Dealing with Diastaticus

Brewers use diastatic strains intentionally when making saison, but if a diastatic strain gets somewhere it doesn’t belong, the hyper-attenuation and off flavors it could produce can spoil your beer — or worse, turn it into a ticking time bomb.

With a threat of that gravity, it’s well worth looking at the accuracy of your contamination detection methods. Recent experimentation by Omega Yeast suggests standard testing methods for diastatic yeast aren’t working as well as you’d think.

1. The first step for better coverage is understanding the strengths and weaknesses of the detection methods we commonly use. Using existing methods in combination is crucial for reliable results.
2. Next, verifying that the methods you’re using are actually working, and working the best for your brewery’s conditions, is important. You might be shocked to learn what commercial LCSM is missing. In short, optimize and validate.
3. Finally, there are also some newly developed detection methods that look for diastatic yeast differently; they measure actual diastatic activity, rather than copper resistance. That has some big advantages, like eliminating many of the false positives and negatives that are inherent in those other tests. These new methods also give more information — like how diastatic the contamination is. That’s something entirely new.

These few improvements could save you enormous hassle and expense.

Brewery QC programs rely on two main methods of detection when looking for diastatic contamination: 

• selective microbial growth media, like LCSM 
• and molecular detection of DNA, like PCR. 

These methods are strongest when used in combination because of the false negatives and positives they are prone to.

Selective Microbial Growth Media

LCSM plates are pretty standard in diastatic testing. Plates are relatively easy to make and use, and they are inexpensive. LCSM contains cupric sulfate, a copper-based compound, which selects for diastatic yeast growth because diastatic yeast have a tendency to be copper resistant, whereas most non-diastatic yeast are not. 

If a yeast strain grows on one of these plates, it’s likely to be an active diastatic threat to your beer. However, there are important exceptions that can leave beer vulnerable. For example: 

• Non-conforming copper resistance
  • A known diastatic strain could go undetected by LCSM if it happens to be copper sensitive (false negative). Confirmed non-diastatic strains can seem diastatic if they are more highly copper resistant than other non-diastatic strains (false positive). In the case of false positives, colonies that grow on LCSM must be followed up with PCR to confirm if a true STA+ contaminant.
• The age of the plates you’re using (and the recipe)
  • LCSM is very good at detecting diastatic yeast in very small amounts — but only when plates are in their prime. If the plate medium is, or has become, either overly restrictive or overly permissive, you’re likely to get an alarming number of false negatives and positives. You can lower the chances of unreliable results through careful media validation and optimization. This is a step many breweries may not know has such importance. Plate age and recipe matter a lot.
• The test’s natural limits of detection.
  • Remember that testing depends on the sample you select containing diastatic yeast. If levels of diastatic contamination were so low at the time you sampled, you may have missed snagging any, even if there was some there.

Molecular Detection, Like PCR

The DNA from the STA1 gene is how diastatic yeast cells are generally identified by PCR (though, recent research has made an additional correlation with intensity of diastatic activity and a promoter sequence for the STA1 gene called UAS, Upstream Activating Sequence). PCR’s advantage is that it’s extremely fast and very sensitive. PCR with the DNA from one suspected STA+ colony on an LCSM plate can provide results within an hour or two. Liquid enrichments work similarly to LCSM plating, and have very low limits of detection. They can be used to grow up only a few cells of STA+ yeast.

• False Positives
  • You can get a positive hit on the presence of diastatic DNA from dead cells, which would be no threat to your beer. With any positive STA1 PCR result, it is critical to follow up with microbial plating to confirm the presence of a living STA+ contaminant in your sample.
  • With DNA methods there are a couple of other types of false positives that are possible, too: Overloading PCR with too much DNA can give a false positive result, even without any STA1 DNA in the sample. That’s because PCR doesn’t really ​“read” DNA. PCR primers are able to bind to specific sequences and result in their amplification (in this case STA1). The amplification is then detected by several different methods, some of which are more prone to false positive results. Just a lot of DNA would cause non-specific amplification when a different sequence in the sample’s DNA is close enough to be a near match for the PCR primers. The primer binds to the near-sequence and results in non-specific amplification as if it were the DNA we were looking for. That would be a false positive result by PCR and no real risk of a STA+ contaminant, for example.

• False Negatives
  • False negatives could result from some other kind of user error, and/or from something coming in under limits of detection, like if there’s too little DNA to get amplification going enough, or an error was made in the PCR setup.
  • Including controls in your PCR will help rule out false positives or false negatives. Use known STA+ and STA- strains to confirm the PCR setup was correct, and that there is not any contaminating DNA in your PCR reagents.

One of the ways we make the testing we already have more accurate is via optimization and validation. Microbial plating is relatively quick, easy, and cheap. Also not to be underestimated, it indicates the presence of live yeast, unlike DNA-based tests. Interestingly, however, when Omega Yeast’s researchers tested the strains in their collection on the common commercial LCSM formula, they found alarmingly spotty coverage.

That points out the extreme importance of optimization and validation. To be effective, brewery’s lab teams should be making sure their media is catching the diastatic strains that they know are around the brewhouse, in addition to other known diastatic strains.

• Optimization: by varying proportions in the recipe for LCSM, Omega Yeast turned up a new recipe that improved the accuracy of screening results when considering all of the diastatic strains in their catalog.
• Validation: The key to validation is setting up good positive and negative controls with known diastatic strains and known non-diastatic strains that behave reliably. 

Watch out for high background strains — strains that appear diastatic but aren’t. Positive hits should be verified with DNA-based testing to confirm before destroying beer.

More important tips:

• Include controls each time you’re plating samples so you know your plates are doing what you expect.
• Remember LCSM isn’t testing for diastatic activity directly. It correlates diastatic yeast with copper resistance. Not all diastatic yeast are copper resistant or as copper resistant as others.
• Multiple methods are best. Coverage is improved when methods are used in combination.
• Be rigorous in maintaining the same way of doing things once you find the conditions that work best for you. Consistency is key.
• Understand plates’ usable window and plan for it. Plates are too permissive on the day they’re poured. The sweet spot is 24 – 48 hours after. Discard unused plates after five days. They’ll be too stringent. You’re looking for the ​“goldilocks” window to eliminate as many false negatives and positives as possible

Note: there’s some evidence autoclave time can play a role in how effective LCSM is, so always autoclave for the same amount of time (Omega Yeast does 30 minutes on liquid cycle). For cupric sulfate, Omega Yeast filter-sterilizes a 10% stock solution. This is never heated, and never autoclaved. The solution is added to the plates after they’ve come out of the autoclave.

There’s plenty of information about the source of diastatic activity in yeast that is still under investigation (there are other STA genes that may influence things). There is also a helpful growing awareness of variation in diastatic activity. The more methods we have that give us different snapshots of what’s going on — and the more we understand our tests’ advantages and limitations — the more accurate and informative our detection is. Testing for diastatic activity directly, which would mean somehow monitoring the attenuation of dextrin would give even more useful info to brewers. 

Omega Yeast’s researchers wondered if they could devise a practical test that didn’t rely on either copper resistance or DNA. The idea was that a test like this could eliminate the perils of false positives and negatives. They came up with three, actually. Each performs clearly, accurately, consistently and without false negatives and positives. And they all use pretty accessible equipment and ingredients. Importantly, unlike existing methods, they indicate how diastatic a strain is. That’s something that could tell you a lot about what a contamination could mean in terms of actual risk to your beer.

If getting a better grip on diastaticus this way is not enough, you could also try ditching it altogether.