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Diss Stephan Wullschleger090619b Research Collection Doctoral Thesis Biodiversity and microbial safety of artisanal Malian sour milk fènè and development of adapted starter cultures for controlled production Author(s): Wullschleger, Stephan M. Publication Date: 2009 Permanent Link: https://doi.org/10.3929/ethz-a-005831274 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH NO. 18287 Biodiversity and microbial safety of artisanal Malian sour milk fènè and development of adapted starter cultures for controlled production A dissertation submitted to ETH Zurich For the degree of Doctor of Sciences Presented by Stephan Markus Wullschleger Dipl. Lm-Ing., ETH Zurich Born February 4, 1980 Citizen of Aarburg (AG) Accepted on the recommendation of Prof. Dr. Christophe Lacroix, examiner Prof. Dr. Leo Meile, co-examiner Prof. Dr. Bassirou Bonfoh, co-examiner Zurich, 2009 2/156 Preface Ancient and extensively practiced agricultural traditions in Mali and concomittant high demand on milk and sour milk for the daily diet are the main forces behind sustainable development of the local dairy market and related research projects. The chance of success of applied research projects in developing countries is greater if such projects are executed in collaboration with local organizations and leading research partners combining basic research and local application. Local organizations should propose research questions where they have the detailed knowledge of local needs and conditions indicating local limits for state of the art basic research and application of achieved results in the field. Their research partner(s) may then contribute with state of the art knowledge and infrastructure to address the research question and finally achieve high impact publications and local application of the results leading to globally recognized research projects. Exchange programs of committed researchers are the key to guaranteeing long term application, knowledge transfer and trust to collaborating organizations favoring follow-up or new research projects. I am very happy that we got the full support of all collaborating organizations behind this research project, the serendipitous exchange program between the Laboratoire Central Vétérinaire and ETH Zurich and final application experiments at small-scale production. This project is a good example of international collaboration combining basic research and application on the field of achieved results leading to new research projects - current under evaluation and local business opportunities ready to apply new knowledge. 3/156 Table of contents Preface............................................................................................................................2 Table of contents.............................................................................................................3 Abbreviations ..................................................................................................................7 List of Tables...................................................................................................................9 List of Figures................................................................................................................11 Summary.......................................................................................................................13 Zusammenfassung........................................................................................................17 Résumé.........................................................................................................................21 1 Introduction............................................................................................................25 1.1 Mali: economy, demographic and its pastoral tradition.......................................25 1.2 Milk Fermentation ..............................................................................................26 1.2.1 Historical Background....................................................................................26 1.2.2 Development and safety of fermented dairy products in Africa.......................27 1.2.3 Fènè a traditional fermented milk of Mali........................................................28 1.3 Starter culture....................................................................................................29 1.3.1 Lactic acid bacteria in fermented milk products..............................................29 1.3.2 Typing of lactic acid bacteria from complex food matrices..............................30 1.3.3 Development of starter cultures for small-scale sour milk production .............32 1.4 Antibiotic resistant bacteria in fermented food....................................................33 1.5 Objectives of this study......................................................................................35 1.5.1 Background....................................................................................................35 1.5.2 Hypotheses....................................................................................................36 1.5.3 General objectives .........................................................................................36 1.5.4 Specific objectives .........................................................................................36 2 Quantitative identification and seasonal fluctuations of microbial diversity in fènè , a spontaneously fermented Malian sour milk....................................................................37 2.1 Abstract .............................................................................................................38 2.2 Keywords...........................................................................................................39 2.3 Introduction........................................................................................................39 2.4 Materials and methods.......................................................................................40 2.4.1 Sample collection...........................................................................................40 2.4.2 Microbiological analysis .................................................................................41 2.4.3 Bacterial DNA extraction and molecular typing strategy to strain level ...........42 4/156 2.4.4 Typing at strain level ......................................................................................44 2.4.5 Statistical analysis..........................................................................................45 2.5 Results ..............................................................................................................47 2.5.1 Bacterial composition and pH of fènè .............................................................47 2.5.2 Microbiological analysis during spontaneous fènè fermentation processes....47 2.5.3 Molecular typing at genus, species and strain level........................................50 2.5.4 Strain diversity of spontaneous fènè fermentation processes.........................53 2.6 Discussion .........................................................................................................56 2.7 Acknowledgement .............................................................................................58 3 Seasonal-dependent antibiotic resistance prevalence of enterococci from the production of fènè , a spontaneously fermented milk of Mali ..........................................59 3.1 Abstract .............................................................................................................60 3.2 Introduction........................................................................................................60 3.3 Material and methods ........................................................................................61 3.3.1 Bacterial strains .............................................................................................61 3.3.2 Phenotypic antibiotic resistance profiling........................................................61 3.3.3 Genotypic antibiotic resistance profiling .........................................................62 3.3.4 Transferability of antibiotic resistance genes..................................................62 3.4 Results ..............................................................................................................64 3.4.1 Phenotypic antibiotic resistance profiling and seasonal differences ...............64 3.4.2 Genotypic antibiotic resistance profiling .........................................................65 3.4.3 Transferability of antibiotic resistance genes..................................................65 3.5 Discussion .........................................................................................................69 3.6 Acknowledgement .............................................................................................70 4 Antibiotic resistance of non-Enterococcus lactic acid bacteria from spontaneous sour milk (fènè) production in Mali.................................................................................72 4.1 Abstract .............................................................................................................73 4.2 Introduction........................................................................................................73
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