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Thread: Refermentation in Packaged beer due to Diastaticus

  1. #31
    Join Date
    Mar 2013
    Location
    Milwaukee WI
    Posts
    25

    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
    Join Date
    Mar 2013
    Location
    Milwaukee WI
    Posts
    25

    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
    Posts
    4
    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
    Posts
    25

    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
    Join Date
    Mar 2013
    Location
    Milwaukee WI
    Posts
    25
    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.

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