Diastaticus An Expos of Everyones Favorite Explosive Yeast Matt - - PowerPoint PPT Presentation

Demystifying Diastaticus

An Exposé of Everyone’s Favorite Explosive Yeast

Matt Linske

Manager & Lead Microbiologist

Brewing and Distilling Analytical Services, LLC BDAS Denver

What is Saccharomyces cerevisiae var. diastaticus?

• Saccharomyces = Sugar fungus
• cerevisiae = from beer
• diastaticus = separation (of starch)

• Andrews & Gilliland
• Describe a “novel

species” in 1952

• Later genomic

research (~1985) reclassified as

• S. cerevisiae variant

Variant: taxonomic designation below species and subspecies

When Yeast Goes Variant

Super Attenuation

• f Beer

Maltotriose Maltotetraose Dextrin Starch

STA1 STA2 STA3

Extracellular Glucoamlyase

Starch degradation It’s in their Genes Molecular Machines

What makes diastaticus different?

• Signal within cell

activates DNA (gene)

• DNA transcribed

into mRNA

• RNA converted to

protein by Ribosome

Information transfer within cells

The Central Dogma:

How it works:

STA Gene family is highly homologous

The Glucoamylase Multigene Family in Saccharomyces cerevisiae var. diastaticus: An overview. Pretorius et al. 2008.

DEX allelic to STA genes

Allelism within the DEX and STA gene families in Saccharomyces diastaticus. Erratt JA, Nasim A. 1986

Rapid Methods for Detecting Saccharomyces diastaticus, a Beer Spoilage Yeast, Using the Polymerase Chain Reaction

• H. Yamauchi, H. Yamamoto, Y. Shibano, N.

Amaya, and T. Saeki, JASBC 1998

Structural analysis of glucoamylase encoded by the STA1 gene of Saccharomyces cerevisiae (var. diastaticus)

Adams et al, Yeast 2004; 21: 379–388.

Enzymatic Action!

Brewing with diastaticus

1.000 1.005 1.010 1.015 1.020 1.025 1.030 1.035 1.040 1.045 1.050 1 2 3 4 5 6 7 8

Apparent Gravity vs Time

BS1 A18 WLP566 Voss Kveik

Strains:

• Brewing Science Institute

A-18 London Ale III (STA1-)

• White Labs WLP566

Belgian Saison II (STA1+)

• Voss Kveik (STA1-)

STA1 positive yeast took longest to reach terminal gravity, lower FG than STA1 negative yeast!

Not all diastaticus Fermentations are the same

Saccharomyces cerevisiae variety diastaticus friend or foe?—spoilage potential and brewing ability of different Saccharomyces cerevisiae variety diastaticus yeast isolates by genetic, phenotypic and physiological characterization. Tim Meier-Dörnberg et al, 2018

Differences in Fermentation Related to Different Gene Expression

A deletion in the STA1 promoter determines maltotriose and starch utilization in STA1+ Saccharomyces cerevisiae strains. Krogerus et al 2019

Differences in Fermentation Related to Different Gene Expression

A deletion in the STA1 promoter determines maltotriose and starch utilization in STA1+ Saccharomyces cerevisiae strains. Krogerus et al 2019

Diastaticus as a Contaminant

Locations, Effects, & Detection

What happens when diastaticus is present?

Over-attenuation

• Final gravity 1.004 or less
• Flavor imbalance
• Package failure
• Bottle bombs or Can shrapnel =

SAFTEY ISSUE!!!

Phenolic Off Flavor

• Not all strains POF+

Sediment/haze

Potential Impacts

Where does contamination occur?

• Brewhouse
• Pipework
• Pitching yeast
• Fermentation cellar
• Packaging lines

Incidence of Saccharomyces cerevisiae

• var. diastaticus in the Beverage Industry:

Cases of Contamination, 2008–2017 Tim Meier-Dörnberg et al 2017

Low Cell numbers can still cause problems! Best practice: Zero Tolerance

The Viability of Minimal Numbers of Saccharomyces diastaticus in Beer, Robert P. Greenspan (1966)

What is the level of risk?

Total Plate Counts

Polymerase Chain Reaction

Methods of Detection

0.0% 20.0% 40.0% 60.0% 80.0% 100.0% 120.0% 140.0%

% RECOVERY NBB VS. YPD + CuSO4

250 PPM 300 PPM 350 PPM

168.49% 155.47% 140.85% 128.43% 115.49% 114.57% 104.41% 102.39% 102.38% 100.00% 100.00% 98.13% 97.24% 96.88% 96.15% 95.08% 93.53% 86.49% 59.39% 53.01% 42.14% 38.59% 32.48% 29.90% 11.38% 8.45% 5.67% 4.46% 0.59% 0.00% 0.00% 0.00% 59.42% 141.82% 116.67% 0.00% 81.73% 117.14% 100.00% 109.62% 131.43% 151.11% 0.00% 103.13% 13.89% 26.53% 52.17% 30.83% 83.33% 116.00% 50.00% 78.05% 103.70% 0.00% 5.71% 103.03% 58.70% 62.95% 0.00% 48.00% 82.00% 0.00% 0.00% 0.00%

LCSM: 2018 VS 2019

% Recovery 2019 LCSM vs. NBB % Recovery 2018 LCSM vs. NBB

0.00% 20.00% 40.00% 60.00% 80.00% 100.00% 120.00% 140.00% 160.00%

24 48 72 96

IOYB64, % RECOVERY VS NBB

FPDM-U FPDM-W LCSM-B LCSM-W

Optimizing LCSM For Diastaticus

0.00% 20.00% 40.00% 60.00% 80.00% 100.00% 120.00% 100 200 300 400 500 600

% Recovery CuSO4, ppm

WLP 565, % Recovery on LCSM

Yeast #4: 0.5g (NH4)2-SO4 Yeast #4: 0.75g (NH4)2-SO4 0.00% 20.00% 40.00% 60.00% 80.00% 100.00% 120.00% 100 200 300 400 500 600

% Recovery CuSO4, ppm

STA1+ #20D-0206, % Recovery on LCSM

Yeast #206: 0.5g (NH4)2-SO4 Yeast #206: 0.75g (NH4)2-SO4

• Enzoklop - CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=32003643

A Primer

• n

Primers

• Short, single-stranded nucleic acid utilized in

the initiation of DNA synthesis

• Both Forward and Reverse Needed to Amplify

Gene of interest

• By Zephyris - CC BY-SA 3.0,

https://commons.wikimedia.org/w/index.php?curid=26794032

Real Time PCR

• DNA amplification detected by

fluorescent dyes

• Can be visualized in Real Time!

End Point Detection

Thank you!

• Team BDAS
• Chuck Skypeck and BA Staff
• Justin Levaugn
• Jess Caudill
• David Bryant
• Lance Shaner & Laura Burns
• Karen Fortmann & Kara

Taylor

• Spencer Weeks
• Tom Boudreau & Dr.

Matthew J. Farber