Hey Escarpment Blog readers, this is the second instalment of our diastatic yeast post started by Alex, where he talks about diastaticus detection testing with PCR. Many of you are already familiar with diastaticus, but for those who aren’t, let’s review what happens when it gets into beer cans:
All kidding aside, Saccharomyces cerevisiae var. diastaticus is beer yeast that can break down longer-chained carbohydrates (dextrins and starches) that regular yeast can’t. It doesn’t actually consume them directly, but it secretes an enzyme (glucoamylase) that breaks down dextrins outside the cell into smaller sugars that the yeast can then metabolize. Diastatic yeast is often problematic for brewers, causing unintended hyperattenuation and secondary fermentation post-packaging, which can lead to over-carbonated product and the dreaded exploding cans.
Because this is such an important issue, a lot of research has gone into detection testing for diastaticus. There are several ways to do this, and we’ve been looking into the most accurate and dependable ways to get results. Here at Escarpment Labs, we use 2 methods: PCR and agar plating. The PCR method tests for STA1, a gene which encodes a secreted glucoamylase enzyme.
There are several different plate media that are used to test for diastaticus, and we tested all of our strains using 2 methods: starch agar plates (method adapted from Meier-Dörnberg et al., 2018), and LCSM (aka Lin’s Cupric Sulfate Medium). The starch agar plates contain a dye which when incubated with diastatic strains that can break down starch, change colour from purple to yellow. This incubation method is anaerobic and takes approximately 3 weeks to complete, with positive results showing up in as early as 4-5 days. Results of the starch plate incubation tests with our strains correlated really well with PCR results, and gave us a more complete picture of how our diastatic yeasts behave.
Some of our strains after 5 days of incubation on starch agar plates
From the plates we see hints that there are three different “strengths” of diastatic yeast, ranging from weakly to strongly diastatic, based on how many days’ incubation was necessary to cause noticeable colour change. The type of PCR testing we use can tell us about the presence of the gene but not the strength of gene expression, so we only get a positive or negative result. It also doesn’t tell us if the DNA came from dead cells or from live active cells. The correlation between starch agar plates and PCR for this reason is not perfect – one of our strains for example, tested positive for STA1 but did not grow on the plates. This means the STA1 gene was present but that the yeast did not secrete glucoamylase (see table below, Old World Saison 2/2). In the future we may find other genes and markers that are related to glucoamylase production and can give us more information about STA1 expression and yeast diastatic activity.
The plates by themselves can be an accessible, affordable alternative to PCR for detecting diastaticus contamination, though will certainly not replace the need for PCR entirely, since the long incubation time will not be suitable for those requiring more immediate results. This plate method can detect as low as 0.1% contamination (see photo below). One great advantage that these plates have is that it is specific for Saccharomyces cerevisiae var. diastaticus. Brettanomyces strains, despite also having the ability to break down dextrins and starches, will not grow or display colour change using this plate with anaerobic incubation - we tested this with a one-month incubation!
Colour change indicating presence of diastatic yeast in cell mixes with a range of diastaticus additions (indicated on the plates)
Another common medium that many breweries and labs are using to detect diastaticus is LCSM, or Lin’s Copper Sulfate Medium. This was specially formulated to detect non-Saccharomyces wild yeast, but several Saccharomyces cerevisiae strains, including the diastaticus variant, grow on this medium as well. This medium contains copper sulfate, which many wild yeasts are resistant to, enabling them to grow.
There is no actual genetic correlation between resistance to copper sulfate and ability to produce glucoamylase – it just so happens that the “wilder” Sacch that are resistant to copper sulfate often also happen to have diastatic abilities. However, there are examples of copper resistant yeasts which do not have diastatic activity.
The table below includes some examples of our strains which we tested using STA1 PCR, starch agar plates, and LCSM. It includes two strains (Trappist Ale and Scotia Sauvage) that, despite being able to grow on LCSM, did not test positive for STA1 nor showed growth on starch agar plates. Ultimately, being able to determine whether a strain will grow on starch or not is the most accurate way to determine diastatic ability on Sacch strains. Trials are ongoing in our lab and others to optimize a medium testing true diastatic activity, with a faster detection time.
This testing of STA1-positive and LCSM-positive strains in our main strain collection is summarized below:
We are continually improving our testing and knowledge base on diastaticus, and our ongoing research means we can provide the most up to date and accurate methods available for breweries interested in testing for diastaticus. Please contact us if you have any questions.