Microbiological approaches to reduce the sulphite addition in oenology

215 Pages·2013·4.01 MB·English

Sede Amministrativa: Università degli Studi di Padova
Dipartimento di Agronomia Animali Alimenti Risorse Naturali e Ambiente

DOTTORATO DI RICERCA IN
Viticoltura, Enologia e Marketing delle Imprese Vitivinicole
CICLO XXV

Microbiological approaches to reduce the
sulphite addition in oenology
---
Approcci microbiologici per la riduzione dei
solfiti in enologia

Coordinatore : Ch.mo Prof. Viviana Corich
Supervisore :Ch.mo Prof. Viviana Corich

Dottoranda : Chiara Nadai

Table of contents
TABLE OF CONTENTS

ABSTRACT ......................................................................................................... 7
1. Introduction .................................................................................................... 11
1.1 Sulphur dioxide properties........................................................................ 11
1.2 Cellular uptake of SO .............................................................................. 13
2
1.3 Toxic impact of SO2 on the yeast cell ...................................................... 16
1.4 Resistance to SO in wine yeasts ............................................................. 17
2
1.5 Cellular and molecular response of yeast cells exposed to sulphur dioxide
...................................................................................................................... 18
1.5.1 Reduction of sulphur dioxide ................................................................. 19
1.5.2 Oxidation ............................................................................................... 21
1.5.3 Production of acetaldehyde ................................................................... 21
1.5.4 Sulphitolysis .......................................................................................... 22
1.5.5 Intracellular accumulation and active efflux ........................................... 23
1.5.6 Global view of sulphite resistance at cellular level ................................. 26
1.6 Project outline .......................................................................................... 29
2. Characterization of sulphite production and sulphite resistance in
Saccharomyces cerevisiae isolated from vineyard ............................................. 31
2.1 Introduction .............................................................................................. 31
2.1.1 Ecology of wine yeasts .......................................................................... 31
2.1.1.1 Origin of wine yeasts ....................................................................... 32
2.1.1.2. Use of selected yeasts for oenological purposes............................ 33
2.1.1.3. Genomic characteristics of wine yeast ........................................... 34
2.2 Selection of yeasts in oenology: commercial and autochthonous wine
strains ............................................................................................................ 35
2.2.1. Selection of ecotypical yeast strains ..................................................... 37
2.2.1.1. Screening methods based on fitness traits ..................................... 38
2.2.1.2. Screening methods based on quality traits ..................................... 40
2.3. Selection of autochthonous wine yeasts isolated from vineyards in Veneto
region ............................................................................................................. 40
2.4 Materials and methods ............................................................................. 43
2.4.1 Yeasts ................................................................................................... 43
2.4.2 Culture media and growth condition ...................................................... 43
Media.......................................................................................................... 43
Growth conditions ....................................................................................... 45
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2.5 Results and discussion ............................................................................ 46
2.5.1 Sulphuric acid production ...................................................................... 46
2.5.2 Sulphur dioxide production .................................................................... 47
2.5.3 Sulphite resistance ................................................................................ 48
2.7 Conclusions ............................................................................................. 51
3. Genomic features of yeasts with sequenced genome .................................... 53
3.1 Introduction .............................................................................................. 53
3.1.1 Genetic Characteristics ......................................................................... 53
3.1.2 Chromosomal Rearrangements and SNPs ............................................ 54
3.1.3 Finishing and gene prediction ................................................................ 55
3.2. From genotype to phenotype................................................................... 56
3.3 Next generation sequencing technology ................................................... 57
3.3.1 Phylogenetic Relationship ..................................................................... 58
3.4 Materials and methods ............................................................................. 60
3.4.1 Strains ................................................................................................... 60
3.4.2 DNA isolation ........................................................................................ 64
3.4.3 Real-time analyses performed to verify translocations........................... 64
3.4.5 Real-time analyses performed on strain-specific genes and high
resolution melting analyses on SNPs. ............................................................ 65
3.5 Results and discussions ........................................................................... 67
3.5.1 Single nucleotide variations in enological strains ................................... 67
3.5.2 Gene finding and annotation ................................................................. 72
3.5.3 Genome finishing and structural variations ............................................ 75
3.6 Conclusions ............................................................................................. 79
4. Phenotypic characterization of yeasts with sequenced genome ..................... 81
4.1 Introduction .............................................................................................. 81
4.1.1 Fitness traits .......................................................................................... 82
4.1.1.1 Main fermentation properties ........................................................... 82
4.1.1.2. Main technological properties ......................................................... 83
4.1.2. Quality traits ......................................................................................... 85
4.1.2.1. Flavour characteristics ................................................................... 85
4.1.2.2. Metabolic properties that influence wine safety .............................. 86
4.2 Yeast sequencing..................................................................................... 87
4.3 Materials and methods ............................................................................. 90
4.3.1 Yeasts ................................................................................................... 90
4.3.2 Culture media and growth condition ...................................................... 90
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4.3.3 Fermentation surveys on Synthetic Nutrient Medium (NSM) (Delfini,
1995) ............................................................................................................. 92
4.3.3.1 Yeasts inoculum preparation ........................................................... 92
4.3.3.2 Test preparation .............................................................................. 93
4.3.4 Ethanol production ................................................................................ 93
4.3.5 Chemical analysis on fermented must ................................................... 94
4.3.6 Statistical analysis ................................................................................. 94
4.4 Results and discussions ........................................................................... 95
4.4.1 Fermentative performance and technological strains characterization in
synthetic must ................................................................................................ 95
4.4.1.1 Fermentation kinetics and ethanol production ................................. 95
4.4.1.2 Fermentative vigour ........................................................................... 96
4.4.1.3 Glycerol production ......................................................................... 98
4.4.2 Fermentative power .............................................................................. 99
4.4.3 Sulphite metabolism ............................................................................ 101
4.4.3.1 Hydrogen sulphide and sulphur dioxide production ....................... 101
4.4.3.2 Sulphite tolerance ......................................................................... 103
4.4.3.3 Acetaldehyde production ............................................................... 105
4.4.3.4 Sulphur dioxide production ............................................................ 106
4.5 Conclusions ........................................................................................... 109
5. Effects of SO on yeast metabolism and changes in the transcriptional profiles
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........................................................................................................................ 111
5. 1 Introduction ........................................................................................... 111
5.1.1 RNA Sequencing ................................................................................ 111
5.1.2 Gene expression studies of yeasts in the presence of sulphites .......... 112
5.2 Matherials and methods ......................................................................... 114
5.2.1 Yeasts ................................................................................................. 114
5.2.2 Culture media .................................................................................. 114
5.2.3 Fermentation surveys on Synthetic Nutrient Medium (NSM) (Delfini,
1995) ........................................................................................................... 117
5.2.3.1 Yeasts inoculum preparation ......................................................... 117
5.2.3.2 Test preparation ............................................................................ 117
5.2.4 Chemical analysis on fermented must ................................................. 118
5.2.5 Statistical analysis ............................................................................... 118
5.2.6 Fermentation in Controlled Bioreactors ............................................... 118
5.2.6.1 Yeasts inoculum preparation ......................................................... 118
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5.2.6.2 Fermentation preparation ................................................................. 119
5.2.7 Cellular pellet sampling ....................................................................... 119
5.2.8 Total RNA extraction ........................................................................... 119
5.2.9 RNA quantification and gel electrophoresis ......................................... 120
5.2.10 rRNA Subtraction .............................................................................. 121
5.2.11 SOLiD Libraries preparation .............................................................. 121
5.2.11.1 Emulsion PCR and beads enrichment ......................................... 122
5.2.12 Sequencing with the SOLiD system .................................................. 123
5.2.13 Hierarchical Clustering using TMEV .................................................. 125
5.2.13 Gene Ontology .................................................................................. 126
5.3 Results and discussion .......................................................................... 128
5.3.1 Evaluation of fermentation kinetics in the presence of different
concentrations of sulphites ........................................................................... 128
5.3.1.1 Fermentative vigour ...................................................................... 131
5.3.1.2 Total sulphur dioxide production ................................................... 133
5.3.1.3 Acetaldehyde production ............................................................... 135
5.3.2 Strains selection and fermentations in MS300 synthetic must ............. 137
5.3.3 RNA-seq results .................................................................................. 141
5.3.4 GO Classes Enriched .......................................................................... 143
6. Selection of reference genes for quantitative real-time PCR studies in
Saccharomyces cerevisiae during alcoholic fermentation in presence of sulphite
........................................................................................................................ 155
6.1 Introduction ............................................................................................ 155
6.1.1 Reference genes as golden standard for normalization ....................... 155
6.2 Materials and methods ........................................................................... 159
6.2.1 Yeasts ................................................................................................. 159
6.2.2 Culture media ...................................................................................... 159
6.2.3 Fermentation in Controlled Bioreactors ............................................... 161
6.2.3.1 Yeasts inoculum preparation ......................................................... 161
6.2.3.2 Fermentation preparation .............................................................. 161
6.2.4 Cellular pellet sampling ....................................................................... 161
6.2.5 Total RNA extraction ........................................................................... 162
6.2.6 RNA quantification and gel electrophoresis ......................................... 162
6.2.7 Reverse transcription (RT-PCR) .......................................................... 163
6.2.7.1. DNAse treatment ......................................................................... 163
6.2.7.2 Synthesis of cDNAs for PCR amplification .................................... 163
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6.2.8 Polymerase Chain Reaction and gel electrophoresis .......................... 164
6.2.9 Real-time analyses .............................................................................. 165
6.3 Results and discussion .......................................................................... 166
6.3.1 Determination of the reference gene candidates ................................. 166
6.3.2 Design of primers ................................................................................ 170
6.3.3 Fermentation trials .............................................................................. 171
6.3.4 Real-time PCR amplification of reference genes ................................. 172
6.3.5 Data analysis ...................................................................................... 173
6.3.5.1 geNorm ......................................................................................... 174
6.3.5.2 NormFinder ................................................................................... 177
6.3.5.3 BestKeeper ................................................................................... 178
6.4 Conclusions ........................................................................................... 183
7. CONCLUSIONS .......................................................................................... 185
References ...................................................................................................... 189
Acknowledgements .......................................................................................... 213
Table of contents
Abstract
ABSTRACT

Sulphite is widely used in winemaking for its antimicrobial and antioxidant
properties, although its toxic effect on human health is proven. For this reason
strategies for reducing chemical preservatives in winemaking is strongly
demanded. Wine yeasts can cope with SO by different systems, such as
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acetaldehyde and S-amino acids production or SO export.
2
In this study a first screening of SO resistance and on plate production of SO
2 2
and H S have been performed for autochthonous strains, isolated in Veneto
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vineyards to be used as starter of fermentation in the production of Prosecco di
Valdobbiadene DOCG and DOC Piave wines, compared to commercial strains.
Then the oenological characteristics of 11 S. cerevisiae strains of have been
evaluated. These strains are 4 autochthonous strains isolated during local
selection projects in DOCG Conegliano - Valdobbiadene and DOC Piave areas,
together with 6 commercial strains, which genome have been recently
sequenced, and relative informations are available in the principal genomic
databases, and the laboratory strain S288c, the first one that has been
sequenced. Main technology and quality characters have been evaluated to
determine the suitability of strains for winemaking process. In particular have
been studied the production of ethanol and glycerol, the glucose consumption at
2 and 7 days, the production of hydrogen sulphide, acetaldehyde and sulphur
dioxide and the resistance to various concentrations of free sulphur dioxide in
synthetic must. Sulphite response in yeast has been investigated in order to
elucidate factors that affect sulphite production during vinification. Moreover
acetaldehyde, another compound produced by yeast, linked with sulphite
metabolism or detoxification, has been analysed since it affects wine quality.
Genetic characteristics identified after genome sequencing of 4 autochthonous
strains (2 from Prosecco area and 2 from Raboso area), such as oenological
SNPs, strain-specific genes and important translocations, have been analyzed in
Real-time PCR for a large number of autochthonous strains.
Then the behaviour towards sulphite of 4 wine yeasts has been investigated,
and transcriptome analysis during fermentation has been performed by
means of next generation sequencing. For all strains, fermentation rate was
monitored together with sulphite production in synthetic must supplemented
with different doses of SO (0 mg/l and 25 mg/l).
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Abstract
Finally, a selection of reference genes for Real-time PCR has been made, and a
set of genes suitable for such conditions has been identified.
Results point out the importance of verifying strain attitudes towards sulphite at
different sulphite concentrations. This study tries to clarify the complex regulative
mechanisms of sulphites during fermentation, thus giving new guidelines for critic
control of these fermentation parameters in order to maximize effect of sulphite
added thus limiting the dose employed during vinification.

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