Università di Pisa, Scuola di Dottorato in Scienze Agrarie e Veterinarie
Programma di “Produzioni Vegetali” (26° ciclo)
AGR/16
Tesi di Dottorato di Ricerca- PhD dissertation
“Wine yeast biodiversity during spontaneous
fermentation in response to environmental stress”
“Biodiversità di lieviti vinari nella fermentazione spontanea
in risposta a stress ambientali”
Candidate: Tilde Labagnara
(tilde.labagnara@for.unipi.it)Supervisors Dr. Annita Toffanin
Prof. Giancarlo Scalabrelli
PhD Coordinator Prof. Alberto Pardossi
Grande è la fortuna di colui che possiede una buona bottiglia,
un buon libro, un buon amico.
Molière
Ai miei familiari
perché sono contenta di poter dare loro questa gioia, perché ci provo di continuo, non sempre riuscendoci purtroppo, a essere la figlia, sorella, nipote e compagna perfetta che si meritano; con grande sostegno mi hanno permesso di raggiungere questo importante obiettivo, infondendo la passione del vino sin da piccola con il loro duro lavoro. Li ringrazio non solo per il loro sostegno morale, ma soprattutto per il loro impegno materiale, e per tutti i sacrifici che sono serviti a portarmi davanti a questo traguardo.
Ringrazio mio papà, per i suoi insegnamenti e per la voglia di trasmettere quotidianamente la passione e l’infinito amore per la vigna e il vino.
Un grazie di cuore alla mia incredibile super-mamma, che mi ha insegnato ad avere tanta forza di volontà per superare le piccole e grandi difficoltà della vita, come ha straordinariamente fatto lei.
Ringrazio il mio Marcello, che con estrema “pazienza” ha sopportato i miei sbalzi di umore e le mie paranoie quando, sotto stress, non avevo altra valvola di sfogo che lui; mi ha sempre incoraggiato dicendomi che potevo farcela. Grazie di cuore per il suo prezioso sostegno, garantendomi sempre uno spiraglio di luce anche quando intorno avevo solo buio.
RIASSUNTO 8
AIM OF THE WORK 9
Chapter 1
Identification and molecular characterization of wine yeasts
INTRODUCTIONIntroduction to organic and biodynamic farming systems 10
Biodynamic agriculture 10
Natural wine versus organic wine 11
Natural wine 11
Biodiversity of wine yeasts 12
Non-Saccharomyces yeasts in wine 13
Spontaneous fermentation 14
Origin of Saccharomyces spp. and wine 15
Saccharomyces cerevisiae 16
The genome of S. cerevisiae 17
Genetic identification of yeast species 18
Ribotyping 19
Intra-specific differentiation of yeast strains 19
Minisatellite analysis as discriminating tool 20
The genes containing minisatellite-like sequences 21
Microsatellite-like sequences as selective tool for S. cerevisiae strains 22
The genes containing microsatellite-like sequences 23
MATERIALS AND METHODS
Sample collection 24
Yeast isolation 24
Composition of agar medium for the growth of microorganisms 24
Colonies growth in WL solid substrate 26
Estimation of population density 26
How to get pure isolates 27
Dna extraction 27
Species and strain determination 28
Amplification of nucleic acids 28
Amplification of ITS1-5,8S-ITS2 ribosomal DNA region 28
RFLP with HaeIII enzyme for discriminating S. cerevisiae vs S. bayanus 29 Characterization of S. cerevisiae strains through Mini-satellites amplification 30 Characterization of S. cerevisiae isolates using Microsatellite amplification multiplex PCR 32
Electrophoresis of nucleic acids on agarose gel 33
RESULTS AND DISCUSSION
Sample collection 34
Yeast isolation 34
Amplification of ITS1-5,8S-ITS2 ribosomal DNA region 35
RFLP with HaeIII enzyme for discriminating S. cerevisiae 37 Characterization of S. cerevisiae strains through Mini-satellites amplification 38 Characterization of Saccharomyces cerevisiae isolates using Micro-satellite amplification multiplex PCR
Technological characterization of S. cerevisiae wine yeasts
INTRODUCTIONGrape must composition and yeast nutritional requirements during the alcoholic fermentation 43
Yeast response to stress factors 44
Technological characterization of wine yeasts 44
Fitness traits 45
Principal fermentation properties 45
Main technological properties 46
Quality traits 47
Flavor characteristics 47
Introduction to the use of SO2 in winemaking 48
The chemistry of SO2 in wine 49
Effect of SO2 on growth of wine yeasts and Saccharomyces cerevisiae in wine 50
Mechanism of SO2 as antimicrobial action 51
Microbial SO2 resistance mechanisms 51
Molecular response to SO2 53
Nitrogen sources in grape must 54
Nitrogen compounds: mechanisms and assimilations 55
Ethanol stress affects microorganisms 56
MATERIALS AND METHODS
Commercial yeast strain 58
Biotypes isolated during the harvest of 2009 58
Medium of ordinary propagation 58
Medium composition for the phenotypic characterization of biotypes 59
Fitness trait: Killer activity test 60
Quality trait: Low sulphite formation 60
Preparation of starter cultures for fermentation trials in MNS 61 Preparation of starter cultures for fermentation test in natural must 61
Fermentation and weight loss of flasks 61
Chemical analysis of fermentation product 61
Determination of Volatile acidity through Gibertini method 62
Determination of ethanol 63
Determination of residual glucose at the end of fermentation 64
Determination of total sulphite in the final product 65
Determination of free sulphite content in the final product 66
Determination of Yeast Available Nitrogen (YAN) 67
Oxidation and assimilation tests through Biolog 68
Ethanol resistance 69
Resistance to sulphur dioxide 69
Statistical analysis 70
RESULTS AND DISCUSSION
Fitness trait: Killer activity test 71
Quality trait: Low sulphite formation 72
Biotypes fermentation and weight loss of flasks in MNS 73
Principal fermentation properties: fermentation (vigor) 75
Principal fermentation properties: fermentative power 76
Chemical analysis of fermentation product 77
Chemical analysis of fermentation product: Total and Volatile acidity 77 Chemical analysis of fermentation product: Residual glucose 78 Chemical analysis of fermentation product: Ethanol production 79 Chemical analysis of fermentation product: Nitrogen consumption 80 Chemical analysis of fermentation product: Sulphur compound (total SO2) 81 Chemical analysis of fermentation product: Sulphur compound (free SO2) 82
Principal fermentation properties (natural grape must): fermentative power 87
Chemical analysis of wines 88
Chemical analysis of wines: Total acidity 88
Chemical analysis of wines: Volatile acidity 89
Chemical analysis of wines: Residual glucose 90
Chemical analysis of wines: Ethanol production 91
Chemical analysis of wines: Nitrogen consumption 91
Chemical analysis of wines: Sulphur compound (total SO2) 92
Chemical analysis of wines: Sulphur compound (free SO2) 93
Ethanol resistance of biotypes 94
Resistance to sulphur dioxide 95
Oxidation and assimilation tests through Biolog 101
Chapter 3
Dynamics of S. cerevisiae during inoculated and spontaneous
fermentations in wine farms
INTRODUCTION
Caiarossa wine farm 103
Stefano Amerighi wine farm 104
Dominance behavior of Saccharomyces cerevisiae 104
Syrah grapevine and relative wine 105
Sensorial analysis of wine 106
Sensory analysis evaluation 107
Scientific methods for the Sensory Evaluation of Wine 108
Triangle and Duo-Trio test 108
Statistical analysis: PCA in sensorial analysis 109
MATERIALS AND METHODS
Fermentation trial of biotype 3A in wine farm Caiarossa 111
Preparation of starter culture of biotype 3A and inoculation in wine farm 111
Sample collection 111
Yeast isolation 111
Molecular characterization of isolates in grape must 111
Dominance of Saccharomyces cerevisiae 112
Fermentation trials in wine farm Az. Ag. Stefano Amerighi 113 Preparation of pre-starter culture with biotype 3B for fermentation in wine farm 113
Composition of enriched medium 114
Preparation of final starter culture of biotype 3B and inoculation in tank 114
Sensory analysis 115
Statistical analyses and Principal Component Analysis 115
RESULTS AND DISCUSSION
Fermentation trial of biotype 3A in wine farm Caiarossa 117
Selection of biotype for fermentation in wine farm 120
Fermentation trials in wine farm Stefano Amerighi 121
Biotypes presence during the alcoholic fermentation in wine farm Stefano Amerighi 122
Principal fermentation properties: fermentative Vigor 123
Principal fermentation properties: fermentative power 123
Chemical analysis of wines: Total acidity 124
Chemical analysis of wine: Volatile acidity 125
Chemical analysis of wine: Residual glucose 126
Chemical analysis of wine: Ethanol production 127
Chemical analysis of wine: Color intensity 127
Dominance of Saccharomyces cerevisiae 132
Sample collection 132
Yeast isolation 132
Molecular characterization of isolates in grape must 133
CONCLUSIVE REMARKS 136
PUBBLICATIONS 138
“Natural wines”, nowadays, could appear an evocative recall to what wine was like before. During the ages, science allowed winemakers to produce wines with the full control of vinification condition, obtained mainly by addition of exogenous components, supplements and microorganisms. Contrarily, “natural wine” come back to basics and simplicity as invasive operations are avoided; but, on the other hand, a deeper knowledge and monitoring of winemaking process is required, in order to obtain high quality products without exogenous substances. Natural wine has been produced by spontaneous fermentation of must by yeasts originated from grapes and winery equipment. The wide variety of natural yeasts reflects the biodiversity, which is still under-exploited despite the large use of commercial Saccharomyces cerevisiae in most grape musts. During fermentation, several strains compete in the same fermenting must, and the dominance of Saccharomyces cerevisiae takes place when it overcomes all the others. The aim was to investigate Saccharomyces cerevisiae diversity and its technological behavior in two biodynamic wine farms in Tuscany. Autochthonous S. cerevisiae from Syrah fermentations were isolated and molecularly characterized in 2009 and 2013 harvests. Saccharomyces cerevisiae strains were isolated starting from Syrah musts with zero or low levels of sulphite added, according with the tendency of natural wines producers to abait every invasive operation during wine making. Samples were collected in different steps of winemaking. ITS-PCR method confirmed isolates belong to Saccharomyces sensu stricto complex. The multiplex PCR amplification of microsatellite loci (SC8132X, YOR267C and SCPTSY7) discriminated S. cerevisiae strains. Genetically diverse S. cerevisiae strains were subsequently subjected to technological characterization. Micro-fermentations were set up to study fitness and quality traits of biotypes characterized. Weight loss kinetics were measured and chemical analyses were performed. Collected Saccharomyces cerevisiae strains were tested also in order to verify the effect of low sulphite additions on biodiversity pursuing information about stress-adaptation mechanisms that allow the survival in the challenging environment of fermenting must. Sulphite addition may act as a selective factor to induce the presence and the activity of strains with diverse features about sulphite metabolism. The addition of different dose of sulphite induced a considerable variability among strains. However, some biotypes exhibited tolerance to certain concentration and only one biotype was classified as resistant to a determinate concentration. In addition, biotypes were subjected to an other environmental stress factor such as ethanol. The increasing of levels of ethanol limited considerably the activity of selected strains, but some of them highlighted a positive tolerance to various ethanol concentrations. During the harvest of 2013, the alcoholic fermentation was conducted inoculating a biotype characterized previously, which expressed good fermentative performance and a multi-strains culture of other S. cerevisiae strains characterized in the same harvest (2009) in a biodynamic farm from a workgroup of Edmund Mach Foundation of San Michele all'Adige (TN). The results of 2009 highlighted peculiar behaviors of 13 biotypes (characterized from four hundred isolates) that dominate the spontaneous fermentations. Not surprisingly, some biotypes were also found in fermentations of 2013, demonstrating yeasts are ubiquitous in that environment. Sensorial analyses of wine, originated by biotypes fermentation, revealed certain diversity in aroma and flavor traits, but also in the structure. Overall, this work suggests the consolidate relationship existing between autochthonous yeasts and the terroir, where yeasts are ubiquitous. Furthermore, autochthonous yeasts surely contribute to the unique identity of wine. In addition, this work suggests that the use of isolated and characterized autochthonous yeast strains could help the winemaking process, where the vintage could be inadequate to develop a particular product such as the wine of a specific wine farm.
I "vini naturali”, oggi, potrebbero apparire un richiamo evocativo al vino del passato. Durante i secoli, la scienza ha reso possibile la produzione di vini ottenuti con il pieno controllo del processo di vinificazione, grazie principalmente ad aggiunte di componenti esogene, additivi e lieviti commerciali. Al contrario, la filosofia dei "vini naturali" riporta alla semplicità nella produzione, evitando operazioni invasive. D'altra parte, in queste condizioni, la conoscenza e il monitoraggio del processo fermentativo diventano basilari per ottenere prodotti di elevata qualità, pur senza l’ausilio di sostanze esogene. I “vini naturali” sono ottenuti grazie all’attività fermentativa dei lieviti presenti sulle uve e sulle attrezzature di cantina. Durante la fermentazione spontanea, molti ceppi di lievito competono nello stesso ambiente, e la dominanza di uno o più ceppi di Saccharomyces
cerevisiae si evidenzia quando questi prendono il sopravvento su altre specie o ceppi,
portando a completamento il processo fermentativo. Lo scopo del presente lavoro è lo studio della biodiversità di Saccharomyces cerevisiae, relative qualità enologiche e tecnologiche in due aziende biodinamiche in Toscana. Lieviti Saccharomyces cerevisiae sono stati isolati e quindi caratterizzati a partire dalle vendemmie 2009 e 2013. Gli isolati sono stati ottenuti da mosti Syrah fermentati in presenza di bassi o assenti livelli di solfiti aggiunti, secondo la tendenza dei produttori di vini naturali di eliminare o ridurre l’aggiunta di additivi. La tecnica molecolare ITS-PCR ha confermato l’appartenenza di almeno 100 ceppi al gruppo Saccharomyces sensu stricto. L’amplificazione di tre loci micro satelliti (SC8132X, YOR267C e SCPTSY7) ha consentito di discriminare diversi biotipi appartenenti alla specie Saccharomyces cerevisiae. I biotipi geneticamente diversi sono stati sottoposti a caratterizzazione enologica e sono state allestite micro - fermentazioni per studiarne le abilità fermentative e altre caratteristiche enologiche. La fermentazione è stata seguita valutando il calo in peso dovuto alla produzione di CO2 e i prodotti finali sono
stati sottoposti ad analisi chimiche. I biotipi di Saccharomyces cerevisiae sono stati successivamente sottoposti a diversi fattori di stress ambientale per verificare l’incidenza di SO2 ed etanolo sulla biodiversità in un ambiente complesso quale il mosto in
fermentazione. L’aggiunta di quantità diverse di solfiti ha evidenziato una variabilità considerevole tra i diversi biotipi. Ad ogni modo, alcuni biotipi hanno mostrato una certa tolleranza alle concentrazioni considerate e solamente un biotipo è stato classificato come resistente alla più alta concentrazione. L’aumento della concentrazione di etanolo ha limitato notevolmente l'attività dei biotipi selezionati, ma alcuni hanno mostrato un certo grado di tolleranza. Durante la vendemmia del 2013, sono state allestite prove fermentative in azienda, inoculando uno dei biotipi selezionati, a confronto con una cultura multi - ceppo messo a disposizione dalla “Fondazione Edmund Mach di San Michele all’Adige (TN) ”. I risultati della vendemmia del 2009 hanno evidenziato comportamenti particolari di tredici biotipi diversi di S. cerevisiae (caratterizzati partendo da almeno quattrocento isolati) che hanno dominato le fermentazioni spontanee. Alcuni dei biotipi precedentemente caratterizzati sono stati ritrovati nella fermentazione spontanea relativa alla vendemmia 2013. Le analisi sensoriali sui vini ottenuti dalle fermentazioni dei biotipi hanno rivelato una certa diversità nei tratti aromatici e sensoriali, ma anche nella struttura e nel corpo del vino. In conclusione, questo lavoro evidenzia la profonda relazione che esiste tra i lieviti autoctoni e l’ambiente in cui si trovano. L'utilizzo di biotipi autoctoni potrebbe rivelarsi utile in vendemmie problematiche o in condizioni inadeguate per sviluppare un vino particolare di una realtà vitivinicola. L’identificazione di lieviti autoctoni con caratteristiche enologiche d’interesse specifico alla produzione di “natural wines” apre la possibilità a interazioni positive nei confronti di realtà aziendali che stanno ottenendo un grande interesse da parte del mercato e del consumatore.
The objectives of this work are associated with the following question:
How S. cerevisiae relates to environment change during spontaneous fermentation in natural wines?
The main aim of this work was to analyze the diversity of wild Saccharomyces cerevisiae isolated from spontaneous fermentations of Syrah grapes in two wine farms that produced Natural wines. The first objective was to find out diverse biotypes belonging to the
Saccharomyces sensu stricto and select some S. cerevisiae to subject further analyses.
To reach the aims the following steps were planned:
• Isolation and molecular characterization of S. cerevisiae wine yeasts during the spontaneous fermentation in the harvest of 2009.
• Analyses of the main fitness and quality traits of selected S. cerevisiae; the main enological characterisation involved the test of killer activity, production of H2S and
principal fermentative abilities, such as the vigor and fermentation power. In addition, chemical analyses were performed at the end of fermentation trials in laboratory including the residual glucose, the nitrogen consumption and the sulphur compound residual in the fermentation product of a synthetic must (MNS) and in biotypes micro-vinifications in natural grapes must.
• Environmental factors of stress were tested in order to study the ability of S.
cerevisiae biotypes to grow and tolerate diverse concentrations of the main factors
stress such as ethanol and SO2.
• Sensorial evaluations were performed on natural wines originated from some S.
cerevisiae biotypes fermentations in a seòected wine farm.
• Dominance of S. cerevisiae biotypes, estimation during the harvest of 2013 and presence of selected biotypes in the spontaneous fermentation of 2013 were also studied.
Introduction to organic and biodynamic farming systems
Nutrients and metabolites in food and beverages, generally, depend on their processing and storage methods, but the farming system makes a deeply difference. The manners of raw materials are produced express the character of a wine and link wine with the place in which it was born. Broadly speaking, consumers perceive organic products as healthier and safer than those that are produced through conventional methods. The heavy-handed use of synthetic fertilizers, weed-killers, fungicides, pesticides and inappropriately applied heavy metals like copper have destroyed soil life in most environments. Even vineyards are changed in recent times. This is problematic for wine.
In order to create balance and health in a vineyard and relative wines, various agricultures farming systems have spread in the last century. Organic and biodynamic farming systems have bloomed since 1900.
Organic viticulture is very similar to the organic farming of other foodstuffs; no pesticides, no herbicides, no fungicides and no synthetic fertilizers are allowed.
The Soil Association in United Kingdom defines what organic farmers can and cannot do: “In organic farming, pesticides are severely restricted and artificial chemical fertilizers are prohibited...". “ These are standards that apply to most organic food certifying bodies around the world.”
A specific section is dedicated to wine, but without any clarity: “For wine it's a bit trickier. There are dozens of certifying bodies around the world, (Nature & Progress, Demeter, Eco-cert, Australian Certified Organic...) each with varying regulations and standards declared. So far, it seems that the USA has the toughest criteria for a wine to be called an 'organic' wine. In Europe, if grapes are cultivated and harvested organically, then no specific regulations will relate to the process in the winery, different rules are applied and all sorts of things can be added.” This is the principal issue in our times.
Biodynamic agriculture
Starting from the year 1924, Whitsun Rudolf Steiner originated a “new” holistic approach in agriculture showing eight lectures in a conference entitled "Spiritual foundations for the renewal of Agriculture" at Koberwitz, Silesia. The Experimental Circle of Anthroposophical farmers immediately tested Steiner’s indications in daily farming practice. Steiner's teachings promoted farming methods that encouraged self-sufficiency in the farm by helping plants and animals strengthen their own immune systems rather than rely on synthetic chemicals to combat disease. The idea of biodiversity in nature was the starting point to develop biodynamic agriculture. Biodynamic agriculture is a form of organic cultivation. Similarly, to organic agriculture, biodynamic farmers use composting and cover cropping instead of mineral fertilizing, and ban pesticides, herbicides, hormones and other chemicals. The main difference from organic agriculture, apart from philosophical and historical point of view, lies in the use of biodynamic preparations. Normally, distinctive herbs or minerals, treated or fermented with animal organs are utilized after the dynamization. The dynamization is a process that ensures the diffusion of the "cosmic forces" inside biodynamic preparations before the application, generally, as sprays especially in vineyards. In addition, biodynamic preparations are conceived to increase the microbiological life of the growing environment. Briefly, biodynamic agriculture is based on a deeply understanding of the human being, plant, animal, earth and cosmos. One of the international labels of certification, founded in 1932 in Germany, is surely Demeter that is the brand for products derived from biodynamic agriculture. The brand is utilized by partners who are strictly controlled. A comprehensive verification process insures strict compliance with the International Demeter production and processing standards, as
applicable organic regulations in the various countries from agricultural production to processing and final product packaging. In the end, whether one product is called organic or biodynamic, indeed perhaps by no name at all, it is not the label that counts. Any truly 'green', sustainable agricultural practice fit together with the real understanding and fundamental principles that support nature rather than fight against it. Naturally, wines from vineyards that apply these principles quite honestly speak for themselves.
Natural wine versus organic wine
Sometimes, it is very difficult to understand the meaning of a new phenomenon. Natural wine and ‘organic wine’ are not the same thing. People are often confused about the differences between organic and natural wines. Natural wine is about farming your vineyard organically and then following the same philosophy in the winery not adding anything, whereas organic producers can add SO2 and tartaric acid. Natural wines are
about respecting nature from the beginning to the end without using chemical products or additions. Sometimes biodynamic winemakers add a little bit of sulphur to wines. Otherwise, some residual sulphur is found in wine. It is due to yeasts because they are living organisms who use sugar to produce alcohol and, during this process, there is a production of a little amount of sulphur compounds. The problem is that no official regulations are available for winemakers or consumers. Certifying bodies may guarantee slightly lower levels of SO2 allowed in the wine, but lack strict parameters in terms of what
happens inside the cellar. Natural wine
'Natural' has become the latest tendency in wine. The natural wine movement, however, is a relatively new trend. No single individual generated this movement; instead, dozens of people contributed to the consciousness that winemaking and viticulture became increasingly industrialized and the phenomenon of standardization has crowded the world wine. Among the earliest visionaries were agriculturalists such as Austria’s Rudolph Steiner and Japan’s Masanobu Fukuoka, and Burgundy’s Jules Chauvet. They were followed more recently in the 1970s and ’80s by producers Favard (Bordeaux), Gravner (Friuli), Joly (Loire), Lapierre (Beaujolais), Laughton (Australia), Maule (Veneto) and Overnoy (Jura). Their philosophies inspired others, causing an increasing interest in this new approach to wine.
Natural wines, nowadays, could appear an evocative recall to what wine was like before. Besides, during the ages, science has able winemakers to produce wines with the full control over every single aspect of growing grapes and winemaking. On the contrary, natural wines are going to basics and simplicity. No official definition of “natural wine” comes out all over the countries, but “natural” surely originates from grapes obtainedt from organic and biodynamic cultivation. No treatments are allowed in vineyard, with the exception for organic or biodynamic products. The harvest is exclusively manual. The main difference is in the fermentation process, where no adding or removing is made in natural grape must. Nowadays, the vast majority of wines are fermented using lab-bred inoculated, industrial yeasts, which are used around the world to control the winemaking process in order to manage the alcoholic fermentation, but also for giving impact to wine styles and flavors. The widespread use of pesticides, weed-killers and fungicides have involved changes in indigenous yeast populations, often they are not able to complete and compete to commercial yeasts in fermentation of the must, so the use of commercial yeasts becomes inevitable. For this reason, wines are increasingly monotone, standardized and predictable. They are not the result of intricate yeast populations created
by nature. Environmental yeasts are part of terroir. They are important to produce authentic, natural wine. Natural wine takes origin from natural fermentation of grapes in cellar.
In fact, the cellar is the place where occurs natural events; the alcoholic fermentation in natural wines is made by indigenous yeasts, naturally present in the environment. Traditionally, farmers relied on the cellar environment and wild indigenous yeasts to carry out the fermentation process and make wine. These yeasts exist naturally in a vineyard and cellar. Natural wine producers are respectful of the land and have a philosophic approach on it. They are great observers of nature with good knowledge of natural environment. Natural wines result healthy and complex, but also a dynamic reflection of biodiversity and natural balance of vineyard. All the nutritive elements, such as yeast nitrogen, enzyme, vitamins etc are present in grapes and soil, so natural wines comes out in a self-sufficient way. Sustainability is preserved with also no irrigation, no-heavy machinery, no pollution from spraying pesticides, herbicides or fungicides. Natural wine becomes sustainable and eco-friendly, an added values for costumers, who prefers natural wine to a conventional one. The other side of the coin, regarding natural wines, is that good amounts of this kind of wines result in organoleptic deficiency or a bad smell and taste. This is probably due to the lack of attention in processing grapes or storage problems. Bad natural wines are widespread. Unfortunately, it results in confusing consumers and making a strong penalization of winemakers who make good natural wines. Consumers are oriented through publicity and sometimes information could not be correct. In conclusion, “Natural” does not mean good or bad wine; consumers have to keep attention to wine farms and their specific products.
Biodiversity of wine yeasts
Biodiversity, according to United Nations Convention, represents the variability among living organisms from different sources such as terrestrial, marine and other ecosystems in a complex environment in which they exist. The alcoholic fermentation is a complex process in which occur interactions between yeasts and bacteria. Studies of the key role of yeasts in conducting the fermentation started since the pioneering investigations of Lois Pasteur. Yeasts, responsible for the alcoholic fermentation originate mainly from two different environments: the surface of grapes and surfaces of winery equipment. Many studies on yeasts isolations from grapes revealed that a total yeasts population in crushed grapes is around 103 and 105 UFC/mL. The genre Kloekera and Hanseniaspora are predominant on the surface of grapes. In fact, they represent a 50-75% of total yeast population. Other species are found in grapes even if their presence is lower than
Kloekera and Hanseniaspora. Candida, Rhodotorula, Pichia and Hansenula are also
native in fresh grapes. The fermentative Saccharomyces spp. is rarely found (less than 50 CFU/mL) in grapes, but they are the best brewers in the alcoholic fermentation. The species S. cerevisiae is prevalent on winery surfaces. This is due to the selective role of grape must and wine as growth substrates and changing environments. Some yeast populations, such as Candida, Pichia, Hansenula and Brettanomyces are also observed in that location. Many authors, with the aim to trace the origin of S. cerevisiae, revealed that winery surfaces are the main source of indigenous S. cerevisiae in fermentations (Rosini, 1984), (Martini and Martini, 1990). All the species of yeasts found in the grape surface and in the first steps of alcoholic fermentation are called non-Saccharomyces yeasts. Yeasts that conduct the primary fermentation (Saccharomyces) are sometimes called “fermentative yeasts” based on their metabolisms.
Non-Saccharomyces yeasts in wine
Grapes must naturally contains a combination of various yeast species and wine fermentation is not a ‘single-species’ fermentation (Fleet, 1990). The indigenous
non-Saccharomyces yeasts, already found in the must, and often in greater numbers than S. cerevisiae, are adapted to the specific environment and in an active growth state, which
gives them a competitive edge (Crayet al. 2013). The presence of high numbers (106 and 108 UFC/mL) of non-Saccharomyces yeasts in modern wine fermentations, results in wine a new complexity allowing winemakers and microbiologists to revisit the role of these yeasts. Consequently, their role is changing in wine production (Fleet et al., 1984; Heard and Fleet,1985; Fleet, 1990, 2003; Herraizet et al., 1990; Longo et al.,1991; Romano et al., 1992; Todd, 1995; Gafner et al., 1996; Gil et al., 1996; Lema et al., 1996; Granchi et al., 1998; Henick-Kling et al., 1998; Lambrechts and Pretorius, 2000; Rementeria et al., 2003; Combina et al., 2005; Xufre et al., 2006; Varela et al., 2009; Ciani et al., 2010; Ciani and Comitini, 2011).
Many external factors affect populations both on grapes and in must. During crushing, non-Saccharomyces yeasts on the grapes, winery equipment and in the cellar environment (air and insect borne) are carried over to the must. However, winery surfaces play a smaller role than grapes as a source of non-Saccharomyces yeasts, as S.
cerevisiae is the predominant yeast inhabiting such surfaces. Despite, all the variables in
grape-harvest and wine production, the yeast species generally found on grapes and in wines are similar throughout the world, but the population profile of yeasts in various regions shows many differences. The contribution by non-Saccharomyces yeasts to wine flavor will depend on the concentration of metabolites formed. The specific environmental conditions in the must, such as high osmotic pressure, the presence of SO2, non-optimal
growth temperature, increasing alcohol concentrations and anaerobic conditions and also the decreasing nutrients, all play a role in determining what species can survive and grow (Bisson and Kunkee, 1991; Longo et al., 1991).
Non-Saccharomyces yeasts, found in grapes must and during fermentation, can be divided into three groups: yeasts that are largely aerobic, (for example, Pichia spp.,
Debaryomyces spp., Rhodotorula spp., and Candida spp.), apiculate yeasts with low
fermentative activity (for example, Hanseniaspora uvarum (Kloeckera apiculata),
Hanseniaspora guilliermondii (Kloeckera apis), Hanseniaspora occidentalis (Kloeckera javanica); and yeasts with fermentative metabolism, (for example, Kluyveromyces marxianus, Torulaspora delbrueckii, Metschnikowia pulcherrima (Candida pulcherrima)
and Zygosaccharomyces bailii.
Other species of yeasts are located in final wine after the alcoholic fermentation; they could have positive and negative effects to the wine. In fact, storage of wines in tanks and barrels prior to packaging is a critical point. Wine that is exposed to air, as in incompletely filled tanks or barrels, quickly develops a surface flora of weakly fermentative or oxidative yeasts, usually species of Candida and Pichia (Pichia membranifaciens). These species oxidize ethanol, glycerol and acids, giving, sometimes, high levels of acetaldehyde, esters and acetic acid to wine. Fermentative species of Zygosaccharomyces, Dekkera (anamorphic Brettanomyces), Saccharomyces and Saccharomycodes are also detected in tanks or bottle of wines. The species of Dekkera/Brettanomyces are also associated with the production of unpleasant mousy and medicinal taints, because they can form tetrahydropyridines and volatile phenolic substances such as 4-ethylguaiacol and 4-ethyl phenol (Du Toit and Pretorius, 2000). However, some yeasts interactive phenomena may be relevant. Yeast autolysis, after alcoholic fermentation, could be a significant source of micronutrients for the growth of these spoilage species and for the ageing of a wine, giving some particular traits to final wine.
Spontaneous fermentation
Spontaneous fermentation, wild yeast fermentation or natural fermentation is a method of wine production, which does not use the addition of commercial yeasts to start and control alcoholic fermentation of the must. Many winemakers conducting spontaneous fermentations (comprising mixed and sequential dominance of non-Saccharomyces and
Saccharomyces yeasts), have the consciousness that indigenous yeasts are fundamental
part to the authenticity of their wines by imparting desired and distinct superior regional characteristics (Amerine et al., 1972). Grapes are crushed and allowed to ferment on their own. In spontaneous fermentation, there is a sequence of dominance by various
Non-Saccharomyces yeasts, followed by relatively alcohol tolerant S. cerevisiae that complete
the alcoholic fermentation (Fleet, 2003). Indigenous yeasts have been reported to contribute either positively or negatively to the overall sensory characteristics of wine. This suggests extensive diversity among all wine yeasts. Indigenous yeasts make a positive contribution to wine through several mechanisms. They utilize grapes must constituents and produce ethanol and other solvents that help to extract flavor components from grapes solids. In addition, they produce enzymes that transform neutral grapes compounds into flavor active compounds. Many hundreds of flavor active, secondary metabolites (acids, alcohols, esters, polyols, aldehydes, ketones, volatile sulphur compounds) are the results of their metabolism (Cole and Noble, 1997; and Lambrechts and Pretorius, 2000). These reactions, especially the production of secondary metabolites, vary within the species and within strains from the same species. In the recent times, an increasing number of winemakers have come back to traditional winemaking methods with the aim to obtain unique attributes that could differentiate their products from others. Spontaneous fermentation can surely improve wine quality, and increase the variety of complex flavours (Diaz et al., 2013).
Spontaneous fermentation is a complex process influenced by many factors, starting from indigenous microorganisms, the grape variety, climatic conditions, and the winemaking process. Initially, species of Hanseniaspora (Kloeckera), Rhodotorula, Pichia, Candida,
Metschnikowia are found at low levels in the first steps of alcoholic fermentation. Most of
non-Saccharomyces yeasts grow during the first days, whereas the central stage of the process is dominated by strains of S. cerevisiae. The dynamics of a spontaneous fermentation is often unpredictable and some non-Saccharomyces yeasts could produce undeliverable compounds. When non-Saccharomyces yeasts limits their activity, due to the adverse changing environment, the species of Saccharomyces cerevisiae take over the fermentation, predominating until its completion. Non-Saccharomyces yeasts disappear because of their weaker ethanol tolerance and their inability to survive within the increasing concentration of ethanol made by Saccharomyces species. Nutrient availability and nutrient limitation during the alcoholic fermentation are factors that modulate yeasts ecology. All the factors contribute to create an evolutionary environment that changes the micro-flora in wine and surely spontaneous fermentation gives more complexity to wine than the conventional fermentation conducted with the use of commercial yeasts. Finally, the variability of microorganisms allows winemakers to create diversified wines, which become a real expression of the terroir.
Origin of Saccharomyces spp. and wine
Yeast was discovered many years ago by Louis Pasteur as the cause of fermentation, the process that converts sugar into ethanol and carbon dioxide. People of ancient times had no understanding of this biochemical process, even if fermentation was used for thousands of years as an effective way to preserve the quality and safety of beverages and foods. Archaeologists have found molecular evidence for the production of a fermented beverage dated back to 7000 b.c from the Neolithic village of Jiahu in China. Similar evidence dated to 5400-5000 b.c comes from the site of Hajji Firuz Tepe in the northern Zagros Mountains in Mesopotamia. In ancient Egypt and Mesopotamia, the most used beverages were made from fruits such as grapes, from honey and from malted cereals (beer). According to many authors, grapes have been domesticated between the Black Sea and Iran during the 7000-4000 b.c period (McGovern, et al., 1996; Arroyo-Garcia et al. 2006; and This et al., 2006). The first evidence of winemaking comes from the presence of tartaric acid in an ancient jar dated from 5400-5000 b.c. in the Neolithic site of Tepe in Mesopotamia and from the remains for grape must extraction from 5000 b.c in the Neolithic site of Dikili Tash in Greece. Archaeologists suggest that wine was made as picking grapes and placed in vats; the grapes were pressed and put in open jars where the fermentation process took place. Wine was principally drunk and offered to gods during religious ceremonies or it was used in medicine. It is expected that starting from those times, beverages fermentation technologies expanded from Asia, Mesopotamia and Egypt throughout the Old World.The cultivation of grapevines and the production of wine spread all over the Mediterranean Sea towards Greece (5000 b.c.), Italy (900 b.c.), France (600 b.c.), Northern Europe (100 a.d.) and much later to the Americas (1500 a.d.). Consequently, the alcoholic fermentation of grapes was widespread in the past and yeasts were largely ubiquitous. The most important species involved in alcoholic beverages belong to the Saccharomyces sensu stricto complex. The fundamental physiological characteristic is growing and degrade carbohydrate, usually six-carbon molecules such as glucose to two carbon components, particularly ethanol, without completely oxidizing them to CO2. The Saccharomyces sensu stricto complex (Fig. 1.1) is composed by different
known species, with the special trait described before. Species phylogeny is shown below.
Saccharomyces bayanus was derived from multiple hybridization events.
S. bayanus species complex includes two natural species (S. uvarum and S. eubayanus)
and two hybrids that had been given species names (Saccharomyces pastorianus and S.
bayanus). Saccharomyces species have been isolated from different substrates. S. cerevisiae, S. bayanus and S. pastorianus, and a hybrid of S. cerevisiae and S. bayanus
(Hyma et al., 2011), are associated with anthropic environments. S. paradoxus, S.
kudriavzevii and S. arboricolus are mainly isolated in natural environments. Most of strains
that make the alcoholic fermentation are linked to the species of S. cerevisiae. Sometimes, the fermentation is made by S. bayanus var. uvarum for the winemaking at low temperature. Some S. paradoxus are isolated in vineyard, but they give occasionally a low contribute to winemaking (Dequin and Casaregola, 2010).
Saccharomyces species have a number of unique traits not found in other yeast species.
In fact, the majority of species from Saccharomyces complex can survive without oxygen (using fermentation process), but other yeasts could not have this ability. They are Crabtree effect positive, which means that they produce ethanol (alcohol) aerobically in the presence of high external glucose concentrations rather than producing biomass, the usual process occurring aerobically in most other yeasts. The fermentation and respiration in S.
cerevisiae is shown in Fig 1.2. In S. cerevisiae, in presence of high sugar concentration,
the alcohol dehydrogenase 1 enzyme (fermentation) is more activated rather then the alcohol dehydrogenase 2 enzyme, which utilize ethanol as substrate for respiration and producing biomass.
Fig. 1.2: The make-accumulate-consume strategy of Saccharomyces yeasts (Adh1: Alcohol dehydrogenase 1, Adh2: Alcohol dehydrogenase 2) (Piskur et al., 2006)
In S. cerevisiae, two genes encodes for the activity of alcohol dehydrogenase in the cytoplasm. Many authors revealed that the alcohol dehydrogenase 1 (ADH1) is normally expressed, whereas the alcohol dehydrogenase 2 (ADH2) is expressed only when the internal sugar concentration drop (Piskur et al., 2006). Consequently, ethanol is normally the product of the reaction, whereas it became a substrate when sugar concentration falls. Accordingly, the ancestor of Saccharomyces spp. had an important advantage during the past times. Saccharomyces spp kills rivals by producing ethanol, which is toxic for other species of microorganisms. In this way, Saccharomyces spp. had spread in the entire world. In the end, several unique properties such as fast growth, efficient glucose repression, ability to produce and consume ethanol and tolerance for several environmental stress make Saccharomyces the best brewer.
Saccharomyces cerevisiae
It is well established that, within S. cerevisiae, different strains have different effects on wine flavor. The variation in production of glycerol, acetic acid, hydrogen and sulphur, are attributed to diverse biotypes of S. cerevisiae (Henschke, 1997). Important characteristics of S. cerevisiae strains include the following: low production of volatile acidity, tolerance to high ethanol concentration, production of ethanol according to sugar content in the must, complete fermentation of sugar in the must, high fermentation rate, ability to grow at high temperatures and high sugar concentrations, tolerance to sulphur dioxide, low production of SO2 and H2S, easy settling after fermentation, low foam production, possession of a
killer or neutral phenotype, low acetaldehyde production, adequate glycerol production and a limited production of higher alcohols (Esteve-Zarsozo et al., 2000; Nikolaou et al., 2006). Strains of S. cerevisiae collected from ecological and geographical diverse sources, typically demonstrate a genetic difference related to the habitat in which they are isolated.
Biotypes of S. cerevisiae correlate to vineyard and wine production in a defined territory, often are genetically groups that are separated from wild yeasts, isolated from soil or other habitat or from other fermentation. The genetic difference from wine and not wine yeasts suggests wine yeast were domesticated from wild S. cerevisiae. The evidence of domestication is observed in phenotypical traits of wine S. cerevisiae, such as the resistance to copper, sulphite, growth and fermentation parameters, freeze/thaw tolerance and efficiency of sporulation (Gerke et al. 2006). Other particular evidence of domestication phenotypes relates to the ability of producing wine aroma, flavor and sensory attributes to the wine and they are influenced by yeasts metabolites. Not considering the influence of grapes variety and fermentation condition, it is demonstrated that the flavor profile in a wine is affected by the production of flavor-active metabolites from different wine yeasts (Mendes-Ferreira et al., 2009; Estevez et al., 2004 and Barbosa et al., 2009). Studies on this topic are widespread in the entire world, this is probably due to the necessity to maintain the biological patrimony of wine areas or region and give a surplus to the terroir in which they are ubiquitous. Moreover, this is a consequence to the intense use of commercial S. cerevisiae yeasts during the last times that may impact on the microbiological biodiversity.
The genome of S. cerevisiae
The use of yeasts for fermentation process has guided the selection of specialized strains for winemaking. S. cerevisiae has a relatively small genome, a large number of chromosomes, little repetitive DNA and few introns. Haploid strains contain approximately 12-13 megabases of nuclear DNA, distributed along 16 linear chromosomes whose sizes vary from 250 to 2000 kb (Barre et al., 1992). Mitotic crossing over and gene conversion promotes a faster adaptation to environmental changes than spontaneous mutations, which occur at comparatively very low rates. Each strain displays specific phenotypic traits. It is largely demonstrated that wine yeasts have a high level of chromosomal length polymorphism. This is the result of cross-chromosomal rearrangements due to translocations, deletions and amplification of chromosomal regions (Bidenne et al., 1992; Carro et al., 2003). The role of retrotransposon mobility contributed also to the variability in wine strain genome. An example of evolution in wine yeasts genome is revealed by the reciprocal translocation between the chromosome VIII and XV, which increase the expression of the gene SSU1. This gene encodes for a protein located in the plasma membrane involved in sulphite anion extrusion, supplying for the sulphite resistance in yeasts. Up to now, the DNA divergence between strains has been highlighted. In fact, many authors revealed that one to one point, four substitutions per kilobase occurs for vineyard isolates, whereas five to six substitution per kilobase occurs between wine strains and S. cerevisiae strains (Benavides et al., 2005; Liti et al., 2009 and Schacherer et al., 2009). The introgression, such as the acquisition of genes by DNA transfer indicates the adaptation of strains to winemaking environment. Some introgressions are related to nitrogen and carbon metabolism, but the function of some genes is still unknown. An other important process that involves the variability in DNA genome of S. cerevisiae is the hybridization. Generally, the hybridization of two genomes is an advantage because the hybrid gets some characteristics from each parental strains. S. pastorianus has an allopolyploid nature, the hybrid was discovered many years ago, but it is shown recently that the two contributors are S. cerevisiae and S. bayanus var. bayanus. Quantitative genetics approaches should help to identify polymorphisms affecting the technological or sensorial properties of S. cerevisiae.
Genetic identification of yeast species
The improving of new molecular techniques about genetics has provided more reliable methods for taxonomic studies. The employments of these techniques in wine characterization allow to deep understanding the microbiology of winemaking. Monitoring of induced fermentations let to have deep knowledge of the evolution of the entire microorganisms in winemaking. This understanding coupled with the possibility of correlating genetic patterns of strains (Nadal et al., 1996), it is based on the new ability to have an overview to the molecular structure of yeast strains, employing techniques such as restriction or amplification of specific or random polymorphic DNA regions. The comparison of ribosomal RNA (rRNA) and its template, generally, permit to achieve differentiation between the taxonomy of yeasts ribosomal DNA (rDNA): this technique has been used extensively in recent years. It allows finding the relationships among many yeast species identification. Some of these molecular technologies are based on sequence analysis; one of the first was the amplification of the 26S rDNA D1/D2 domain (Kurtzman et al., 1998) and then the amplification of the 18S subunit (James, Collins et al., 1996). However, these methods now are impractical for the routine screening concerning the identification of yeast isolates. In 1999, Esteve-Zarzoso et al. proposed an easy and rapid method for identification at species level. This method was based on PCR amplification and restriction analysis of the 5.8S rRNA gene and the internal transcribed spacers (ITS1 and ITS4). A restriction profile database was also created and improved to allow identification of more than 300 yeast species. Similar methodology was utilized by Dlauchy D. et al. in the same year. The techniques consisted in the amplification of a different region, 18S rRNA and ITS1. They also constructed a database of restriction fragment patterns of 128 species associated mostly with food and fermented drinks. During the times, many authors focused on species attribution of yeasts belonging to the
Saccharomyces genus, which has undergone innumerable changes during the 150 years
of his history, including the division in sensu stricto for species associated with alcoholic fermentation (Vaughan-Martini et al., 1993). The species within the Saccharomyces sensu stricto group (S. cerevisiae, S. bayanus, S. pastorianus, S. paradoxus, S. mikatae, S.
kudriavzevii and S. cariocanus) according to Kurtzman and Robnett (1998) cannot be
easily distinguished by traditional microbiological analysis, for this reason molecular techniques have recently developed for species characterization of these yeast strains. In 1994, Hansen and Kielland-Brandt (1997) proposed PCR amplification of the gene MET2 and restriction analysis to differentiate S. cerevisiae from S. bayanus. This gene encodes for the homoserine acetyl transferase. The two yeasts are discriminating through differences in profiling fragments originated from PCR. Then, the PCR-RFLP technique has been developed and adapted for rapid analyses. De Barros Lopez et al. (1996) developed a remarkable technique using PCR primers based on the conserved intron splicing sites (ISS), generating amplification fragments of different lengths within the
Saccharomyces species. This method, however, can be troubled by intra-specific
polymorphism. More recently, the discrimination among the Saccharomyces sensu stricto group, has been performed by two techniques: the Denaturing Gradient Gel Electrophoresis (DGGE) and the Temperature Gradient Gel Electrophoresis (TGGE). The protocol was elaborated by Cocolin, Manzano et al., (2005). The advantage from these two methods consists in different mobility properties to amplification fragments, due to the substitution of one or few nucleotides in the rDNA sequences.
Ribotyping
Ribo-typing refers to the amplification of ribosomal genes. These areas or regions would include the 5.8S, 18S and 26S ribosomal genes, which are grouped in tandem to form transcription units. These transcription units are repeated between 100-200 times in the genome. Other regions include the internal transcribed spacer (ITS) and external transcribed spacers (ETS), which are areas that are transcribed, but not processed. Intergenic spacers called (IGS) separate also the transcription units. These ribosomal regions have become the tools for identifying phylogenetic relationships between all living organisms (Kurtzman et al., 2011) and between yeasts (Kurtzman and Robnett, 1998). The transcribed units are more likely to be similar for strains of the same species than for different species. In general, the specific regions on the subunits commonly referred to the domain D1/D2, on the 18S (James et al., 1996) and 26S (Kurtzman and Robnett, 1998) have been sequenced. According to Kurtzman & Robnett (1998) when assigning unknown yeast or yeast strains to a specific species, the nucleotide sequences in these regions may be used to measure homology to known or related yeasts. Furthermore, the amplification and restriction profiling of these regions have yielded notable results in identifying species (Kurtzman and Robnett, 1998). Dlauchy et al. (1999) used specific primers NS1 and ITS1 to amplify regions of the 18S gene, which was then digested with enzymes (AluI, HaeIII, MspI and RsaI). White et al. (1990) used primers ITS1 and ITS4 to amplify regions of the 5.8S gene, which was exclusively used for the identification of yeast strains in wine. The non-transcribed areas, 18S gene, ITS region and 26S gene have been widely used by various authors to identify species in the Saccharomyces sensu stricto group (Baleiras-Couto et al., 1996; Tornai-Lehoczki and Dlauchy, 2000; Caruso et al., 2002 Capece et al., 2003). The internal transcribed regions (ITS) has also been targeted by restriction analysis with DraI and HaeIII to identify and characterize yeast populations with enological significance, as well as species in the larger Saccharomyces sensu stricto group (Esteve-Zarzoso et al., 1999; Granchi et al., 1999).
Intra-specific differentiation of yeast strains
Saccharomyces cerevisiae species includes a huge number of biotypes that show various
technological properties. For this reason, the discrimination among them is necessary to understand the evolution and the properties of each strain in winemaking. The ability to characterize different individuals is perceived in ecological studies of spontaneous and starter-guided fermentation, with the purpose to select strains presenting the greatest enological qualities and to explain the dominance capability of selected yeasts. (Briones et al., 1996; Guerra et al., 2001). Recently, discrimination among yeast strains has been developed with the use of several typing methodologies based on DNA polymorphisms. These techniques have permitted to study the population dynamics of Saccharomyces
cerevisiae strains in vineyards or wineries (Guillamon et al., 1996). The study of yeast and
the possibility to test various strains for their enological properties allowed optimizing also the use and the study of ecology of wild strain isolates (Lilly et al., 2006). The first application of mitochondrial DNA restriction profiling was utilized for yeast characterization in food and beverage, specifically to brewing yeast. Many several improvements of the technique of restriction fragment length polymorphism analysis (RFLP) of mitochondrial was applied as an easy fingerprinting method. Fleet et al., (2003) applied this method for the fists time to wine yeasts. The digestion of mtDNA with restriction enzymes like HinfI or RsaI is associated to a highest polymorphism, and it was used to study the authenticity of commercial wine yeast strains (Fernandez et al., 2001).
Mitochondrial DNA (mtDNA) of S. cerevisiae is a small molecule between 65-80 kb in length and it shows high variability when subjected to restriction, which make it very polymorphic (Fernandez-Espinar et al., 2006). MtDNA is rich in A, T and in part G and C, and it is the GC content difference, between nuclear and mtDNA that can be exploited by total fungal DNA digests. When nuclear DNA is digested, a number of smaller fragments are noticeable, but cannot be detected by normal agarose gel electrophoresis. However, the mtDNA will be superimposed on the shadow of the nuclear DNA. Once the mtDNA has been isolated, it can be digested with restriction enzymes (e.g. HinfI, Hae III and RsaI) and the restriction patterns can be analyzed by agarose gel electrophoresis (Guillemin et al., 1994; Fernandez-Espinar et al., 2001). This technique can be used to characterize and identify indigenous and commercial wine strains. However, it is an impractical long protocol, with a difficult DNA extraction method.
During the times, several PCR-based methods have been proposed to discriminate different strains of S. cerevisiae. The genome of S. cerevisiae contains repetitive DNA sequences, such as the N (regions) sequences that are frequently associated with the Ty1 transposon (Cameron, Loh and Davis, 1979). Polymerase chain reaction profile analysis of these sequences (also known as interdelta) has a good level of discrimination for analyzing commercial strains; but, on the other hand, it seems to be less powerful when used to identify indigenous strains isolated from natural environments (Lavellee et al., 1994 and Masneuf et al., 1995). Recently, new powerful approach to identification of S.
cerevisiae strains has been developed. Amplification of mini-satellite like sequences in
several genes of the S. cerevisiae and microsatellite based technique has been performed for the discrimination among S. cerevisiae strains (Hennequin et al., 2001). These techniques allow differentiating yeast from various origins (worldwide different environments) through the analysis of several loci. The potential of this technique for the characterization of enological strains is a promising tool, providing accurate and unequivocal results that can be even quantify as base pair number (or number of repeats).
Minisatellite analysis as discriminating tool
Minisatellites are tandem repetition units of 10–100 bp in the genome. These regions are dispersed throughout the eukaryotic genome and may be as large as 30 kb in total length (Marinangeli et al., 2003). Minisatellites, frequently, show substantial variability in tandem repeat number among individuals of a population. They are useful genetic markers in biology, medicine, and forensics. Despite this widespread application, however, the nature of variability at minisatellite loci is not well understood. Minisatellite variation is typically scored exclusively based on DNA fragment length, and consequently, the homology of repeat length variants and the mutational mechanism underlying repeat variability remain largely unknown. Because of their length polymorphism, which results from variations in the number of repeats, and the ability of some of these arrays to cross-hybridize with tens of other similar loci throughout the genome, minisatellites have opened the way to DNA fingerprinting for individual identification (Jeffreys et al., 1985). However, minisatellites has provided the first highly polymorphic, multiallelic markers for linkage studies. The polymorphic minisatellites (also called VNTRs for variable number of tandem repeats) represent a molecular targets for the characterization of wine yeasts.
The genes containing minisatellite-like sequences
All the genes listed below encodes for cell wall protein. The cell wall of Saccharomyces
cerevisiae is made up of complex polymers of glucose (β1,3- and β1,6-glucan), chains of
N-acetylglucosamine (chitin) and mannoproteins (Fleet, 1991). The β1,3-glucan accounts for about 55% of the wall dry weight and, together with chitin, which represents only 1-2% of the cell wall, it determines the shape and strength of the cell wall (Fleet, 1991). β 1,6-glucan is a highly branched molecule, representing about 10% of the cell wall. Recently,
β1,6-glucan has been demonstrated to have a ‘cementing’ function, interconnecting β 1,3-glucan, chitin and mannoproteins (Kapteyn et al., 1996). All these genes contain minisatellite-like sequences that allow discriminating S. cerevisiae strains. (Marinangeli et al., 2004)
SED 1
The SED1 gene (YDR077W) is a structural constituent of cell wall; it contributes to the structural integrity of the cell wall of S. cerevisiae. In fact, it encodes for the major cell wall glycoprotein in the stationary-phase cells and contains two blocks of tandem repeat units located within two distinct regions of the nucleotide sequence.
AGA 1
The gene AGA1 encodes for anchoring subunit of a-agglutinin of a cells; a protein of 725 amino acids with high serine and threonine content, a putative N-terminal signal sequence, and a C-terminal hydrophobic sequence similar to signals for the attachment to glycosyl phosphatidylinositol anchors to cell wall. (de Nobel et al., 1995). In the Fig. 1.3 is shown the structure of the AGA1 gene according to the corresponding S288C sequences available on-line (http://www.yeast-genome.org). Boxes represent the regions containing repeat units. Inside each box are indicated the length (bp) and the number of each repeat unit, respectively. Arrows indicate the primer sites of annealing.
Fig. 1.3: The gene AGA1 DAN 4
The gene DAN4 encodes for cell wall mannoprotein. It is expressed under anaerobic conditions (only after 1 hour of anaerobic shift). This gene is completely repressed during aerobic growth. Its specific role is still unknown, moreover, it takes part of the group of DAN/TIFF genes; generally the DAN proteins are more stringently repressed by oxygen and insensitive to cold (Abramova et al., 2001). In the Fig 1.4 is shown the structures of the DAN4 gene according to the corresponding S288C sequences available on-line (http://www.yeast-genome.org). Boxes represent the regions containing repeat units. Inside each box are indicated the length (bp) and the number of each repeat unit, respectively. Arrows indicate the primer sites of annealing.
Fig. 1.4: The gene DAN4 HSP150
The gene HSP150 encodes for a secreted mannosylated heat shock protein; it is secreted and covalently attached to the cell wall via beta-1,3-glucan and disulfide bridges; the protein is required for cell wall stability: it is induced by heat shock, oxidative stress, and nitrogen limitation. In the Fig. 1.5 is shown the structure of the HSP150 gene according to the corresponding S288C sequences available on-line (http://www.yeast-genome.org). Boxes represent the regions containing repeat units. Inside each box are indicated the length (bp) and the number of each repeat unit, respectively. Arrows indicate the primer sites of annealing.
Fig. 1.5: The gene HSP150
Microsatellite-like sequences as selective tool for S. cerevisiae strains
Microsatellite analysis is the genetic tagging by synthesized oligonucleotides complementary to single repetitive sequences, present in the genome of the organisms. These repetitive sequences are generally referred to microsatellites (Fernandez-Espinar et al., 2006). Microsatellites (also known as Short Tandem Repeats) are a well-known class of tandem repeats that are composed of units 1-14bp in length. These repeat units are typically composed of non-variable mono, di or tri nucleotide sequences. Microsatellites, composed of mono or di nucleotide repeats, are primarily localized at intergenic regions while, those composed of tri nucleotide repeats, are located within or adjacent to coding sequences. The analysis of microsatellite differs from RAPDs method. It utilizes a higher annealing temperature of 55°C instead of 37°C, which enhances specific oligonucleotide hybridization and coincides with a higher resolution and reproducibility (Stephan et al., 2002; Dalle et al., 2003; Fernandez-Espinar et al., 2006). These techniques have been useful in the identification of Saccharomyces cerevisiae strains (Hennequin et al., 2001). The utilization of microsatellites also includes the study of microsatellite loci that are scattered throughout the genome of the microorganism, which is made possible by whole genome sequencing. The complete sequence of S. cerevisiae genome allows for the identification of these regions. Some of the most frequently utilized loci include YOR267C, SC8132X and SCPTSY7 (Techera et al., 2001). These loci can also be used for multiplex-PCR reactions where two or more loci are amplified (Vaudano & Garcia-Moruno, 2008; Richards et al., 2009). Results are expressed as a number of repeats of the loci. These loci have been identified and used in studies to successfully discriminate between S.
cerevisiae strains (Techera et al., 2001) and evaluated to distinguish between
The genes containing microsatellite-like sequences
In the Fig. 1.6 are listed the primers constructed for the amplification of three
Saccharomyces cerevisiae loci. The gene YOR267C belong to a subgroup of yeast protein
kinases, its activity is dedicated to the regulation of plasma membrane transporters. It plays a role in ion homeostasis; the increasing of proteins encoded by YOR267C is the response to DNA replication stress (Goossens et al., 2000).
The gene, in which the locus SC8132X is located (YPL009c), has not been associated with any function in the S. cerevisiae genome, but the deletion mutant is viable, assuming the non-essential function of this gene, its variability amongst strains of S. cerevisiae could be expected (Howell et al., 2004).
