Rapid Profiling of Beer Spoilage Micro-organisms and Yeast Health Dr Jon Meneses Brewing & Supply Chain Manager Coopers Brewery Limited Asia Pacific Convention 2018 | Wellington, New Zealand
Rapid Profiling of Beer Spoilage
Micro-organisms and Yeast Health
Dr Jon Meneses
Brewing & Supply Chain Manager
Coopers Brewery Limited
Asia Pacific Convention 2018 | Wellington, New Zealand
Non-Saccharomyces Brettanomyces spp.
Pichia spp.
Candida spp.
Rhodotorula spp.
Saccharomyces S. cerevisiae
S. diastaticus
S. bayanus
S. pastorianus
Gram Positive Lactobacillus spp.
Pediococcus spp.
Bacillus spp.
Micrococcus spp.
Gram Negative Acetobacter spp.
Zymomonas spp.
Pectinatus spp.
Megasphaera spp.
IMPACTS • Off Flavours
• Haziness
• Stuck Fermentation
• Over attenuation
• Damaged reputation
• Financial loss
Beer Spoilage Micro-organisms
0 500 1000 1500 2000 2500 3000
0
2000
4000
6000 Inte
ns.
[a.u
.]
320 330 340 350 360 370 380 390 400 410 420 m/z
Identification Of Micro-organisms
Selective Agar Plates
Real Time PCR MALDI-TOF MS
Light Microscope
0 500 1000 1500 2000 2500 3000
0
2000
4000
6000 Inte
ns.
[a.u
.]
320 330 340 350 360 370 380 390 400 410 420 m/z
ADVANTAGES • Characterise proteins,
lipids and other
organic macro
molecules.
• High throughput
• Cost effective Vs PCR
• Accurate and rapid ID
DIS-ADVANTAGES • Cost of instrument
• Requires pre-
enrichment
• Suited to pure
cultures
MALDI-TOF Mass Spectroscopy (Matrix Assisted Laser Desorption Ionisation Time Of Flight)
How MALDI-TOF Analysis Work (Video Show)
Asia Pacific Convention 2018 | Wellington, New Zealand
Prepare cell
culture
1
2x water wash
1x ethanol wash
3
Dried-droplet onto
MALDI target
5
Biotyper acquisition 1 biological replicate,
8 technical replicates
Acquire 3X (24 Spectra)
6
Extract with 70%
formic acid
4
Harvest 150,000
cells
2
7
Automated identification Review and Validation
(5,643 MSP in library)
Agar plate
Protein Extraction and MALDI-TOF Analysis
Asia Pacific Convention 2018 | Wellington, New Zealand
Mass Spectra Profile (MSP) Creation For Database
STRAIN
Dekkera/Brettanomyces bruxellensis
Dekkera/Brettanomyces bruxellensis
Weihenstephan Weizen
Kölsch
Munich Lager
Czech Pils
Lactobacillus brevis
Lactobacillus lindneri
Pediococcus damnosus
Megasphaera cerevisiae
Pectinatus frisingensis
Wild yeast isolate from Coopers Brewery Ltd.
Top-fermenting brewing yeasts,
commercially sourced
Bottom-fermenting brewing yeasts,
commercially sourced
Wild yeast isolate, commercially sourced
Facultative anaerobic bacteria,
commercially sourced
Obligate anaerobic bacteria,
commercially sourced
(20 biological replicates of each strain to generate reference spectrum)
MALDI-TOF Mass Spectra Profile Of Samples
Identification From Spiked Beer Samples
Prepare cell
culture
1
2x water wash
1x ethanol wash
3
Dried-droplet onto
MALDI target
5
Biotyper acquisition 1 biological replicate,
8 technical replicates
Acquire 3X (24 Spectra)
6
Extract with 70%
formic acid
4
Harvest 150,000
cells
2
7
Automated identification (5,643 MSP + 16 Generated MSP)
Agar plate
Bacteria or Yeast
Asia Pacific Convention 2018 | Wellington, New Zealand
Strain MSP Score Against Database (5,643) & Internal Database (16)
Range Interpretation Colour
2.00 - 3.00 High Confidence Identification
Genus and species Green
1.70 - 1.99 Low Confidence Identification
Species Yellow
< 1.70 No Organism Identification Possible Red
STRAIN
Dekkera/Brettanomyces bruxellensis
Weihenstephan Weizen
Kölsch
Munich Lager
Czech Pils
Lactobacillus brevis
Lactobacillus lindneri
Pediococcus damnosus
Megasphaera cerevisiae
Pectinatus frisingensis
SCORE
2.195 / 2.179 / 2.190 / 2.120
2.080 / 2.243 / 2.132 / 2.120
2.463 / 2.386 / 2.492 / 2.471
2.369 / 2.422 / 2.377 / Czech Pils (N = 1)
detected as Munich Lager (N = 4)
2.318 / 2.096 / 2.359 / 2.044
2.400 / 2.252 / 2.209 / 2.298
2.248 / 2.309 / 2.332 / 2.273
2.377 / 2.439 / 2.484 / 2.512
2.573 / 2.589 / 2.525 / 2.450
Accuracy
4/4
4/4
4/4
3/4
0/4
4/4
4/4
4/4
4/4
4/4
Brewery Vs Commercial Dekkera/Brettanomyces Wild Yeast MSP
Asia Pacific Convention 2018 | Wellington, New Zealand
Identification of Brewery Isolates
Analysed 29 agar-plates from routine
microbiological plating of samples from
brewery and malt extract plant.
50 distinctive colonies were selected and
analysed by MS profiling.
Five colonies produced distinctive spectra, but
could not be identified (score below threshold)
Analysis was rapid taking only 1/2 day to
complete.
90% (45 colonies) could be identified using
Biotyper of which 25 microorganisms were
unique.
Asia Pacific Convention 2018 | Wellington, New Zealand
Identification Of Unknown Contaminants By LC-MS/MS Sample
Plate 4
Plate 5
Plate 11
Plate 12
Plate 13
Colony Morphology
Small green colonies
Green viscous growth
Green viscous growth
Green viscous growth
Green viscous growth
Microorganism
Identified as Acidomonas methanolica
Several microorganisms identified
Several microorganisms identified
Several microorganisms identified
Several microorganisms identified
Plate 5 – green viscous growth Plate 11 – green viscous growth
Plate 12 – green viscous growth Plate 13 – green viscous growth
Isolates
Lager brewing yeast
Brettanomyces bruxellensis
Burkholderia cepacia
Burkholderia vietnamiensis
Candida guilliermondii
Candida inconspicua
Candida krusei
Candida pelliculosa
Enterococcus gilvus
Exophiala dermatitidus
Lactobacillus brevis
Lactobacillus paracasei
Lactococcus lactis
Pandoraea apista
Pichia manshurica
Staphylococcus capitis
Staphylococcus hominis
Isolate Impact
Bottom-fermenting brewing yeast Lager yeast
Burkholderia cepacia BCC organisms are typically found in water and soil, human pathogen, affects lungs
Burkholderia vietnamiensis BCC organisms are typically found in water and soil, human pathogen, affects lungs
Candida guilliermondii Oppotunistic fungal pathogen
Candida inconspicua Oppotunistic fungal pathogen
Candida krusei Oppotunistic fungal pathogen
Candida pelliculosa Oppotunistic fungal pathogen
Dekkera/Brettanomyces bruxellensis Wild yeast
Enterococcus gilvus Large genus of Lactic acid bacteria
Exophiala dermatitidus Respiratory pathogen
Lactobacillus brevis Lactic acid producers
Lactobacillus paracasei Lactic acid producers
Lactococcus lactis Lactic acid producers
Pandoraea apista Respiratory pathogen
Pichia manshurica Produce volatile phenols
Rhodotorula mucilaginosa Oppotunistic fungal pathogen
Staphylococcus capitis Associated with endocarditis
Staphylococcus hominis Produce thioalcohol (rotten eggs)
Asia Pacific Convention 2018 | Wellington, New Zealand
Part II Profiling of Yeast Health
Asia Pacific Convention 2018 | Wellington, New Zealand
Monitoring Yeast Health (Project Aims)
To determine if the yeast proteome during
fermentation and re-pitching produces
different mass spectrum profiles (MSP).
To identify the proteins that change and how
they relate to off-flavours.
To assess how these changes affect the
overall flavour profile of the product, in
conjunction with metabolite profiling.
To establish a database that allows monitoring
of these changes.
To determine the applicability of the database
by analysing samples from the brewery
Asia Pacific Convention 2018 | Wellington, New Zealand
Prepare cell
culture
1
2x water wash
1x ethanol wash
3
Dried-droplet onto
MALDI target
5
Biotyper acquisition 1 biological replicate,
8 technical replicates
Acquire 3X (24 Spectra)
6
Extract with 70%
formic acid
4
Harvest 150,000
cells
2
7
Automated identification Review and Validation
Agar plate
Protein Extraction and MALDI-TOF Analysis
Issues for High-Throughput sampling and obtaining
consistent profiles:
Extraction
Not enough protein Too few cells
Formic acid extraction not consistent
good for bacterial cells but poor for yeast cells
Asia Pacific Convention 2018 | Wellington, New Zealand
Protein Extraction Optimisation
Coomassie stain (µg) Silver stain (ng)
FA: Formic Acid
TCA: Trichloro Acetic Acid
SDS: Sodium Dodecyl Sulfate
Biorupter®: Sonicator of cells
Asia Pacific Convention 2018 | Wellington, New Zealand
TCA/Biorupter®
&
Acetone precipitation
Prepare cell
culture
1
Dried-droplet onto
MALDI target
5
Biotyper acquisition 1 biological replicate,
8 technical replicates
Acquire 3X (24 Spectra)
6
Harvest 150,000
cells
2
7
Automated identification Review and Validation
Agar plate
Alternative Extraction Protocols (Trichloroacetic acid (TCA)/ acetone precipitation)
20% TCA
Biorupter
3
Acetone
precipitation
4
?
Asia Pacific Convention 2018 | Wellington, New Zealand
Optimisation Of Cell Number For Database MSP Generation
Pair-wise correlations
Spotted 8 technical replicates for each yeast cell concentration
Acquired 3 x spectra per replicate ( 24 spectrum)
This was repeated 5X
3,000,000 cells
88% spectral resolution
1,000,000 cells
60% spectral resolution
Correlation above 0.90 Correlation above 0.95
2,000,000 cells
92% spectral resolution
Asia Pacific Convention 2018 | Wellington, New Zealand
(Value of 1 means complete agreement, Value of -1 means complete dis-agreement)
Prepare cell
culture
1
Dried-droplet onto
MALDI target
5
Biotyper acquisition 1 biological replicate,
8 technical replicates
Acquire 3X (24 Spectra)
6
Harvest 2,000,000
cells
2
7
Automated identification Review and Validation
Agar plate
Optimised Extraction Protocol (Trichloroacetic acid (TCA)/ acetone precipitation) & 2,000,000 yeast cells
20% TCA
Biorupter
3
Acetone
precipitation
4
Asia Pacific Convention 2018 | Wellington, New Zealand
Validation Of Changes To Yeast Proteins Exposed To Stress
Normal (Yeast-peptone D-
glucose media)
Nutrient deprivation (Yeast-
peptone media)
Oxidative stress (1% (v/v) and
2mM hydrogen peroxide)
Osmotic stress (15% (w/v)
mannitol)
Ethanol stress (10% (v/v)
ethanol)
Cold (4C)
Asia Pacific Convention 2018 | Wellington, New Zealand
Profile Changes After Cold and Ethanol Stress
Cold
(4C)
Ethanol
(10%)
Asia Pacific Convention 2018 | Wellington, New Zealand
Profile Changes After Oxidative Stress
1% H202
2mM
H202
Asia Pacific Convention 2018 | Wellington, New Zealand
Profile Changes After Osmotic and Nutrient Deprivation
Osmotic
(15% Mannitol)
Nutrient
deprivation
Asia Pacific Convention 2018 | Wellington, New Zealand
Preliminary Data of Lager Fermentation & Yeast Storage MSP Profile
Day 1:
Fermentation
Day 2:
Fermentation
Day 8:
Fermentation
Day 5:
Yeast Storage
Day 1:
Yeast Storage
0 500 1000 1500 2000 2500 3000
0
2000
4000
6000 Inte
ns.
[a.u
.]
320 330 340 350 360 370 380 390 400 410 420 m/z
Conclusions
Eliminate pre-enrichment by
direct testing of beer
Optimised accuracy of detection
in mixed cultures utilising
Microfluidics
Identifying the proteins that
change when exposed to
different stresses
Suitable for the identification of
beer spoilage microorganisms
Accurate and rapid
Can detect protein changes
in yeast under stress
Future Research
Asia Pacific Convention 2018 | Wellington, New Zealand
Acknowledgements
Professor Peter Hoffmann Dr Florian Weiland Dr Mark Condina Dr Michelle Turvey
Brooke Dilmetz Erica Keller
Asia Pacific Convention 2018 | Wellington, New Zealand