MALDI-TOF MS M ATRIX -A SSISTED L ASER D ESORPTION /I ONIZATION T IME - - PDF document

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MALDI-TOF MS M ATRIX -A SSISTED L ASER D ESORPTION /I ONIZATION T IME - - PDF document

Oct 05, 2022 162 likes •338 views

MALDI-TOF MS for Brewing Microbiology B EER F ERMENTATION P ROCESSES AND MALDI-TOF MS F OR B REWING M ICROBIOLOGY Anita Van Landschoot & Anneleen Decloedt Anneleen.decloedt@ugent.be B EER F ERMENTATION P ROCESSES The 4 fermentation processes

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MALDI-TOF MS for Brewing Microbiology 1

BEER FERMENTATION PROCESSES AND MALDI-TOF MS FOR BREWING MICROBIOLOGY

Anita Van Landschoot & Anneleen Decloedt

Anneleen.decloedt@ugent.be 2

Spontaneous fermentation Top fermentation Bottom fermentation Mixed fermentation

BEER FERMENTATION PROCESSES

The 4 fermentation processes

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SLIDE 2

MALDI-TOF MS for Brewing Microbiology 2

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BEER FERMENTATION PROCESSES

Top and Bottom fermentation Micro-organisms: pure brewers yeast

pH 5.6 5.2-5.6 4.2-4.6 T (°C) 50-78 78 > 100 20 4 time 2h 2h 2h 2h > 5d > 5d

4

pH 5.6 5.2-5.6 3.2-3.8 T (°C) 50-78 78 > 100 20 10-15 time 2h 2h 2h 2h > 5d weeks

BEER FERMENTATION PROCESSES

Mixed fermentation Micro-organisms: bacteria and top fermented yeasts

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SLIDE 3

MALDI-TOF MS for Brewing Microbiology 3

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pH 5.6 5.2-5.6 3.2-3.8 T (°C) 50-78 78 > 100 100 20 10-15 time 2h 2h 2h

  • vernight

months/years

Coolship Fermentation/Maturation Lambic

BEER FERMENTATION PROCESSES

Spontaneous fermentation Micro-organisms: bacteria, wild yeasts and top fermented yeasts

Current microbial detection / identification methods

– Using selective growth media/conditions – Specific oligonucleotide probes – PCR-based detection and/or identification – Ribotyping – Immuno-based – …

MALDI-TOF MS as a rapid, specific, inexpensive and high-throughput

identification/differentiation tool for brewing microbiology Laborious, time-consuming, lack specificity and sensitivity

6

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SLIDE 4

MALDI-TOF MS for Brewing Microbiology 4

MALDI-TOF MS

MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY

Time-Of-Flight Detector Mass Spectrometer +20kV

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Acetobacter pasteurianus Pediococcus damnosus Lactobacillus brevis

MALDI-TOF MS

OUTPUT

Relative intensity (%) Signal intensity

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SLIDE 5

MALDI-TOF MS for Brewing Microbiology 5

References for MALDI-TOF MS database Brewing Sample Defined strains cultured at species-specific conditions Isolations (+ Modified Extraction Protocol) Preparing Cell Extract – Standard Protocol REFERENCE IDENTIFICATION DATABASE COMPARE

MALDI-TOF MS

DATA

MALDI-TOF MS

TOOL FOR BREWING MICROBIOLOGY

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1000s of good quality mass spectra 100s of strains representing >50 brewing species Validation of the reference database Polyphasic approach – 16S rRNA gene sequence analysis – Species specific gene sequence analysis(e.g. pheS) – FAFLP (Fluorescent amplified fragment length polymorphism) – DNA-DNA hybridisations

MALDI-TOF MS IN-HOUSE IDENTIFICATION DATABASE

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SLIDE 6

MALDI-TOF MS for Brewing Microbiology 6

MALDI-TOF MS DATA ANALYSIS OF MASS SPECTRA

4000 Series Explorer Software

  • Data Explorer Software

(.t2d-files .txt-files)

  • BioNumerics Applied Maths

(Database)

  • Quality Control

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Microbiology Lambic Beer Fermentation Differentiation of Brewing Yeast Strains Identification of Microorganisms MALDI-TOF MS FOR Identification Beer spoilage Bacteria Follow up Fermentation

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SLIDE 7

MALDI-TOF MS for Brewing Microbiology 7

Identification Beer spoilage Bacteria Isolated Contaminants Direct Detection and Identification MALDI-TOF MS FOR

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Gram positive bacteria

Lactobacillus spp.

  • Lb. backii
  • Lb. brevis
  • Lb. brevisimilis
  • Lb. buchneri
  • Lb. casei
  • Lb. coryniformis
  • Lb. curvatus
  • Lb. lindneri
  • Lb. malefermentans
  • Lb. parabuchneri
  • Lb. plantarum

Pediococcus spp.

  • P. acidilactici
  • P. claussenii
  • P. damnosus
  • P. inopinatus

Kocuria spp.

  • K. kristinae

Gram negative bacteria

Megasphaera spp.

  • M. cerevisiae
  • M. paucivorans
  • M. sueciensis

Acetic Acid Bacteria Acetobacter spp. Gluconobacter spp.

Enterobacteriaceae Pectinatus spp.

  • P. cerevisiiphilus
  • P. frisingensis

Selenomonas sp. Zymomonas sp. Zymophilus sp.

>75% L A B L A B A A B

Identification Beer spoilage Bacteria

14

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MALDI-TOF MS for Brewing Microbiology 8

14 Spoiled Beer and Brewery samples 348 isolates Enrichment Isolation Dereplication of mass spectra (assessment of diversity) 15 clusters Cluster representatives (1-6 isolates/cluster) MALDI-TOF MS-based identification Peak-based cluster analysis Confirmation of ID results using protein coding gene sequence analysis

Media AAM MRS NBB

Wieme et al., 2014, Int. J. Food Microbiol.

Isolated Contaminants Identification Beer spoilage Bacteria

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Pearson correlation (Opt:0.01%) [0.0%-100.0%]

100 80 60 40

R-49153 R-49164 R-49156 R-49163 R-49166 R-49151 R-49152 R-49157 R-49143 R-49155 R-49159 R-49154 R-49147 R-49162 R-49160 R-49161 R-49158 R-49149 R-49165 R-49148 R-49144

75.4

Lactobacillus brevis Example of cluster

  • f isolates

Wieme et al., 2014 Int. J. Food Microbiol.

Identification Beer spoilage Bacteria Isolated Contaminants

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SLIDE 9

MALDI-TOF MS for Brewing Microbiology 9

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5 10 15 20 25 30 35 40 45 S S S S S S S S S S S S S S

  • A. cerevisiae/A. malorum
  • A. fabarum
  • A. indonesiensis
  • A. orleanensis
  • A. persici

Gluconobacter cerevisiae sp Gluconobacter sp. nov.

  • G. cerinus
  • G. japonicus
  • G. oxydans
  • Lb. backii
  • Lb. brevis
  • Lb. malefermentans
  • P. claussenii
  • P. inopinatus
  • A. cerevisiae/A. malorum
  • A. fabarum
  • A. indonesiensis
  • A. orleanensis
  • A. persici

Gluconobacter cerevisiae Gluconobacter sp.

  • G. cerinus
  • G. japonicus
  • G. oxydans
  • Lb. backii
  • Lb. brevis
  • Lb. malefermentans
  • P. claussenii
  • P. inopinatus

AAB # isolates LAB

7x 3/7 single sp

Acetobacter Gluconobacter Lactobacillus Pediococcus 1 2 3 4 5 7 8 9 10 11 12 13 14

7/14 single species

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2. Few cell numbers present in spoiled samples: 1 - 50 CFU/100-250 mL sample 5 - 10×107 CFU/mL needed for MALDI-TOF MS analysis 1. Beer matrix peak suppression effect

Easy removal of beer matrix using washing steps Enrichment step

  • Presence of moulds or yeast cells?
  • Different protocols were tested
  • Filtration step prior to enrichment = best-suited

Identification Beer spoilage Bacteria Direct Detection and Identification

18

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SLIDE 10

MALDI-TOF MS for Brewing Microbiology 10

DIVERSITY CHECK UNDER MICROSCOPE (if possible) MALDI-TOF MS ANALYSIS ENRICHMENT IN LIQUID MEDIUM ONLY BACTERIA? No Yes FILTRATION (5-8-0.45 µm) RETAIN 0.45 µm FILTER CELL EXTRACTION ISOLATION OF CONTAMINANTS CELL EXTRACTION

Identification Beer spoilage Bacteria

Isolated Contaminants VERSUS Direct Detection and Identification

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Microbiology Lambic Beer Fermentation Differentiation of Brewing Yeast Strains Identification of Microorganisms MALDI-TOF MS FOR Identification Beer spoilage Bacteria Follow up Fermentation

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MALDI-TOF MS for Brewing Microbiology 11

  • Beers of spontaneous fermentation
  • Spontaneous = no starter cultures
  • Acidic beverage
  • Basis for other beers e.g. Gueuze, Kriek
  • Malted barley, wheat, old hop bells and water
  • Traditional, artisan product:
  • Aged in wooden barrels for 2 to 3 years
  • production “only” in Senne river valley

Coolship or cooling tun

Microbiology Lambic Beer Fermentation

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PCR-DGGE / MALDI-TOF MS

Wort not pH adjusted Wort pH 4

Saccharomyces Brettanomyces

Enterobacteriaceae Yeasts Pediococcus damnosus AAB

Spitaels et al., 2014, PlosOne

Microbiology Lambic Beer Fermentation

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MALDI-TOF MS for Brewing Microbiology 12

Microbiology Lambic Beer Fermentation Differentiation of Brewing Yeast Strains Identification of Microorganisms MALDI-TOF MS FOR Identification Beer spoilage Bacteria Follow up Fermentation

23 24

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MALDI-TOF MS for Brewing Microbiology 13

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Microbiology Lambic Beer Fermentation Differentiation of Brewing Yeast Strains Identification of Microorganisms MALDI-TOF MS FOR Identification Beer spoilage Bacteria Follow up Fermentation

26

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MALDI-TOF MS for Brewing Microbiology 14

37 old beers

+ 1 chang Nepal ferment Enrichment in Wort Broth and Modified Broth Base Isolation on 3 different media DYPA, MRS, AAM 28 isolates MALDI-TOF MS Data MALDI-TOF MS-based identification Peak-based cluster analysis

9 ≠ species

Identification of Microorganisms

Beer Analysis Microscopy

27 28

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SLIDE 15

MALDI-TOF MS for Brewing Microbiology 15

Identification of Microorganisms

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Identification of Microorganisms

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MALDI-TOF MS for Brewing Microbiology 16

Microbiology Lambic Beer Fermentation Differentiation of Brewing Yeast Strains Identification of Microorganisms MALDI-TOF MS FOR Identification Beer spoilage Bacteria Follow up Fermentation

31 1 day t = 0 2 days 4 days

4 time points fermentation → 36 spectra/Eme point

Discriminant analysis

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MALDI-TOF MS for Brewing Microbiology 17

QUALITY CONTROL TOOL FLEXIBILITY DIVERSE GROUP OF MICROORGANISMS EASY SAMPLE TREATMENT

MALDI-TOF MS

AS A NOVEL TOOL FOR DETECTION, IDENTIFICATION AND DIFFERENTIATION OF MICROBIOTA IN THE BREWING INDUSTRY

… AND MUCH MORE

RELIABLE & ACCURATE DEREPLICATION OF LARGE DATASETS LOW CONSUMABLE COSTS CULTURE- DEPENDENT DATA COMPATIBILITY THROUGHPUT CAPACITY RAPID HIGH INVESTMENT COSTS FERMENTATION CONTROL TOOL CULTURE- DEPENDENT

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MALDI-TOF MS

FOR BREWING MICROBIOLOGY

Thanks to:

  • Dr. Anneleen Wieme, Dr. Freek Spitaels,
  • ing. Jolien Koek, ing. Freya Van Alboom
  • Dr. Koenraad Van Hoorde & Prof. Dr. Peter Vandamme