Microbial Consortia from Smear-Ripened Cheese: Biodiversity, Incidence of Commercial Starter Microorganisms and Anti-Listerial Activity of Yeasts
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ABTEILUNG MIKROBIOLOGIE ZENTRALINSTITUT FÜR ERNÄHRUNGS- UND LEBENSMITTELFORSCHUNG WEIHENSTEPHAN TECHNISCHE UNIVERSITÄT MÜNCHEN Microbial consortia from smear-ripened cheese: Biodiversity, incidence of commercial starter microorganisms and anti-listerial activity of yeasts STEFANIE GOERGES 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 Haushalts- und Ernährungswissenschaften (Dr. oec. troph.) genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. G. Cerny Prüfer der Dissertation: 1. Univ.-Prof. Dr. S. Scherer 2. Univ.-Prof. Dr. K. Heller (Christian-Albrechts-Universität Kiel) Die Dissertation wurde am 18.08.2005 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 12.10.2005 angenommen. Acknowledgments The present work has been performed at the “Abteilung Mikrobiologie, Zentralinstitut für Ernährungs- und Lebensmittelforschung (ZIEL) Weihenstephan, TU München“ under supervision of Prof. Dr. Siegfried Scherer. Special thanks go to my supervisor Siegfried Scherer for his excellent support, for giving me the possibility to follow my own ideas and to continue research on anti-listerial properties in yeast although this task was canceled ahead of schedule in the SCM project. I am very grateful to Prof. Dr. Knut Heller (Christian-Albrechts-Universität, Kiel) for being my second examiner. Furthermore, I am very thankful to Dr. Barbara Silakowski and Ulrike Aigner for their contribution to the section on anti-listerial yeast. My cordial thanks also go to Dr. Roberto Gelsomino (BCCM/LMG Bacteria Collection, University of Ghent, Belgium), Jérôme Mounier (Dairy Products Research Centre, Teagasc, Fermoy, Co. Cork, Ireland) and Mary Rea (Biotechnology Department, Moorepark Food Research Centre, Teagasc, Fermoy, Co. Cork, Ireland). Their reliability enabled an excellent cooperation. Sincere thanks are given to all of my colleagues, especially to these with whom I shared office and labs, for providing a pleasant working atmosphere. The freely given technical assistance of Lisa Rieder and Andreas Bischof is also greatly appreciated. Special thanks are addressed to Dr. Kinga Jakob and Dr. Mareike Wenning for fruitful discussion, helpful advices and critical reading of my manuscript. Finally, I would like to cordially thank my parents and especially Sebastian Müller for their interest in my Ph.D. work and their constant, infinite encouragement and support throughout this time. This work was partially supported by the EU, project QLK1-CT-2001-02228: Biodiversity and anti-listerial activity of surface microbial consortia from Limburger, Reblochon, Livarot, Tilsit and Gubbeen cheese; Acronym: SCM (Smear cheese microflora), and by the “Vereinigung zur Förderung der Milchwissenschaftlichen Forschung an der Technischen Universität München in Freising - Weihenstephan e.V.”. Table of contents II Table of contents Acknowledgments.......................................................................................I Table of contents........................................................................................II List of figures ............................................................................................IV List of tables..............................................................................................VI List of abbreviations ............................................................................... VIII Summary ..................................................................................................XI Zusammenfassung .................................................................................. XIV 1 Introduction .....................................................................................1 1.1 Smear-ripened cheese .............................................................................1 1.2 Taxonomy and identification of ripening organisms .........................................5 1.3 Listeria ................................................................................................8 1.4 Biological preservation of food................................................................. 10 1.5 Anti-bacterial, in particular, anti-listerial activity of smear-ripening organisms ...... 12 1.6 Objective .......................................................................................... 15 2 Materials and methods ..................................................................... 16 2.1 Determination of yeast populations from different European smear-ripened cheeses ............................................................................................. 16 2.2 Production of Limburger cheese............................................................... 17 2.3 Isolating microorganisms from the surface of Limburger cheeses ....................... 18 2.4 Isolating microorganisms from a commercial smear starter culture..................... 19 2.5 Cultivation and maintenance ................................................................... 20 2.6 Fourier transform infrared (FTIR) spectroscopy ........................................... 21 2.7 Physiological tests on yeasts .................................................................... 22 2.8 Sequence analysis of the D1/D2 domain of the 26S rDNA............................... 25 2.9 Screening for anti-listerial activity in food-borne yeast.................................... 26 3 Results ............................................................................................ 32 3.1 Characterization of yeast populations from different European smear-ripened cheeses ............................................................................................. 32 3.2 Biodiversity of surface microbial consortia from Limburger cheese..................... 33 3.2.1 Cell counts......................................................................................... 33 3.2.2 Identification and differentiation of yeast isolates by FTIR spectroscopy............... 34 3.2.3 Phenotypic characterization of yeast isolates ................................................ 37 3.2.4 Identification and differentiation of bacterial isolates by molecular based methods ............................................................................................ 37 3.2.5 Identification and differentiation of coryneform isolates by FTIR spectroscopy ...... 40 3.2.6 Pulsed field gel electrophoresis on coryneform isolates ................................... 41 Table of contents III 3.3 Incidence of commercial surface starter microorganisms during ripening of a Limburger cheese ................................................................................ 42 3.3.1 Detailed analysis of the smear starter organisms............................................ 46 3.3.2 Incidence of the smear starters in isolates of a further Limburger batch................ 49 3.3.3 Typing of yeast isolates by molecular based methods ...................................... 52 3.3.4 Typing of bacterial isolates by molecular based methods.................................. 55 3.4 Anti-listerial activity of food-borne yeast .................................................... 59 3.4.1 Agar-membrane screening assay............................................................... 59 3.4.2 Inhibition of L. monocytogenes in a yeast-co-cultivation assay .............................. 64 3.4.3 Test for killer activity............................................................................ 65 4 Discussion....................................................................................... 66 4.1 Characterization of yeast populations from different European smear-ripened cheeses ............................................................................................. 66 4.1.1 Species diversity on the individual cheeses................................................... 66 4.1.2 Intraspecies variability ........................................................................... 69 4.2 Biodiversity of surface microbial consortia from Limburger cheese..................... 72 4.2.1 Yeast and bacterial cell counts ................................................................. 72 4.2.2 Biodiversity within the yeast flora ............................................................. 73 4.2.3 Biodiversity within the bacterial flora......................................................... 74 4.2.4 Characterization of the microflora by a polyphasic approach............................. 76 4.3 Incidence of commercial surface starter microorganisms during ripening of a Limburger cheese ................................................................................ 76 4.3.1 Incidence of the yeast starters .................................................................. 77 4.3.2 Incidence of the bacterial starters.............................................................. 80 4.3.3 Congruence and suitability of the phenotypic and genotypic methods used ........... 81 4.3.4 Development and application of defined starter cultures ................................. 82 4.4 Anti-listerial activity of food-borne yeast .................................................... 84 4.4.1 Inhibition of L. monocytogenes in the agar-membrane based screening assay ............ 84 4.4.2 Inhibition of L. monocytogenes in the co-cultivation