ALMA MATER STUDIORUM – UNIVERSITÀ DI BOLOGNA FACOLTA’ DI AGRARIA DOTTORATO DI RICERCA IN SCIENZE E BIOTECNOLOGIE DEGLI ALIMENTI Triennio Accademico 2007/2009 - XXII Ciclo di Dottorato Settore scientifico-disciplinare: AGR/15 Study of the components of quality in SO 2-free wines obtained by innovative vinification protocols Evaluation of the pre-fermentative addition of lysozyme and oenological tannins Tesi presentata da Francesca Sonni Coordinatore: Tutor: Prof. Claudio Cavani Prof. Claudio Riponi Co-Tutor: Dott. Fabio Chinnici Esame finale anno 2010 I Table of Contents INTRODUCTION...................................................................................................................... 1 CHAPTER 1 . Interactions between hen egg white lysozyme and some of the principal macro- components of musts and wines 1. Introduction ...................................................................................................................... 3 1.1 Activity of lysozyme ................................................................................................ 8 1.2 Effect of lysozyme on some bacterial strains........................................................... 9 1.3 Lysozyme as a preservative in food ....................................................................... 10 1.3.1 Meat............................................................................................................... 10 1.3.2 Cheese ........................................................................................................... 12 1.3.3 Fermented beverages..................................................................................... 13 1.4 Lysozyme activity in winemaking.......................................................................... 13 1.4.1 Effect on musts and wine bacterial strain...................................................... 13 1.4.2 Effect of some must and wine components in model solution...................... 14 1.4.3 Effect of fining agents and adjuvants on lysozyme activity.......................... 15 1.4.4 Legal framework ........................................................................................... 17 2. Experimental .................................................................................................................. 18 2.1 Wine model solutions............................................................................................. 18 2.2 Chemicals and Standards........................................................................................ 19 2.3 Quantification of the residual protein..................................................................... 19 2.4 Quantification of the enzyme activity .................................................................... 20 3. Results and Discussion................................................................................................... 21 3.1 Ethanol.................................................................................................................... 22 3.2 Sulphur dioxide ...................................................................................................... 22 3.3 Tannins ................................................................................................................... 23 3.4 Pectins.................................................................................................................... .24 3.5 Polyphenolic extract ............................................................................................... 26 4. Conclusions .................................................................................................................... 27 5. References ...................................................................................................................... 28 I CHAPTER 2. Replacement of sulphur dioxide by lysozyme and oenological tannins during fermentation: influence on volatile composition of white wines 1. Introduction .................................................................................................................... 35 2. Experimental .................................................................................................................. 38 2.1 Fermentations......................................................................................................... 38 2.2 Oenological parameters.......................................................................................... 39 2.3 Chemicals and Standards ....................................................................................... 39 2.4 HPLC Analysis....................................................................................................... 40 2.5 GC Analysis ........................................................................................................... 40 2.6 GC-MS Analysis .................................................................................................... 40 2.7 Sensorial Analysis.................................................................................................. 41 2.8 Statistical Analysis ................................................................................................. 41 3. Results and Discussion................................................................................................... 41 3.1 General parameters of final wines.......................................................................... 41 3.2 Volatile characterisation of final wines.................................................................. 42 3.2.1 Alcohols ........................................................................................................ 44 3.2.2 Esters ............................................................................................................. 45 3.2.3 Acids.............................................................................................................. 46 3.2.4 Principal component analysis........................................................................ 50 3.2.5 Sensory evaluation ........................................................................................ 50 4. Conclusions .................................................................................................................... 53 5. References ...................................................................................................................... 53 CHAPTER 3. Fermentation of sulphite-free white musts with added lysozyme and oenological tannins: nitrogen consumption and biogenic amines composition of final wines 1. Introduction .................................................................................................................... 58 2. Experimental .................................................................................................................. 60 2.1 Chemicals and Standards ....................................................................................... 60 2.2. Vinification............................................................................................................ 60 2.3. Assimilable nitrogen and ratio of nitrogen compounds ........................................ 61 2.4. Analysis of free amino acids and biogenic amines ............................................... 61 2.4.1. Reaction of Derivatization ........................................................................... 61 2.4.2. HPLC Analysis............................................................................................. 62 2.5. Statistical Analysis ................................................................................................ 62 II 3. Results and Discussion................................................................................................... 64 3.1 Amino acid composition of must ........................................................................... 64 3.2 Yeast Assimilable Nitrogen (YAN) of must.......................................................... 64 3.3 Nitrogen consumption during fermentation ........................................................... 66 3.4 Biogenic amines content of final wines ................................................................. 72 4. Conclusions .................................................................................................................... 73 5. References ...................................................................................................................... 73 CHAPTER 4. Replacement of sulphur dioxide by lysozyme and oenological tannins during fermentation: influence of bottle storage on the evolution of volatile compounds of white wines 1. Introduction .................................................................................................................... 78 1.1 Primary and Secondary Aroma .............................................................................. 79 1.2 Aroma compounds formed during fermentation.................................................... 80 1.2.1 Volatile Acids................................................................................................ 81 1.2.2 Alcohols (Higher alcohols, Ethanol)............................................................. 82 1.2.3 Esters ............................................................................................................. 85 1.2.4 Carbonyl compounds..................................................................................... 90 1.2.5 Sulphur compounds (Sulfides,