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Understanding of Stuck Fermentations: Investigations on the Relationship Between the Microbial Diversity and Chemical Composition of Must

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Understanding of Stuck Fermentations: Investigations on the Relationship Between the Microbial Diversity and Chemical Composition of Must Institut für Mikrobiologie und Weinforschung Understanding of stuck fermentations: investigations on the relationship between the microbial diversity and chemical composition of must Dissertation Zur Erlangung des Grades Doktor der Naturwissenschaften Am Fachbereich Biologie Der Johannes Gutenberg-Universität Mainz vorgelegt von Eva Christ geboren am 21.12.1981 in Frankfurt am Main Mainz, im Januar 2015 Erklärung Erklärung Die Experimente dieser Arbeit wurden von Mai 2010 bis September 2014 am Institut für Mikro- biologie und Weinforschung der Johannes Gutenberg-Universität Mainz durchgeführt. Ich erkläre, dass ich die Arbeit selbstständig angefertigt und nur die angegebenen Hilfsmittel benutzt habe. Alle Stellen, die dem Wortlaut oder dem Sinn nach anderen Werken, gegebenen- falls auch elektronischen Medien, entnommen sind, sind von mir durch Angabe der Quelle als Entlehnung kenntlich gemacht. _____________ ________________ Ort, Datum Eva Christ Dekan: 1. Berichterstatter: 2. Berichterstatter: Tag der mündlichen Prüfung: 19.02.2015 Vineyard `Winninger Uhlen`of a winery from the upper Moselle in Germany Abbreviations Abbreviations % percent °C degree Celsius µg microgram µl microliter µM micromolar Ala alanine Arg arginine AS amino acid Asp aspartic acid BLAST Basic Local Alignment Search Tool bp base pair CAT8 zinc cluster-encoding gene CBS Centraalbureau voor Schimmelcultures, Utrecht cf. compare CFU colony forming units cm centimeter COX2 gene for the subunit II of the cytochrome c oxidase CRP Centre de Recherche Public Gabriel Lippmann CYR1 gene for the adenylate cyclase d days DNA desoxyribonucleic acid dNTP desoxy nucleotide triphosphate d.o.f. day of fermentation DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig/Germany (German Collection of Microorgansims and Cell Cultures) eH2O deionized water et al. and others Fig. figure FMOC-Cl fluorenylmethyloxycarbonyl chloride g gravity g gram Glu glutamic acid GPYA glucose - peptone - yeast extract - agar Abbreviations GSY1 gene for the glycogen synthase GYP glucose - yeast extract - peptone h hour H. Hanseniaspora His histidine HPLC high performance liquid chromatography IMW Institute for Microbiology and Wine Research, Mainz/Germany ITS internal transcribed spacer kg kilogram L liter Lb. Lactobacillus Lc. Leuconostoc M molar MALDI-TOF-MS matrix assisted laser desorption/ionization – time of flight – mass spectrometry Met methionine MET6 gene for the cobalamin-independent methionine synthase mg milligram min minute ml milliliter mM millimolar NCBI National Center of Biotechnology Information n.d. not detected nm nanometer O. Oenococcus OD optical density OPY1 overproduction-induced pheromone-resistant yeast gene P. Pediococcus PDA potato – dextrose – agar PCR polymerase chain reaction rDNA ribosomal desoxyribonucleic acid resp. respectively RFLP restriction fragment length polymorphism rpm revolutions per minute S. Saccharomyces SAPD specifically amplified polymorphic DNA Abbreviations SCAR sequence characterized amplified region Ser serine T type strain Tab. table Taq Thermophilus aquaticus TBE TRIS – Borate – EDTA Thr threonine TJ tomato juice medium Trp tryptophan TSA tryptone – soja – agar UV ultra violet V volt var. variety YAN yeast assimilable nitrogen YPM yeast extract – peptone – mannitol Contents Contents 1 Introduction ................................................................................................................................................................ 1 1.1 Spontaneous fermentation versus other fermentation procedures .......................................... 1 1.1.1 Succession of yeasts during spontaneous fermentation ....................................................... 3 1.1.2 Succession of bacteria during spontaneous fermentation ................................................... 4 1.2 Chemical composition of must and wine .............................................................................................. 5 1.3 Reasons for stuck fermentation ................................................................................................................ 5 1.4 Identification of Saccharomyces species in winemaking ................................................................ 7 1.5 Goals of the investigations .......................................................................................................................... 8 2 Material and methods ............................................................................................................................................. 9 2.1 Organisms .......................................................................................................................................................... 9 2.2 Biochemicals, kits and enzymes ............................................................................................................... 9 2.3 Equipment and accessories ..................................................................................................................... 10 2.4 Sampling .......................................................................................................................................................... 11 2.5 Cultivation of microorganisms ............................................................................................................... 11 2.6 Culture media ................................................................................................................................................ 12 2.6.1 Media for cultivation of yeasts ..................................................................................................... 12 2.6.2 Media for cultivation of bacteria .................................................................................................. 12 2.6.3 Synthetic medium for growth experiments ............................................................................ 13 2.7 Micromanipulation...................................................................................................................................... 15 2.8 DNA isolation ................................................................................................................................................. 16 2.8.1 DNA isolation of yeasts .................................................................................................................... 16 2.8.2 DNA isolation of bacteria ................................................................................................................ 16 2.9 Polymerase chain reaction (PCR) ......................................................................................................... 16 2.9.1 Amplification ........................................................................................................................................ 16 2.9.2 Multiplex PCR ...................................................................................................................................... 17 2.9.3 Specifically amplified polymorphic DNA (SAPD)-PCR........................................................ 17 Contents 2.10 Restriction enzyme digestion ................................................................................................................. 19 2.11 Yeast assimilable nitrogen ....................................................................................................................... 19 2.12 Sugar, alcohol and organic acid analysis ............................................................................................ 19 3 Results ........................................................................................................................................................................ 21 3.1 Succession of yeasts .................................................................................................................................... 21 3.1.1 Differentiation of Hanseniaspora uvarum and Saccharomyces bayanus...................... 21 3.1.2 Succession of yeasts in the first vintage 2011/2012 ........................................................... 22 3.1.3 Succession of yeasts in the second vintage 2012/2013..................................................... 23 3.2 Succession of bacteria ................................................................................................................................ 31 3.2.1 Succession of bacteria in the first vintage 2011/2012 ....................................................... 34 3.2.2 Succession of bacteria in the second vintage 2012/2013 ................................................. 34 3.3 Identification of lactic acid bacteria via multiplex PCR ............................................................... 35 3.4 Characterization of the isolated yeasts ............................................................................................... 39 3.4.1 Sugar consumption and growth curves at a glucosee:fructose ratio of 1:1 ............... 39 3.4.2 Growth and sugar consumption in relation to the ammonium concentration ......... 41 3.4.3 Growth and sugar consumption in relation to amino acid concentrations ............... 46 3.4.4 Growth and sugar consumption in relation to lacking amino acids ............................. 50 3.5 Chemical composition of must and young wine ............................................................................. 56 3.5.1 Concentration of yeast assimilable nitrogen .........................................................................
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