strains of Diastaticus Yeast out there, and not all of them are similar, for example, Belle Saison is a confirmed Diastaticus variant, and also produces tons of fruity esters (its a saison strain). In fact, the large majority of Diastaticus strains seems to be from the Belgian/French Family, but there are definitly strains that act and look and perform and taste IDENTICALLY to ale yeast strains (up to and including being inhibited by cupric sulfate, even though the belgian/french diastaticus strains ARE NOT inhibited) , with the exception of containing glucoamylase enzymes. Secondarily, because of the precise action of glucoamylase secretion, we found that even a low threshold of this organism (.1CFU/ml) was enough to cause otherwise uninfected beer to referment.

    Since the glucoamylase is secreted outside the cell (it HAS to be, becuase anything larger than a trisaccharide is too large to transport across the cell membrane). Its starts the work of breaking down sugars, and ANY residual yeast in solution (including your house strain of yeast) is capable of fermenting those sugars and refermenting beer.

    And if you do have a low level of Diastaticus Infection, it can sometimes take a few months for it to show out at market with room temp product because it takes that long for the enzyme to breakdown residual sugars and for any remaining yeast in there to eat it. We saw almost identical results to what you are reporting for refermented canned and bottled product.

    In other news we have finally concluded our investigation into a low tech method of detection, its not 100% foolproof on its own the way a PCR Machine results are, but if you are small or start up brewery with little to no lab resources this might be a good option to incorporate into testing. I have included a link to the copy of the poster, and if anyone has any further questions or is interested in the protocol, please let me know, as i will only devote time to building one out based on actual interest from folks on here.

    https://drive.google.com/file/d/1nU1...ew?usp=sharing

  5. #35
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    Mar 2013
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    Milwaukee WI
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    Also keep in mind that this poster was designed to be presented to a low level college Biology Class, and so a lot of the more technical aspects have been simplified or dumbed down to make it easier to understand to laypeople.

  6. #36
    Join Date
    Mar 2018
    Location
    Athens, GA USA
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    3

    Any further updates?

    Thanks for putting together such detailed info. We've been having a lot of the same issues you've described, and plan to implement some changes hot side. That said, I'd love to see your procedure for the low budget assays.

    Cheers

    Mark

    Quote Originally Posted by mobcraftbeer View Post
    Conclusions:
    • Through the above analysis of re-fermenting beers we have arrived at an extremely alarming conclusion that had been corroborated by other experts in the brewing industry interpreting the same data: That a heretofore undetected strain of wild yeast (also undetected by our 3rd party testing lab) was the root cause of our refermenting issues and that this strain of wild yeast had found its way into our brewery VIA one specific strain of house yeast we were using from our new supplier. WE HAVE SINCE SWITCHED BACK TO OUR ORIGINAL YEAST SUPPLIER.
    • This means that even though we were testing for wild yeast this specific strain would not show up on any of the typical medias used to look for this organism.
    • This problem was limited to multiple pitches of the SAME STRAIN bought from our supplier.
    • Not every batch of beer fermented with this specific strain of yeast had this problem.
    • Light, highly attenuated beers have not had this issue to the same extent as darker beers with less attenutation
    • Importantly, there is a precedent for this as well. Earlier this year a Bells QA tech gave a presentation in which they had encountered a similar problem and were not able to solve it through typical Microbiological testing. Rather, they had to use GENOMIC ANALYSIS in order to determine that there was an additional yeast strain in there.
    • Their conclusion was also that a wild yeast, S. Diastaticus, had piggy backed from yeast coming from their supplier and was causing refermentation in packaging.
      PCR Genomic Results
      We were able to get our Beers tested on a GeneDisc PCR, 4 of them fermented with the strain of yeast suspected of causing our problems, two of them that were not. Result were conclusive, much, much higher levels of diastaticus found in beers fermented with our problem yeast than those that were not. Picture comparison of two beers below, to show you the difference.
      Attachment 51590
      Attachment 51591

      Saccharomyces Diastaticus is a saccharomyces wild yeast that does not give off a lot of off flavors and produces little-no acid. All it does is break down the complex sugars that conventional yeast leaves behind because it cannot metabolize them.
      Recommendations Moving Forward:
      Using a broader spectrum no-rinse sanitizer in our cleaning procedures such as Ecolabs Vortex (common in the juice and dairy industry) as opposed to our current use of a narrow spectrum sanitizer PAA (Peracetic Acid, a strong oxidizing agent). It is of note that PAA is a very common sanitizer found in the brewing industry.
      Using a wider spectrum sanitizer allows us to kill a wider variety of both spoilage and non spoilage bacteria.
      Begin acid washing yeast prior to use.
      Acid washing with either sanitizer and/or acid is a technique used to reduce the viability of any other organisms in our yeast strains OTHER than our desired yeast strain.
      We did not have the capability, until very recently, to be able to properly perform this procedure due to lack of dedicated yeast brinks in our brewery.
      Modify existing testing protocols to INCLUDE
      PCR genomic analysis and validation on all beers out to market to verify that they are free of significant levels of microbial contaminants.
      Extended refermentation testing and wider testing of all retain beers to cast as wide a net as possible (within reason) to look for any defects.

  7. #37
    Join Date
    Mar 2018
    Location
    Chicago, IL
    Posts
    10
    I finally got going with the Pall Yeast disks and decided to throw that local brewery's problem can on a run... Voila! All the signs were pointing to it, just didn't have a way to prove it till now. Since these problems arose they have ditched their French Saison strain, did a very deep clean with hose replacement, heat exchange cleaning, etc, and they have not had any issues since then. Hopefully this little bugger is completely eradicated, but I agree that even the smallest brewery can take steps to prevent these issues in forms of basic plating or even starch conversion rapids.

    This was the first run and I had to tweak a few parts of the SOP, not the prettiest curves but under the cutoffs so should be valid data. Still working on getting my DNA dilutions optimized for the samples I'm working from.


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  8. #38
    Join Date
    Jan 2014
    Location
    RALEIGH, NC, USA
    Posts
    29

    Diastaticus detection PDF

    Hi, Caroline from Lallemand here. We have just completed a simple best practices sheet that contains information on diastaticus detection options that you may find useful. Please send me an email to cparnin@lallemand.com if you would like a copy.
    Cheers,
    Caroline

  9. #39
    Join Date
    Mar 2013
    Location
    Milwaukee WI
    Posts
    28

    Diastaticus Detection Protocol (Baseline)

    Diastaticus Detection Protocol Using Malto-Media
    By Andrew Gierczak, Mobcraft Beer Inc.

    Required Materials

    Autoclave
    40-50ml Screw Cap Test Tubes
    2-5 Liter Erlenmeyer Flask
    Incubator/Hot stir plate
    Distilled, Filtered or Purified Water
    8-10ml Test tubes (must be able to fit inside 40-50ml tubes)
    M040 Maltodextrin
    US-05 Dry Yeast
    Water
    Gram Scale
    1-10ml Pipet(s)

    Experimental Design Theory
    Sacc var Diastaticus secretes glucoamylase enzymes that allow it to externally break down complex sugars in beer. Since glucoamylase production is the defining characteristic of Diastaticus, it would follow that any assay design to detect this organism must revolve around detecting its ability to ferment complex sugars. The gold standard for detection of this organism revolves around using PCR to detect the genes responsible for glucoamylase production. Since genetic methods are highly specific and also very expensive, an attempt was made to develop a low cost assay for this organism that would bypass the need for Genetic detection and simply test for the presence of Glucoamylase activity itself, as opposed to the genes. The idea of this assay is to create a pure solution of long chain “unfermentable” sugars for a standard Durham Tube fermentation test (more info here: https://microbeonline.com/carbohydra...edure-results/) that would allow us to detect the presence of glucoamylase in beer or yeast samples.

    Commercial Maltodextrins are available that are made up to 95% complex sugars and 5% fermentable sugars. Because of the mechanics of maltodextrin production, it is impossible to achieve a 100% Maltodextrin, as there will ALWAYS be a small portion that is made up of simple fermentable sugars. To get around this we simply make up a solution of Maltodextrin to roughly 12 degrees Plato and first ferment the solution with a known NON-Diastaticus Strain, we usually use US-05. We use this yeast to remove the bulk of simple fermentable sugars, incubating at roughly 90F, which then gives us a relatively pure maltodextrin solution. Once the yeast has done its job, we re-autoclave the solution to kill the yeast and any other organisms in the beer, and then innoculate a sample of beer from any point in the process (Ferm, BBT, Package). The bright side is that the autolyzed yeast also acts as a source of nutrition for organisms contained in the beer sample. Once inoculate the media with our beer sample, we incubate all Durham Tubes @ 90F for 2-5 Days to ensure full enrichment. Once this is done, you simply look at the durham tubes for any gas production. If you see gas in the tubes/active fermentation, you know you have something in your tube capable of fermenting complex fermentable sugars.



    Protocol

    Malto-Media Preparation/Sample Innoculation

    Bring 1 L of Water to a boil in erlenmeyer flask , add 70 grams of Maltodextrin M040 to water and stir frequently to dissolve. Once dissolved, autoclave at 250F @ 15PSI for 15-30 minutes. Allow to cool to room temperature
    Once cool, pitch 1 homebrew sachet of US-05 into the mixture and allow to ferment in incubator @ 90F for 24-48 Hours. (if no incubator available, use a heated stir plate).
    Once solution appears to be done fermenting, shake or stir vigorously to ensure solution is well mixed, and proceed to pour evenly into 40-50ml tubes (the total amount doesnt really matter as long as its consistent across each tube). In each 40-50ml tube, place the smaller 10ml test tube inverted into the solution, cap, and proceed to invert entire 40-50ml tube to get solution up inside the inverted tube without any air or gas being trapped inside the tube.
    Autoclave tubes to 250F @ 15PSI for 15-30 minutes and allow to cool. Once cooled, the samples are ready to be innoculated with between 10-20mls of sample (whatever your tubes can accomodate). Ensure that two tubes are reserved for a positive control and a negative control to ensure preparation was correct. I use US-05 as my negative control and Belle Saison from Lallemand as my positive Diastaticus control because they are relatively easy to obtain.
    Incubate for a period of up to 5 days but no less than 2, checking progress of samples against negative and positive controls daily.

    Results Interpretation
    If you see gas production in any of the tubes, you have a strong indication of an organism capable of fermenting complex sugars in your sample. If no signs of fermentation (no bubbles, no gas in inverted tube), then no diastaticus organisms are present.

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