a new pcr-based method for the early detection and identification of spoilage yeast...
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Better Lives. Better Planet.SM
This presentation is the Confidential work product of Pall Corporation and no portion
of this presentation may be copied, published, performed, or redistributed without
the express written authority of a Pall corporate officer
© 2016 Pall Corporation
A New PCR-based Method
for the Early Detection and
Identification of Spoilage Yeast
in Beer, Wine and Alcohol Free
Beverages
Institute of Brewing and Distilling
Asia Pacific Section
March 17th, 2016
Vincent Ulvé
2
Yeast: a Double Microorganism?
with a complex family affiliation.
Need to be characterized and controlled
L. Salichos et al. Nature 497, 327–331 (16 May 2013)
Yeasts can do good and evil…
3
Traditional Method for Yeast Monitoring
Slurry / tanksamples
Unfilterable products
Filterable products
Filtration
Platingselective / not selective conditions
No filtration
Action Low riskPresence Absence
No universal method
Time to results: 3 – 7 days
4
The Pall GeneDisc® System at a glace
– Step 3 - Run the Real-Time PCR test
– Step 1 - extract DNA
– Step 2 - fill the GeneDisc Plate
Real time PCR based system for the detection of bacteria and
yeast in only three simple steps from sample to result:
5
Pall GeneDisc Method for Yeast: Flexible Workflow
Slurry / tank& Colony
Action Low risk
Unfilterable products
Filterable products
EnrichmentSample filtration
Yeast lysis
Enrichment filtration
Yeast IdentificationYeast Screening
Presence Absence
6
Sector• One sector receives one extracted sample
• A 6 sector GeneDisc Plate allows for 6
different samples to be tested
Well• Each sector is connected to 6 peripheral wells
by microchannels
• Each well contains all necessary reagents (primers and probes) for a specific PCR reaction
Barcode• Traceability of the product
• Recognized by the GeneDisc Cycler for automatic setup of the correct PCR program and results interpretation
The GeneDisc Plate
The Pall GeneDisc Plate
7
Pall GeneDisc Plate for Yeast
Well FAM detection
1 Inhibition control
2 Yeast Screening
Yeast Screening GeneDisc plates allowing the detection of spoilage yeast
Yeast ID GeneDisc plates allowing the identification of the main yeast spoilers
Well FAM detection ROX detection
1 Inhibition control Saccharomyces cerevisae
diastaticus
2 Saccharomyces cerevisiae Saccharomyces spp.
3 Brettanomyces bruxellensis Saccharomycodes spp.
4 Brettanomyces spp. Saccharomyces pastorianus
5 Schizosaccharomyces spp. Zygosaccharomyces spp.
6 Candida-Pichia spp. Zygosaccharomyces bailii
8
The Pall GeneDisc System: From Sample to Result
The GeneDisc Plate is positioned in
the GeneDisc Cycler
One run is completed in an hour
9
GeneDisc Plate for Yeast Results
10
GeneDisc Plate for Yeast Results
11
GeneDisc Plate for Yeast Results
12
A Validation Study on Beer Samples
Validation of the performances of the GeneDisc method
vs. traditional method
13
A Validation Study on Beer Samples
Inoculation of yeast at low level in:
Lager, wheat and mixed beers Slurry yeast
Spiked with:Brettanomyces bruxellensis, Brettanomyces custersianus, Candida stellata,
Saccharomycodes ludwigii, S. cerevisae, S. cerevisae diastaticus, S. eubayanus,
S.pastorianus, Schizosaccharomyces japonicus, Pichia fermentans, Pichia
membranifaciens, Zygosaccharomyces bisporus
14
Yeast Screening on Beer Samples
Results of GeneDisc Yeast Screening and culture methods for artificially spiked beer samples with one yeast spoiler.
Shorter time to result
Sample Spiked with GeneDisc result
(28 h)
Culture Method
(up to 3 days)CFU/sample
Lager Beers
(filterable)
Control 0 Absence Absence
B. bruxellensis 1-10 Presence Presence
S. c. diastaticus 10-100 Presence Presence
Mixed Beers
(filterable)
Control 0 Absence Absence
S. pastorianus 1-10 Presence Presence
Wheat Beers
(unfilterable)
Control 0 Absence Absence
S. c. diastaticus 1-10 Presence Presence
B. bruxellensis 1-10 Presence Presence
15
Yeast Identification on Beer Samples
Results of GeneDisc Yeast ID and culture methods for artificially spiked beer samples with one spoilage yeast.
Sample
Spiked with
GeneDisc result Culture method
CFU/sample
Lager
Beers
Control 0 Absence Absence
S. c. diastaticus1-10
Saccharomyces spp. / S. cerevisiae /
S. c. diastaticusPresence
B. bruxellensis1-10 Absence Absence
10-100 B. bruxellensis Presence
Mixed
Beers
Control 0 Absence Absence
S. pastorianus 1-10 Saccharomyces spp. / S. pastorianus Presence
Wheat
White
Beers
Control 0 Saccharomyces spp., / S. cerevisiae Presence
S. c. diastaticus1-10
Saccharomyces spp., S. cerevisiae and
S. c. diastaticusPresence
B. bruxellensis 1-10Saccharomyces spp., / S. cerevisiae,
Brettanomyces spp. / B. bruxellensisPresence
Enable identification of the spoiler yeast
16
Yeast Identification on Yeast Slurry sample
Results of the GeneDisc Yeast ID method for spoilage yeast detection/ identification in an artificially spiked yeast propagation sample containing 109 CFU/ml.
Yeast StrainSpiking dose
(CFU/mL)GeneDisc Method result
Brettanomyces bruxellensis 2.5 102Presence of Brettanomyces spp. / B.
bruxellensis
Pichia membranifaciens 2.5 103 Presence of Candida-Pichia spp.
Saccharomycodes ludwigii 2.1 103 Presence of Saccharomycodes spp.
S. cerevisiae diastaticus 4.0 103 Presence of S. cerevisiae diastaticus
Schizosaccharomyces
japonicus8.6 102 Presence of Schizosaccharomyces spp.
S. pastorianus 2.1 102 Presence of S. pastorianus
Time to result < 2 h
17
Yeast Screening on Co-spiked Beer Samples
Results of GeneDisc Yeast Screening and culture methods for artificially co-spiked beer samples with two spoilage yeasts.
SampleSpiked with GeneDisc Method
result
Culture
method CFU/sample
Lager Beers
Control 0 Absence Absence
S. pastorianus &
S. japonicus1-10 Presence Absence
C. stellata &
B. bruxellensis
1-10 Presence Absence
10-100 Presence Presence
Mixed Beers
Control 0 Absence Absence
S. eubayanus &
P. fermentans1-10 Presence Absence
S. ludwigii &
S. cerevisiae1-10 Presence Presence
Mixed Wheat
Beers
Control 0 Presence Absence
B. custersianus &
Z. bisporus1-10 Presence Absence
18
Yeast Screening on Co-spiked Beer Samples
Enrichment from the GeneDisc method was confirmed as positive
Results of GeneDisc Yeast Screening and culture methods for artificially co-spiked beer samples with two spoilage yeasts.
SampleSpiked with GeneDisc Method
result
Culture
method CFU/sample
Lager Beers
Control 0 Absence Absence
S. pastorianus &
S. japonicus1-10 Presence Absence
C. stellata &
B. bruxellensis
1-10 Presence Absence
10-100 Presence Presence
Mixed Beers
Control 0 Absence Absence
S. eubayanus &
P. fermentans1-10 Presence Absence
S. ludwigii &
S. cerevisiae1-10 Presence Presence
Mixed Wheat
Beers
Control 0 Presence Absence
B. custersianus &
Z. bisporus1-10 Presence Absence
19
Yeast Screening on Co-spiked Beer Samples
SampleSpiked with GeneDisc Method
result
Culture
method CFU/sample
Lager Beers
Control 0 Absence Absence
S. pastorianus &
S. japonicus1-10 Presence Absence
C. stellata &
B. bruxellensis
1-10 Presence Absence
10-100 Presence Presence
Mixed Beers
Control 0 Absence Absence
S. eubayanus &
P. fermentans1-10 Presence Absence
S. ludwigii &
S. cerevisiae1-10 Presence Presence
Mixed Wheat
Beers
Control 0 Presence Absence
B. custersianus &
Z. bisporus1-10 Presence Absence
Results of GeneDisc Yeast Screening and culture methods for artificially co-spiked beer samples with two spoilage yeasts.
High sensitivity
20
Yeast Identification on co-spiked Beer Samples
Results of the GeneDisc Yeast ID and culture methods for artificially co-spiked beer samples with two spoilage yeasts.
MatrixSpiked with GeneDisc result at Additional result at
CFU/sample 28 h 72 h
Lager Beers
Control 0 Absence -
B. bruxellensis &
C. stellata
1-10 Candida-Pichia spp.B. spp. and B.
bruxellensis
10-100 Candida-Pichia spp.B. spp. and B.
bruxellensis
S. pastorianus &
S. japonicus1-10
S. spp, / S. pastorianus and
Schizosaccharomyces spp. -
Mixed Beers
Control 0 Absence -
S. eubayanus &
P. fermentans1-10 S. pastorianus
S. spp and
Candida-Pichia spp.
S. ludwigii &
S. Cerevisiae1-10
S. spp, / S. cerevisiae and
Saccharomycodes spp. -
Mixed Wheat
Beers
Control 0 Absence -
B. custersianus &
Z. bisporus1-10 Absence Z. spp
Sensitivity related to the enrichment time
21
Sensitivity management
Time to results obtained by both methods according to the spiked
yeast strain.
GeneDisc Method
28 h 72 h Not detected
Culture
Method
2 days
S. c. diastaticus
B. bruxellensis (high level)
S. cerevisiae (co-spiking)
S. ludwigii (co-spiking)
3 days
S. c. diastaticus
S. pastorianus
S. pastorianus (high level, co-spiking)
S. eubayanus (co-spiking)
C. stellata (high level, co-spiking))
B. bruxellensis (low level)
S. japonicus (high level, co-spiking)
Not
detect
ed
C. stellata (low level, co-spiking)
S. pastorianus (low level, co-spiking)
Pichia fermentans (co-spiking)
B. bruxellensis (co-spiking)
Z. bisporus (low level, co-spiking)
S. japonicus (low level, co-spiking)
B. bruxellensis (low level)
B. custersianus (co-spiking)
22
Conclusions
Flexible and rapid method
– Time to result down to 2 h
– Dedicated to each sample types without PCR inhibition
– Assessment of the presence of yeast
– Simultaneous identification of the 12 major spoilage yeast
Highly sensitive method, able to detect 1-10 CFU/ sample
23
Conclusions – Application to Breweries
Accelerated decision-making
Early preventive controls (e.g. yeast slurry control) to reduce risk of product spoilage and speed up batch release
Fast corrective actions implementation
Reduce negative financial impact of spoilage with rapid root cause analysis
Tailor-made informative method
Two GeneDisc options for various testing strategies
Reduced total test cost
Ease of use simplifies testing workflows and on-site implementation.
24
The Pall GeneDisc Project Team
Aurore Besson,
Carine Tessier,
Christelle Nahuet,
Danielle Wedral,
Gaëlle Leborgne,
Guillaume Piquet,
Helène Beaupied,
Marilyne Rummelhard,
Nathalie Kerriguy,
Nigel Cook,
Richard Revel,
Sarah Jemmal,
Sébastien Bouton,
Sirine Assaf,
Sylvie Hallier Soulier
Better Lives. Better Planet.SM
This presentation is the Confidential work product of Pall Corporation and no portion
of this presentation may be copied, published, performed, or redistributed without
the express written authority of a Pall corporate officer
© 2016 Pall Corporation
Pall Corporation has offices and plants throughout the world. For Pall representatives in
your area, please go to www.pall.com/contact
Please contact Pall Corporation to verify that the product conforms to your
national legislation and/or regional regulatory requirements for water and
food contact use.
Because of technological developments related to the products, systems, and/or services
described herein, the data and procedures are subject to change without
notice.
Please consult your Pall representative or visit www.pall.com to verify that this information
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© Copyright 2016, Pall Corporation. Pall, , and GeneDisc are trademarks of Pall
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