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Spoilage-Associated Psychrotrophic and Psychrophilic Microbiota on Modified Atmosphere Packaged Beef

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Spoilage-Associated Psychrotrophic and Psychrophilic Microbiota on Modified Atmosphere Packaged Beef TECHNISCHE UNIVERSITÄT MÜNCHEN Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Lehrstuhl für Technische Mikrobiologie Spoilage-associated psychrotrophic and psychrophilic microbiota on modified atmosphere packaged beef Maik Hilgarth Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Prof. Dr. Horst-Christian Langowski Prüfer der Dissertation: 1. Prof. Dr. Rudi F. Vogel 2. Prof. Dr. Siegfried Scherer 3. Prof. Dr. Jochen Weiss Die Dissertation wurde am 08.08.2018 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 22.11.2018 angenommen. Spoilage-associated psychrotrophic and psychrophilic microbiota on modified atmosphere packaged beef Maik Hilgarth ‘Everything is everywhere, but, the environment selects’ - Lourens Gerhard Marinus Baas Becking, inspired by Martinus Willem Beijerinck Doctoral thesis Freising, 2018 - This thesis is dedicated to my beloved parents - IV Abbreviations °C degree Celsius (centrigrade) µ micro (10-6) A ampere ANI average nucleotide identity aw water activity B. Brochothrix BHI brain-heart infusion medium BLAST basic local alignment search tool bp base pairs C. Carnobacterium CFC cephalothin-fucidin-cetrimide medium CFU colony forming units contig contiguous consensus DNA sequence Da dalton diH2O deionized water DNA desoxyribonucleic acid dNTP desoxy nucleoside triphosphate DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen EDTA ethylenediaminetetraacetic acid g gram H. Hafnia h hour HCA hierarchical cluster analysis L liter LAB lactic acid bacteria LB lysogenic broth Lc. Lactococcus Le. Leuconostoc M molar (mol/L) m milli (10-3), meter M molarity MALDI-TOF MS matrix-assisted laser desorption-ionisation time of flight mass spectrometry MAP modified atmosphere packaged Mb mega base pairs MB marine broth MEGA Molecular Evolutionary Genetics Analysis min minute (') MSL minimum shelf life MSM meat simulation medium MSP mass spectrometry profile n nano (10-9) NCBI National Center for Biotechnology Information nri not reliable identified nt nucleotides OD590 optical density at 590 nm wavelength P. Photobacterium p probability value PB Photobacterium broth PCR polymerase chain reaction PGAP Prokaryotic Genome Annotation Pipeline pH negative decimal logarithm of hydrogen ion activity ppbv parts per billion by volume Ps. Pseudomonas V PTR-MS proton transfer reaction mass spectrometry RAPD randomly amplified polymorphic DNA RAST Rapid Annotations using Subsystems Technology s second ('') S. Serratia SE standard error SMRT single molecule real time SSO specific spoilage organism(s) STAA streptomycin-thallous acetate-actidione agar Taq Thermus aquaticus TBE Tris-HCl, boric acid, EDTA (buffer) TCA cycle tricarboxylic acid cycle TCBS thiosulfate citrate bile salts sucrose medium TMW Technische Mikrobiologie Weihenstephan TSA tryptic soy agar TVC total viable count V volt v/v volume per volume VP vacuum packaged W watt w/v weight per volume w/w weight per weight WGS whole genome shotgun VI Table of contents 1 Introduction .............................................................................................................................1 1.1 Background and economic relevance ..................................................................................1 1.2 Modified atmosphere packaging ...........................................................................................1 1.3 Ecosystem of cold stored MAP beef .....................................................................................2 1.4 Spoilage-associated microbiota on meat..............................................................................4 1.5 Identification and differentiation of spoilage associated microbiota .....................................5 1.6 Controversial role of LAB in meat spoilage ..........................................................................6 2 Hypotheses .............................................................................................................................8 3 Material and methods .......................................................................................................... 10 3.1 Microorganisms ................................................................................................................. 10 3.2 Isolation and cultivation of spoilage-associated microbiota............................................... 11 3.2.1 Meat samples, sampling and sensory evaluation ...................................................... 11 3.2.2 Cultivation of bacteria ................................................................................................ 13 3.2.3 Cultivation and growth evaluation of meat spoilers and photobacteria ..................... 14 3.2.4 Antibiotic susceptibility testing ................................................................................... 15 3.2.5 Cultivation media ....................................................................................................... 15 3.3 Matrix Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI- TOF MS) ............................................................................................................................ 18 3.3.1 Sample preparation ................................................................................................... 18 3.3.2 MALDI-TOF MS analysis and identification ............................................................... 19 3.3.3 Reference MSPs ........................................................................................................ 19 3.3.4 Data analysis and processing .................................................................................... 19 3.4 Proton-transfer-reaction mass spectrometry (PTR-MS) .................................................... 20 3.5 Molecular biological methods ............................................................................................ 20 3.5.1 Isolation of genomic DNA .......................................................................................... 20 3.5.2 PCR amplification ...................................................................................................... 21 3.5.3 Analytical agarose gel electrophoresis ...................................................................... 22 3.5.4 Purification and Sequencing ...................................................................................... 22 3.6 Sequence and fingerprinting analysis................................................................................ 23 3.6.1 Phylogenetic sequence analysis ............................................................................... 23 3.6.2 RAPD-PCR fingerprinting .......................................................................................... 23 3.7 Genome analysis ............................................................................................................... 23 3.7.1 Single Molecule Real Time (SMRT) sequencing ....................................................... 24 3.7.2 Whole genome shotgun (WGS) sequencing ............................................................. 24 3.7.3 Annotation and metabolic pathways .......................................................................... 24 3.7.4 Average nucleotide identity ....................................................................................... 25 VII 3.8 Intraspecies assertiveness and interaction of Lactococcus piscium strains with spoilers in vitro ................................................................................................................................ 25 3.9 Chemotaxonomic analysis of photobacteria ...................................................................... 26 3.10 Phenotypic characterization of photobacteria ................................................................... 26 4 Results .................................................................................................................................. 28 4.1 Monitoring of spoilage-associated microbiota on MAP beef ............................................. 28 4.1.1 Sensorial evaluation .................................................................................................. 28 4.1.2 Identification of spoilage microbiota and monitoring of spoilage-associated parameters ................................................................................................................. 28 4.1.3 Cultivation of potentially psychrophilic spoilage-associated microbiota .................... 32 4.1.4 Comparison of microbiota on top and bottom of beef steaks .................................... 33 4.1.5 Diversity assessment of beef steak isolates below species level ............................. 34 4.1.6 Discriminatory power of MALDI-TOF MS on strain level ........................................... 35 4.1.7 Random retail beef samples ...................................................................................... 36 4.2 Monitoring of spoilage-associated microbiota on MAP minced beef ................................. 39 4.2.1 Sensorial evaluation .................................................................................................. 39 4.2.2 Identification
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