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Thread: Refermentation in Packaged beer due to Diastaticus

  1. #1
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    Refermentation in Packaged beer due to Diastaticus

    Posting this up here to see if anyone else has had similar issues, and what they have done about it. I was ripping my hair out over this issue for the last six months, and finally have the data to back up my conclusions. I apologize for the Novel like length of this post, but being a man of science, data is always required to support my conclusions, if i have missed something, someone please tell me.

    We have had some recalls over the last 6 months or so related to over-fermented product in cans and bottles. This has only been an issue for some of our brands of beer, but not all them. It was not until recently that we were able to definitively pinpoint the cause of these issues. This document will hopefully serve as reassurance that we had (at the time) done everything in our power to ensure quality product out to market, and that, ultimately, this issue will cease moving forward. The underlying cause was very difficult to pinpoint without specialized equipment and/or laboratory analysis.
    Background:
    Upon startup of our new facility every batch of beer was tested in house AS WELL AS through a third party laboratory to ensure the beer was free of microbial contaminants. We have copies of these analysis’ for every batch of beer sold to market. Each and every single batch tested that went out to market showed that the beers were FREE AND CLEAR of the most common microbial contaminants found in the brewery using ASBC or EBC standard methods of detection. (American Society of Brewing Chemists & European Brewing Convention)
    Upon startup of our new facility we switched house yeast strains and suppliers to find strains of yeast that would create a more desirable profile, had better flocculation (easing the strain put on by our filter during filtration), and were much more efficient at fermenting than our previous house strains.
    Very early on this year we began to a notice an issue we had never before encountered in our canned products. Cans (and some bottles) had begun to referment to the point where the cans were exploding/becoming dangerous. It was at this point that we started down the long road of looking for the root cause of this issue.
    Analysis:
    Tank Cleaning/Sanitation Procedures and Validation
    • Tank cleaning/sanitation procedures were validated through ATP Bioluminescence and found to be within the very tightest allowances for cleanliness and sanitation. Zero lumens were found, which means there was an undetectable amount of biomaterial on the inside of the tanks.
    • This combined with visual inspection of the inside of each tank after every cleaning validated the issue was not from tank cleanliness/sanitation.
    • Every time a tank or piece of equipment is cleaned or sanitized in the brewery chemical concentrations and strengths are validated with test strips/titrations to ensure proper concentrations within industry standards.


    Transfer Lines/Filter Sanitation Validation
    • All transfer lines and filters that are used to pump beer/wort from one place to another are pasteurized with hot water to 175F for 20 minutes just prior to use. When beer is being transferred to a bright beer tank or other aging vessel the transfer lines are also blown out with sanitary CO2 from our bulk tank. When we begin to transfer wort from our brewhouse to the fermenter we pack the lines/heatex with uncooled 200F and sit for 2-3 minutes to ensure everything is as sanitary as possible. (this is in addition to a pasteurization we run from our WP vessel through our Heat ex through our xfer lines to fermenter that runs between 170-180F for AT LEAST 20 minutes)
    • This process was validated in house and externally by checking microbial stability of water inside transfer lines both during pasteurization and post blowdown of the lines/filter (there is some residual water in lines post blowdown, enough to pull a sample for microbial stability). No contaminants were found.
    • For each batch we pull a wort stability sample to check for contaminants between the brewhouse and the fermenter. To date none of the samples have tested positive for common spoilage organisms.


    Exterior and Interior Testing for Microbial Contaminants
    We use a variety of different microbiological media to look for spoilage organisms in our beers.
    • We currently test for the most common strains of Wild Yeast, Lactobacillus and Pediococcus found in most breweries using HLP, UBA, NBB and LWYM
    • Our exterior lab tested for the same organisms using different media, MRS, UBA+A, LWYM & Lysine.
    • All tests performed for all beer released to market came back negative for Microbial Contamination PRE AND POST PACKAGING.

    Packaging Line Cleaning and Sanitation Validation
    • Interior and exterior cleaning and sanitation procedures were validated through ATP Bioluminescence and found to be within the very tightest allowances for cleanliness and sanitation (again zero lumens).
    • Packaging lines are cleaned and sanitized immediately before every use.
    • The entire exterior of the packaging lines are foamed with a foaming chlorinated caustic cleaner, which simultaneously cleans the exterior of the line while the chlorine sanitizes the cleaned surfaces.

    Yeast Health and Management
    • We validated our oxygenation procedures via DO Meter to ensure proper oxygenation.
    • We perform cell counts and adjust pitching rates for every batch of beer, and plot the ferm data out on a graph to look at the fermentation curve data
    • We began performing forced wort fermentation samples to determine proper finishing gravity of Flagship beers. We found that the data collected from the forced fermentation samples lined up perfectly with the brew log data collected over the last year. This continues to be a vital QA check for us and ensures our yeast are happy, healthy and viable so they can fully ferment beers as well as they ought.
    • We evaluate (via microscopy) that yeast is free of pediococcus and lactobacillus because their shapes make them easy to differentiate from conventional yeast strains. This method DOES NOT work for wild yeast strain detection.

    Analysis of Re-fermenting Beers
    • Microbial testing after refermentation - the tests revealed the same results as before the beer went to market, the beer was clean of the microbial contaminants we were testing for. (These tests were conducted inhouse AND third party.)
    • Refermented beers exhibited finishing gravities up to 1Plato below final finishing gravity prior to packaging, even though in some cases beers sat for three weeks at terminal gravity @ fermentation temperature, and theoretically the beer should have finished fermenting.
    • Every beer that went out to market underwent refermentation testing (not just ones that exhibited refermentation). Method and analysis are detailed below.
    • Post packaging, beers sit 1-2 weeks (depending on the beer) in an 85F incubator to simulate the worst possible abuse beers can take out at market. After this beers are gravity tested for refermentation and for microbial contamination. The 85F temperature also falls within the ideal growth range of MOST COMMON spoilage organisms found in breweries.
    • All beers tested in this fashion showed no signs of re fermentation or microbial contamination.
      Continuous regular testing for refermentation of warm retains (setup to simulate at-market conditions) for the FIRST MONTH of the beers packaged life showed no significant signs of refermentation.
    • We have since learned that for this particular issue our protocols were insufficient, as the issue took between 2.5-3 months out at market JUST TO BEGIN TO MANIFEST
    • Secondly, warm retains kept from the SAME BATCH that were affected displayed varying levels of refermentation with some showing none at all and some exploding. This contributed to the level of difficulty in noticing this problem with our testing regimens and procedures. We assumed that every can would be uniform in showing this issue AT THE SAME TIME, this was not the case, some cans, under the exact same conditions, appeared to referment much more quickly than others.
    • pH testing on all of the refermented beers found that the pH was the same as it was when the product was packaged. This indicates that ACID PRODUCING BACTERIA WERE NOT THE CULPRIT.
      Had acid producing bacteria been the culprit, even if acid could not be detected by taste, we would have seen a significant drop in the pH.
    • Taste panel on all batches of refermented beers revealed no significant taste difference other than standard flavor degradation due to warm storage as well as dryness and over carbonation when compared to the cold retains. No off flavors such as diacetyl, phenolics or acidity were present.
      MOST spoilage organisms will show the above off flavors in the beer if they are present, providing an easy qualitative measure for determining source/cause of issue to a trained palate.
    • Filtration had no impact on whether a batch would referment or not, one batch that was recalled had been filtered, another one had not, but the same issue manifested in both. We use a fairly loose filter sheet that is designed to remove 95% of yeast in solution. This looser filter set allows us to remove the majority of yeast while still maintaining flavor profile pre and post filtration.
    • One beer that had this issue used 1st generation yeast STRAIGHT FROM OUR SUPPLIER. This was our first red flag.

  2. #2
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    Refermentation in Packaged beer due to Diastaticus pt2

    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.
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      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.

  3. #3
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    This is above and beyond. If I may ask, why are you showing us this? These data should be supplied to the yeast lab in question. It seems pretty clear that one of their freezer stocks of the strain in question is contaminated, for two reasons:

    1) it was only that strain (implying not an equipment issue)
    2) not every new pitch of that same strain contained contaminants (indicating only some stocks are contaminated)

    If you get in contact with them, and they take your data as valid, their records combined with yours should allow them to destroy the contaminated freezer stocks in question.

    It is also possible that they use one set of equipment to grow this strain, but that isn't a typical usage of resources.

    This would be pretty damaging to any commercial yeast supplier if this was made public, so you can certainly help them.

  4. #4
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    Quote Originally Posted by theomuller View Post
    This is above and beyond. If I may ask, why are you showing us this? These data should be supplied to the yeast lab in question. It seems pretty clear that one of their freezer stocks of the strain in question is contaminated, for two reasons:

    1) it was only that strain (implying not an equipment issue)
    2) not every new pitch of that same strain contained contaminants (indicating only some stocks are contaminated)

    If you get in contact with them, and they take your data as valid, their records combined with yours should allow them to destroy the contaminated freezer stocks in question.

    It is also possible that they use one set of equipment to grow this strain, but that isn't a typical usage of resources.

    This would be pretty damaging to any commercial yeast supplier if this was made public, so you can certainly help them.
    I would suggest they are posting this information to help others with similar problems. Most likely to tell them it is in fact possible for a fresh pitch from a supplier to be contaminated (which many do not believe is possible).

    I would disagree that this (definitively) shows a contaminated freezer stock. In my mind that is jumping to a very large conclusion. It could be cross contamination in the production lab. Or even potentially in the brewery. I only say this, because I believe poster said that this data was from the re-fermented beers, and not from the original pitch of yeast straight from the supplier. Without seeing direct correlating data on the initial pitch, we are left to assume it was present before the beers had re-fermented. If not all beers on the shelf are exhibiting the same conditions, that leaves a question in my mind at least.

    Questions: Have you seen any of this activity in (or tested) kegged product? Have you tested the direct pitch from the supplier? Have you tested any samples (from suspect strain) in the fermentation vessel, prior to packaging?

    I am not suggesting this is happening in the brewery, merely that it is still a possibility based on the data I see. Since growth is usually started from slant, there is many chances during production for cross contamination due to poor handling. If your beer is sitting for multiple weeks in tank without signs of the continued activity, that would suggest to me a potential contamination in packaging. More data would be helpful in a defined conclusion (at least for me).

    I would agree that this could be very damaging to the producer and should absolutely be referenced back to them. There are a lot of newer yeast producers out there, some with more book knowledge than practical experience. Not to say this is a newer supplier, but new people often make mistakes in lab procedures and anything is possible. If they (supplier) are smart, they would do some PCR's on inventory of the suspect strain to come to a reasonable conclusion. If they concluded to me similar PCR results, I would certainly feel there is a strong case for contaminated stock.

    To the point at hand, if a new (or in your case old) supplier has eliminated the issue, that is what counts in the end. But thank you for all the details, I find it a quite useful post!

  5. #5
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    Thank you ,mobcraftbeer, for this audacious post.
    Since early July, when I discovered a recent batch of a brew had gone bad, definitely due to re-fermentation, I've been de-stablelized, unable to fathom out why and thought I was alone. A lab analysis did reveal some yeast and mold but also some lactobacillus. With this new information I'm going back to the lab for strain and species identity. Thank you.

  6. #6
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    Refermentation in Packaged beer due to Diastaticus

    The main reason for this post was to inform, and also curious if anyone else has been having these issues, and how they deal with them.

    We (like many other breweries) assumed that yeast from the supplier would have a rare chance of being contaminated straight at the source. The Brewery that helped us with getting our Beers Tested was a regional brewery that had had the same issue of contaminated yeast and had a massive recall off the market last year due to infected product, many other large (either regional or national) breweries have also had this same issue. All of them have started doing the same thing, PCR testing incoming slurries and all throughout the propagation and fermentation stages. If you have the funds to invest in advanced analytics machines and the staff to manage it, awesome. But if you dont have access to an actual machine or the resources for one you would have to send it out for outside lab testing for analysis. This means i would have to (A) Sanitarily open my yeast pack to collect a sanitary sample for analysis and send it out, and (B) sit on this opened yeast pack for a week while i wait for results to come back.

    So i posted up here to see if anyone had figured out a better way, I'd hate to tie up propogator for a week and delay brewing if the incoming slurry was shit anyway, the only other feasible option it seems is invest in the laboratory upgrades (a hell of a lot cheaper than a PCR machine, btw) to propogate up from a single cell in house, my background is in food science and microbiology so i have the skill to do it, just would need the equipment.

    I have reached out to the supplier with the lot numbers for the yeasts we used that had problems with, and they will run PCR of thier own, the interesting part is, even though they have a PCR machine, they don't use it for thier COAs, thier COA's all use microbiological media and i was curious about that, bc if you had a PCR machine, why wouldnt you use it for your COA's?

    We have so far had no reports of this happening for draught product, but i would assume thats because our draught product moves WAY faster than our cans sitting out at retail, and the kegs are refrigerated and, even if they arent refrigerated out at market, they are usually put on tap before the issue has a chance to manifest, i would assume.

    As far as it being a contamination issue on our packaging line, two of the beers that tested negative were also run out on that same packaging line, which doesnt completely preclude it from being packaging line issue but certainly precludes it from being a systemic issue with our cleaning sanitizing/protocols

    The same brewery that ran the first set of tests is coming by for a 2nd round of samples this week, and i will definitly be looking at the difference (if any) in kegged product versus canned (if i can find any kegs from older batches, that is) as well as fermentor and BBT samples. My biggest fear is that we have essentially spent the last six months propogating a spoilage organism in our brewery and that it might have gotten a toe hold somewhere.
    Last edited by mobcraftbeer; 08-22-2017 at 11:21 AM.

  7. #7
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    I do use Ecolab-

    Difference in ACTIVE INGREDIENTS:

    Vortex
    Peroxyacetic Acid
    Hydrogen Peroxide.......................................... .................6.9%
    Peroxyacetic Acid.............................................. ................4.4%
    Octanoic Acid.............................................. ........................3.3%
    INERT INGREDIENTS:...................................... ........... 85.4%
    TOTAL:............................................ ..................................100.0%

    Oxonia
    Hydrogen Peroxide.......................................... ..............27.5
    Peroxyacetic Acid.............................................. .............5.8

    So what is Octanoic Acid?

    http://www.fao.org/fileadmin/templat...microbials.pdf

    "Based on the above technical assessment on the efficacy of octanoic acid at target concentration during
    typical use of the mixtures, it could be suggested that perhaps the antimicrobial action of octanoic acid be
    attributed more to its surface-active properties. As a surfactant, octanoic acid may possibly modify surface
    properties of the mixtures to aid in wetting of hydrophobic food surfaces such as meat and thus enhance
    spreading and encourage retention of other more active antimicrobial agents at effective concentration" (Clark
    2003

    Although Vortex has much less Hydrogen Peroxide and therefore less oxidation power. Might be worth contacting my rep about.
    Brewmaster, Fitger's Brewhouse
    tbriggs@justtakeaction.com
    "Your results may vary"

  8. #8
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    I think you are approaching this in the best way possible. Your data and conclusions seem logical to me.

    I think an in house propagation from slant is always your best bet - As long as you know what you are doing, which it sounds like you do.

    You will still be subjected to the same micro-media forms of testing your own propagation though, and they may or may not provide the same "clean" results. If your contaminant has indeed found a place to live in your brewery, this could still be a significant risk of repeat.

    I know for a fact that some major suppliers have seen double-digit contamination rates in production, and these are very well skilled laboratory operators. Hopefully they are catching it all before it goes out. It is absolutely possible this is not from your brewery, but keep in mind how easy it is to foul your own propagation.

    Cost savings on your own propagation will be significant.

    My guess is they do not run the PCR on every batch due to labor cost and time constraints. Although PCR is relatively quick, the key word is relatively. DNA extraction and the sequencing takes longer than plating on agar, and must be performed by a more capable lab staff. Cross contamination is a very real risk in PCR too.

    Example, I may need to centrifuge a dense DNA extraction sample for up to 10mins, but I can swab about 10 plates in that time. When you have hundreds or thousands of samples, this could be a significant factor.

    I would agree PCR is a better approach overall.

    Ted - If you want a good contact, talk to Dana Johnson at Birko corp. I know him personally, he knows his Chems better than most any suppliers I have ever met, and I know my chems pretty well too. He is also a home brewer, and just wrote a great article in the July/August edition of New Brewer. I've been following his methods for a few years now, and they have yet to steer me wrong. He's been doing brewery specific chemicals since the mid 90's. - I like the Birk-Ox myself.

  9. #9
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    Diastaticus

    I would disagree that this (definitively) shows a contaminated freezer stock. In my mind that is jumping to a very large conclusion. It could be cross contamination in the production lab. Or even potentially in the brewery. I only say this, because I believe poster said that this data was from the re-fermented beers, and not from the original pitch of yeast straight from the supplier. Without seeing direct correlating data on the initial pitch, we are left to assume it was present before the beers had re-fermented. If not all beers on the shelf are exhibiting the same conditions, that leaves a question in my mind at least.
    I could agree too, and I'm glad the conclusion wasn't jumped to

    Given you had said this
    Secondly, warm retains kept from the SAME BATCH that were affected displayed varying levels of refermentation with some showing none at all and some exploding. This contributed to the level of difficulty in noticing this problem with our testing regimens and procedures. We assumed that every can would be uniform in showing this issue AT THE SAME TIME, this was not the case, some cans, under the exact same conditions, appeared to referment much more quickly than others.
    which may implicate the packaging line as the culprit.

    Another question with PCR is if the target contaminant is going to be amplified. If it's not in the primer then well you won't find it anyway. Especially since I do belive it's actually S. Cerevisiae var. Diastaticus and there's no such thing as a distinct species called S.Diastaticus but I might be wrong about that.
    I hope I encouraged you!

  10. #10
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    Same boat

    First of all, WOW. If more people were this diligent and concise, my job would be a lot easier. Well done!

    Secondly, I was excited to finally see that someone was having a problem so similar to mine. Whether the issue came from our yeast supplier, some yeast that we borrowed (now an extinct practice here), or from a cross contamination with a Saison strain we are using (which has been confirmed to contain Sacc. Diastaticus) we have yet to figure out. Our issues are similar in these ways: 1)Everything performs normally in the fermenter, with a usual final gravity, 2)There is zero indication of contamination with any common spoiler, 3)The effects are showing up a month or two after packaging (overcarbed, bulging cans, explosions).

    There is, however, one difference here: We see the problem only with our kettle sour beers. After souring, the wort has been FULLY STERILIZED with a 60 min boil, and the finished beers show NO signs of any spoiler contamination.
    I've learned that Sacc. Diastaticus, when under conditions of stress, secretes diastatic enzymes to break down dextrins. Could the lower pH of these beers be the stress factor that is triggering diastase production in the Diastaticus?

    As an extra QC check, I've been force aging (@35C) six cans/bottles from each run, and testing the FG once per week to get an early warning of refermentation. So far (after 8 weeks of this), the only beers to referment have been our kettle sours.

    Now, all new yeast come into the lab first for a PCR test for Diastaticus before they go to the brewhouse.

    Anyone else having this issue with low pH beers?

  11. #11
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    Have you tested the re-fermented packages themselves to conclude definitively that they contain the Sacc. Diastaticus vs live lacto, ect? Have you tested the post kettle wort to confirm the same is or isn't present before yeast pitch?

    I am only asking since you state you check the initial yeast pitches, but don't say about definitive lab testing of later process. It may be possible your issue is coming from somewhere other than the yeast, although it would seem unlikely. The fact you only see this in kettle soured beers, and now PCR your yeast pitches leaves me a small amount of doubt between Diastaticus and Lacto, ect.

    You claim FULLY STERILIZED, however that is not absolutely positive with a 60min boil under normal conditions. Usually you need pressure and temperatures above 100*C (or other methods) to achieve STERILITY (autoclave, pressure cooker). It is possible for certain spores to live through autoclaving as well, but very unlikely when you have ideal germination conditions prior to heating, such as your kettle sour would provide. Your boil should be enough to sanitize, but not necessarily enough to disinfect, or sterilize.

    I would think that lower pH levels could certainly contribute to more stress and the breakdown of dexterins provided you do in fact have S. Diastaticus, but other "bugs" can cause the issues you are seeing as well. I would suggest more testing to isolate the strain definitively, and then work on where it may be getting introduced.

    PCR is a GREAT tool, but since it replicates DNA, there is still some room for false results. For instance if there is not S. Diastaticus in your sample being run, it will not show. There still may be some present in the pitch, but not enough to get into the test sample. Testing again post pitch might at least give you a second chance to sample and check, and testing known bad packages would help confirm the contaminant. Then you can backtrack to find the source.

    I had a similar issue with this in regular beers (showing up three months post), but eventually traced it to a lacto. We didn't use lacto and had very stringent practices prior (including malt handling) but never actually isolated its cause. I strongly believe it was a contaminated initial pitch, but had no means to confirm positively as it was after the pitch, and we did not test initial pitches at the time. It must have been an extremely low count as the beers held steady in the ferm for many days (all were source from same yeast, as no issue was found at the time of harvest). It is possible it was sourced during packaging, however problem did not surface again.

  12. #12
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    Sorry, I should have clarified. By "no signs of spoiler contamination" what I mean is, we run PCR for lacto and pedio at fermentation, BBT and post-packaging. There have been no positive results on any product with this refermentation issue. I've also run HLP tubes on these ones, just in case something's not right with my thermocycler.
    Yes, there are a few microbes that can survive 60 min at 100C atmospheric, but certainly not lacto. Poor choice of words on my part.
    What also leads me away from lacto as a suspect are two clues: 1) there is no pH change after refermentation in package, and 2) the FG of the referments is always in the 1.0 - 1.3 plato range. It's never gone below 1.0, just like Diastaticus (or if you'd prefer, 'French Saison yeast') would do. I think these two in combination with negative PCR across the board rule out lacto.

    I should mention another similarity with mobcraftbeer, which is the lack of uniformity. From the same batch, some cans are refermenting and other aren't.

    Has anyone else had this issue?
    Last edited by MakeBeerNotWar; 09-20-2017 at 06:26 AM. Reason: clarification

  13. #13
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    All, i thought i would share some information and updates on our current situation.

    We are hopefully through our last and final round of recalls, and I have sent samples from the most recent batches of beer for additional PCR testing, unfortunately the lab we sent them to, thier PCR thermocycler broke down not long after our samples arrived so we had to rely on good ole fashioned plating to see what we had in there in terms of contaminants. More on that in a bit.

    One of the most important things i learned that I'd figured i'd share is that certain strains of belgian/french yeasts (mostly saison strains from what i have been able to research) HAVE BEEN CONFIRMED TO BE DIASTATICUS. This is because they contain the genes for Glucoamylase enzyme production, and thus are considered to be Diastaticus. This means that if you have been using those strains confirmed to have glucoamylase genes in them than this is more than likely the source of your infection. This is apparently the reason why saisons are able to ferment beer out so dry and attenuate so well.

    One of the other important things i have learned is that Diastaticus can be effective at extremely low concentrations, this is based on anecdotal evidence only and my own conjecture, but other breweries i have talked to have seen problems show up very early in propogation, indicating the organism is present but effective at below typical detectable thresholds. The way they were able to even find it was through a Cupric Sulfate/Wort enrichment and plating the subsequent enrichment on LCSM for detection. Even though the original slurry did not show any signs of Diastaticus, the enrichment revealed the presence of wild yeast (most likely diastaticus by the colony morphology). The main reason i beleive diastaticus can be effective at such low concentrations is because it is capable of secreting the glucoamylase enzymes OUTSIDE THE CELL. This would make sense as most of the dextrins and complex sugars that glucoamylase are capable of cleaving are simply to big for most single celled organisms to transport across the cell wall (this is the main reason why culture yeast can only ferment simple sugars). What this means is that if you have active diastaticus in solution, the glucoamylase in solution will break down the complex sugars at a relatively constant rate, and any active yeast still in solution will be capable of fermenting those simpler sugars into alcohol and CO2, thus resulting in gushing and overattenuation.

    Based on the reasoning above, we have lowered out Wild Yeast Contamination threshold (the point at which we determine go no go for packaging and selling) from 10 Colony Forming Units per Milliliter down to 1 Colony Forming Unit per Millilter.

    Alcolyzer or densitometer data can be extremely effective at telling if your beer is okay or not, since these instruments are more sensitive than the hydrometers most of us use, they are able to tell more precise changes in residual sugars and alcohol content. If you have alcolyzer data from day of packaging and from 1-2 months out at market, if you see a decrease in residual sugar content AND an increase in Alcohol content, this is a very strong indicator of diastaticus presence (this would have to be coupled with a forced fermentation in order to determine proper final finishing gravity to make sure your commercial batch attenuates to where it should be).

    Looking at our records, we did IN FACT have a French saison strain in our brewery during the time we were having these problems. Checking our suppliers website, apparently this strain has been genetically typed and confirmed to contain the STA1 gene, however the website assures that it is not considered a contaminant and "will not cause over attenuation in finished beer by fermenting dextrins". We brought 1L pitchable quantities each of the ale strain and the french saison strains at the same time and propagated them in 2 separate 15bbl fermenters splitting a 30bbl batch two ways off of our brewhouse. This batch was the 3rd batch of beer to be brewed at our new brewery on start up, with the previous two brews being Hefewiezens using the WB-06 dry yeast. Even though the strains were propogated at the same time in separate fermenters, chances of cross contamination between these two are minimized ( I beleive) for the following reasons:

    The ale strain was pitched into the fermenter BEFORE the Saison strain.

    The ale strain finished out first and was pitched 10 days ahead of the saison yeast in the other propogator being pitched.

    The method of pitching was cone to cone for both propogation, and protocol is to heat kill with 170-180F water for 20 minutes prior to any beer touching the hoses. Same goes for beer transfers to BBT's.

    Ale beer had already fully attenuated by the time the saison was pitched from propagator into full fermenter again using cone to cone and heat kill protocols.

    All of the tanks that were used had been freshly passivated (including a thorough CIP) on startup of the new facility. CIP logs confirm caustic and PAA levels to spec for propagator, fermenter and BBT.

    Ale beer had been packaged 20 days prior to the saison beer even being transferred.

    Hope this helps anyone out there, and if my conjecture is flawed above, please correct, also if anyone could think of a possible way these two beers could have been cross contaminated id love to entertain questions and thoughts!

  14. #14
    Join Date
    Mar 2013
    Location
    Milwaukee WI
    Posts
    12

    Diastaticus Detection

    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.

  15. #15
    Join Date
    Oct 2013
    Location
    Dallas, Bangalore and soon Goa
    Posts
    195
    Quote Originally Posted by mobcraftbeer View Post
    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.
    No experience isolating S. diastaticus, however I have made many types of agar in the past. I would suggest to culture it you could make your own un-malted barley agar in varying ratios. The starch agar is a good idea for identifying and you could add some iodine after incubation to confirm if the S. diastaticus has shown exoenzymes that hydrolyze the starch.

    Let us know what you choose to do and the results, I find this thread to be quite interesting.

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