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Preventing and Controlling Diastaticus Yeast
- 1. Preventing and Controlling Diastatic Yeast Richard Preiss, Escarpment Laboratories Ontario Craft Brewers Conference Co-presented by the MBAA District Ontario October 29, 2019
- 2. Our focus: harnessing the science of microbial cultures Escarpment Labs Team – Feb 2019 Who am I? • MSc. Molecular Biology & Genetics, University of Guelph • Cofounder Escarpment Laboratories, 2015-present Who is Escarpment Labs? • liquid beer yeast and quality/consulting services • focus on quality, knowledge and supporting the beer community Guelph, Ontario, Canada
- 3. UP-FRONT ACKNOWLEDGEMENTS – Alex Mitro and Isabelle Netto (method development, yeast screening and research) – Shelby Stein, Jill Yuen (PCR characterization) – Nate Ferguson (prior slide decks)
- 4. WHO HAS SEEN THIS?
- 5. POTENTIAL CAUSES OF BURSTING • Physical failure of can seams • Beer packaged before terminal gravity with yeast present • Beer packaged with added sugar (fruit juice or purée) and with yeast present • Hops-derived amylase causing refermentation (“hop creep”) • Beer packaged at stable terminal gravity but with a S. cerevisiae var. diastaticus contamination
- 6. GENERAL PROACTIVE SUGGESTIONS • Sterile filter your beer • Pasteurize your beer • Implement micro QC program
- 7. DIASTATICUS IN THE NEWS
- 8. WHY IS IT A PROBLEM NOW? • Higher usage of diastatic Saison yeasts in breweries • Rapid growth of breweries may leave QC and SOPs catching up • Canning/packaging lines that cannot be heat-sterilized
- 9. OVERVIEW • Diastatic yeast has emerged as a high-risk contaminant in craft beer • These yeasts can cause significant losses and safety risk due to their ability to ferment sugars and starches beyond the capabilities of normal brewing yeasts. • Can be wild yeasts or commercial Saison yeasts • Detected with agar plating and/or PCR
- 10. KEY QUESTIONS • What is diastatic yeast? • Are some diastaticus yeasts higher risk than others? • How can I detect it? • How can I prevent it? • What do I do if I have it? • What are yeast suppliers doing about this problem?
- 11. WHAT IS DIASTATICUS? • Saccharomyces cerevisiae var. diastaticus • Historically thought to be a wild yeast • It’s regular yeast, with a nifty new glucoamylase (produces glucose from starch) SGA1 + FLO11 = STA1 Glucoamylase Inside the cell Cell surface glycoprotein/flocculin Breaks down carbs Inside the cell Helps with sporulation What is it? What does it do? Gene name Helps cells flocculate Glucoamylase Outside the cell Breaks down carbs Outside the cell
- 12. WHAT IS DIASTATICUS? SGA1 + FLO11 = STA1 Glucoamylase Inside the cell Cell surface glycoprotein/flocculin Breaks down carbs Inside the cell Helps with sporulation What is it? What does it do? Gene name Helps cells flocculate Glucoamylase Outside the cell Breaks down carbs Outside the cell SGA1FLO11- STA1-negative yeast Yeast cell
- 13. WHAT IS DIASTATICUS? SGA1 + FLO11 = STA1 Glucoamylase Inside the cell Cell surface glycoprotein/flocculin Breaks down carbs Inside the cell Helps with sporulation What is it? What does it do? Gene name Helps cells flocculate Glucoamylase Outside the cell Breaks down carbs Outside the cell SGA1FLO11- STA1-positive yeast Yeast cell - STA1
- 14. WHERE DID DIASTATICUS COME FROM? • Modern DNA sequencing reveals it is not so wild • Mostly part of the “beer 2” family which includes Saison yeasts Krogerus, K., Magalhães, F., Kuivanen, J., and Gibson, B. (2019). A deletion in the STA1 promoter determines maltotriose and starch utilization in STA1+ Saccharomyces cerevisiae strains. Appl. Microbiol. Biotechnol.
- 15. VISUAL DIFFERENCES DO NOT EXIST STA1 negative STA1 positive
- 16. HOW DOES PCR WORK? • Polymerase Chain Reaction ”PCR” • Gene amplification • Is the STA1 gene present?
- 17. WHAT DO PCR RESULTS LOOK LIKE? • In this example, samples 2 and 4-7 are STA1 positive • This only tells us the gene is present Example – yeast DNA is run on a gel to separate it (1. Is a size marker) • Commercially available solutions work this way too • Sensitivity to ~1:10^8 (1 in 100 million)
- 18. SOME STRAINS DEGRADE STARCH FASTER • Which STA1+ strains consume starch? • Agar plates containing starch and a pH indicator predict diastatic activity • Takes ~3 weeks for incubation Meier-Dörnberg, T., Kory, O. I., Jacob, F., Michel, M., and Hutzler, M. (2018). Saccharomyces cerevisiae variety diastaticus friend or foe?.
- 19. SENSITIVITY OF STARCH AGAR • Sensitive to 1:1000 (low) Meier-Dörnberg, T., Kory, O. I., Jacob, F., Michel, M., and Hutzler, M. (2018). Saccharomyces cerevisiae variety diastaticus friend or foe?.
- 20. LCSM AGAR IS A USEFUL QC TOOL FOR DIASTATICUS • Lin’s Cupric Sulfate Medium • Used to identify wild yeasts (resistant to copper) • So are all the STA1 positive yeasts we have tested Meier-Dörnberg, T., Kory, O. I., Jacob, F., Michel, M., and Hutzler, M. (2018). Saccharomyces cerevisiae variety diastaticus friend or foe?. LCSM (copper sulfate) WLN (general purpose) • Risk of false positives • Some Belgian strains are STA1 negative but grow on LCSM • Growth in 3 days • Sensitivity to ~1:10^8 (1 in 100 million) + -
- 21. VARIATION AMONG DIASTATIC YEASTS
- 22. Kristoffer Krogerus VTT, Finland WHY ARE SOME DIASTATICUS MORE AGGRESSIVE? August 2018: Escarpment Labs starch plate results sent to VTT
- 23. WHY ARE SOME DIASTATICUS MORE AGGRESSIVE? STA1 gene Upstream activation sequence weak deletion strong What they found: There is a deletion in the promoter region of the weak strains Yeast genome
- 24. Strains with deletion grow much slower in beer This is due to lower gene expression WHY ARE SOME DIASTATICUS MORE AGGRESSIVE?
- 25. NEW QC TOOL: STA1 ACTIVATION SEQUENCE PCR primers developed to detect upstream activation sequence
- 26. NEW QC TOOL: STA1 ACTIVATION SEQUENCE • Samples 1 and 9 are STA1 negative • Samples 5 and 8 contain the activation sequence • Other samples are likely to be weakly diastatic
- 27. NEW QC TOOL: STA1 ACTIVATION SEQUENCE • Multiplexing – two in one PCR • This can be used in QC analysis to determine risk • Takes <1 day (vs. starch plates)
- 28. UAS + ACTIVATION SEQUENCE CORRESPONDS TO STARCH CONSUMPTION NOTE: UAS- strains can still consume starch/dextrins, just slower.
- 29. SUGGESTED DIASTATICUS MONITORING PROGRAM LCSM agar plating (0.1mL yeast slurry or 1 mL beer) UAS+ High risk UAS- Lower risk Clear Good work! Monitor product for continued fermentation Total time for monitoring 8 FVs or packaging runs: ~8 hours per week colony PCR Results in 2-4 days
- 31. BARRIERS • “We’re too busy to run a lab” • “We’re too small to run a lab” • “We don’t have the money to buy lab equipment” • “We tried _insert system here_ and it didn’t work” • “We’ve never had an infection” • “Our SOPs are rock-solid”
- 32. These are excuses. Not barriers.
- 33. COST OF IMPLEMENTATION • Agar plates can be purchased pre- poured ($1.50-3 each) • Bunsen Burner $100 • Pipettes $150 • PCR Thermocycler $650 and up • Gel system $350 • Startup reagents and misc. tools $250 Plate pouring automation @Escarpment Labs
- 34. You can set up a lab that can properly screen for diastaticus for less than $2000 in equipment and less than 8 hours of work per week.
- 35. HOW CAN I PREVENT DIASTATICUS? • Ask your supplier how they prevent diastaticus • Run QC on yeast and beer • Use a proactive approach to manage risk
- 36. Dedicated tanks/lines are ideal A PROACTIVE RISK MANAGEMENT APPROACH
- 37. QUALITY CONTROL IN THE YEAST LAB * Colony PCR as needed * * * Not released until triple all-clear
- 38. HOW CAN I MAKE SAFE SAISONS? Problem • Saisons are a popular beer style, especially in summer • Nearly all commercial Saison strains are STA1+ • STA1 is beneficial for Saisons, to help achieve dryness Solution • Screen of yeast bank Saison-origin strains for STA1- • Found one from a bottle of Belgian Saison • Average attenuation • Requires high nutrients and highly fermentable wort • Can dose enzyme (amyloglucosidase) for dryness
- 39. • Not all diastaticus are the same • It’s important to know which is in your brewery • Limit use of diastaticus in packaging breweries • You can detect it for a surprisingly low price TAKE HOME FOR THE BOSS