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Title Dairy-Met: compositional metagenomic analysis of milk and cheeses Author(s) Quigley, Lisa Publication date 2013 Original citation Quigley, L. 2013. Dairy-Met: Compositional metagenomic analysis of milk and cheeses. PhD Thesis, University College Cork. Type of publication Doctoral thesis Rights © 2013, Lisa Quigley. http://creativecommons.org/licenses/by-nc-nd/3.0/ Item downloaded http://hdl.handle.net/10468/1406 from Downloaded on 2018-08-23T19:26:10Z Dairy-Met: Compositional metagenomic analysis of milk and cheeses A Thesis Presented to the University College Cork for the Degree of Doctor of Philosophy (PhD) October 2013 By Lisa Quigley BSc, MSc 1Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland 2Department of Microbiology, University College Cork, Co. Cork, Ireland Research Supervisors: Dr Paul D. Cotter1, Dr Orla O’ Sullivan1, 2 Professor Gerald Fitzgerald This thesis is dedicated to the loving memory of Amelia, our little angel. Table of Content Declaration………………………………………………………………………………... i Abstract…………………………….…………………………………………………....... ii Publications………….…………………………………………………………………… vi Glossary of Terms……………………………………………………………………….. vii List of Figures…………………………………………………………………………..... ix List of Tables..……………………………………………………………………………. xi Literature Review I 1 Molecular approached to analysing the microbial composition of raw milk and raw milk cheese Abstract……………………………………………………………………………….......... 2 1. Introduction……………………………………………………………………………… 3 2. Culture-independent analysis…………………………………………………………. 4 2.1 Differentiating between the DNA live and dead microorganisms………… 4 2.2 Nucleic acid extraction………………………………………………………... 5 2.3 Target region…………………………………………………………………… 5 2.4 Choice of culture-independent technique…………………………………... 6 3. Application of culture-independent methods to study the microbiota and diversity of milk and cheese………………………………………………………………………… 7 3.1 Evaluation of the microbial diversity of milk………………………………… 7 3.2 Evaluation of the microbial diversity of whey starters……………………... 9 3.3 Evaluation of the microbial diversity of different cheeses………………… 10 3.4 Comparison of outcomes when culture-dependent and culture-independent approaches are used to assess the microbiota of milk and cheese……... 12 3.5 Investigation of microbial composition and succession during cheese 14 manufacture…………………………………………………………………..... 3.6 Application of molecular biology for pathogen and spoilage related 18 investigations of milk and cheese products………………………………… 4. Conclusion and future prospects……………………………………………………… 22 References…………………………………………………………………………………. 24 Literature Review II 44 The complex microbiota of raw milk Abstract…………………………………………………………………………………….. 45 1. Introduction……………………………………………………………………………… 46 2. Methods employed to determine the microbial composition of milk………………. 47 3. Sources of milk microorganisms……………………………………………………… 49 4. The microbial composition of different milk types…………………………………… 51 4.1 Cows milk…………………………………………………………………..…... 51 4.2 Goats milk…………………………………………………………………..….. 53 4.3 Sheep milk…………………………………………………………………..…. 54 4.4 Buffalo milk……………………………………………………………….....…. 55 4.5 Milk from other animal sources………………………………………………. 56 Box 1. Human milk………………..…………………………..…………………… 57 5. Technologically relevant bacteria of raw milk……………………………………….. 69 5.1 Lactococcus……………………………………………………………...…..… 60 5.2 Lactobacillus…………………………………………………………….....….. 62 5.3 Streptococcus………………………………………………………………..... 65 5.4 Propionibacterium………………………………………………………….….. 68 5.5 Leuconostoc……………………………………………………………...…..... 69 5.6 Enterococcus…………………………………………………………….....…. 70 6. Other microorganisms with potential technological relevance…………………….. 71 6.1 Gram positive microorganisms…………………………………………….... 71 6.2 Gram negative microorganisms……………………………………………… 73 6.3 Fungal populations………………………………………...……………...…... 74 7. Impact of storage conditions and downstream treatments on the microbiology of raw milk……………………………………………………………...……………………….. 76 7.1 Cold storage………………………………………………………….…....…... 76 7.2 Pasteurisation………………………………………………………..……....... 77 7.3 Bacteriophage……………………………………………………………......... 80 7.4 Biopreservative potential of raw milk microorganisms…………………….. 82 8. Human health association……………………………………………………………... 81 8.1 Pathogenic bacteria associated with raw milk……………………………… 81 8.2 Antibiotic residues and antibiotic resistant bacteria in milk…………..…… 85 8.3 Health promoting microbes…………………………………………………... 87 9. Conclusion……………………………………………………………...……………….. 90 References……………………………………………………………...…………………. 91 Supplementary Data……………………………………………………………...………. 127 Chapter I 130 A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese Abstract……………………………………………………………...……………………... 131 1. Introduction……………………………………………………………...………………. 132 2. Materials and Methods……………………………………………………………...…. 134 2.1 Food sources…………………………………………………………………... 134 2.2 DNA extraction methods…………………………………………………….... 134 2.3 DNA quantification measurement……………………………………………. 135 2.4 PCR amplification of the microbial community 16S rRNA gene…………. 135 2.5 Purification and amplification of DNA from pathogen-spiked milk…..….... 136 3. Results…………………………………………………………………………………… 138 3.1 Comparison of the yield and purity of DNA extracted from raw milk and a raw milk cheese using a variety of extraction methods………………………… 138 3.2 Amplification of DNA extracted from raw milk and raw milk cheese…….. 139 3.3 Recovery of Gram-negative and Gram-positive bacteria as assessed by conventional PCR……………………………………………………………... 139 3.4 Improvement of DNA yield and PCR efficiency……………………………. 140 4. Discussion………………………………………………………………………………. 142 5. Conclusion………………………………………………………………………………. 145 References……………………………………………………………………………….… 146 Chapter II 154 High-throughput sequencing detects subpopulations of bacteria not previously associated with artisanal cheeses Abstract…………………………………………………………………………………….. 155 1. Introduction……………………………………………………………………………… 156 2. Materials and Methods……………………………………………………………….... 157 2.1 Cheese collection and nucleic acid extraction……………………………... 157 2.2 PCR amplification of the microbial community 16S rRNA gene…………. 157 2.3 High-throughput sequencing and bioinformatics analysis………………… 158 3. Results…………………………………………………………………………………… 160 3.1 Sequencing and bioinformatics analysis……………………………………. 160 3.2 The microbial composition of artisanal cheese as revealed by pyrosequencing……………………………………………………………….. 160 3.3 Revealing the microbial composition of the rind of artisanal cheeses…… 162 4. Discussion………………………………………………………………………………. 164 5. Conclusion………………………………………………………………………………. 167 References…………………………………………………………………………………. 168 Supplementary Data………………………………………………………………………. 177 Chapter III 182 The microbial content of raw and pasteurised cows milk as determined by molecular approaches Abstract…………………………………………………………………………………….. 183 1. Introduction……………………………………………………………………………… 184 2. Materials and Methods………………………………………………………………… 186 2.1 Strains and culture collections……………………………………………….. 186 2.2 Flow cytometry……………………………………………………………….... 186 2.3 Collection and treatment of milk samples…………………………………... 187 2.4 High-throughput sequencing and bioinformatics analysis………………… 188 2.5 Real-time quantitative PCR-based analysis………………………………... 190 3. Results…………………………………………………………………………………… 192 3.1 Flow cytometry highlights the proportion of viable microorganisms in pasteurised milk. ……………………………………………………………… 192 3.2 High-throughput sequencing reveals the presence of taxa not traditionally regarded as components of the milk microbiota…………………………… 193 4. Discussion………………………………………………………………………............ 197 5. Conclusion............................................................................................................. 200 Acknowledgements……………………………………………………………………….. 201 References……………………………………………………………………………….… 202 Supplementary Data………………………………………………………………………. 209 Chapter IV 214 Thermus thermophilus and its role in the pinking defect in cheese Abstract…………………………………………………………………………………….. 215 1. Introduction……………………………………………………………………………… 216 2. Materials and Methods………………………………………………………………… 218 2.1 DNA extraction from cheeses………………………………………………... 218 2.2 Generation of 16S rRNA amplicons for high-throughput sequencing…… 218 2.3 High-throughput sequencing and bioinformatic analysis………………….. 219 2.4 Culturing of Thermus………………………………………………………….. 220 2.4.1 Culture-based Method……………………………………………..…… 220 2.4.2 PCR and qPCR-based detection of Thermus………………...……… 220 2.5 Cheese spiking studies………………………………………………..……… 222 2.5.1 Cheese manufacture and analysis.................................................... 222 2.5.2 Visual detection of pinking…………………………............................ 223 2.5.3 Statistical Analysis…………………………………………................... 223 2.6 Environmental Sampling……………………………………………………… 223 3. Results…………………………………………………………………………………… 225 3.1 High-throughput 16S sequencing of samples extracted from cheeses with and without a pink defect………………………………………………………………. 225 3.2 Identification of bacteria by pyrosequencing……………………………….. 225 3.3 Detection of Thermus in cheese……………………………………………... 226 3.4 Formation of “pinking” in spiked cheese……………………………………. 227 3.4.1 Starter, PAB and NSLAB viability during cheese ripening………….. 228 3.4.2 Survival of Thermus thermophilus throughout cheese manufacture and ripening……………………………………………………………………..…… 228 3.4.3 Composition of cheeses………………………………………………… 229 3.4.4 Visual examination
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  • George Charimba

    George Charimba

    THE TAXONOMY AND SIGNIFICANCE OF Chryseobacterium ISOLATES FROM POULTRY GEORGE CHARIMBA January 2012 THE TAXONOMY AND SIGNIFICANCE OF Chryseobacterium ISOLATES FROM POULTRY by GEORGE CHARIMBA Submitted in fulfilment of the requirements for the degree of PHILOSOPHIAE DOCTOR In the Faculty of Natural and Agricultural Sciences Department of Microbial, Biochemical and Food Biotechnology University of the Free State Promoter: Prof. C. J. Hugo Co-promoter: Prof. P. J. Jooste January 2012 DECLARATION I George Charimba, declare that the thesis hereby submitted by me for the Ph.D. degree in the Faculty of Natural and Agricultural Sciences at the University of the Free State is my own independent work and has not previously been submitted by me at another university/faculty. I furthermore cede copyright of this thesis in favour of the University of the Free State. G. Charimba January, 2012 To my wife Eunice, my children Millicent, Tariro and George Jr. TABLE OF CONTENTS Chapter Title Page TABLE OF CONTENTS i ACKNOWLEDGEMENTS vii LIST OF TABLES ix LIST OF FIGURES xii LIST OF ABBREVIATIONS xvi 1 INTRODUCTION 1 1.1 Background to the study 1 1.2 Purpose, hypotheses and objectives of the study 3 2 LITERATURE REVIEW 5 2.1 Introduction 5 2.2 Taxonomy of the Flavobacteriaceae family 6 2.2.1 Historical Overview 6 2.2.2 Current Taxonomy 9 2.2.3 Phylogeny 9 2.2.4 Description of the family Flavobacteriaceae 12 2.2.5 Methods to study the taxonomy of the Flavobacteriaceae 14 2.2.6 Procedure for Polyphasic Taxonomy 30 2.3 The genus Chryseobacterium 31 i 2.3.1