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 Spontaneous fermentation Top fermentation Bottom fermentation Mixed fermentation 2 1
MALDI-TOF MS for Brewing Microbiology B EER F ERMENTATION P ROCESSES 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 3 B EER F ERMENTATION P ROCESSES Mixed fermentation Micro-organisms: bacteria and top fermented yeasts 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 4 2
MALDI-TOF MS for Brewing Microbiology B EER F ERMENTATION P ROCESSES Spontaneous fermentation Micro-organisms: bacteria, wild yeasts and top fermented yeasts Lambic Coolship Fermentation/Maturation 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 overnight months/years 5 Current microbial detection / identification methods – Using selective growth media/conditions – Specific oligonucleotide probes – PCR-based detection and/or identification – Ribotyping – Immuno-based – … Laborious, time-consuming, lack specificity and sensitivity MALDI-TOF MS as a rapid, specific, inexpensive and high-throughput identification/differentiation tool for brewing microbiology 6 3
MALDI-TOF MS for Brewing Microbiology MALDI-TOF MS M ATRIX -A SSISTED L ASER D ESORPTION /I ONIZATION T IME - OF -F LIGHT M ASS S PECTROMETRY Time-Of-Flight Mass Spectrometer Detector +20kV 7 MALDI-TOF MS OUTPUT Acetobacter pasteurianus Relative intensity (%) Signal intensity Pediococcus damnosus Lactobacillus brevis 8 4
MALDI-TOF MS for Brewing Microbiology MALDI-TOF MS T OOL F OR B REWING M ICROBIOLOGY Brewing References for MALDI-TOF MS database Sample Defined strains cultured Isolations at species-specific conditions (+ Modified Extraction Protocol) Preparing Cell Extract – Standard Protocol COMPARE MALDI-TOF MS REFERENCE IDENTIFICATION DATABASE DATA 9 MALDI-TOF MS I N -H OUSE I DENTIFICATION D ATABASE 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 10 5
MALDI-TOF MS for Brewing Microbiology MALDI-TOF MS D ATA ANALYSIS OF MASS SPECTRA 4000 Series Explorer Software - Data Explorer Software (.t2d-files � .txt-files) - BioNumerics Applied Maths (Database) - Quality Control 11 MALDI-TOF MS F OR Identification Beer spoilage Bacteria Microbiology Lambic Beer Fermentation Differentiation of Brewing Yeast Strains Identification of Microorganisms Follow up Fermentation 12 6
MALDI-TOF MS for Brewing Microbiology MALDI-TOF MS F OR Identification Beer spoilage Bacteria Isolated Contaminants Direct Detection and Identification 13 Identification Beer spoilage Bacteria Gram positive bacteria Gram negative bacteria >75% Lactobacillus spp. Acetic Acid Bacteria A Acetobacter spp. A Lb. backii Pediococcus spp . B Gluconobacter spp. Lb. brevis L P. acidilactici Lb. brevisimilis L Megasphaera spp . A Enterobacteriaceae P. claussenii A Lb. buchneri M. cerevisiae B P. damnosus B Pectinatus spp . Lb. casei M. paucivorans P. inopinatus P. cerevisiiphilus Lb. coryniformis M. sueciensis P. frisingensis Lb. curvatus Lb. lindneri Selenomonas sp . Kocuria spp. Lb. malefermentans K. kristinae Zymomonas sp . Lb. parabuchneri Zymophilus sp. Lb. plantarum 14 7
MALDI-TOF MS for Brewing Microbiology Identification Beer spoilage Bacteria Isolated Contaminants 348 isolates Media 14 Spoiled Enrichment AAM Beer and Isolation MRS Brewery NBB samples Dereplication of mass spectra (assessment of diversity) MALDI-TOF MS-based identification 15 clusters Peak-based cluster analysis Confirmation of ID results using Cluster representatives protein coding gene sequence analysis (1-6 isolates/cluster) Wieme et al. , 2014, Int. J. Food Microbiol. 15 Identification Beer spoilage Bacteria Isolated Contaminants Pearson correlation (Opt:0.01%) [0.0%-100.0%] Wieme et al. , 2014 Int. J. Food Microbiol. 100 40 60 80 R-49153 R-49164 R-49156 R-49163 R-49166 R-49151 R-49152 R-49157 R-49143 Lactobacillus brevis R-49155 75.4 Example of cluster R-49159 of isolates R-49154 R-49147 R-49162 R-49160 R-49161 R-49158 R-49149 R-49165 R-49148 R-49144 16 8
MALDI-TOF MS for Brewing Microbiology 0 5 10 15 20 25 30 35 40 45 # isolates 1 S 2 S A. cerevisiae / A. malorum A. cerevisiae/A. malorum 3 S A. fabarum A. fabarum Acetobacter 4 A. indonesiensis S A. indonesiensis AAB A. orleanensis 5 A. orleanensis S A. persici A. persici 6 S Gluconobacter cerevisiae Gluconobacter cerevisiae sp 7 S Gluconobacter sp. Gluconobacter sp. nov. 7/14 8 G. cerinus S Gluconobacter G. cerinus single species G. japonicus 9 S G. japonicus G. oxydans 10 S G. oxydans Lb. backii Lb. backii 11 Lactobacillus S Lb. brevis 7x 3/7 single sp Lb. brevis Lb. malefermentans 12 LAB S Lb. malefermentans P. claussenii Pediococcus 13 P. claussenii S P. inopinatus P. inopinatus 14 S 17 Identification Beer spoilage Bacteria Direct Detection and Identification 1. Beer matrix � peak suppression effect Easy removal of beer matrix using washing steps 2. Few cell numbers present in spoiled samples: 1 - 50 CFU/100-250 mL sample 5 - 10×10 7 CFU/mL needed for MALDI-TOF MS analysis Enrichment step • Presence of moulds or yeast cells? • Different protocols were tested • Filtration step prior to enrichment = best-suited 18 9
MALDI-TOF MS for Brewing Microbiology Identification Beer spoilage Bacteria Isolated Contaminants VERSUS Direct Detection and Identification DIVERSITY CHECK ONLY BACTERIA? No UNDER MICROSCOPE (if possible) Yes FILTRATION (5-8-0.45 µm) RETAIN 0.45 µm FILTER ENRICHMENT IN LIQUID MEDIUM CELL EXTRACTION MALDI-TOF MS ANALYSIS CELL EXTRACTION ISOLATION OF CONTAMINANTS 19 MALDI-TOF MS F OR Identification Beer spoilage Bacteria Microbiology Lambic Beer Fermentation Differentiation of Brewing Yeast Strains Identification of Microorganisms Follow up Fermentation 20 10
MALDI-TOF MS for Brewing Microbiology Microbiology Lambic Beer Fermentation • Beers of spontaneous fermentation • Spontaneous = no starter cultures • Acidic beverage • Basis for other beers e.g. Gueuze, Kriek Coolship or cooling tun • 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 21 Microbiology Lambic Beer Fermentation PCR-DGGE / MALDI-TOF MS Spitaels et al. , 2014, PlosOne Wort pH 4 Wort not pH adjusted Yeasts Pediococcus damnosus AAB Enterobacteriaceae Saccharomyces Brettanomyces 22 11
MALDI-TOF MS for Brewing Microbiology MALDI-TOF MS F OR Identification Beer spoilage Bacteria Microbiology Lambic Beer Fermentation Differentiation of Brewing Yeast Strains Identification of Microorganisms Follow up Fermentation 23 24 12
MALDI-TOF MS for Brewing Microbiology 25 MALDI-TOF MS F OR Identification Beer spoilage Bacteria Microbiology Lambic Beer Fermentation Differentiation of Brewing Yeast Strains Identification of Microorganisms Follow up Fermentation 26 13
MALDI-TOF MS for Brewing Microbiology Identification of Microorganisms Enrichment in 37 old beers Wort Broth and + 1 chang Modified Broth Base Nepal ferment Isolation on 3 different media DYPA, MRS, AAM Beer Analysis 28 isolates MALDI-TOF MS-based identification Microscopy Peak-based cluster analysis MALDI-TOF MS Data 9 ≠ species 27 28 14
MALDI-TOF MS for Brewing Microbiology Identification of Microorganisms 29 Identification of Microorganisms 30 15
MALDI-TOF MS for Brewing Microbiology MALDI-TOF MS F OR Identification Beer spoilage Bacteria Microbiology Lambic Beer Fermentation Differentiation of Brewing Yeast Strains Identification of Microorganisms Follow up Fermentation 31 4 time points fermentation 1 day → 36 spectra/Eme point t = 0 2 days 4 days Discriminant analysis 32 16
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