a new pcr-based method for the early detection and identification of spoilage yeast...

Better Lives. Better Planet.SM

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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


11


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)


S. pastorianus 1-10 Presence Presence

Wheat Beers

(unfilterable)


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


S. c. diastaticus1-10

Saccharomyces spp. / S. cerevisiae /

S. c. diastaticusPresence

B. bruxellensis1-10 Absence Absence

10-100 B. bruxellensis Presence

Mixed

Beers


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


S. pastorianus &

S. japonicus1-10 Presence Absence

C. stellata &

B. bruxellensis

1-10 Presence Absence

10-100 Presence Presence

Mixed Beers


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


Enrichment from the GeneDisc method was confirmed as positive



result

Culture

method CFU/sample

Lager Beers


S. pastorianus &


C. stellata &

B. bruxellensis



Mixed Beers


S. eubayanus &


S. ludwigii &


Mixed Wheat

Beers


B. custersianus &


19



result

Culture

method CFU/sample

Lager Beers


S. pastorianus &


C. stellata &

B. bruxellensis



Mixed Beers


S. eubayanus &


S. ludwigii &


Mixed Wheat

Beers


B. custersianus &



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

remains valid.

© Copyright 2016, Pall Corporation. Pall, , and GeneDisc are trademarks of Pall

Corporation.

® Indicates a trademark registered in the USA.

Better Lives. Better Planet. is a service mark of Pall Corporation.

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a new pcr-based method for the early detection and identification of spoilage yeast...

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