Environmental and genetic factors affecting Saccharomyces cerevisiae performance during second fermentation

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fermentation

Maria Marti Raga

To cite this version:

Maria Marti Raga. Environmental and genetic factors affecting Saccharomyces cerevisiae performance during second fermentation. Food engineering. Université de Bordeaux; Universitat Rovira i Virgili (Tarragone, Espagne), 2015. English. �NNT : 2015BORD0185�. �tel-01271050�

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THÈSE PRÉSENTÉE POUR OBTENIR LE GRADE DE

DOCTEUR DE L’UNIVERSITÉ DE BORDEAUX

ET DE L’UNIVERSITÉ ROVIRA I VIRGILI

ÉCOLE DOCTORALE : SCIENCES DE LA VIE ET LA SANTÉ SPÉCIALITÉ : OENOLOGIE

Par Maria Martí Raga

Environmental and genetic factors affecting

Saccharomyces cerevisiae performance during second

fermentation

Sous la direction de : Philippe MARULLO (co-directeur : Gemma BELTRAN)

(co-directeur : Albert MAS)

Soutenue le 19/10/2015

Membres du jury :

M. GONZÁLEZ, Ramon. Directeur de recherche, ISVV, CSIC. Président. M. LITI, Gianni. Chargé de recherche HDR, IRCAN, CNRS. Examinateur. M. GUILLAMON, José Manuel. Directeur de recherche, IATA, CSIC. Examinateur.

Mme. DEQUIN, Sylvie. Directrice de recherche, INRA. Rapporteuse. M. Warringer, Jonas. Maitre de conférences, University of Gothenburg. Rapporteur.

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,

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fermentation

Résumé :

La méthode traditionnelle utilisée pour produire des vins mousseux (comme cava et champagne) est caractérisée par une seconde fermentation qui a lieu à l'intérieur de la bouteille. Cette deuxième fermentation présente des caractéristiques très spécifiques tels que une teneur élevée en éthanol, la pression de CO2, basse température et une faible disponibilité des nutriments. Dans cette thèse, on a tout d'abord analysé l'effet des facteurs environnementaux sur la cinétique de fermentation de Saccharomyces cerevisiae au cours de la seconde fermentation. Deuxièmement, on a analysé l'effet de pratiques communes telles que l'ajout de nutriments à le vin de base, sur la composition finale du vin mousseux. Enfin nous avons cherché à identifier la base génétique de la variabilité phénotypique observé en la seconde fermentation en utilisant un Quantitative Trait Locus (QTL) approche. Les résultats obtenus ont permis de déterminer la température, le vin base utilisé, la souche de levure et la source d'azote utilisée dans l'acclimatation de levure comme les facteurs qui ont la plus grande incidence dans la cinétique de la seconde fermentation. Deuxièmement, par rapport à la composition finale de vin mousseux, nous avons trouvé que l'addition d'azote dans le vin base favorise la libération des acides aminés. Bien que l'ajout de levure sèche inactive, favorise la libération des polysaccharides et favorise les propriétés moussantes du vin mousseux. Enfin, on a pu identifier quatre gènes dont la variation allélique explique la variation phénotypique observée parmi les souches.

Mots clés :

levure, azote, QTL

Title : Environmental and genetic factors affecting

Saccharomyces cerevisiae performance during second

fermentation

Abstract :

The traditional method used to produce sparkling wines (such as cava and champagne) is characterized by a second fermentation that takes place inside the bottle. This second fermentation has very specific characteristics such as a high ethanol content, increasing CO2 pressure, low temperature and low nutrient availability. In this thesis, we have firstly analyzed the effect of environmental factors on fermentation kinetics of Saccharomyces cerevisiae during the second fermentation, by monitoring the second fermentation development using

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capacity (mosalux) of the sparkling wine. Finally we aimed to identify the genetic basis of the second fermentation using Quantitative Trait Locus (QTL) mapping and validation approach.

The results obtained enabled us to identify the temperature, the base wine used, the yeast strain and source of nitrogen used in the acclimatization of yeast as the factors that have the highest impact in the second fermentation kinetics. Secondly, with respect to the final composition of sparkling wine, we have found that the addition of nitrogen to the wine base favors the release of amino acids. While the addition of inactive dry yeast, promotes the release of polysaccharides and favors the foaming properties of the sparkling wine. Finally, could identify four genes whose allelic variation explains the phenotypic variation observed among strains.

Keywords :

yeast, nitrogen, QTL,

Unité de recherche

Université Bordeaux Institute des Sciences de la Vigne et du Vin, EA4577, Unité recherche Œnologie (210 Chemin de Leysotte, 33140 Villenave-d'Ornon, France) Universitat Rovira i Virgili, Departament de bioquímica i biotecnologia, Biotecnologia enològica (c/ Marcel·lí Domingo, nº 1, 43007, Tarragona, España)

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E

NVIRONMENTAL AND

G

ENETIC

F

ACTORS

A

FFECTING

S

ACCHAROMYCES

CEREVISIAE

P

ERFORMANCE DURING

S

ECOND

F

ERMENTATION

C

ONTEXTE

B

IBLIOGRAPHIQUE

:

La production des vins effervescents (Champagne, Cava, Spumante) est un secteur important économique et culturellement en Espagne et la France. L'utilisation de la méthode traditionnelle pour produire le vin effervescent (Cava, le Champagne) implique deux processus de fermentation. Le premier consiste dans la transformation du raisin en vin. Tandis que la deuxième étape implique une fermentation secondaire qui a lieu à l'intérieur d'une bouteille fermée, suivie à un temps vieillissant pendant lequel autolysis aura lieu (Carrascosa et d'autres. 2011).

Cette seconde la fermentation présente des plusieurs contraintes environnementales comme, la haute concentration d'éthanol, la pression de CO2 croissante, la

disponibilité de substances nutritives basse et la température basse. Ainsi, les levures doivent être acclimatées avant leur inoculation dans le vin de base pour assurer l'achèvement de la deuxième fermentation.

La recherche conduite en relation avec le deuxième étape a été surtout concentrée dans le processus d'autolysis et son effet avantageux sur le le profil organoleptique du vin pétillant (Alexandre et 2006 Guilloux-Benatier). Cependant, peu de recherche a été conduite pour évaluer les facteurs environnementaux et génétiques qui affectent la progression de la deuxième fermentation. Ainsi, la thèse présente a eu pour but de répondre à ces questions. Le travail a été effectué dans le groupe de recherche de Biotechnologie de Vin, au Departament de Biochimie et la Biotechnologie (URV) et dans l'Unité de Recherche d'Oenologie dans l'institut la Science de la Vigne de la et Vin (de l'ISVV, Université Bordeaux) dans le cadre de deux projets.

Le premier projet, CENIT DÉMETER, à la sous-ligne liée à des vins effervescents ("Aspectos que influyen en la calidad de los vinos espumosos elaborados mediante el método tradicional") a été concentré sur la meilleure compréhension de quels facteurs (la température, le vin de base, des compléments (additions) d'azote, la tension

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utilisée) peuvent affecter la progression de la deuxième fermentation et les caractéristiques organoleptiques du vin effervescent. En outre, en raison de la importance de l'azote dans la fermentation alcoolique (Bell and Henschke 2005), le besoin de déchiffrer le rôle de l'azote dans la deuxième fermentation a été souligné. Deuxièmement, en raison de l'intérêt de Bio Laffort de meilleure comprendre le déterminisme génétique de la deuxième fermentation, un de projet ayant droit : "l'Étude génétique des paramètres de la deuxième fermentation" a été financée. Son but principal était d'identifier les variantes génétiques responsables des différences phénotypiques exposées pendant la deuxième fermentation par des souches de différentes. En tout, tant le projet a partagé le but double d'augmenter la connaissance de base liée à la deuxième fermentation que répondre aux questions industriellement pertinentes.

H

YPOTHESE ET OBJECTIVES

:

L'hypothèse de travail de la thèse était comme suit : la performance de S. cerevisiae pendant la production de vin effervescent dépend lourdement de le background génétique de la levure et de la disponibilité nutritionnelle.

Pour démontrer cette hypothèse, l'objectif général était d'étudier comment les facteurs environnementaux et génétiques impliqués dans la deuxième fermentation modulent la performance de S. cerevisiae pendant la deuxième fermentation et affectent la qualité du vin effervescent. Trois objectifs ont été tirés du général : • Objectif 1 : Identifier les exigences d'azote de S. cerevisiae pendant la deuxième fermentation. • Objectif 2 : Caractériser l'effet de différentes additions d'azote dans les vin base en la composition de vin effervescent finale.

• Objectif 3 : Déchiffrer la base génétique des différences phénotypiques en la performance fermentaire de S. cerevisiae.

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M

ÉTHODOLOGIE ET

R

ÉSULTATS

Objectif 1: Identifier les exigences d'azote de S. cerevisiae pendant la deuxième fermentation. La performance de S. cerevisiae a été analysée pendant la deuxième fermentation et l'effet de plusieurs facteurs environnementaux (T°, le vin de base, l'addition d'azote au vin de base, la source d'azote dans la phase d'acclimatation) a été quantifié. Pour atteindre ce but, plusieurs essais de fermentation (tirages) ont été exécutés et l'évolution de la deuxième fermentation a été suivie en mesurant la pression à l'intérieur de la bouteille avec un aphrometer. En outre, nous avons évalué la croissance de levure (OD600) pendant la phase d'acclimatation utilisant des microplaques et la viabilité de levure pendant la deuxième fermentation utilisant la cytometrie de flux.

Les principaux résultats obtenus étaient :

• La température de fermentation et le vin de base a utilisé avait un effet important sur la deuxième cinétique de fermentation.

• L'addition d'azote au vin de base n'a pas affecté la cinétique de la deuxième de fermentation, cependant la source d'azote utilisé pour acclimater les cellules a eu un effet important sur la performance fermentaire de la levure pendant la deuxième fermentation.

• La source d'azote utilisée dans la phase d'acclimatation affecte la viabilité de levure pendant la deuxième fermentation.

Objectif 2 : Caractériser l'effet de différentes additions d'azote dans les vin base en la

composition de vin effervescent finale.

Vins effervescents ont été produits utilisant trois souches de levure, deux vins base différents, naturels et synthétiques et deux additions d'azote différents à chacun du vins de base. Les bouteilles ont été ouvertes à la fin de la deuxième fermentation et après 9 et 18 mois de vieillissement. La composition du vin effervescents a été évaluée

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en mesurant son contenu en acide aminés et polysaccharides (analyse HPLC) et mesurant ses propriétés moussantes (Mosalux). L'utilisation de vin base synthétique nous a permis de quantifier la contribution de la cellule de levure à la composition de vin pétillant. Les principaux résultats obtenus étaient : • L'addition de phosphate diammonium au vin base a promu la libération d'acide aminé, tandis que, l'addition de levure sèche inactive a favorisé le contenu de polysaccharide et les propriétés moussantes du vin pétillant. • La cellule de levure a particulièrement contribué au contenu de polysaccharide du vin pétillant. • La souche de levure avait un effet significatif sur la composition de vin pétillant.

Objectif 3: Déchiffrer la base génétique des différences phénotypiques en la

performance fermentaire de S. cerevisiae pendant la deuxième fermentation. Pour déduire quelles sont les variations génétiques naturelles causant les différences phénotypiques entre des souches de S. cerevisiae, nous avons conduit un programme de QTL. Deux souches avec de capacité fermentaire distincte ont été choisies comme des souches parentales (précédemment analysées, l'objectif 1).

Leur hybride et leur progéniture F1 (117 segregants) on été phénotypes pour leur comportement fermentaire pendant la deuxième fermentation. On a sequencé leur génome complet utilisant la technologie de Séquencement de Nouvelle Génération (NGS), ce que nous a permis de construire une carte de marqueur génétique.

La cartographie de QTL et la validation, nous a permis d’identifier la variation allélique dans quatre gènes comme le responsable de la divergence phénotypique quantifiée dans la cinétique de la deuxième de fermentation. Ces gènes sont surtout liés au maintien de homéostasie cellular.

Une explication moléculaire plausible du heterosis et homeostasis observé dans la souche hybride a été trouvé.

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C

ONCLUSIONS

En tout, ces résultats confirment l'hypothèse que la performance de S. cerevisiae

pendant la production de vin effervescent dépend lourdement du background génétique de la levure et de la disponibilité nutritionnelle.

En outre, cette thèse ouvre les portes aux nouveaux aspects qui sont pertinents pour l'industrie de vin pétillant :

- Le rôle essentiel de la préparation du pied-de-cuve, qui est souche et nutritif-dépendant. Ainsi, des formulations appropriées pour chaque souche devraient être avancées.

- Le manque d’importance de l’addition d'azote dans le vin base pour la deuxième fermentation. Seulement dans des vins base avec des niveaux de concentration d'azote extrêmement bas (qui est tout à fait rare), l'addition d'azote au vin base pourrait améliorer la cinétique de la deuxième fermentation.

- La pertinence du supplément de vin de base avec levure sèche inactive en raison de son effet positif dans la composition de vin pétillant finale.

- Les variations alléliques dans les gènes qui expriment ATPases sont responsables des différences phénotypiques exposées pendant la deuxième fermentation par des différentes souches de levure. Probablement, lié au pH bas dans des vins base. Ainsi, on recommanderait particulièrement des souches avec ces "mieux" variantes alléliques pour être utiliser en la production de vins pétillants. Alexandre H, Guilloux-Benatier M. Yeast autolysis in sparkling wine - a review. Aust J grape wine Res 2006;12:119–27. Bell SJ, Henschke PA. Implications of nitrogen nutrition for grapes, fermentation and wine. Aust J grape wine Res 2005;11:242–95.

Carrascosa A V, Martínez-Rodríguez A, Cebollero E et al. Saccharomyces Yeast II: Secondary fermentation. In: Carrascosa A, Munoz R, Gonzalez R (eds.). Molecular Wine Microbiology. San Diego: Academic Press, 2011, 33–49.

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“Aprendre!que!l’esperança!és!mentida!si!no!hi!ha!cada!dia!un!esforç!pel!nou!demà.”

APRENDRE.,LLUÍS,LLACH.,

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En!primer!lloc,!moltes!gràcies!als!meus!directors!de!tesi.!Al!Dr.!Albert!Mas,!a!la!Dra.! Gemma!Beltran!i!al!Dr.!Philippe!Marullo,!moltes!gràcies!per!donardme!la!oportunitat!de! participar! en! aquesta! aventura! i! per! la! confiança! que! heu! depositat! en! mi.! Moltes! gràcies!per!l’entusiasme,!l’atenció!i!el!temps!que!heu!dedicat!a!aquesta!tesi.!Em!sento! molt!afortunada!d’haver!treballat!amb!vosaltres.!Només!espero!que!un!cop!acabat!el! viatge,!no!hagueu!quedat!decebuts.!

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“Welcome!to!the!jungle!we've!got!fun!and!games! We!got!everything!you!want!honey,!we!know!the!names”!

!WELCOME,TO,THE,JUNGLE.,GUNS,N’,ROSES

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A!tots!els!professors,!tècnics,!secretàries,!conserges!i!vigilants!del!departament!de! bioquímica!i!biotecnologia!de!la!URV.!Moltíssimes!gràcies!a!totes!les!integrants!de! la! secretaria! del! departament,! per! la! vostra! accessibilitat! i! disponibilitat.! Moltíssimes!gràcies!als!tècnics!(Rosa,!Niurka,!Santiago),!sempre!disposats!a!ajudar! i!anar!una!mica!més!enllà.!!

Mil! gràcies! a! la! Chus! i! al! Braulio,! pels! mil! somriures! i! les! mil! converses! compartides.! Gràcies! per! escoltardme! en! els! moments! difícils,! pels! ànims! i! pels! consells.!M’emporto!la!vostra!amistat.! ! ! !“Más!de!cien!pupilas!donde!vernos!vivos,!! Más!de!cien!mentiras!que!valen!la!pena.!”! MÁS,DE,CIEN,MENTIRAS.,SABINA !

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converses!imprescindibles!per!superar!la!tesis.!Moltes!gràcies!Claudio,!Flor,!Maria! José,!Núria,!Mari!Ángeles,!Josep,!Isa,!Judit,!Mar,!Mireia,!Elena,!Olga,!Laura,!María,! Nikos,! ChunXiao! (Many! thanks! darling!! !),! Imma,! Jessica,! Valentina,! Gemma,! Anna,!Gemma,!Bea!P.,!!Bea!G.,!David!i!Zara.!Moltes!gràcies!Meri!per!les!estones!i! els!riures!compartits.!Moltes!gràcies,!Jenny!i!Àngela!per!compartir!tants!moments! amb!mi,!heu!estat!el!meu!oxigen!aquest!darrer!any.!Moltes!gràcies!Mariona,!per! totes! les! birres,! per! acompanyardme! a! fumar! fins! i! tot! quan! ja! no! fumaves! i! per! totes!les!converses,!les!de!quan!encara!erets!a!la!mateixa!franja!horària!que!jo!i!les! d’ara,! transatlàntiques.! Moltes! gràcies! per! la! teva! amistat,! alegria! i! paciència.! Moltes!gràcies!Marta,!per!ensenyardme!tant!a!tots!els!nivells,!bona!part!d’aquesta! tesi!és!teva.! ! ! “Juste!moi,!mes!délires! Je!n'ai!rien!d'autre!à!offrir! Mais!je!sais!qu'en!vrai!c'est!déjà!ça.”

SOULMAN.,BEN,L’ONCLE,SOUL!

! Merci!à!tous!les!gens!de!l'ISVV.!Je!me!suis!senti!comme!chez!moi!grâce!à!vos!sourires! accueillants.!Je!voudrais!spécialement!remercier!mes!collègues!et!amis!du!laboratoire! des!levures:!Margaux,!Warren,!Laura,!Marina,!Emilien,!Elodie,!Cécile,!Guillaume,!Lucas,! Isabelle!et!Philippe.!Entre!bières,!chansons!de!Grease,!sauvetages!de!chats,!déjeuners! au!soleil!et!fuites!dans!les!fermenteurs!on!a!beaucoup!travaillé!et!c'était!toujours!dans! la!bonne!humeur.!Merci!à!tous!pour!m'avoir!accueilli!comme!vous!l'avez!fait,!et!pour! m'avoir!intégré!au!groupe!dès!le!premier!jour.!Je!ne!vous!oublie!pas.!! ! “Sabes,!quisiera!darte! siempre!un!poco!más!de!lo!que!te!pido.”!

POR,LA,BOCA,VIVE,EL,PEZ.,FITO,Y,FITIPALDIS!

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escoltar! les! meves! cabòries! sobre! la! tesis! i! sobre! la! vida.! Disculpeu! les! meves! absències,! les! llargues! temporades! sense! respondre! els! vostres! mails,! la! poca! disponibilitat.!Sapigueu!que!sovint!em!trobo!pensant!en!vosaltres.!Sapigueu!que!feu! que!la!meva!vida!sigui!més!bonica!i!millor.!Sou!per!mi!absolutament!imprescindibles.! Moltes!gràcies.! “Sempre!quan!es!pensa!amb!aquells!anys,!quan!regnava!sols!l’amor! entre!fum!i!el!sentiment!de!llibertat.! Tornen!les!imatges!del!passat!i!el!record!dels!quatre!amics,!disposats!a!compartir!la!realitat.! I!quan!la!distància!els!separa!per!complet!els!uniran!velles!històries,!vells!acords.! I!recordaran!aquelles!nits!d’hivern!passades!junts!al!foc!rient!i!composant!cançons”! QUATRE,AMICS.,JOAN,ROVIRA.!

!

Al!nucli!dur.!Amb!els!que!he!compartit!tants!moments!que!es!impossible!enumerardlos.! Als!que!m’heu!vist!créixer!i!als!que!sens!dubte,!heu!contribuït!a!definir!la!persona!que! ara!sóc.!Als!que!m’heu!animat,!m’heu!escoltat,!m’heu!aconsellat!i!m’heu!fet!costat!en! els!moments!més!difícils.!Sou!amb!els!que!he!celebrat!tot!el!bo!i!el!dolent.!Sou!els!que! malgrat! la! distància,! sempre! heu! estat! ben! a! prop! meu.! Mil! gràcies! per! tots! els! moments!i!mil!disculpes!per!les!absències.!Mil!gràcies,!Oriol,!Quim,!Nani,!Neus,!Núria,! Magri,! Anna,! Àlex.! Sou! els! millors.! No! us! ho! dic! prou,! però! ho! sou.! Per! mi,! imprescindibles.!! ! ! “Mar!i!marona!que!amb!el!teu!alè!de!vida!! mous!les!onades!i!els!batecs!dels!nostres!dies.” MARONA.,LLUÍS,LLACH! ! Finalment!a!la!meva!família.!A!la!meva!àvia!i!a!mon!iaio!que!tant!em!va!ensenyar!i!que! encara!avui!segueix!sent!ben!present!a!la!meva!vida.!Als!meus!germans!(i!nebots)!i!al! meu! pare.! Però! molt! especialment,! a! la! meva! mare.! Font! inesgotable! de! paciència! i! sensibilitat.! Model! de! dona! i! de! persona.! Suport! constant.! Sense! tu! no! hi! hagués! arribat,! sense! tu! no! seria! qui! sóc.! No! trobo! les! paraules! adequades! per! expressar! el! meu!agraïment!i!l’amor!que!sento!per!tu,!i!sempre!em!quedo!amb!la!sensació!que!mai! et!podré!tornar!tot!el!que!has!fet!per!mi.!Moltes!gràcies,!mama,!t’ho!dec!tot.! ! !

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I

NDEX

,

O

BJECTIVES,

&

,

O

UTLINE,OF,THESIS,...,13,

I

NTRODUCTION,...,19, 1! WINE!YEAST!PHYSIOLOGY!...!21! 1.1! YEAST!UTILIZATION!IN!FERMENTED!GOODS.!...!21! 1.2! GENERAL!CHARACTERISTICS!OF!S.%CEREVISIAE.!...!21! 1.2.1! CELLULAR!ARCHITECTURE!...!21! 1.2.2! REPRODUCTION!AND!CELL!CYCLE!...!24! 1.3! S.%CEREVISIAE!SUGAR!AND!NITROGEN!METABOLISM.!...!26! 1.3.1! RESPIRATION!AND!FERMENTATION!...!26! 1.3.2! NITROGEN!METABOLISM.!...!28! 1.3.2.1! NITROGEN!CATABOLITE!REPRESSION!...!29! 1.3.2.2! NITROGEN!SOURCE!CLASSIFICATION!...!31!

2! SPARKLING!WINE!PRODUCTION!...!33!

2.1! INTRODUCTION!...!33! 2.2! THE!TRADITIONAL!METHOD!IN!SPARKLING!WINE!PRODUCTION!...!34! 2.2.1! OBTENTION!OF!BASE!WINE!...!34! 2.2.2! PRISE!DE!MOUSSE!...!35! 2.2.2.1! TIRAGE!AND!YEAST!ACCLIMATION!...!35! 2.2.2.2! SECOND!FERMENTATION!...!38! YEAST!RESPONSE!TO!STRESS!...!40! ETHANOL!STRESS!...!40! LOW!PH!...!41!

INTRACELLULAR!PH!CONTROL!IN!S.%CEREVISIAE!...!42!

LOW!TEMPERATURE!...!43!

INCREASING!CO2!PRESSURE!...!43!

2.2.2.3! AGING!...!44!

2.2.2.4! RIDDLING,!DISGORGING!AND!DOSAGE!...!48!

3! STUDYING!YEAST!NATURAL!GENETIC!VARIATION!...!49!

3.1! S.!CEREVISIAE!NATURAL!DIVERSITY.!...!49! 3.1.1! GENETIC!DIVERSITY!...!49! 3.1.1.1! ORIGINS!OF!YEAST!GENETIC!VARIATION.!...!51! 3.1.2! S.%CEREVISIAE!PHENOTYPIC!VARIATION!IN!WINE!TRAITS.!...!52! 3.2! MAPPING!GENES!UNDERLYING!PHENOTYPIC!VARIATION!...!54! 3.2.1! FORWARD!GENOMICS!–!FROM!PHENOTYPIC!TRAIT!TO!GENOTYPE!...!54!

3.2.1.1! LINKAGE!ANALYSISd!FAMILY!RELATED!QTL!MAPPING!...!55!!

QTL!MAPPING!IN!THE!OENOLOGY!FIELD!...!58! 3.2.1.2! QTL!VALIDATION!...!61! 4! REFERENCES!...!63!

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HAPTER,

1:

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,

OBJECTIVES

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&

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OUTLINE

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OF

,

THESIS,

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The! present! work! aimed! to! obtain! a! PhD! degree! has! been! performed! from! 2011! to! 2015! in! co;direction! between! the! Rovira! i! Virgili! University! (URV)! and! the! Bordeaux! University! (UB).! This! work! has! been! developed! in! the! Wine! Biotechnology! research! group,! at! the! Departament! of! Biochemistry! and! Biotechnology! (URV),! and! in! the! Oenology!Research!Unit!in!the!institute!de!la!Science!de!la!Vigne!et!du!Vin!(ISVV,!UB).! During! this! period! I! held! a! grant! FI;2012! from! the! AGAUR! council! (Catalonia! government).!!

This!thesis!was!carried!out!within!the!framework!of!two!projects.!Firstly,!the!project! CENIT! DÉMETER,! in! the! sub;line! related! to! sparkling! wine! (cava):! “Aspectos( que(

influyen( en( la( calidad( de( los( vinos( espumosos( elaborados( mediante( el( método( tradicional”.! Secondly,! due! to! the! interest! of! Bio! Laffort! in! better! understanding! the!

genetic!determinism!of!the!second!fermentation,!a!project!entitled:!“Etude(génétique(

des(paramètres(de(la(seconde(fermentation”!was!funded.!The!main!points!of!these!two!

projects! were! to! study! the! environmental! factors! of! the! second! fermentation! that! affect! S.( cerevisiae( performance,! with! emphasis! in! the! determination! of! the! yeast! nitrogen! requirements! during! the! second! fermentation! and! to! decipher! the! genetic! basis!of!the!second!fermentation.!!!

!

Within!this!framework,!the!working!hypothesis!was!as!follows:!The*performance*of*S.#

cerevisiae* during* sparkling* wine* production* is* heavily* dependent* on* the* yeast* genetic*background*and*the*nutritional*availability.!

In!order!to!demonstrate!this!hypothesis,!the!general!objective!was!to!study!how!the! environmental! and! genetic! factors! involved! in! the! second! fermentation! modulate! S.(

cerevisiae! performance! during! the! second! fermentation,! and! affect! sparkling! wine!

quality.!Three!objectives!were!derived!from!the!general!one:! !

OBJECTIVE*1:!To!identify!the!nitrogen!requirements!of!S.(cerevisiae(during!the!second!

fermentation.((

S.(cerevisiae!performance!was!analysed!during!the!second!fermentation,!for!sparkling!

wine! production,! and! the! effect! of! several! environmental! factors! (T°,! base! wine,! nitrogen! addition! to! the! base! wine,! nitrogen! source! in! the! acclimation! phase)! was!

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quantified.! To! reach! this! purpose,! several! fermentation! trials! (tirages)! were! performed,!and!the!evolution!of!the!second!fermentation!was!followed!by!measuring! the!pressure!inside!the!bottle!with!an!aphrometer.!

o The!first!tirage!consisted!in!acclimating!three!different!yeast!strains,!using!two! different! nitrogen! sources! in! the! acclimation! media.! Those! strains! were! then! used!for!fermenting!three!different!natural!base!wines,!supplemented!with!two! different!nitrogen!combinations,!at!two!different!temperatures.!!

o The!second!tirage!involved!the!use!of!three!yeast!strains,!a!natural!base!wine! (NW)! and! a! synthetic! base! wine! (SW).! To! each! base! wine,! two! nitrogen! additions!were!considered.!!

o The!third!tirage!involved!the!use!of!only!one!natural!base!wine,!five!different! nitrogen!sources!in!the!acclimation!phase,!and!eight!different!yeast!strains.!The! growth! during! the! acclimation! phase! of! every! strain! acclimated! with! each! nitrogen! source! was! assessed! by! monitoring! their! growth! using! a! microplate! reader.! Furthermore,! yeast! viability! during! the! second! fermentation! was! assessed!by!flux!cytometry.!! ! The!first!objective!is!described!in!the!chapter!1!and!chapter!2!of!the!present!thesis.!! Chapter!1:*The*effect*of*nitrogen*addition*on*the*fermentative*performance* during*sparkling*wine*production.!Results!published!in!Food!Research.!International.! (2015),!67,126–135.! Chapter!2:*Nitrogen*modulation*of*yeast*fitness*and*viability*during*sparkling* wine*production.!Results!submitted!to!Food!Microbiology.! *

OBJECTIVE* 2:! To! characterize! the! effect! of! different! nitrogen! additions! in! the! final!

sparkling!wine!composition.!!

Sparkling! wine! was! produced! using! three! yeast! strains,! two! different! base! wines,! natural!and!synthetic,!and!two!different!nitrogen!additions!to!each!of!the!base!wine.! Bottles!were!opened!at!the!end!of!the!second!fermentation!and!after!9!and!18!months! of!aging.!The!composition!of!the!sparkling!wine!was!evaluated!by!measuring!its!amino! acid!and!polysaccharides!content!and!its!foaming!properties.!The!use!of!synthetic!base!

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wine! allowed! us! to! quantify! the! contribution! of! the! yeast! cell! to! the! sparkling! wine! composition.!!! ! This!objective!is!described!in!the!third!chapter!of!this!thesis.!! Chapter!3:*Contribution*of*yeast*and*base*wine*supplementation*on*sparkling* wine*composition.*Results!submitted!to!Journal!of!Agricultural!and!Food!Chemistry* * *

OBJECTIVE* 3:! To! decipher! the! genetic! basis! of! the! fermentative! performance! of! S.(

cerevisiae.(!

To! infer! which! are! the! natural! genetic! variations! causing! the! phenotypic! differences! between! S.( cerevisiae! strains! we! conducted! a! QTL! program.! Two! phenotypically! distinct!wine!strains!were!chosen!as!parental!strains!(previously!analysed,!objective!1,! chapter!2).!Their!hybrid!and!their!F1!progeny!(117!segregants)!were!phenotyped!for! their! fermentative! behaviour! during! the! second! fermentation.! The! whole! genome! sequencing! using! NGS! technology! of! the! 117! segregants! allowed! us! to! construct! a! genetic!marker!map.!!

!Through! QTL! mapping! and! validation,! the! allelic! variation! within! four! genes! was! identified! as! the! responsible! for! the! phenotypic! divergence! quantified! in! the! second! fermentation!kinetics.!

!

This!objective!is!described!in!the!forth!chapter!of!this!thesis.!!

Chapter!4:*QTL*mapping*of*second*fermentation*performance*of*wine*yeast*

identifies* genes* involved* in* hybrid* vigor* and* pH* homeostasis.* Manuscript! in!

preparation.! !

A! schematic! overview! of! the! thesis! is! represented! in! Figure! 1,! which! includes! each! objective!and!the!experimental!plan!that!has!been!followed.!!

!! !

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!

*

! Figure*1.*Thesis!overview.* NW:!Natural!base!wine;!SW:!synthetic!base!wine;!N:!ammonium;!Act:!preparation!of!inactive!dry!yeast;!Mu:!nitrogen!mixture!similar!to! that!of!grape!must;!BW:!nitrogen!mixture!similar!to!that!of!base!wine;!P:!organic!nitrogen!mixture;!Ar:!organic!nitrogen!mixture,!noN:!no! nitrogen!added;!DAP:!diammonium!phosphate;!IDY:!inactive!dry!yeast.!!

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!

INTRODUCTION!

!

! ! ! ! ! ! ! ! !

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1 WINE!YEAST!PHYSIOLOGY!

1.1 Yeast!utilization!in!fermented!goods.!

Throughout!history!and!around!the!world,!human!societies!at!every!level!of!complexity! discovered! how! to! make! fermented! beverages! from! sugar! sources! available! in! their! local! habitats.! Fermentation! helps! to! preserve! and! enhance! the! nutritional! value! of! foods!and!beverages!and!has!a!combined!analgesic!and!disinfectant!effect.!Moreover,! fermented!beverages!have!played!key!roles!in!the!development!of!human!culture!and! technology,!contributing!to!the!advance!and!intensification!of!agriculture,!horticulture,! and! food@processing! techniques! (McGovern! et# al.! 2004).! In! that! sense,! yeasts! have! been!used!to!produce!fermented!goods!since!ancient!times.!But!it!was!only!in!1863! when!Pasteur!recognized!the!yeasts’!key!role!as!catalyst!in!wine!fermentation.!Since! then,!the!application!of!yeast!starters!has!become!a!standard!practice!in!the!industrial! processes!such!as!fermentations!or!production!of!other!products!made!by!yeasts!or! from!yeast!cells.!Among!all!yeasts!species,!only!about!a!dozen!are!currently!being!used! at!industrial!level!(Deak!2009).!Among!these!species!Saccharomyces#cerevisiae#has!an! outstanding! role,! in! several! process! such! as! bakery,! brewery! and! winery.! As! an! example,! its! association! with! wine! goes! back! to! ancient! times,! as! suggested! by! the! finding!of!DNA!matching!that!of!S.#cerevisiae!in!a!wine!jug!from!3150!BC!(Cavalieri!et# al.!2003).!Because!of!its!key!role!on!the!winemaking!process,!S.#cerevisiae!is!referred! also!as!“the!wine!yeast”!(Pretorius!2000).! !

1.2 General!characteristics!of!S.#cerevisiae.!

1.2.1 Cellular!architecture! As!a!eukaryote,!S.#cerevisiae!contains!membrane@bound!organelles,!its!chromosomes! are! located! in! the! nucleus,! and! it! uses! mitochondria! to! conduct! cellular! respiration! (Figure!1A).!Looking!from!the!inside!out,!the!yeast!cytosol!is!surrounded!by!the!plasma! membrane,!the!periplasmic!space,!and!the!cell!wall.!The!cell!wall!is!a!rigid!structure!

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about!100–200!nm!thick!that!constitutes!about!25%!of!the!total!dry!mass!of!the!cell.! The!cell!wall!is!composed!by:!highly!glycosylated!glycoproteins!(mannoproteins),!two! types!of!β@glucans,!and!chitin!(Figure!1B).!Its!composition!and!function!evolve!during! the!life!of!cell,!in!response!to!environmental!factors.!Although!its!principal!function!is! to!protect!the!cell,!it!participates!in!other!processes!such!as!sexual!agglutination!and! flocculation.! Flocculation! occurs! when! cells! adhere,! reversibly,! to! one! another,! which! leads! to! the! formation! of! macroscopic! flocs! sedimenting! out! of! suspension.! The! phenomenon! is! due! to! specific!cell!wall!lectins!in!yeast!(flocculins)! –!surface!glycoproteins!capable!of!directly! binding! mannoproteins! of! adjacent! cells.! Different!FLO!genes!genetically!determine! yeast! flocculation! behaviour! (Bester,! Jacobson!and!Bauer!2012).!Flocculation!is! a! sought@after! characteristic! for! yeast! producing!sparkling!wine!or!lager!beers.! !!

Like! other! biological! membranes,! the! plasma! membrane! (PM)! of! yeast! can! be! described! as! a! lipid! bilayer,! which! harbours! proteins! serving! as! cytoskeletal! anchors,! and! enzymes! for! cell! wall! synthesis,! signal! transduction,! and! transport! (Figure! 1C).! The! lipid! components!comprise!mainly!phospholipids!as!well!as!sterols!(ergosterol).!Like!the!cell! wall,!the!plasma!membrane!changes!both!structurally!and!functionally!depending!on! the!conditions!of!growth.!The!primary!functions!of!the!yeast!plasma!membrane!are:! o Physical!protection!of!the!cell.! o Control!of!osmotic!stability.! Figure!1.!(A)!S.#cerevisiae!cell.!(Feldmann!2012)(B)! Yeast!cell!wall.!(McClanahan!2009)!(C)!Plasma! membrane!(University!of!Regina)!

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o Control!of!cell!wall!biosynthesis.! o Anchor!for!cytoskeletal!compounds.!

o Selective! permeability! barrier! controlling! compounds! that! enter! or! that! leave! the!cell.!Of!prime!importance!in!active!transport!of!solutes!is!the!activity!of!the! plasma!membrane!proton@pumping!ATPase.! o Transport@related!functions!in!endocytosis!and!exocytosis.! o Location!of!the!components!of!signal!transduction!pathways.! o Sites!of!cell–cell!recognition!and!cell–cell!adhesion.! !

Enclosed! by! the! plasma! membrane,! like! in! all! other! cellular! organisms,! the! yeast! cytoplasm!is!the!site!for!many!cellular!activities!and!the!space!for!intracellular!traffic.! In!yeast,!it!is!an!aqueous,!slightly!acidic!colloidal!fluid!that!contains!proteins,!glycogen,! and! other! soluble! macromolecules.! Larger! macromolecular! entities! like! ribosomes,! proteasomes,! or! lipid! particles! are! suspended! in! the! cytoplasm.! The! cytosolic! (nonorganellar)! enzymes! include! the! glycolytic! enzymes,! the! fatty! acid! synthase! complex,!and!the!components!and!enzymes!for!protein!biosynthesis.!Many!functions! essential!for!cellular!integrity!are!localized!in!the!cytoplasm!(e.g.,!the!components!that! form!and!control!the!cytoskeletal!scaffold).!!

Various! compartments! surrounded! by! individual! membranes! are! located! within! the! yeast! cytoplasm! such! as:! the! endoplasmic! reticulum! (ER),! the! Golgi! apparatus,! the! vacuole,! the! mitochondria,! peroxisomes! and! the! yeast! nucleus.! The! endoplasmic! reticulum! and! the! Golgi! complex! are! key! organelles! for! all! processes! controlling! the! stability,!modification!and!transport!of!proteins.!The!yeast!vacuole!is!mainly!involved! in! intracellular! protein! trafficking! and! non@specific! intracellular! proteolysis.! Furthermore,!the!vacuole!serves!as!a!storage!compartment!(e.!g.!basic!amino!acids)! and!is!implied!in!the!regulation!of!the!cytosolic!pH.!The!mitochondria!is!the!organelle! responsible!for!cellular!respiration!and!is!involved!in!several!other!processes!such!as! synthesis!and!desaturation!of!fatty!acids,!Krebs!cycle,!biosynthesis!of!ergosterol.!It!will! adopt!different!morphologies,!depending!on!the!conditions!under!which!S#cerevisiae!is! grown.!The!main!characteristic!of!peroxisomes,!is!the!occurrence!of,!at!minimum,!one!

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hydrogen! peroxide@producing! oxidase! and! a! detoxifying! catalase,! an! enzyme! that! is! capable!of!metabolizing!hydrogen!peroxide.!

The!nuclear!structure!in!yeasts!is!a!nearly!round!organelle!of!about!1.5!mm!diameter! located!in!the!centre!of!the!cell!or!slightly!eccentrically.!The!nucleoplasm!is!surrounded! by!a!double!membrane!bilayer!(inner!and!outer!nuclear!membrane),!thus!separating! the! nucleoplasm! from! the! cytoplasm.! The! nucleoplasm! harbors! the! nuclear! chromosomes!packed!into!chromatin!structure.!S.#cerevisiae!has!16!chromosomes!and! its!genome!size!is!of!12.1!Mbp.!!

1.2.2 Reproduction!and!cell!cycle!

Yeast!can!follow!two!modes!of!reproduction:!(i)!asexual!budding,!the!most!common! mode! of! vegetative! reproduction! in!

yeasts,! or! (ii)! sexual! mating! of! haploid! cells! of! opposite! mating@type! that! can! propagate! vegetatively! or! –! under! starving! conditions! –! sporulate.! In! budding! cells,! the! chromosomes! are! duplicated! in! a! mitotic! cycle,! and! distributed! between! mothers! and! daughters! followed! by! cell! separation,! while! sporulation! involves! meiosis! to! generate! four! (haploid)! ascospores.! S.#

cerevisiae! cell! cycle! is! represented! in!

Figure!2).! !

Budding! occurs! in! both! haploid! and! diploid! yeast! cells.! Haploid! cells! exhibit! an! axial! budding! pattern! (the! mother! cell! buds! immediately! adjacent! to! its! last! daughter),! whereas! diploid! cells!

exhibit! a! bipolar! budding! pattern! (the! mother! cell! can! bud! at! or! near! either! of! its!

budding

budding budding

Figure!2.!S.#cerevisiae!life!cycle!adapted!from!(Sun!and!

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poles)! (Figure! 3A).! Budding! progresses! accordingly! with! the! cellular! cycle,! as! represented!in!Figure!3B.!

In!S.#cerevisiae,!sexual!mating!consists!in! the! fusion! of! two! haploid! cells! of! opposite!mating@type!(a!and!α)!to!form!a! diploid! cell.! The! mating! type! is! defined! by! the! MAT! locus.! Based! on! their! capacity! to! switch! their! mating! types,! yeast!can!be!classified!in!heterothallic!or! homothallic.! Heterothallic! haploid! cells! retain! their! mating! types! as! they! go! through! rounds! of! mitosis.! In! contrast,! homothallic! cells! frequently! switch! mating! type.! Thus,! the! progeny! of! a! single! homothallic! spore! colony! can! mate! with! each! other! to! form! diploids,! while! those! of! a! single! heterothallic! spore!colony!cannot.!! The!process!of!mating!type!switching!is!a!gene!conversion!event!initiated!by!the!HO! gene.!The!HO!gene!is!a!tightly!regulated!haploid@specific!gene!that!is!only!activated!in! haploid!cells!during!the!G1!phase!of!the!cell!cycle.!The!protein!encoded!by!the!HO!gene! is!a!DNA!endonuclease,!which!physically!cleaves!DNA!at!the!MAT!locus.!Once!HO!cuts! the!DNA!at!MAT,!exonucleases!begin!to!degrade!the!DNA!on!both!sides!of!the!site.!The! resulting!gap!in!the!DNA!is!repaired!by!copying!in!the!genetic!information!present!at! either!HML!or!HMR,!filling!in!a!new!allele!of!either!the!MATa!or!MATα!gene!(Haber! 2012).!!

The! lifespan! of! S.# cerevisiae! cells! is! divided! in! two! main! stages.! The! first! is! the! replicative! lifespan! (RLS),! and! is! measured! by! the! finite! number! of! divisions! that! a! particular! cell! has! undergone.! The! second,! the! chronological! lifespan! (CLS),! refers! to! the!total!lifespan!of!a!given!cell!and!is!the!sum!of!the!replicative!lifespan!and!the!time! a!cell!remains!viable!in!a!non@dividing!state!(Herman!2002;!Sinclair!2002).!Genetic!and! Figure!3.!(A)Budding!patterns!of!S.#cerevisae#! (Slaughter,!Smith!and!Li!2009).!(B)!Cellular!cycle! (Schroer!2005)! A! B!

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environmental! factors! will! determine! yeasts! lifespan.! In! that! sense,! several! studies! have!been!focused!on!the!deciphering!of!the!genetic!and!environmental!basis!of!the! aging!process.!As!an!example,!the!work!Powers!et#al.!(2006)!(Figure!4)!illustrates!how! the! removal! of! preferred! amino! acids! such! as! asparagine! and! glutamate! from! the! media!extends!the!CLS,!even!though!total!nitrogen!content!is!held!constant.!! !

1.3 S.#cerevisiae!sugar!and!nitrogen!metabolism.!

1.3.1 Respiration!and!fermentation! Yeasts!preferred!carbon!source!are!sugars.!S.#cerevisiae!can!metabolize!diverse!sugars,! such!as!glucose,!fructose,!galactose!or!mannose,!disaccharides!as!maltose!or!sucrose;! yet,!glucose!and!fructose!are!their!preferred!substrates.!The!metabolic!routes!for!the! dissimulation!of!hexoses!and!disaccharides!share!the!same!pathways!and!differ!only!in! the!initial!basic!steps!of!metabolism.!For!the!sugar!utilization,!yeast!has!primarily!to! sense!the!presence!of!glucose!or!fructose!in!the!environment!and!then!to!transport!it! across!the!plasma!membrane.!Once!inside!the!cell!it!will!enter!the!glycolysis!pathway! and!will!be!transformed!into!pyruvate.!Two!principal!modes!of!the!use!of!pyruvate!in! further! energy! production! can! be! distinguished:! respiration! and! fermentation! (Faria@ Oliveira!et#al.!2013).!

During! fermentation,! yeasts! produce! ethanol! via! a! two@step! reaction! from! pyruvate,! which! is! first! decarboxylated! to! acetaldehyde,! followed! by! the! reduction! of!

Figure!4.!CLS!extension!due!to!changes!in!the!nutrient!

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acetaldehyde!catalysed!by!alcohol!dehydrogenase,!forming!ethanol!and!regenerating! NAD!molecules!(Figure!5).!At!the!same!time,!and!in!addition!to!the!2!molecules!of!CO2!

and!of!ethanol,!formed!per!molecule!of!glucose,!the!carbon!squeleton!is!incorporated! into! other! by@products! such! as! acids! (pyruvic,! acetaldehyde,! ketoglutaric,! lactic)! and! also!glycerol.!

Although! fermentation! usually! happens! in! the! absence! of! oxygen,! when! the! concentration!of!glucose!is!high!(above!9!g/l),!even!in!the!presence!of!high!levels!of! oxygen,! S.# cerevisiae! will! metabolize! the! glucose! by! the! fermentative! pathway.! This! phenomenon!is!called!the!Crabtree! effect,!defined!as!the!inhibition!of! aerobic! metabolism! when! glucose! is! highly! concentrated! in! the! medium,!which!occurs!both!in!the! presence! or! absence! of! oxygen! (recently! reviewed! Pfeiffer! and! Morley! (2014).! For! instance,! S.#

cerevisiae! is! known! as! Crabtree!

positive! yeast,! since! is! able! to! produce!ethanol!aerobically!in!the! presence! of! high! external! glucose! concentrations.!

!

In! respiration,! pyruvate! enters! the! mitochondrial! matrix! where! it! is! oxidatively! decarboxylated!to!acetyl@CoA!by!the!pyruvate!dehydrogenase!multienzyme!complex.! This!reaction!links!glycolysis!to!the!citric!acid!cycle!(tricarboxylic!acid!(TCA)!cycle),!in! which!the!acetyl@CoA!is!completely!oxidized!to!yield!two!molecules!of!carbon!dioxide! and!reductive!equivalents!in!form!of!NADH!and!FADH2.! Figure!5.!Alcoholic!fermentation,!enzymatic!steps!in!S.# cerevisiae!(Faria@Oliveira!et#al.!2013)!

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1.3.2 Nitrogen!metabolism.!

Of! all! the! nutrients! assimilated! by! yeast! during! the! fermentation! of! grape! must,! nitrogen!is,!quantitatively,!the!most!important,!after!carbon@compounds.!In!fact,!the! nitrogen! composition! of! grapes! affects! the! growth! and! metabolism! of! yeast,! the! fermentation!rate!and!the!completion!of!fermentation!(Monteiro!and!Bisson!1991).! Yeasts! are! capable! of! utilizing! a! range! of! different! inorganic! and! organic! sources! of! nitrogen!for!incorporation!into!the!structural!and!functional!components!of!the!cell,! such! as! amino! acids! (and! consequently! peptides! and! proteins),! polyamines,! nucleic! acids,!and!vitamins.!!

Essentially,! all! nitrogen! compounds! accumulated! inside! the! cell! will! be! degraded! to! one!of!two!end!products,!ammonium!or!glutamate.!The!interconversion!of!ammonia,! glutamate! and! glutamine! is! called! Central! Nitrogen! Metabolism! (CNM).! From! these! two! amino! acids! all! other! nitrogen! containing! compounds! in! the! cell! are! produced! (Figure! 6).! Glutamate! and! glutamine! can! be! synthesized! using! ammonia! as! amino! donor.! Ammonium! and! α@ketoglutarate! will! be! used! by! the! NADPH! dependent! glutamate!dehydrogenase!(GDH1)!to!produce!glutamate!(reaction!1),!and!ammonium! and! glutamate! will! be! used! to! synthesize! glutamine! by! the! glutamine! synthetase! (GLN1)! (reaction2)! (recently! reviewed! by! Ljungdahl! and! Daignan@Fornier! (2012).! The! rest! of! nitrogen! compounds! will! be! synthesized! using! the! ammonia! from! either! glutamate!or!glutamine!as!indicated!in!the!Figure!6.!!

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! !

1.3.2.1 Nitrogen!catabolite!repression!

The! nitrogen! catabolite! repression! (NCR)! primarily! functions! to! ensure! that! cells! selectively!use!preferred!nitrogen!sources!when!they!are!available.!In!the!absence!of!a! preferred!nitrogen!source,!the!general!derepression!of!NCR@regulated!genes!enables! cells!to!use!alternative,!nonpreferred!nitrogen!sources.!The!NCR!operates!at!different! levels.!Firstly!at!protein!level,!it!will!inactivate,!internalise!and!degrade!permeases!that! transport! poor! nitrogen! sources,! is! the! case! for! example! of! the! general! amino! acid! permease!Gap1p.!In!presence!of!good!nitrogen!sources,!Gap1p!is!dephosphorylated,! ubiquinated!and!later!degraded.!Other!permeases!such!as!Hip1p!(histidine!permease)! or!Can1p!(basic!amino!acid!permease)!are!expressed!constitutively,!thus!not!regulated!

Figure! 6.! Schematic! diagram! of! the! main! pathways! of! nitrogen! metabolism.! The! entry! routes! of!

several!nitrogen!sources!into!the!central!core!reactions!are!shown.!The!class!A!preferred!and!class! B!nonpreferred!nitrogen!sources!are!in!green!and!red!text,!respectively.!The!nitrogen!of!preferred! nitrogen! sources! is! incorporated! into! glutamate.! Nitrogen! from! branched@chain! amino! acids,! aromatic! amino! acids,! and! methionine! (within! box)! is! transferred! to! a@ketoglutarate! by! transaminases! forming! glutamate.! The! rest! of! nitrogenous! compounds! are! synthesized! with! nitrogen!derived!from!glutamate!or!glutamine!as!indicated!(blue!arrows)!(Ljungdahl!and!Daignan@ Fornier!2012).!

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by!NCR!system!(Ter!Schure,!Van!Riel!and!Verrips!2000).!Secondly,!NCR!operates!at!the! transcriptional!level.!At!this!level,!five!main!players!participate!in!the!NCR.!Four!GATA! transcriptions!factors,!two!positives!(activators):!Gln3p,!Gat1p/Nil1p!and!two!negative! (repressors):! Dal80p! and! Deh1/Gzf3p.! With! the! notable! exception! of! GLN3,! the! expression! of! genes! of! the! other! three! GATA! factors! (GAT1,! GZF3,! and! DAL80)! is! sensitive! to! NCR.! The! fifth! player! is! the! regulatory! protein! Ure2p! (Ljungdahl! and! Daignan@Fornier! 2012).! Under! nitrogen@rich! conditions! the! general! transcription! activator! Gln3p! is! found! in! the! cytoplasm! in! a! complex! with! Ure2p,! which! prevents! Gln3p! to! enter! in! the! nucleus.! Under! nitrogen@limiting! conditions,! Gln3p! is! dephosphorylated,! enters! the! nucleus! and! increases! the! expression! of! NCR@sensitive! genes,!such!as!genes!encoding!some!permeases!such!as!GAP1,!MEP2!and!also!of!the! genes! of! the! central! nitrogen! metabolism! GDH2,! GLN1! (Ter! Schure,! Van! Riel! and! Verrips! 2000;! Magasanik! and! Kaiser! 2002;! Ljungdahl! and! Daignan@Fornier! 2012).! A! schematic!representation!of!the!NCR!circuit!is!shown!in!Figure!7!

The!effect!of!NCR!during!wine!fermentations!has!been!studied,!revealing!that!the!NCR! system! controls! the! different! patterns! of! ammonium! and! amino! acid! consumption! during!the!alcoholic!fermentation!which!in!turn!can!modify!the!secondary!metabolites! produced!by!the!yeast!and!affect!the!organoleptic!characteristics!of!the!wine!(Beltran!

Figure!7.!The!NCR!circuit.!Green!arrows!indicate!up@regulation;!blunted!

red! arrows! represent! down@regulation! at! the! level! of! transcription.! Dashed!lines!indicate!a!weaker!response.!Blue!lines!indicate!repression! that!is!not!at!the!transcriptional!level!(Boczko!et#al.!2005).!

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et#al.!2004).!As!an!example,!the!expression!level!of!GAP1#and!MEP2#(both!controlled! by!NCR)!during!the!fermentation.!In!high!nitrogen!condition!(HNC)!the!expression!of! both!genes!is!inhibited,!however,!in!low!nitrogen!condition!(LNC),!once!the!ammonium! concentration!decreases,!the!gene!expression!of!both!genes!increases.!(Figure!8).! 1.3.2.2 Nitrogen!source!classification! The!various!nitrogen!sources!used!by!S.#cerevisiae!are!often!qualitatively!referred!to!as! being!preferred!(good)!or!non@preferred!(poor).!This!classification!has!been!empirically! based!on!two!criteria.!The!first!criterion!is!how!well!the!individual!compounds!support! growth! when! present! as! sole! source! of! nitrogen.! The! second! criterion! reflects! the! finding! that! preferred! nitrogen! sources! generally! repress! processes! required! for! the! utilization! of! non@preferred! nitrogen! sources! (reviewed! in! Cooper! 1982;! Magasanik! 1992;!ter!Schure,!van!Riel!and!Verrips!2000;!Magasanik!and!Kaiser!2002;!Ljungdahl!and! Daignan@Fornier!2012).!!

The!classification!of!nitrogen!sources!is!not!absolute,!and!their!repressive!effects!can! vary!significantly!between!different!yeast!strain!backgrounds.!For!example,!ammonium! and,!to!a!lesser!extent!glutamate!are!repressing!nitrogen!sources!for!S1278b@derived!

Figure! 8.! Relative! gene! expression! of! ammonia! permeases! (MEP1,!

MEP2,!MEP3)!and!general!amino!acid!permease!(GAP1)!at!time!zero! (before! inoculation)! and! at! several! fermentation! points! in! all! nitrogen!conditions!studied!(Beltran!et#al.!2004).!

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strains,! whereas,! for! many! S288c@derived! strains,! they! are! not,! even! though! these! nitrogen!sources!promote!high!growth!rates!(Magasanik!and!Kaiser!2002).!The!analysis! of! the! transcriptome! of! S.# cerevisiae! growing! on! 21! individual! sources! of! nitrogen! revealed!that!the!expression!pattern!of!the!yeast!is!highly!correlated!with!the!growth! rate!exhibited!(Godard!et#al.!2007).!Furthermore,!several!of!the!nitrogen!sources!could! unambiguously!be!classified!as!follows:!class!A,!preferred!nitrogen!sources,!supporting! fast!growth—!nitrogen@sensitive!gene!expression!is!repressed!(NCR!is!active)!and!class! B,! nonpreferred! nitrogen! sources,! promoting! slow! growth—nitrogen! sensitive! gene! expression! is! derepressed! (NCR! inactive).! The! amino! acid! classification! based! on! the! yeast! growth! and! the! intracellular! pool! of! glutamate! and! glutamine! is! listed! in! the! Table!1.!!

!

During! wine! fermentations,! different! strains! present! different! growth! and! fermentation!profiles!depending!on!the!nitrogen!source!and!concentration!present!in! the!media.!Recently,!the!growth!of!different!wine!strains!during!the!fermentation!in!

Table!1.!Compilation!of!literature!values:!generation!times!and!glutamate!and!glutamine!pool!sizes!in!cells!

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different!nitrogen!sources!has!been!analysed.!The!study!demonstrates!than!wine!yeast! strains!differ!in!terms!of!their!ability!to!use!a!variety!of!nitrogen!sources!(Gutiérrez!et#

al.! 2012).! Brice! et# al.! (2014)! assessed! the! capacity! to! ferment! under! conditions! of!

nitrogen!deficiency!of!several!yeast!strains.!Important!differences!were!found!in!their! fermentation! capacity! in! a! nitrogen@limited! media,! indicating! those! strains! had! different! nitrogen! requirements,! this! time! in! terms! of! nitrogen! concentration.! These! divergences!were!not!explained!by!differences!in!their!ability!to!store!nitrogen!neither! by! their! protein! synthesis! activity.! However,! the! transcriptome! analysis! showed! specific!expression!patterns!for!the!strains!with!low!or!high!nitrogen!requirements.! !

2 SPARKLING!WINE!PRODUCTION!

2.1 Introduction!

The!international!organization!of!vine!and!wine!(OIV)!includes!sparkling!wines!in!the! category!of!special!wines!and!defines!them!as!follows:!! “Sparkling#wines#(18/73#&#6/79)#

Special# wines# produced# from# grapes,# musts# or# wines# processed# according# to# techniques# accepted#by#OIV,#characterised#on#uncorking#by#the#production#of#a#more#or#less#persistent# effervescence#resulting#from#the#release#of#carbon#dioxide#of#exclusively#endogenous#origin.# […]”# Thus,!establishing!their!most!defining!and!appreciated!characteristic,!its!effervescence! that!is!explained!by!the!presence!of!CO2!bubbles!rising!continuously!through!the!liquid.! Sparkling!wines!are!then!classified!depending!on!the!method!used!to!produce!them.!In! the!traditional!or!Champenoise!method!the!second!fermentation!takes!place!inside!the! bottle,!this!is!the!case!of!Champagne,!produced!in!the!Champagne!region!in!France,! Cava,!produced!in!Spain,!the!Crémant!(France)!or!Franciacorta!(Italy).!An!alternative!is! the!Charmat!method,!where!the!second!fermentation!will!take!place!inside!a!closed! tank,!and!once!the!second!fermentation!has!finished!the!wine!will!be!bottled!under! pressure.!Prosecco!and!Asti!Spumanti!are!sparkling!wines!produced!using!this!method.!!

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2.2 The!traditional!method!in!sparkling!wine!production!

The!main!steps!involved!in!the!traditional!method!are!represented!in!the!Figure!9.! 2.2.1 Obtention!of!base!wine! The!first!stage!of!the!production!of!sparkling!wine!through!the!traditional!method!is! the!obtention!of!the!base!wine.!It!is!important!to!notice!that!the!grape!varieties!that! will!be!used!to!produce!the!base!wine!are!restricted!to!those!specified!by!the!different! regulatory! boards.! In! the! case! of! cava,! the! regulatory! board! (Consell! Regulador! del! Cava)!establishes!that!the!only!grape!varieties!that!can!be!used!to!produce!cava!are:! Parellada,!Xarelolo,!Macabeu,!Chardonnay,!Malvoisie,!Pinot!Noir,!Trepat,!Red!Grenache! and!Monastrell!(Ordre!14.11.91!BOE!Núm.!278!(20.!11.91)).!To!produce!champagne,! the!grape!varieties!permitted!by!its!regulatory!board!(Comité!Interprofessionnel!du!vin! de!Champagne)!are:!Arabanne,!Chardonnay,!Petit!Meslier,!Pinot!blanc,!Pinot!gris,!Pinot! Meunier!and!Pinot!Noir.! OBTENTION'OF'

BASE'WINE' PRISSE&DE&MOUSSE&

TIRAGE' STACKING' RIDDLING' DISGORGING' DOSAGE' acclimated'yeast& base'wine' sucrose,'bentonite' SECOND' FERMENTATION' AGING' AUTOPHAGY' AUTOLISIS' Figure!9.!Schematic!representation!of!the!traditional!method!to!produce!sparkling!wine!

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Usually,!to!produce!the!base!wine!each!grape!variety!will!be!fermented!separately!in! tanks!with!controlled!temperatures!between!15º@18º.!The!desired!base!wine!will!have! moderate!alcohol!content!(≤11.5%!vol.)!and!a!certain!level!of!acidity!(Ribéreau@Gayon!

et#al.!2006;!Carrascosa!et#al.!2011).!The!aromatic!profile!of!the!base!wine!will!define!

the!organoleptic!properties!of!the!sparkling!wine!produced,!thus!yeast!will!be!selected! to! conduct! the! primary! fermentation! mostly! due! to! their! impact! in! organoleptic! properties! of! the! base! wine! (Vanrell! et# al.! 2007).! Once! the! fermentation! has! been! completed!the!base!wines!will!be!clarified,!raked,!filtered!and!fined.!The!next!step!is! the! assembling! or! coupage,! which! consists! on! the! blending! of! base! wines! produced! from! different! varieties,! different! origins! or! even! different! vintages.! The! assembling! stage! will! result! in! the!

obtention! of! the! cuvée.# Once! the!cuvée!has!been!obtained,!it! will! be! cold! stabilized! to! prevent! tartrate! precipitation! (Ribéreau@Gayon! et# al.! 2006;! Carrascosa! et# al.! 2011).! The! optimal! characteristics! of! the! base! wine! used! to! produce! cava!are!listed!in!Table!2.!

2.2.2 !Prise!de!mousse!

The!second!stage!of!sparkling!wine!production!is!the!prise#de#mousse,!during!which!the! second! fermentation! and! yeast! autolysis! will! take! place.! Several! technological! operations! take! place! during! this! stage:! ! tirage,! stacking,! riddling,! disgorging! and! dosage!(Carrascosa!et#al.!2011).!!

2.2.2.1 Tirage!and!yeast!acclimation!

The! tirage! consists! in! the! bottling! of! the! base! wine! and! the! liqueur# de# tirage.! The!

liqueur# de# tirage! is! a! solution! of! yeast! cells,! sugar! (usually! in! form! of! sucrose,! but!

concentrated!must!may!also!be!used)!and!riddling!agent,!usually!bentonite!(~3g/hl),!

Table!2.!Optimal!characteristics!of!the!base!wine!used!to!

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used!to!facilitate!the!precipitation!and!the!subsequent!removal!of!yeast!cells.!Before! being! inoculated! into! the! base! wine,! yeast! cells,! usually! selected! strains! of!

Saccharomyces# cerevisiae,! need! to! be! acclimated! in! order! to! obtain! an! industrial!

inoculum! apt! to! endure! and! overcome! the! harsh! conditions! of! the! second! fermentation!(low!pH,!high!ethanol!content,!increasing!CO2!pressure)!(Carrascosa!et#al.!

2011).!This!process,!known!as!piedadeacuve,!mainly!consists!on!culturing!the!yeast!cells! in! a! media! with! increasing! ethanol! concentration,! as! it! has! been! proved! to! be! most! successful! way! to! acclimate! yeast! and! ensure! the! completion! of! the! second! fermentation! (Kunkee! and! Ough! 1966;! Juroszek,! Feuillat! and! Charpentier! 1987).! As! illustrated! in! the! Figure! 10! the! adaptation! of! yeast! in! base! wine! results! in! faster! glucose!consumption.!

!

In! the! acclimation! procedure! yeast! cells! are! first! rehydrated,! and! then! cultured! in! a! media!composed!at!least!by!diluted!base!wine,!supplemented!with!sugar,!this!stage!is! known!as!acclimation!or!adaptation!phase.!Once!the!adaptation!phase!is!completed! (usually! between! 6! to! 48h),! diluted! base! wine! and! sugar! are! added! to! the! yeast! culture,! in! order! to! increase! its! volume! and! its! ethanol! concentration.! This! second! stage!is!known!as!proliferation!phase,!and!will!produce!enough!biomass!to!inoculate! the!base!wine.!Usually,!the!inoculum!from!the!piedadeacuve!will!only!represent!2!to!4%! of!the!final!volume,!thus,!if!the!initial!population!for!the!second!fermentation!should! be!around!1x106!cells/ml,!at!the!end!of!the!piedadeacuve!the!biomass!produced!should! Figure!10.!Fermentation!of!glucose!in!wine!under!CO2!pressure! by!yeast!propagated!in!base!wine!and!propagated!under!5!atm! of!CO2!pressure!in!grape!juice!(Kunkee!and!Ough!1966).!

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be!at!least!of!5x107!_{cells/ml!(Laurent!and!Valade!2007;!Carrascosa!et#al.!2011).!Valade!}

et!al!(2007)!proposed!the!acclimation!protocol!as!illustrated!in!the!Figure!11:!

Some!protocols!include!a!last!stage!that!consists!in!slowly!decreasing!the!temperature! of!the!culture,!in!order!to!adapt!yeast!to!the!low!temperatures!at!which!the!second! fermentation! will! be! carried! out.! It! has! been! demonstrated! that! the! acclimation! of! yeast! cells! can! modify! their! autolytic! process,! thus! the! compounds! released! to! the! media!and!the!final!flavour!of!the!sparkling!wine!(Perrot!et#al.!2002).!!

The!concentration!of!sugar!added!through!the!liqueur#de#tirage!into!the!base!wine!will! determine!the!pressure!inside!the!bottle,!thus!the!concentration!of!sucrose!should!be! adjusted!to!achieve!the!desired!pressure.!It! is! estimated! that! 4.00@4.30! g! of! sucrose! produce!one!atmosphere!of!pressure!inside! the! bottle! at! 10ºC! (Torresi,! Frangipane! and! Anelli!2011).!Table!3!indicates!the!quantities! of! sugar! required! to! produce! the! desired! pressure! in! the! bottle! after! the! second!

Figure!11.!Yeast!acclimation!protocol!for!inoculating!1000!Hl!of!base!wine!as!

proposed!by!(Laurent!and!Valade!2007)!

Table!3.!Sugar!concentration!of!the!base!wine!

according!to!the!desired!pressure!(Ribéreau@ Gayon!et#al.!2006).!

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fermentation.!!

Once!the!base!wine!and!liqueur#de#tirage!have!been!introduced!into!the!bottles,!those! are!hermetically!sealed!with!plastic!cup@shaped!seals!(bidule)!and!metal!crown!caps.! Bottles!are!then!stored!in!a!horizontal!position!in!box!pallets!or!stacks!in!a!relatively! cold!room!(12º@18º),!operation!known!as!stacking!(Ribéreau@Gayon!et#al.!2006;!Torresi,! Frangipane! and! Anelli! 2011).! Inside! the! bottle,! yeasts! go! through! the! second! fermentation,!followed!by!autophagy!and!autolysis.!

2.2.2.2 Second!fermentation!

Second! fermentation! begins! after! the! tirage.! During! the! second! fermentation,! yeast! will!consume!the!sugar!added!to!the!base!wine!and!transform!it!to!CO2!and!ethanol.!

The!second!fermentation!will!take!place!at!low!temperatures!between!12@18º!and!its! length!takes!from!15!to!45!days.!!

The!growth!pattern!of!S.#cerevisiae!during! the! second! fermentation! is! similar! to! its! growth!in!a!traditional!wine!fermentation.! However,! due! to! the! fewer! carbon! and! nitrogen!sources!present!in!the!base!wine,! yeast! growth! will! be! slower! and! more! limited! (Carrascosa! et# al.! 2011).! The! growth! and! yeast! viability! during! the! second! fermentation! (Figure! 12)! are! strictly! strain! dependent,! some! strains! do! growth! during! the! second! fermentation,! achieving! populations! of! 1x107_{! cel/ml,! while! others! do! not! growth.! Similarly,! yeast!}

viability!through!the!second!fermentation!last!between!40!to!90!days!after!the!tirage,! depending!on!the!strain!used!(Martínez@Rodríguez!et#al.!2002).!

!

In!sparkling!wine!fermentation!S.#cerevisiae!encounters!several!stress!sources,!among! them:! high! ethanol! concentration,! low! pH,! low! temperature! and! increasing! CO2!

pressure.!Thus,!the!ability!of!a!yeast!strain!to!overcome!these!stress!sources!has!been! recently!proposed!as!selection!method!for!yeast!to!conduct!the!second!fermentation!

Figure!12.!Yeast!population!through!the!second!

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(Borrull,!Poblet!and!Rozès!2015).!The!study!of!the!transcriptomic!profile!of!S.#cerevisiae! during! the! second! fermentation! revealed! that! the! genes! more! expressed! are! those! related! to! respiratory! metabolism,! oxidative! stress! response,! autophagy,! or! peroxisomal!function!(Penacho,!Valero!and!Gonzalez!2012).!This!expression!pattern!is! consistent! with! ethanol! being! the! main! environmental! factor! influencing! transcriptional! responses! to! winemaking! conditions.! Other! stress! factors! influencing! the! expression! patter! during! the! second! fermentation! are! the! low! fermentation! temperature,!and!to!a!lesser!extent!by!low!nitrogen!availability.!!

Kunkee! and! Ough! (1966)! demonstrated! how! some! stress! factors! found! in! sparkling! wine!production!affect!the!growth!of!the!yeast!strain!during!the!second!fermentation! in! an! additive! way! (Table! 4)! and! determined! that! the! CO2!pressure! was! the! main!

inhibitor!factor!for!yeast!growth.!In!addition,!the!stress!sources!present!in!the!second! fermentation!also!affect!the!fermentation!kinetics,!i.e.!sugar!consumption!rate!(Table! 5).!

!!

The! low! temperature! of! the! second! fermentation! also! slows! down! the! fermentation! activity! of! yeast,! as! well! as,! inhibits! its! growth!(Kunkee!and!Ough!1966),! similarly! to! the! situation! observed! in! the! cold! fermentation! of! white! wines! (Torija!et#al.!2003;!Beltran!et#al.!

Table!5.!Effect!of!pressure,!pH!and!nutrients!on!!glucose!

consumption!during!the!second!fermentation!(Kunkee!and! Ough!1966)!

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2008).! In! white! wine! fermentations,! the! decrease! in! the! fermentative! temperature! causes!a!prolongation!on!yeast!viability!(Beltran!et#al.!2006,!2008),!fact!that!may!be! occurring!also!in!the!second!fermentation.!

Thus,! due! to! the! limited! growth! and! the! restrictive! conditions! of! the! second! fermentation,! the! size! of! the! inoculum! should! be! between! 1x106_{! cells/ml! and! 2x10}6_!

cells/ml!to!ensure!the!completion!of!the!second!fermentation!(Kunkee!and!Ough!1966;! Ribéreau@Gayon!et#al.!2006;!Carrascosa!et#al.!2011).!! ! Yeast!response!to!stress!! The!cellular!stress!response!is!evolutionarily!conserved!in!all!living!organisms!–!a!major! role!being!attributed!to!the!induced!heat@shock!proteins!(Hsps)!and!other!molecules! that! confer! stress! protection.! The! molecular! responses! elicited! by! the! cells! dictate! whether!the!organism!adapts,!survives,!or,!if!injured!beyond!repair,!undergoes!death.!! Many!different!stresses!in!yeast!provoke!a!similar!set!of!responses.!The!terms!“general! stress! response”! (GSR)! or! “environmental! stress! response”! (ESR)! illustrate! these! molecular!events.!As!physiological!results,!yeast!cells!exposed!to!mild!stress!develop! tolerance! not! only! to! higher! doses! of! the! same! stress,! but! also! to! stress! caused! by! other!agents.!This!phenomenon,!known!as!cross@protection,!suggests!the!existence!of! an!integrating!mechanism!that!senses!and!responds!to!different!forms!of!stress!(Gasch! et#al.!2000).!! ! Ethanol!stress! Yeasts!produce!a!number!of!compounds!during!fermentation!that!hinder!its!ability!to! ferment,!among!them!ethanol.!Some!of!the!effects!of!ethanol!in!yeast!are!similar!to! those! caused! by! high! temperature.! Ethanol! modifies! PM! fluidity! and! the! yeast! cells! respond! to! this! by! increasing! the! concentration! of! unsaturated! fatty! acids! and! ergosterol,!and!by!decreasing!the!protein!content!in!the!membrane!(Bisson!1999).!The! ethanol@disrupted!membrane!becomes!more!permeable!and!H+_{!ions!leak!into!the!cell,!}

resulting! in! loss! of! the! proton! gradient! that! powers! proton! symporters! (including! amino! acid! transporters)! and! in! cytosolic! acidification! (Cartwright! et# al.! 1986).! In!