Demystifying Diastaticus (Part 1)

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. The gene encoding this glucoamylase is called STA1. 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.

Diastatic yeast can be beneficial to the brewing process to achieve low final gravities and in producing beers with dry finishes. These characteristics are possible because diastatic yeast is capable of producing glucoamylase, an enzyme that breaks down complex carbohydrates into fermentable sugars to continue the fermentation process. However, this same desirable trait can pose an immense threat to the integrity of a beer if the presence of diastatic yeast is unintentional as it can cause continued fermentation in “finished” products. This continued fermentation results in increased alcohol content, flavour changes, and pressure build up inside packaged products. By screening samples for diastatic yeast using PCR, these unwanted characteristics can be greatly reduced and even mitigated.

PCR is a powerful molecular tool capable of amplifying and detecting the presence of specific regions of DNA. DNA can be thought of as the set of instructions or code found inside of every cell that is required for the organism to live, develop and reproduce. This set of instructions is then read, or translated, in the cell to produce proteins that then carry out specific functions within the organism. Each organism has a different genetic code, or different sets of instructions, and this is true across different strains of yeast. By using molecular techniques such as PCR, testing for genetic differences in DNA between yeast species is possible and this method can be used to determine if a yeast strain is diastatic or not.

Totally accurate depiction of DNA being amplified via PCR

There are a few different PCR systems available on the market, including traditional PCR, qPCR, and RT-PCR, each of which has their own advantages, limitations and capabilities. The fundamental basis of all PCR systems involves specific primers designed to bind a target region of DNA, read and translate the DNA, and create multiple copies of that target region. When the primers attach to the DNA and move along it, nucleotides (or building blocks) are added in the specific and correct order until the DNA sequence is made. Now, this all may sound a bit technical, but it helps to think of the process as assembling a Lego pug. When building a Lego pug, you pick up the instructions (DNA) with your hands (primers) and follow the instructions line by line (primers moving along the DNA). As you follow the instructions, specific Lego pieces are added in the correct sequence (nucleotides) until the pug is completed (DNA copy). This process then repeats multiple times, until you are out of Lego pieces (and energy) and have a small grumble of Lego pugs.

PCR assembles fragments of DNA from individual building blocks

As mentioned, there are a few different PCR systems out there that can be used to screen for STA1. To screen for diastatic yeast, primers designed to only bind the STA1 gene (which encodes the glucoamylase enzyme that digests starch molecules) will amplify the DNA and produce many more copies which can more easily be detected. At Escarpment Labs, we use traditional PCR methods in our routine screening and microbial analyses. This screen is cost effective and is efficient in detecting low level contaminations, which is important to us since a diastatic contamination at any level is too much of a risk. While other systems can provide more information on the samples, show you the PCR reaction in real time, or tell you the specific level of contamination, we choose to use the traditional method for its simplicity and effectiveness. Also below, at the end of this blog post, is our PCR protocol for detecting STA1. 

Although PCR is a powerful tool, it does have its limitations as it is only able to amplify specific DNA regions and does not test for protein functionality. This means that a sample containing dead, non-viable diastatic yeast or yeast that contains non-functional versions of the STA1 gene will test positive in a PCR test, while exerting no net effect on the integrity of the sample. The reason for this is still unclear. Early research identified important components of the genetic code for STA1, such as the domain required for its export from the cell. Slight genetic variations in STA1 could radically alter its function. The public availability of many beer yeast whole genomes may help understand diastatic yeasts in greater detail moving forward. 

When screening samples for active diastatic yeast, PCR is a great place to start to determine if there is a potential contamination in the sample and whether or not further testing is required. If the sample is suspected to be contaminated with diastatic yeast, the use of agar media such as starch agar or LCSM would be beneficial. This secondary test will be able to determine if the contaminant poses a threat to the beer, or if the detected STA1 contaminant is one of the non-functional variants.

As promised above, here is our PCR protocol for detecting STA1. One of our core initiatives at Escarpment Labs is giving back to the brewing community. Stay tuned for Part 2 of this blog to learn more about agar testing for diastaticus!

Materials: 

We have found that MiniPCR is a good source for cost-effective tools and reagants for use in these protocols.

Colony PCR DNA Extraction

1. Place a medium sized yeast colony (can also use small amount of yeast slurry) into 200µL BioRad Instagene Matrix (7326030) in a sterile centrifuge tube.

2. Vortex and let sit at 56°C for 30 minutes.

3. Vortex and boil for 9 minutes, centrifuge at 13000rpm for 3 mins and keep the supernatant.

4. Place 10µL into 15µL of Mini PCR EZ PCR Master Mix (see below).

Colony PCR Setup

1. For each PCR sample to be tested, collect 9µL sterile distilled water (sdH2O), 5µL of Master Mix (which contains salts, nucleotides, dye, DNA polymerase and other PCR essentials), and 0.5µL of each STA1 primer.

   1. Primer 1 (STA1 SD-6B): 5’-GAT-GGT-GAC-GCA-ATC-ACG-A-3’

   2. Primer 2 (STA1 SD-5A): 5’-CAA-CTA-CGA-CTT-CTG-TCA-TA-3’

2. Aliquot 15µL of the above Mix into a PCR tube, repeat for each sample to be tested.

3. Add 10µL of the DNA extracted above to the 15µL of Mix, and place into the PCR thermocycler.

4. Run the following protocol on the PCR thermocycler:

   1. Initial denaturing at 94°C for 15 minutes;

   2. 30 cycles of denaturing at 94°C for 1 minute, annealing at 49°C for 2 minutes, and extension at 72°C for 2 minutes.

   3. Final extension step at 72°C for 10 minutes, then hold samples at 4°C.

Detection

1. Gel electrophoresis and UV detection, using the Mini PCR Gel Electrophoresis system. 

Note from Richard Preiss: We have transitioned to using an Invisible Sentinel PCR system in our lab for beer and slurry samples due to the lower processing time. This is worth it if you're running more than a dozen samples per week, make sure to use relevant controls every time, and have realistic expectations of what PCR can do for you!

Learn more in Part 2.