Saccharomyces en Vinos PDF

UNIVERSIDAD COMPLUTENSE DE MADRID
FACULTAD DE CIENCIAS BIOLÓGICAS

DEPARTAMENTO DE MICROBIOLOGÍA III
TESIS DOCTORAL
Estudio filo-funcional de levaduras de interés

enológico para su aplicación industrial
MEMORIA PARA OPTAR AL GRADO DE DOCTOR
PRESENTADA POR
Ignacio Belda Aguilar
DIRECTORES
Antonio Santos de la Sen

Eva Navascués López-Cordón
Madrid, 2017
© Ignacio Belda Aguilar, 2016

TESIS DOCTORAL
Estudio filo-funcional de levaduras de
interés enológico para su aplicación
industrial
MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR:
IGNACIO BELDA AGUILAR
Directores
Dr. Antonio Santos de la Sen
Dra. Eva Navascués López-Cordón
Madrid, 2016

TESIS DOCTORAL
industrial
IGNACIO BELDA AGUILAR
Madrid, 2016

industrial
Tesis Doctoral presentada por D. Ignacio Belda Aguilar para

optar al grado de Doctor en Biología por la Universidad
Complutense de Madrid
Madrid, 2016
Directores:
Dr. Antonio Santos de la Sen Dra. Eva Navascués López-Cordón

Profesor Titular Directora Técnica
Departamento de Microbiología III Agrovin S.A.
Universidad Complutense de Madrid

A mi familia
“In hard times, imagination is more important than knowledge”
Albert Einstein

Agradecimientos:
Esta Tesis es de mucha gente. Mucha gente que ha puesto medios económicos,
materiales y, sobre todo, humanos para que pueda llevarse a cabo.
Obligatorio comenzar por agradecer a Antonio Santos la dedicación en cuerpo y alma

en estos ya casi 5 años juntos. No ha habido hora ni día en que haya mostrado una negativa a
poner sobre la mesa lo que fuera necesario por nuestro trabajo. Como decía al principio, esta
Tesis es de mucha gente, pero desde luego tuya es. Tirando también del carro de la dirección,
Eva Navascués, los pies en la tierra y la que nos enseño la ciencia que esconde una botella de
vino. Apostó desde el principio por nosotros y, desde luego, este trabajo no sería una realidad
sin su fe en nosotros y en nuestra manera de ver la Microbiología. Finalmente y, aunque no
figura como Director de esta Tesis, suya es la culpa de que yo comenzara a trabajar en este
grupo. De nuevo, una confianza ciega, que me otorgó sin apenas conocerme, o si… yo nunca
lo sabré. Domingo, gracias por ese tiempo y esa puerta abierta que siempre has tenido para lo
que aconteciera.
Ahora van mis compañeros de laboratorio, todos ellos responsables de mis ganas de
venir a trabajar y pacientes de mis manías (que no detallaré). Alex, desde el comienzo, un
buen compañero que se hizo amigo. Hemos compartido muchas horas y faenas para sacar esto
adelante, enhorabuena por lo que te toca. Rocío, ya no está, se fue a la cumbre, pero sigue
presente y ayudando en lo que puede. Fuiste clave en los inicios y sigues siendo muy
importante en este final. Gracias por todo. Patri, a dos laboratorios de distancia y con distinta
área de investigación, conoce al dedillo lo que hago, porque soy muy pesado y ella me
escucha con un poleo delante. Por escuchar siempre, lo científico y lo no, gracias. Javi, llegó
el último, pero arrimó el hombro como el primero. Con todas mis ganas le he trasmitido lo
que sé y ahora ya es todo un científico, quédense con el Ruiz et al. Millones de gracias por tu
dedicación. Albert, sonrisa siempre en la boca, dando vida a un laboratorio que inunda de
anécdotas. Gracias por desenrranciarme siempre que está en tu mano. Y por último, Cris,
nuestra pie en la química y en lo volátil. Gracias porque nunca haya horarios, por vivir con
nosotros las prisas y presiones como si fueran tuyas. Al resto de compañeros que han pasado
por este laboratorio o han paseado por el pasillo de esta planta, gracias por lo que hayáis
podido aportar.

Echando la vista atrás, y unos pisos más abajo, ni más ni menos que responsables de
que yo acabara con decencia esta preciosa carrera de Biología; Pati, Isa, Estela, Gerardo:
gracias por todo lo vivido.
La vida científica sale fuera de las puertas de un laboratorio. Por su constante apoyo y
momentos compartidos, quería agradecer a Víctor J. Cid sus consejos y su visión de la
Ciencia y la vida científica. Un lujo haberme cruzado contigo. En el mismo pack, de los home
idols con vocación cervecera, Ana Alastruey, mil gracias también por muchas horas de trabajo
y consejos. Gobernarás el mundo, lo sé. De nuevo, un placer. A Mª Victoria Moreno-Arribas,
porque la confianza no se paga con nada y tú lo has hecho en mí cada vez que lo he
necesitado. Gracias por cada empujón y segundo dedicado. A Santiago Benito y Fernando
Calderón, un pie en el laboratorio y otro en la bodega. Mucho de esta Tesis es suyo, no sólo
resultados, si no un enorme respeto mío y de los míos por una dedicación admirable.
En el capítulo personal, gracias a mi familia, siempre. Una fe ciega y un cariño

constante responsable de que sea quien soy. Papa, Mamá, Emi, Ani, Ahinoa, Nahia (preciosa)
y Rocio. Gracias por todo. A esta última, Rocío, gracias especiales por todo lo que has puesto
en esta tesis. Lo científico y lo no tanto, leyendo manuscrito tras manuscrito que componen
esta tesis y aportando lo mejor de ti en ellos. Por mucho e impagable, Gracias.
Como decía al principio, esta Tesis es de mucha gente, y por supuesto debo reconocer el
papel de los organismos y empresas que han confiado y financiado este trabajo a través de
distintos proyectos. En primer lugar y, tanto en el plano institucional como al personal de
Laboratorio, gracias a Agrovin S.A. (proyecto ENZIOXIVIN, IDI- 20130192) por su
constante confianza y al Centro para el Desarrollo Técnico Industrial (CDTI, Ministerio de
Economía y Competitividad) por su sustento. Este trabajo ha sido financiado también por
Bodegas Pago de Carraovejas (proyecto IDI-20140448), Bodegas Emilio Moro, quienes con
su valentía han sido personajes fundamentales en la transferencia a la industria del
conocimiento generado en este trabajo.

ÍNDICE

ÍNDICE
ABREVIATURAS ..........................................................................................................IX
RESUMEN .............................................................................................................XIII
SUMMARY .......................................................................................................... XVII
1. INTRODUCCIÓN........................................................................................................ 3
1.1. Microbiología, enología e industria enológica ................................................ 3
1.2. Las levaduras en enología................................................................................. 4
1.2.1. Saccharomyces cerevisiae............................................................................. 8
1.2.2. Levaduras no-Saccharomyces ..................................................................... 10
1.2.2.1. Aplicaciones actuales............................................................................. 10
1.2.2.2. Perspectivas de investigación y aplicaciones futuras ............................ 14
1.3. Microbiología y elaboración de vino.............................................................. 15
1.3.1. Etapa prefermentativa.................................................................................. 15
1.3.2. Fermentación alcohólica.............................................................................. 16
1.3.3. Etapa post fermentativa .............................................................................. 18
1.4. Incidencia de las levaduras en la composición del vino ............................... 19
1.4.1. Consumo de azúcares y liberación de etanol............................................... 19
1.4.2. Glicerol........................................................................................................ 20
1.4.3. Ácidos.......................................................................................................... 21
1.4.3.1. Ácidos volátiles..................................................................................... 21
1.4.3.2. Ácidos no volátiles................................................................................ 22
1.4.3.2.1. Ácido málico .................................................................................. 22
1.4.3.2.2. Ácido láctico................................................................................... 23
1.4.4. Compuestos nitrogenados............................................................................ 23
1.4.5. Compuestos aromáticos............................................................................... 25
1.4.5.1. Ésteres................................................................................................... 25
1.4.5.2. Alcoholes superiores y ácidos grasos volátiles..................................... 26
1.4.5.3. Terpenos................................................................................................ 27
1.4.5.4. Compuestos azufrados .......................................................................... 29

2. OBJETIVOS .............................................................................................................. 33
3. CAPÍTULO 1 ............................................................................................................. 35
3.1. Estudio de la diversidad de especies de levaduras asociadas a distintas
regiones vitivinícolas y estudio inter- e intraespecífico de la producción de enzimas de
interés enológico. ................................................................................................................ 37
4. CAPÍTULO 2 ............................................................................................................. 81
4.1. Aplicación de levaduras pectinolíticas en maceración prefermentativa para
la mejora tecnológica de vinos tintos ................................................................................ 83
5. CAPÍTULO 3 ........................................................................................................... 113
5.1. Desarrollo de un métodos rápido para la selección de levaduras con elevada
actividad β-liasa................................................................................................................ 115
5.2. Caracterización de la fisiología en fermentación de Torulaspora delbrueckii y
su contribución a la complejidad de vinos tintos........................................................... 143
ANEXO ................................................................................................................ 161
6. CAPÍTULO 4 ........................................................................................................... 165
6.1. Estudio de la incidencia en la calidad de vinos tintos de la crianza sobre lías
de levaduras no convencionales ...................................................................................... 167
7. DISCUSIÓN GENERAL......................................................................................... 199
7.1. Diversidad microbiana y metabólica asociada al proceso de fermentación:
estudio filo-funcional de levaduras de interés enológico............................................... 199
7.2. Aplicación de levaduras pectinolíticas en maceración de vinos tintos........ 204
7.3. Selección y aplicación de levaduras en fermentación para la mejora de las
propiedades sensoriales de los vinos ............................................................................... 207
7.4. Aplicación de levaduras no convencionales en fases postfermentativas de
crianza sobre lías .............................................................................................................. 213
7.5. Perspectivas futuras......................................................................................... 216
8. CONCLUSIONES.................................................................................................... 219
9. BIBLIOGRAFÍA...................................................................................................... 225

Abreviaturas
ABREVIATURAS
AC Contenido en Antocianos / Anthocyanin Content

ADN/DNA Ácido Desoxirribonucleico / Desoxiribonucleic Acid
Ácido Desoxirribonucleico ribosómico / Ribosomal Desoxiribonucleic
ADNr/rDNA
Acid
Ap Aureobasidium pullulans
ARN/RNA Ácido Ribonucleico / Ribonucleic Acid
ARNr/rRNA Ácido Ribonucleico ribosomal / ribosomal Ribonucleic Acid
BLAST Basic Local Alignment Search Tool
CECT Colección Española de Cultivos Tipo / Spanish Type Culture Collection
CFU Unidades Formadoras de Colonias/ Colony Forming Units
CI Intensidad de Color / Color Intensity
CO2 Dióxido de Carbono / Carbon dioxide
C-S Carbono-Azufre / Carbon-Sulfur
cv Variedad Cultivada / Cultivated Variety
CYC Colección Complutense de Levaduras / Complutense Yeast Collection
D.O. Denominación de Origen / Apellation of Origin
Electroforesis en Gel con Gradiente de Desnaturalización / Denaturing
DGGE
Gradient Gel Electrophoresis
EM Bodegas Emilio Moro / Emilio Moro winery
G Bodegas Gordonzello / Gordonzello winery
Cromatografía de Gases-Espectrometría de Masas / Gass Cromatography-
GC-MS
Mass Spectometry
H2 S Ácido sulfhídrico / Hydrogen sulfide
HCl Ácido clorhídrico / Hydrochloric acid
HL Homocigoto para el alelo íntegro / Homozygous full-lenght allele
Cromatografía líquida de alta eficacia / High-Performance Liquid
HPLC
Chromatography
HS Homocigoto para el alelo delecionado / Homozygous Short allele
HTS Cribado de alto rendimiento / High-Throughput Screening
HT Alelo heterocigoto / Heterozygous allele
L. Linneo / Linneaus
Lt Lachancea thermotolerans
DMDS Dimetil disulfuro / Dimethyl disulfide
Mp Metschnikowia pulcherrima
MTL Metanotiol / Methanethiol
NaCl Cloruro Sódico / Sodium chloride
NCBI National Center for Biotechnology Information
NCR Represión Catabólica por Nitrógeno / Nitrogen Catabolite Repression
NTU Unidades Nefelométricas de Turbidez / Nephelometric Turbidity Units
O Bodegas Ossian / Ossian winery

IX

Abreviaturas

OD Densidad Óptica / Optical Density

Organización Internacional para la Vid y el Vino / International
OIV
Organisation of Vine and Wine
PCs Componentes principales / Principal Components
PCA Análisis de Componentes principales / Principal Component Analysis
PDC Bodegas Pago de Carraovejas / Pago de Carraovejas winery
PYCC Colección Portuguesa de Levaduras / Portuguese Yeast Culture Colection
Sc Saccharomyces cerevisiae
SM Coinoculación simultánea / Simultaneous co-inoculation
SMC S-metil-L-cisteína / S-methyl-L- cysteine
SO2 Dióxido de azufre / Sulfur dioxide
sp. Especie / Species
SPME Microextracción en Fase Sólida / Solid Phase Microextraction
SQ Inoculación secuencial / Sequential inoculation
Td Torulaspora delbrueckii
TIC Cromatograma de Iones Totales / Total Ion Chromatogram
TPI Índice de Polifenoles Totales / Total Polyphenol Index
UPGMA Unweighted Pair Group Method with Arithmetic Mean
YCB Base de Carbono para Levaduras / Yeast Carbon Base
YNB Base de Nitrógeno para Levaduras / Yeast Nitrogen Base
YMA Agar extracto de malta-extracto de levadura / Yeast Malt Agar
Agar extracto de malta-azul de metileno / Yeast Malt Agar-Methylene
YMA-MB
Blue
YPD Extracto de levadura-Peptona-Dextrosa / Yeast Peptone Dextrose
Extracto de levadura-Peptona-Glicerol-Etanol / Yeast Peptone Glycerol
YPGE
Ethanol
vs.
Versus
v/v
Volumen/Volumen / Volume/Volume
wt/vol
Peso/Volumen / Weight/Volume
3-MH
3-Mercaptohexanol / 3-Mercaptohexanol
3-MHA
Acetato de 3-mercaptohexilo / 3- mercaptohexyl acetate
4-MMP
4-Mercapto-4metilpentan-2-ona / 4-mercapto-4-methylpentan-2-one

RESUMEN & SUMMARY

XI

XII

Resumen
RESUMEN
La microbiología asociada al proceso de fermentación espontánea de un mosto de uva

consiste en una compleja sucesión de especies de levaduras que establecen una dinámica
poblacional determinada, fundamentalmente, por la presión selectiva que ejerce la creciente
concentración de etanol en el medio. En este contexto, es conocido cómo Saccharomyces
cerevisiae logra dominar el proceso, aún estando en concentraciones relativamente bajas en la
población inicial del mosto, a través de la estrategia de producción-acumulación-consumo de
etanol. Esta estrategia ha permitido a S. cerevisiae hacer, de un ambiente fundamentalmente
antrópico como son las fermentaciones, uno de sus principales nichos ecológicos. No
obstante, el proceso de fermentación vínica no es un proceso axénico y, dado el papel crucial
de S. cerevisiae en el proceso, la bibliografía tradicional ha denominado al resto de especies
de levadura involucradas en la fermentación vínica como levaduras “no-Saccharomyces”.
Así, las levaduras no-Saccharomyces constituyen un grupo heterogéneo de especies

fundamentalmente involucradas en las primeras etapas del proceso de fermentación. Puede
establecerse una clasificación de las mismas en función de su poder fermentativo y, por tanto,
del mayor o menor tiempo que, a priori, podrán mantenerse activas en la fermentación.
Existen levaduras eminentemente oxidativas (Pichia spp., Debaryomyces spp., Rhodotorula
spp., Cryptococcus spp.), levaduras poco fermentativas (Hanseniaspora spp.) y levaduras con
cierta o notable capacidad fermentativa (Metschnikowia spp., Kluyveromyces spp., Lachancea
spp., Torulaspora spp. y Zygosaccharomyces spp.). Su fisiología y su contribución a la
composición y calidad del vino están todavía poco estudiadas, aunque en la actualidad, tanto
científicos como enólogos, son conscientes de su potencialidad en la mejora de la calidad de
los vinos, tanto en aspectos sensoriales como tecnológicos y de seguridad alimentaria.
El presente trabajo abordó, en una primera etapa, el establecimiento de una amplia

colección de levaduras no-Saccharomyces, aisladas de mostos sin fermentar de tres
Denominaciones de Origen diferentes y a lo largo de tres vendimias. En esta colección,
compuesta por un total de 770 aislamientos, fueron identificadas 15 especies distintas, entre
las cuales dominaron notablemente las pertenecientes a los géneros Hanseniaspora,
Lachancea y Metschnikowia. En el estudio de la producción de enzimas de interés enológico
por parte de las levaduras de la colección, se observó como ciertas actividades como β-

XIII

Resumen
glucosidasa o proteasa estaban ampliamente distribuidas en el total de aislamientos y, por el

contrario, actividades como α-L-arabinofuranosidasa, pectinasa y celulasa estaban restringidas
a un bajo número de especies y cepas con, además, baja representación relativa en la
población total. En una posición intermedia se encontraron las actividades β-xilosidasa,
sulfito reductasa y β-liasa, destacando esta última por encontrarse en niveles de intensidad
moderados en la mayoría de especies que la mostraban. Los resultados de este estudio no sólo
permitieron establecer características generales de cada especie, si no observar diferencias
intraespecíficas que, además, respondían a patrones dependientes de origen, por lo que fue
posible establecer clusters de cepas dependiendo del lugar y el año en que fueron aisladas
considerando sus patrones de producción de actividades enzimáticas en el correspondiente
análisis de componentes principales.
Dada la dificultad del estudio de las 8 actividades enzimáticas mencionadas en la

colección de 770 aislamientos con los métodos descritos hasta el momento, este trabajo
requirió de la adaptación de los mismos a un formato de screening de alto rendimiento o del
desarrollo de métodos nuevos para actividades, como la β-liasa, para los que hasta el
momento no existía un método de evaluación directo mas allá de la valoración de los
compuestos volátiles derivados de su acción sobre sus precursores. Así en el presente trabajo
se describe un medio de cultivo diferencial para levaduras con alta actividad β-liasa basado en
el uso de la S-Metil-L-Cisteína como sustrato de dicha actividad análogo a los sustratos
cisteinilados presentes en el mosto y con el amonio derivado de su hidrólisis como única
fuente de nitrógeno. Conocido el gen IRC7 de S. cerevisiae como principal responsable de la
liberación de tioles voláties a partir de sus precursores cisteinilados en el mosto y conocida
también la existencia de dos isoformas del mimo, una activa y una inactiva, el medio de
cultivo descrito permitió la diferenciación, por su crecimiento, de aquellas cepas que
presentaban en homocigosis o heterocigosis la copia de funcional del gen IRC7. De igual
manera, el medio de cultivo permitió identificar algunas cepas de especies como Torulaspora
delbrueckii o Kluyveromyces marxianus como cepas con elevada actividad β-liasa,
comprobándose en fermentaciones en mosto blanco de la variedad verdejo, la gran capacidad
de la cepa T. delbrueckii Viniferm NS-TD para la liberación de los dos principales
compuestos volátiles tiólicos (3-MH y 4-MMP).

XIV

Resumen

Tras el estudio filo-funcional, en el que pudieron establecerse los perfiles de producción

de enzimas de las 15 especies identificadas en la colección, se procedió al estudio de la
repercusión de levaduras con actividad poligalacturonasa sobre el proceso de extracción de
color y mejora del proceso de clarificación de vinos tintos. En este estudio pudo
comprenderse como la especie Metschnikowia pulcherrima, considerada como
moderadamente fermentativa logra incidir más notablemente sobre los citados parámetros que
la otra especie pectinolítica, Aureobasidium pullulans, fundamentalmente oxidativa. Además,
de estos resultados se deriva la importancia de la baja temperatura en los procesos de
maceración prefermentativa, que no sólo mejora la extracción de ciertos pigmentos por
motivos químicos de solubilidad de la matriz acuosa que se mantiene por retraso de aparición
del etanol, si no por favorecer el desarrollo de ciertas especies de levaduras no-
Saccharomyces mas criófilas que S. cerevisiae y por el retraso en el desarrollo de ésta.
Con el objetivo de mejorar la calidad sensorial de los vinos tintos, se llevó a cabo el
estudio de la incidencia de la cepa Torulaspora delbrueckii Viniferm NS-TD mediante
inoculación simultánea y secuencial con S. cerevisiae. Como primera conclusión se deriva
que, a pesar del notable carácter fermentativo de T. delbrueckii, la coinoculación simultánea
de ésta con S. cerevisiae en concentraciones iguales limita notablemente su desarrollo. En el
caso de los vinos producidos con inoculación secuencial de T. delbrueckii Viniferm NS-TD
seguido de S. cerevisiae tras un descenso de 15 g/L en la densidad del mosto, se pudo apreciar
un notable incremento en la calidad de estos en comparación con lo exclusivamente
fermentados con S. cerevisiae. Esta mejora pudo asociarse con un notable incremento en el
contenido en manoproteínas de estos vinos, así como un ligero descenso en la acidez y en la
concentración de alcoholes superiores de los mismos.
Finalmente este trabajo abordó la aplicación de cepas de levadura no convencionales en

crianza sobre lías para la mejora de las propiedades sensoriales de los vinos tintos, analizando,
como factor principal, la liberación de manoproteínas durante el proceso. Los resultados
mostraron que, nuevamente, la cepa T. delbrueckii Viniferm NS-TD logra liberar
concentraciones muy elevadas de manoproteínas también durante la crianza sobre lías,
alcanzando niveles ligeramente superiores a los mostrados por la cepa, superproductora de
manoproteinas, S. cerevisiae Viniferm 3D. Notablemente por debajo, aunque superando las
concentraciones de manoproteínas liberadas por la cepa control S. cerevisiae Viniferm CT007

XV

Resumen
y por la cepa Lachacea thermotolerans NS-G-32, la cepa M. pulcherrima NS-EM-34, mostró

una producción dos veces mayor a la de dicha cepa control. Destacó la mayor liberación de
aminoácidos mostrada por esta cepa de M. pulcherrima, aunque debe mencionarse que esto
no supuso en ningún caso un incremento en la concentración de aminas biógenas en los vinos
tras los 4 meses de crianza.
En resumen, el presento trabajo aborda la línea de investigación en levaduras no-

Saccharomyces desde el punto de vista de sus propiedades enzimáticas de interés en enología.
Los resultados obtenidos no se limitan al establecimiento de los patrones generales de
producción de enzimas en las especies estudiadas, si no que demuestran su incidencia sobre
parámetros determinados de la calidad de los vinos, tanto en ensayos a escala de laboratorio
como a escala industrial. El conocimiento generado sobre el metabolismo y la fisiología en
fermentación de las cepas de la colección establecida en este trabajo, abren una amplia línea
de investigación futura en el estudio de las bases genéticas y los factores transcripcionales que
determinan un correcto desarrollo de estas cepas de levadura no-Saccharomyces facilitando su
su uso exitoso como inóculos en la industria enológica.

XVI

Summary
SUMMARY
Microbiology associated to the spontaneous fermentation process of grape must

comprises a complex series of yeast species that stablish a certain population dynamics,
basically due to the selection pressure exerted by the increasing ethanol concentration present
in the environment. In this context, it is known how Saccharomyces cerevisiae gets to
dominate the process, even when present at relatively low concentrations in the initial must
population, by an ethanol production-accumulation-consumption strategy. This strategy has
allowed S. cerevisiae to turn an essentially antropic environment such as fermentations into
one of its main ecologic niches. Nevertheless, wine fermentation is not a single-species
process and, given the crucial role of S. cerevisiae, traditional bibliography refers to all the
other yeast species involved in wine fermentation as ‘non-Saccharomyces yeasts’.
Thus, non-Saccharomyces yeasts comprise a heterogeneous group of species

largely/mainly involved in the first stages of the fermentation process. These can be classified
according to their fermentative capacity and, therefore, to the amount of time they could
potentially stay active during the fermentation. There are essentially oxidative yeasts (Pichia
spp., Debaryomyces spp., Rhodotorula spp., Cryptococcus spp.), low fermentative yeasts
(Hanseniaspora spp) and notably fermentative yeasts (Metschnikowia spp., Kluyveromyces
spp., Lachancea spp., Torulaspora spp. y Zygosaccharomyces spp). Their physiology and
contribution to wine composition and quality are still poorly studied, although nowadays both
scientifics and enologists are aware of their potential use in the improvement of wine quality,
both in sensorial and technologic and food-safety aspects.
The current work addressed, in a first phase, the establishment of a wide non-
Saccharomyces yeast collection isolated from unfermented musts from 3 different Certificates
of Origin during 3 harvests. In this collection, formed by a total of 770 isolates, 15 different
species were identified, among which the ones belonging to the genera Hanseniaspora,
Lachancea and Metschnikowia largely predominated. In the study of the production of
enzymes with enologic interest by yeasts from the collection, certain activities such as β-
glucosidase or protease activities were largely distributed in all the isolates and, on the
contrary, activities such as α-L-arabinofuranosidase, pectinase y celulase were restricted to a
low number of species and strains with also low relative representation in the total population.

XVII

Summary

β-xilosidase, sulfite reductase and β-lyase activities were in an intermediate situation,

standing out the latter for being in moderate intensity levels in most of the species that
showed this activity. The results of this study allowed us not only to establish general
characteristics of each species, but also to observe intraspecific differences that, in addition,
varied depending on the origin; thus, we were able to establish clusters of strains depending
on the location and year when they were isolated taking into account their enzymatic activity
patterns in the corresponding analysis of principal components.
Given the difficulty of studying the 8 above-mentioned enzymatic activities in the 770
isolates collection with the described methods to date, this work needed to either adapt them
to a high throughput screening format or develop new methods for activities such as β-liase,
which, to date, lack a direct evaluation method other than the valoration of volatile
compounds derived from their action on their precursors. Thus, in the present work we
describe a differentiating culture medium for yeasts with high β-liase activity based on the use
of S-Methil-L-Cysteine as the substrate for this activity, analogue to the cysteinylated
substrates present in the must, and the use of the ammonium resulting from its hydrolysis as
the only nitrogen source. Knowing that the IRC7 gene of S. cerevisiae is the main responsible
for the release of volatile thiols from cysteinylated precursors in must, and the existence of
two isoforms of it, one active and the other one inactive, the described culture medium
allowed for the differentiation by their growing of those strains that were homozygotic or
heterozygotic for the functional copy of the IRC7 gene. In a similar way, the culture medium
allowed us to identify some strains of species such as Torulaspora delbrueckii or
Kluyveromyces marxianus as strains with high β-liase activity, testing in fermentations of
white musts from ‘verdejo’ variety the great ability of the strain T. delbrueckii NS-TD to
release the two main volatile compounds (3-MH and 4-MMP).
After the phylo-funcional study in which we could establish the enzymatic production
profile of the 15 species identified in the collection, we went on to study the incidence of
yeasts with polygalacturonase activity on the color extraction process and on the improvement
of the red wine clarification process. In this study we could elucidate how Metschnikowia
pulcherrima, considered as moderately fermentative, could affect more notably the mentioned
parameters than the other pectinolytic species, Aureobasidium pullulans, mainly oxidative. In
addition, from these results we could highlight the importance of low temperatures during

XVIII

Summary
pre-fermentative maceration processes, which not only improve the extraction of certain
pigments due to chemical reasons of solubility of the aqueous matrix that is maintained due to
the delay in the ethanol appearance, but also favor the development of certain species of non-
Saccharomyces yeasts that are more cryophilic than S. cerevisiae and the delay in the
development of the latter.
With the aim of improving the sensorial quality of red wines, we performed a study
about the incidence of the strain Torulaspora delbrueckii NS-TD by simultaneous and
sequential inoculation with S. cerevisiae. As a first conclusion, we found that despite the
important fermentative role of T. delbrueckii, simultaneous co-inoculation with S. cerevisiae
at the same concentrations notably limits its development. In the case of wines produced by
sequential inoculation of T. delbrueckii NS-TD followed by S. cerevisiae, after a 15 g/L
decrease in must density, we noted a high increase in must quality as compared to the ones
only fermented by S. cerevisiae. This improvement was related to a notable increase in the
mannoprotein content of these wines, as well as to a slight decrease in their acidity and higher
alcohols concentration.
Finally, this work addressed the application of non-conventional yeast strains in wine
ageing over-lees for the improvement of sensorial properties of red wines, analyzing the
release of mannoproteins as the main factor during the process. The results showed that,
again, the strain T. delbrueckii NS-TD was able to release very high concentrations of
mannoproteins also during the ageing over-lees, reaching levels slightly higher than the
mannoprotein-overproducer strain S. cerevisiae Viniferm NS-TD. Considerably below but
also over the concentration of mannoproteins released by the control strain S. cerevisiae
Viniferm CT007 and by the strain Lachancea thermotolerans NS-G-32, the strain M.
pulcherrima NS-EM-34 showed a two-fold production compared to the control strain. The
higher amino acids release showed by this M. pulcherrima strain must be highlighted,
although we it should be mentioned that this did not correlate with an increase in the
concentration of biogenic amines in wine after 4 months ageing.
In summary, this work address the non-Saccharomyces topic in enology industry from
an original enzymatic point of view. These results not only establish the enzymatic properties
of some yeast species of enological interest, but also proving their incidence on wine quality

XIX

Summary

in both laboratory and industrial scale. This metabolic and physiological knowledge allow us
to foresee a large number of future works on the study of the genetic basis and the
transcriptional factors that determine the usefulness and the commercial success of non-
Saccharomyces yeasts in wine industry.

XX
1. INTRODUCCIÓN

Introducción
1. INTRODUCCIÓN
1.1. Microbiología, enología e industria enológica
La enología, como ciencia pluridisciplinar, emplea conocimientos derivados de la

química, la microbiología y la tecnología. Los avances en investigación permiten hoy en día,
mediante su interacción con la industria, anticiparse a las demandas del consumidor
desarrollando herramientas tecnológicas, bioquímicas y biológicas que marcarán el futuro de
la enología (Belda et al., 2015a).
Desde sus inicios, la microbiología enológica ha suscitado el interés de los enólogos

sobre el proceso fermentativo y los determinantes microbiológicos que condicionan de la
calidad final de los vinos que producen. En este campo, los trabajos iniciados a principios del
siglo XIX por el químico y enólogo Müller-Thurgau comenzaron a desarrollar las
herramientas microbiológicas básicas para el control tecnológico y sensorial del proceso de
fermentación mediante el uso de fermentaciones dirigidas inoculadas con cepas seleccionadas
de Saccharomyces cerevisiae (Pretorius, 2000). Sin embargo, la naturaleza inherente al
proceso de fermentación vínica no se restringe únicamente a la presencia de dicha especie, si
no que surge de la participación secuencial de una gran número de especies de levaduras y
bacterias, y de su interacción con las propiedades varietales de la uva (Fleet, 1990). Este
consorcio microbiano, como fruto de sus propiedades metabólicas, será responsable de gran
parte de la calidad sensorial de los vinos (Liu et al., 2015), que es en ultima instancia el objeto
de esta ciencia hedonista. La difícil elección entre la complejidad sensorial asociada a las
fermentaciones espontáneas y la seguridad industrial de las fermentaciones dirigidas ha
llevado en las últimas décadas al desarrollo de una intensa línea de investigación para el
completo entendimiento de la microbiología de ambos procesos para aunar las virtudes de
ambas tendencias, incrementando de forma paralela la calidad organoléptica y la seguridad
higiénica e industrial de los vinos.
Si bien las buenas prácticas vitícolas son determinantes para la llegada a la bodega de
una materia prima de calidad, es labor del enólogo la elección de las herramientas adecuadas
para el desarrollo de un proceso de fermentación que permita la revelación de las propiedades
varietales de cada uva. Para ello es fundamental la comprensión de la fisiología en
Introducción

fermentación de las distintas cepas de levaduras disponibles hoy en el mercado, así como el
desarrollo de nuevas cepas que amplíen el espectro de aplicaciones de las levaduras a lo largo
del proceso de elaboración de vino (Pretorius, 2000; Cordente et al., 2012; Pretorius et al.,
2012; Belda et al., 2016a).
1.2. Las levaduras en enología
Las uvas constituyen uno de los principales reservorios naturales de levaduras, entre
las que se establecen comunidades microbianas muy heterogéneas (Liu et al., 2015). Hasta 93
especies de levadura de 30 géneros distintos han sido descritos en la literatura, considerando
los resultados de estudios que copan 49 variedades de uva distintas procedentes de 22 países
(Barata et al., 2008; 2012; Bisson y Joseph, 2009). Renouf et al. (2007) pudo identificar, en
un solo estudio y mediante técnicas moleculares clásicas de PCR-DGGE, un total de 47
especies de levadura pertenecientes a 22 géneros a partir de la superficie de uvas de 6
variedades diferentes Aureobasidium, Auriculibuller, Brettanomyces, Bulleromyces, Candida,
Cryptococcus, Debaryomyces, Hanseniaspora, Issatchenkia, Kluyveromyces, Lipomyces,
Metschnikowia, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Sporidiobolus,
Sporobolomyces, Torulaspora, Yarrowia, Zygoascus y Zygosaccharomyces. Al margen de
esta gran diversidad microbiana, la densidad poblacional en la superficie de las uvas es baja
en las primeras etapas de maduración de la uva (101-103 UFC/g), incrementándose en varios
órdenes de magnitud (103-106 UFC/g) hasta el momento de la vendimia (Jolly et al., 2003;
Prakitchaiwattana et al., 2004; Renouf et al., 2005; Setati et al., 2012). La disponibilidad de
nutrientes en el hollejo, las condiciones climáticas, la variedad de uva y su estado fitosanitario
van a determinar la distribución y el balance en la población de levaduras asociadas, aunque
la influencia de cada uno de estos factores sobre dicha población no está carente de
controversia (Liu et al., 2015).
La población de levaduras en la superficie de la uva comienza a establecerse durante la

etapa de maduración de las uvas en la que su superficie aumenta conforme aumenta también
la disponibilidad de nutrientes y decrece la acidez (Combina et al., 2005; Cadez et al., 2010).
Las características fisiológicas y bioquímicas de cada variedad de uva pueden contribuir a
Introducción

determinar la población de levaduras que se establezca en su superficie (Guerzoni y

Marchetti, 1987; De La Torre et al., 1999; Sabate et al., 2002; Renouf et al., 2005; Nisiotou et
al., 2007). También las condiciones climáticas, como el grado de humedad generado por la
pluviometría de cada año, muestra en la mayoría de estudios una relación directa con el
incremento de la población de hongos y levaduras sobre las uvas (Longo et al., 1991; De la
Torre et al., 1999; Combina et al., 2005; Cadez et al., 2010). Sin embargo, a pesar de esta
mayor proliferación fúngica, no se ha observado relación entre las condiciones climáticas y la
diversidad de levaduras en un viñedo (Barata et al., 2012). Los tratamientos vitícolas, según
sean orgánicos, convencionales respetuosos o altamente agresivos, van a determinar también
la heterogeneidad de levaduras en un viñedo (Comitini y Ciani, 2008; Cadez et al., 2010;
Cordero-Bueso et al., 2011; Schmid et al., 2011; Tofalo et al., 2011; Tello et al., 2012;
Milanovic et al., 2013; Martins et al., 2014; Belda et al., 2016a). A este respecto, cabe
destacar la influencia del estado fitosanitario de las uvas en la población de levaduras en su
superficie. Por ejemplo, la presencia de Botrytis cinerea en las uvas afecta la composición
nutricional de las uvas y su superficie, por lo que se verá afectada la población de levaduras
en ella (Nisiotou y Nychas, 2007; Barata et al., 2008). A su vez, la capacidad de antibiosis de
ciertas especies del género Metschnikowia por la liberación de ácido pulquerrimínico genera
un efecto inhibitorio del desarrollo de otras levaduras y hongos que hace que estén
particularmente presentes en uvas afectadas por ese tipo de infecciones fúngicas (Sipikzci,
2006). En este sentido, cabe mencionar la producción de toxinas killer por parte de ciertas
levaduras sobre la propia ecología microbiana asociada al proceso fermentativo, así como su
uso en biocontrol (Marquina et al., 2002; Alonso et al., 2015; Velazquez et al., 2015).
Los estudios metagenómicos llevados a cabo por Bokulich et al. (2013, 2014)
permiten concluir que la microbiota asociada a un viñedo no es aleatoria, si no que responde a
unos patrones marcados por la situación geográfica, la variedad de uva y los factores
climáticos.
Si bien la población de levaduras que se establezca en la superficie de las uvas formará

parte de la “materia prima” para el proceso de fermentación, las condiciones y prácticas
enológicas en bodega, antes, durante y después del proceso de fermentación, determinarán la
evolución de la microbiota asociada a ella. El proceso de fermentación espontánea de un
mosto de uva está definido por la sustitución secuencial de una serie de especies de levaduras

5

Introducción
en función de la presión selectiva que ejerce la creciente concentración de etanol en el medio

(Figura 1). Así, en su mayor parte, y en especial en sus últimas etapas, el proceso está
dominado por cepas de la especie Saccharomyces cerevisiae, muy tolerantes al etanol. La
estrategia de “producción-acumulación-consumo” de etanol, característica de las especies del
género Saccharomyces (y compartida con el género Dekkera), confiere la ventaja competitiva
responsable de su dominancia en el proceso (Marsit y Dequin, 2015). Esto es debido al rápido
consumo de los azúcares del mosto, su conversión en etanol que actúa como inhibidor del
crecimiento del resto de especies y el posterior consumo del cierta cantidad del etanol
acumulado tras la dominancia del nicho ecológico (Thomson et al., 2005; Piškur et al., 2006;
Rozpędowska et al., 2011; Dashko et al., 2014). Así, la presencia y prevalencia de S.
cerevisiae en el proceso es un hecho esperado y deseado para la correcta llegada a término del
proceso de fermentación (Jolly et al., 2014).
Figura 1. Esquema representativo de la evolución de la población de levaduras a lo largo del proceso de

fermentación espontánea de un mosto de uva. Se aprecia la dominancia de Saccharomyces cerevisiae en las
etapas finales del proceso, conforme aumenta la concentración de etanol en el medio.
Sin embargo, la coexistencia de S. cerevisiae con el resto de especies presentes en el

mosto de uva y en las primeras etapas de la fermentación parece ser relevante para la
complejidad y calidad final en los vinos. Dado el papel crucial de S. cerevisiae en el proceso,
la bibliografía tradicional ha denominado al resto de especies de levadura involucradas en el
proceso como levaduras “no-Saccharomyces”. La presencia de ciertas especies de levaduras
no-Saccharomyces en las etapas iniciales e intermedias de la fermentación vínica contribuye,
a través de su fisiología y su interacción con S. cerevisiae, a modular las propiedades

6

Introducción
sensoriales de los vinos, dotándolos de una mayor complejidad (Zironi et al., 1993; Gil et al.,
1996; Lema et al., 1996; Toro y Vázquez, 2002; Ciani et al., 2006; Viana et al., 2008). Al
margen de la carga y diversidad microbiana asociada a la uva desde el viñedo, existen
diferentes prácticas que favorecen el desarrollo de las levaduras no-Saccharomyces durante
las primeras etapas fermentativas, retardando en cierta manera el desarrollo de S. cerevisiae y
el consiguiente inicio de la fermentación tumultuosa que acabará con la mayor parte de las
levaduras no-Saccharomyces. Una adición moderada de SO2, muy efectivo frente a la mayoría
de especies no-Saccharomyces, o el desarrollo de etapas de maceración prefermentativa
pueden favorecer el desarrollo de las poblaciones de levaduras no-Saccharomyces en el
mosto, obteniendo una mayor incidencia de sus beneficios. Sin embargo, el desconocimiento
a priori de la microbiota asociada a esas uvas puede provocar la aparición de desviaciones
sensoriales inesperadas. Fleet (2008) comenzó a sentar las bases de lo que hoy se conoce
como fermentaciones combinadas, secuenciales o multistarter controladas y su aplicación en
la industria mediante la producción de inóculos a gran escala de levaduras no-Saccharomyces.
Las primeras aplicaciones de las levaduras no-Saccharomyces para la mejora de las
características organolépticas de los vinos se produjeron a mediados del siglo XX, cuando
Cantarelli (1955) y Castelli (1955) realizaron trabajos de reducción del contenido en ácido
acético de los vinos haciendo uso de cepas seleccionadas de Torulaspora delbrueckii. Sin
embargo, la industria siguió relacionando este grupo heterogéneo de levaduras no-
Saccharomyces con ciertas desviaciones sensoriales, paradas de fermentación, incremento de
la acidez volátil de los vinos o la aparición de especies perjudiciales para el vino como B.
bruxellensis. Los progresivos trabajos de caracterización fisiológica de las distintas levaduras
implicadas en el proceso, ha permitido vislumbrar aquellas que parecen susceptibles de
aplicación en la industria para la mejora de la calidad de los vinos en aspectos de mejora
sensorial (Egli et al. 1998; Esteve-Zarzoso et al. 1998; Fleet y Heard 1993; Gil et al. 1996;
Henick-Kling et al. 1998; Lambrechts yPretorius 2000; Fleet 2003, 2008; Romano et al.
2003; Viana et al. 2008; Belda et al., 2015b; Benito et al., 2015), tecnológica (Belda et al.,
2016b) e incluso de la seguridad nutricional de los mostos y vinos (Oro et al., 2014; Alonso et
al., 2015).
Introducción

1.2.1. Saccharomyces cerevisiae
Como levadura responsable de la mayor parte del proceso de fermentación vínica S.

cerevisiae ha copado el interés de microbiólogos y enólogos en el estudio de sus propiedades
metabólicas que determinan gran parte de las características finales de los vinos. S. cerevisiae
ha acompañado a la humanidad a lo largo de gran parte de su desarrollo, sufriendo, a través de
su uso en fermentaciones, un proceso de “domesticación” (Liti et al., 2009). Se han
establecido dos eventos de domesticación, uno asociado a la elaboración del Sake y el otro
asociado al proceso de vinificación (Fay y Benavides, 2005). Desde el descubrimiento de las
levaduras como responsables del proceso de fermentación (Pasteur, 1860) una gran cantidad
de cepas de S. cerevisiae han sido aisladas, la mayoría de ellas asociadas a ambientes
fermentativos, y estudiadas a nivel genómico y metabólico. Sin embargo, a pesar de lo
antiguo de este proceso de domesticación, determinado por las prácticas fermentativas, no fue
hasta finales del siglo XIX cuando dos circunstancias determinaron el futuro del uso industrial
de S. cerevisiae. A principios de la década de 1880, Emile Christian Hansen, en el Carlsberg
laboratory (Dinamarca), desarrolló el primer inóculo axénico que fue usado en
fermentaciones experimentales pocos años después. En 1890, Müller-Thurgau realizó la
primera inoculación de un mosto de uva con un cultivo puro de levadura, aunque este proceso
no fue adecuadamente desarrollado en la industria hasta la década de los 70 del siglo XX.
Desde ese momento se incrementaron los procesos de selección de cepas de levaduras con
propiedades enológicas óptimas para el desarrollo de procesos de fermentación controlados
(Marsit y Dequin, 2015). La disponibilidad en un mercado global de un número limitado de
cepas de S. cerevisiae provoca que los grandes esfuerzos de las bodegas en su viticultura por
obtener una vendimia de calidad e identificativa de su terroir, queden atenuados o mermados
durante la posterior fermentación por el uso de cepas comerciales de levaduras aisladas de
orígenes distintos al propio entorno de la bodega y comunes para bodegas diferentes. A este
respecto surgen los procesos de selección de levaduras autóctonas adaptadas al entorno
vitivinícola del cual han sido aisladas y donde posteriormente serán utilizadas como inóculos
(Lopes et al., 2002; Capece et al., 2010), incrementando la tipicidad.
El complejo metabolismo de las cepas industriales de S. cerevisiae, debido a su

complejo genoma, deriva en la existencia de una gran diversidad intraespecífica, que permite
dotar al enólogo de un extenso catálogo de cepas de S. cerevisiae comerciales para la
Introducción

elaboración de sus vinos. Las propiedades óptimas y deseables de una cepa de S. cerevisiae
para su uso como inóculo en enología son bien conocidas (Figura 2). Estas propiedades
pueden ser diferenciadas y agrupadas en: propiedades fermentativas, propiedades sensoriales
y propiedades tecnológicas.
Figura 2. Resumen de propiedades tecnológicas y sensoriales deseables en cepas industriales de levaduras

Saccharomyces cerevisiae.
Como se ha comentado anteriormente, las propiedades fermentativas óptimas parecen

ser exclusivas de ciertas especies del género Saccharomyces. Sin embargo, parece claro que la
diversidad de cepas de S. cerevisiae no cubre todas las demandas exigidas por los enólogos y
no permite la revelación completa del potencial sensorial de las distintas variedades de uva.
Por el contrario, las posibilidades que brinda la enorme diversidad de especies de levaduras
asociadas a las primeras etapas del proceso fermentativo permitirán suplir las carencias que
muestran la inmensa mayoría de cepas de S. cerevisiae (Jolly et al., 2014). Estas levaduras,
conocidas en su conjunto como levaduras no-Saccharomyces, constituyen en la actualidad
uno de los focos de interés en I+D para el desarrollo y comercialización de nuevas cepas de
levaduras con propiedades innovadoras (Tabla 1).
Introducción

1.2.2. Levaduras no-Saccharomyces
La taxonomía actual reconoce un total de 149 géneros de levaduras que comprenden

unas 1500 especies (Kurtzman et al., 2011), de las cuales más de 90 han podido ser aisladas
de mostos de uva (Liu et al., 2015).
La prevalencia de las diferentes especies no-Saccharomyces durante el proceso de

fermentación vínica depende de su vigor fermentativo y de su resistencia al etanol, más que
de la concentración celular inicial que presenten en la uva. Así, al igual que ocurre con S.
cerevisiae, levaduras que se encuentran generalmente en bajas concentraciones en la uva pero
que muestran un cierto poder fermentativo, como es el caso de T. delbrueckii, y algunas
especies de los géneros Kluyveromyces y Lachancea, perduran en la fermentación hasta las
etapas más tardías. En función de su poder fermentativo, las levaduras no-Saccharomyces
pueden dividirse en 3 grupos: levaduras estrictamente aerobias (Pichia spp., Debaryomyces
spp., Rhodotorula spp., Cryptococcus spp.), levaduras apiculadas con bajo poder fermentativo
(Hanseniaspora uvarum (y su anamorfo Kloeckera apiculata) y Hanseniaspora
guilliermondii) y levaduras con metabolismo fermentativo (Metschnikowia pulcherrima,
Kluyveromyces marxianus, Torulaspora delbrueckii y Zygosaccharomyces bailii) (Jolly et al.,
2014). En base a esto, las levaduras de este último grupo parecen ser las de mayor potencial
para su uso en cultivos combinados con S. cerevisiae, puesto que serán las únicas capaces de
desarrollar de forma suficiente su metabolismo para que pueda tener una incidencia en la
calidad del vino (Ciani et al., 2010).
1.2.2.1. Aplicaciones actuales
- Grado alcohólico. Uno de los objetivos de mayor interés en la enología actual, y que
está mostrando resultados muy prometedores, es la disminución del contenido en etanol de los
vinos (Contreras et al., 2014; Quirós et al., 2014). Este objetivo se aborda mediante el
aprovechamiento del menor rendimiento alcohólico de ciertas especies no-Saccharomyces.
Dichas levaduras presentan un metabolismo, cuya regulación incluso en condiciones de alta
presión osmótica, les permite consumir parte de los azúcares de forma oxidativa generando
una cantidad de etanol menor. El uso de M. pulcherrima en inoculación secuencial con S.
cerevisiae ha permitido lograr, en condiciones controladas y en ausencia de desviaciones
sensoriales asociadas al metabolismo respiratorio (excesiva liberación de ác. acético) la

10

Introducción
disminución de hasta 4% (v/v) de etanol en los vinos (Morales et al., 2015). Unido al distinto
uso de los azúcares en fermentación, el incremento de la producción de glicerol en los vinos
es una de las virtudes mas destacadas de las fermentaciones espontáneas y corroboradas en el
desarrollo de fermentaciones secuenciales con levaduras no-Saccharomyces. Diversas
especies levaduras no-Saccharomyces en fermentación contribuyen a un incremento
significativo en el contenido en glicerol en los vinos, sin embargo los resultados mas
prometedores a este respecto se han encontrado en el uso de cepas de L. thermotolerans y
Candida zemplinina (Ciani y Ferraro, 1998; Soden et al., 2000; Comitini et al., 2011).
- Acidez de mostos y vinos. En el estudio del metabolismo de las levaduras no-

Saccharomyces, su incidencia sobre la acidez de los vinos ofrece herramientas útiles para su
control. Ha sido ampliamente demostrada la incidencia de T. delbrueckii sobre la acidez
volátil de los vinos logrando una reducción significativa (Moreno et al. 1991; Bely et al.
2008; Renault et al. 2009; Belda et al., 2015b). También, las desviaciones en la acidez total
inicial de los mostos pueden ser corregidas mediante la disminución de la acidez málica o el
incremento de la liberación de ácido láctico durante la fermentación. Benito et al. (2013)
demostró la capacidad de cepas seleccionadas de SchizoSaccharomyces pombe para la
degradación completa del ácido málico del vino. Zygosaccharomyces bailii y T. delbrueckii
presentan también cierta capacidad de degradación del ácido málico en fermentación
(Romano y Suzzi, 1993; Belda et al., 2015b). En cuanto al incremento de la acidez de los
vinos, el uso de L. thermotolerans ha permitido una mejora muy significativa del perfil
sensorial de los vinos mediante la liberación de ácido láctico (Comitini et al., 2011; Gobbi et
al., 2013).
-Perfil aromático. El principal objetivo del uso de levaduras no-Saccharomyces es el

incremento del carácter varietal y la intensidad aromática de ciertas variedades de uva debido
a su potencial enzimático. Si bien las propiedades enzimáticas de distintas especies no-
Saccharomyces han sido estudiadas desde hace mucho tiempo, en especial las glicosidasas
por su efecto sobre el perfil varietal de vinos blancos (Rosi et al., 1994; Manzanares et al.,
1999; McMahon et al., 1999; Mendes-Ferreira et al., 2001; Mateo et al., 2011), la
confirmación de su repercusión sobre las propiedades del vino es una tarea difícil y que
requiere mayor estudio (Belda et al., 2015c). A este respecto, la búsqueda de levaduras con un
determinado perfil enzimático ha permitido identificar nuevas aplicaciones de las levaduras

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Introducción
no-Saccharomyces para la mejora de otras propiedades aromáticas, como el incremento del

carácter tiólico de ciertos vinos blancos por actividades β-liasas alternativas (Belda et al.,
2016c),
Tabla 1. Resumen de las propiedades enológicas a destacar de levaduras no-Saccharomyces. Contribuciones

positivas y negativas a la composición y calidad de los vinos.
Propiedades enológicas
Especie Efectos indeseables Referencias
positivas
Disminución del contenido en Cinética fermentativa
ácido acético. más lenta.
Incremento en la liberación de
Liberación de H2S. Castelli, 1955; King & Dickson, 2000;
manoproteínas.
Torulaspora Renault et al., 2009; Azzolini et al., 2012,
Incremento del carácter varietal
delbrueckii 2015; Taillander et al., 2014; Belda et al.,
(liberación de aromas tiólicos y
2015b, 2016d; Velazquez et al., 2016)
terpénicos).
Disminución de astringencia
(consumo de ácido málico).
Retrasos o paradas de
Intensa liberación de ésteres
fermentación por
aromáticos (fruta blanca).
acción antimicrobiana.
Incremento del carácter varietal
(liberación de aromas tiólicos y Bisson & Kunkee, 1991; Pallman et al.,
terpénicos). 2001; Clemente-Jimenez et al., 2004b;
Metschinkowia
Incremento en la liberación de Rodriguez et al., 2010; Sadoudi et al.,
pulcherrima
manoproteínas. 2012; Oro et al., 2014; Contreras et al.,
Elevada actividad pectinolítica 2015; Belda et al., 2016b
(extracción de color y proceso
de clarificación).
Reducción del grado alcohólico
de los vinos.
Obtención de vinos
Liberación de ácido láctico. con atributos "ácidos" Kapsopoulou et al., 2007; Comitini et al.,
Lachancea o "picantes". 2011; Gobbi et al., 2013; Benito et al.,
thermotolerans Producción elevada de glicerol. 2016)
Liberación de 2-feniletanol.
Efecto poco predecible
Alta producción de glicerol. (cepa-dependiente) en
el perfil aromático
Ciani & Maccarelli, 1998; Soden et al.,
Consumo preferencial de
Candida zemplinina 2000; Andorra et al., 2010; Di Maio et al.,
fructosa.
2012; Sadoudi et al., 2012
é liberación de terpenos.
ê producción de alcoholes
superiores y aldehidos.
Liberación de 2-feniletil
Elevada acidez volatil
acetato.
Liberación de terpenos (alta Caruso et al., 2002; Rojas et al., 2003;
Producción de H2S
actividad β-glucosidasa). Viana et al., 2009; De Benedictis et al.,
Hanseniaspora vineae
Formación de aminas 2011; Belda et al., 2016b; Martin et al.,
biógenas 2016; Lleixá et al., 2016
Retraso en el inicio de
la fermentación
Elevada producción de
ê producción de H2S Y SO2.
ácido acético
Degradación de ácido málico.
Liberación de polisacáridos. Romano & Suzzi, 1993; Domizio et al.,
Zygosaccharomyces
Consumo preferencial de 2011, Loureiro & Malfeito-Ferreira., 2003;
bailii
fructosa. Sütterlin, 2010
Elevado vigor fermentativo
(reactivación de paradas de
fermentación).
Alta producción de
Schizosaccharomyces Degradación de ácido málico y Peinado et al., 2004; Benito et al., 2013,
acetaldehido, propanol
pombe ácido glucónico 2015
y 2,3-butanediol

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Introducción

Finalmente, de la coinoculación de levaduras no-Saccharomyces junto con S.

cerevisiae ha mostrado una repercusión muy significativo sobre el perfil de aromas
fermentativos en los vinos, mediante, por ejemplo, una reducción del contenido en alcoholes
superiores que en ocasiones tienen a monopolizar el aroma de los vinos en detrimento de su
complejidad sensorial (Belda et al., 2015b; Benito et al., 2016). Estudios recientes revelan
que el uso de levaduras no-Saccharomyces en fermentaciones combinadas con S. cerevisiae
no sólo modula el perfil aromático de los vinos por el propio metabolismo de las primeras, si
no que su mera coexistencia provoca variaciones muy significativas en el patrón de expresión
génica, y por tanto en el metabolismo, de la cepa de S. cerevisiae que lleva a cabo la mayoría
del proceso fermentativo (Barbosa et al., 2015). Así, los verdaderos mecanismos que
determinan la variación en el perfil aromático de los vinos en fermentaciones mixtas han de
ser estudiado en profundidad en un futuro próximo.
-Liberación de manoproteínas. Recientemente la liberación de manoproteínas por

ciertas especies no-Saccharomyces ha sido identificada como una característica con gran
repercusión en la calidad de los vinos. Ello es debido a que ciertas levaduras presentan en su
pared una mayor cantidad de manoproteínas y a que dichas levaduras sufren un proceso de
lisado previo a la finalización de la fermentación vínica (Domizio et al., 2014). Este hecho es
especialmente acusado en cepas seleccionadas de T. delbrueckii, posiblemente por el gran
desarrollo y prevalencia que esta levadura alcanza en fermentación (Belda et al., 2015b)
aunque, recientemente, se han demostrado los beneficios de su aplicación durante la crianza
sobre lías para modificar el perfil sensorial de los vinos (Belda et al., 2016d).
-Control biológico y seguridad alimentaria. Por último, el control biológico es una de

las aplicaciones mas prometedoras de las levaduras no-Saccharomyces, aunque todavía en
fases muy preliminares. La producción de toxinas killer en especies como Pichia
membranifaciens, Ustilago maydis y ciertas especies del género Kluyveromyces y de otras
moléculas que generan antibiosis, como el ácido pulquerrimímico de M. pulcherrima, han
sido utilizados en el control biológico en pre- y post-cosecha de especies deteriorantes como
B. bruxellensis (Comitini et al., 2004; Santos et al., 2009, 2011; Mehlomakulu et al., 2014;
Oro et al., 2014) o en el control de la contaminación de mostos concentrados por parte de
especies osmófilas (Alonso et al., 2015) u hongos fitopatógenos como Botrytis cinerea
(Santos et al., 2004). Asimismo, en seguridad alimentaria el uso de inóculos de levaduras

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activas o inactivas para la adsorción de ocratoxina A está siendo estudiada para paliar los
problemas de salud asociados a su consumo (Petruzzi et al., 2014).
1.2.2.2. Perspectivas de investigación y aplicaciones futuras
El desarrollo de las tecnologías ómicas, en especial de la metagenómica han puesto de

manifiesto la verdadera diversidad microbiana asociada a cada una de las etapas del proceso
de vinificación, desde la viña hasta la crianza de los vinos (Bokulich et al., 2014, Liu et al.,
2015). Complementariamente, la metabolómica ha permitido identificar los determinantes
químicos los vinos de distintas variedades de uva y provenientes de regiones vitivinícolas
diferentes, así como la influencia con que las distintas levaduras implicadas en el proceso de
vinificación contribuyen a la composición final del vino (Hong 2011; Alañón et al., 2015). De
esta forma, comienzan a establecerse las bases analíticas de la repercusión que las distintas
técnicas agronómicas y enológicas muestran sobre el perfil sensorial de los vinos y, que desde
hace décadas se vienen observando de forma empírica. Siguiendo con la tendencia de
tecnologías de alto rendimiento, uno de los objetivos abordados en esta tesis doctoral ha sido
el desarrollo y adaptación de los métodos y procesos de selección de levaduras no-
Saccharomyces en base a sus propiedades enzimáticas (Belda et al., 2016a, 2016c). Los datos
genómicos disponibles hasta el momento para la mayoría de especies de levadura no-
Saccharomyces dificulta el establecimiento de las bases genéticas su metabolismo, así como
su respuesta transcripcional en distintas condiciones enológicas. Los numerosísimos estudios
de regulación génica llevados a cabo en S. cerevisiae durante las últimas décadas de S.
cerevisiae hasta la actualidad han permitido un profundo conocimiento sobre los parámetros
importantes en la fermentación para el control y la optimización al máximo de las distintas
posibilidades que S. cerevisiae ofrece a nivel metabólico. De esta forma la tarea a realizar en
un futuro próximo consistirá en estudiar la estructura y función génicas en las especies no-
Saccharomyces para las que existen ya inóculos comerciales (T. delbrueckii, L.
thermotolerans, M. pulcherrima y P. kluyvery, fundamentalmente) así como sus mecanismos
de regulación para controlar y optimizar su uso en bodega. Este será el trabajo que garantizará
el establecimiento real de los no-Saccharomyces en la industria, ofreciendo al enólogo una
opción cada vez mas real en su uso como herramientas para el control y modulación del perfil
sensorial de los vinos.

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Introducción
1.3. Microbiología y elaboración de vino
El enólogo tras el delicado proceso de vendimiado de las uvas y su transporte hasta la

bodega hace uso de su conocimiento y del conjunto de técnicas físicas, químicas y biológicas
para obtener el mejor resultado, en forma de vino, tras el proceso de fermentación.
Centrándonos en el proceso microbiológico de fermentación, se puede dividir el conjunto de
las operaciones de vinificación en 3 grandes etapas. Así, flanqueando al proceso fermentativo,
existirá una etapa prefermentativa y una postfermentativa (Figura 3). Ambas presentan una
duración variable y el conjunto de técnicas que se pueden aplicar en ambas etapas es también
muy amplio y determinará junto con el proceso fermentativo, las propiedades finales de los
vinos.
1.3.1. Etapa prefermentativa.
Comprende desde la llegada de la uva a la bodega hasta el inicio de la fermentación

alcohólica. Durante esta etapa se corrigen posibles defectos que traiga la uva consigo o para la
mejora de las propiedades del mosto de partida para la vinificación.
En zonas cálidas es necesaria la acidificación de los mostos para el mantenimiento de

ciertas características de frescura y longevidad deseadas por los consumidores, especialmente
en vinos blancos. El método más habitual es la adición de ácido tartárico. Por el contrario, en
climas fríos, cuando las uvas no alcanzan una madurez completa su acidez puede ser elevada
y se lleva a cabo una desacidificación, bien química o biológica. La adición de agentes
antimicrobianos como el SO2, que ayuden a estabilizar la microbiota indígena que trae
consigo la uva, es necesario para el disminuir el riesgo de desviaciones microbianas a lo largo
del proceso de elaboración y crianza.
En la elaboración de vinos tintos, se procede al estrujado y despalillado de la uva

previa a su introducción en los depósitos donde se llevará a cabo la fermentación. Una
operación prefermentativa opcional es la técnica de maceración preferemntativa en frío (MPF)
que consiste en el encubado de la uva entera a baja tempertura (10-12ºC) durante un intervalo
de tiempo variable (4-7 días) a fin de incrementar el potencial aromático y colorante de los
vinos. En vinos blancos las operciones prefermentativas son el despalillado, prensado de la
uva, para la extracción de mosto y desfangado o clarificación de los mismos, realizándose la

15

Introducción
fermentación en ausencia de hollejos y otras partes sólidas de la uva. El desfangado consiste

en las eliminación de sustancias pécticas del mosto, localizadas en las paredes de las células
del hollejo. El mosto de uva contiene enzimas pectinolíticas, (pectín metil esterasa y
poligalacturonasa) a la degradación de las pectinas que logrando un descenso en la viscosidad
de los mostos.
Al margen de estas actividades endógenas, es frecuente el uso de preparados

enzimáticos comerciales en enología para abordar distintos procesos de mejora tecnológica y
sensorial de los vinos. En primer lugar, la adición de enzimas pectinolíticas
(poligalacturonasa, pectín metil esterasa y pectín liasa) acelera el proceso de clarificación de
mostos evitando los riesgos de iniciar la fermentación sobre mosto no clarificado. En el caso
de los vinos tintos, estos preparados enzimáticos se emplean durante el proceso de maceración
para la mejora de la extracción de compuestos fenólicos al mosto que contribuyan al color de
los vinos así como a su estructura.
1.3.2. Fermentación alcohólica
La fermentación alcohólica del vino consiste en la transformación de los azúcares

presentes en el mosto en alcohol etílico y CO2. Sin embargo, el proceso de fermentación de un
vino es un complejo proceso bioquímico en el que diversas rutas metabólicas de los
microorganismos implicados en ella y diversos procesos físico-químicos determinan las
propiedades finales del vino. En el caso de los vinos blancos, la fermentación se lleva a cabo
sobre un mosto clarificado en ausencia de hollejos y otras partes sólidas de la uva. En
elaboración de vino tinto, la fermentación transcurre a la vez que la maceración, es decir se
fermenta en contacto con los hollejos, y pepitas.
Aunque sigue habiendo vinificaciones particulares mediadas por la microbiota

espontanea de la uva, lo habitual es la fermentación controlada mediante la adición de un
inóculo seleccionado de levadura S. cerevisiae (Pretorius, 2000). Actualmente el elaborador
dispone de cientos de cepas para elegir en función de las características del vino buscado. En
los últimos cinco años, se están trasladando a la industria el empelo de inóculos secuenciales
de una levadura no-Saccharomyces seleccionada seguida de S. cerevisiae para la finalización
del proceso fermentativo (Fleet, 2008; Jolly et al., 2014).

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Introducción
Como ya se dijo anteriormente, en el proceso de fermentación espontánea de un mosto

de uva se suceden una serie de especies de levaduras siendo S. cerevisiae la encargada de
llevar a término el proceso. Sin embargo, al contrario que en las fermentaciones en las que se
adiciona un cultivo seleccionado de S. cerevisiae, su desarrollo y dominancia en el proceso
espontáneo es más lento. Esta es una de las explicaciones de las virtudes de las
fermentaciones espontáneas, que gozan de una etapa prefermentativa más extensa que
aquellas inoculadas con S. cerevisiae por lo que la flora de levaduras no-Saccharomyces
salvajes presentes en el mosto puede desarrollarse en mayor medida (Constantini et al., 1998;
Egli et al., 1998).
Centrándonos en el desarrollo de fermentaciones controladas, la inoculación de S.

cerevisiae en concentraciones en torno a los 106 células/mL garantiza en la mayoría de casos
la implantación de esta cepa sobre la flora autóctona de la uva, iniciándose rápidamente el
proceso de fermentación de los azúcares del mosto y produciéndose un vino de características
relativamente predichas en función del mosto de partida y la cepa de levadura de elección
(Pretorius, 2000). Por el contrario, el uso de procesos complejos de fermentación secuencial
cuentan con dos momentos de inóculo cuyo objetivo es sacar el mayor fruto de una primera
etapa de menor rendimiento fermentativo llevada a cabo por una cepa no-Saccharomyces y
una segunda etapa de fermentación alcohólica clásica llevada a cabo por S. cerevisiae. Así, de
alguna forma se pueden obtener los beneficios de las fermentaciones espontáneas sin asumir
el riesgo que ellas implican (Fleet, 2008). El intenso conocimiento sobre la fisiología y la
regulación génica de S. cerevisiae ha llevado en la actualidad a la existencia de un enorme
catálogo de cepas comerciales que el enólogo puede emplear en función de sus intenciones.
En inicio, el uso de las levaduras no-Saccharomyces fue meramente empírico (Cantarelli,
1955), sin embargo, los intensos trabajos de investigación llevados a cabo hoy en día permiten
un uso dirigido de las distintas especies con fines de mejora de la calidad de los vinos
ofreciendo productos innovadores al consumidor (Belda et al., 2015a). La fermentación
alcohólica se considera finalizada cuando se han agotado los azúcares existentes en el medio,
de forma que progresivamente las levaduras quedan depositadas en el fondo, deteniéndose su
metabolismo. En el caso de los vinos tintos, tras la fermentación alcohólica es frecuente el
desarrollo de una fermentación maloláctica. Este proceso, llevado a cabo por bacterias de la
especie Oenococcus oeni tiene como objetivo la conversión del ácido málico del vino en

17

Introducción

ácido láctico, disminuyendo la sensación de acidez y ayudando a la estabilidad microbiología

del vino, pues el ácido láctico no es degradado.
Figura 3. Esquema de un proceso clásico de elaboración de vino tinto. Las letras destacadas (*) hacen
referencia a los trabajos presentados en esta Tesis Doctoral con origen o incidencia en los procesos
enológicos sobre los que se sitúan.
1.3.3. Etapa post fermentativa
Finalizada la fermentación alcohólica, y maloláctica en su caso, el vino estaría listo

para su consumo en forma de vino joven tras las operaciones de estabilización, clarificación y
filtración. En el caso de los vinos tintos, tras la fermentación alcohólica, se lleva a cabo el
prensado de los hollejos.
Además de estas operaciones básicas postfermentativas, existen una serie de opciones

que comprenden el proceso de crianza de los vinos que contribuyen a incrementar su
complejidad y tipicidad. Tradicionalmente la crianza de los vinos se ha llevado a cabo en
barricas, generalmente de roble, durante un tiempo variable de tiempo desde los pocos meses
hasta varios años, que conllevaba un efecto positivo sobre las características sensoriales de los
vinos (Pérez-Prieto et al., 2003). La composición del vino cambia de forma continua durante
su crianza incidiendo significativamente sobre su composición en compuestos fenólicos y

18

Introducción

aromáticos. El elevado coste de este método tradicional de crianza por su duración, control y
almacenaje, ha llevado a la aparición de una serie de tecnologías que permiten acelerar dicho
proceso obteniendo resultados beneficiosos similares sin los riesgos y el coste asociados a una
crianza prolongada (Tao et al., 2014). Estas tecnologías pueden resumirse en el uso de
fragmentos o chips de madera que aceleran el efecto de la madera sobre el vino por aumentar
la superficie de contacto con él.
Durante esta fase, tanto en barrica como en depósito, el vino puede someterse al proceso
de crianza sobre lías que permite la extracción de ciertos metabolitos de interés, en especial de
manoproteínas, tras la lisis de las levaduras muertas tras la fermentación.
1.4. Incidencia de las levaduras en la composición del vino
1.4.1. Consumo de azúcares y liberación de etanol
El alcohol etílico es el producto fundamental derivado del metabolismo fermentativo

de S. cerevisiae, con la consecuente producción de CO2 (Figura 4) (Piskur et al., 2006). El
nivel de etanol en los vinos varía en concentraciones entre 8 y 16 % dependiendo de la
variedad de uva, su estado de maduración y el metabolismo fermentativo de las levaduras
empleadas en su fermentación. El etanol juega un papel relevante en el aroma del vino debido
fundamentalmente a su interacción con otros compuestos. Tiene un efecto en la volatilidad de
compuestos aromáticos, cuyo incremento o detrimento puede influir en el aroma global del
vino (Voilley y Lubbers, 1998; Chambers y Koppel, 2013). El etanol provoca también la
modificación en la conformación de las proteínas, reduciéndose la adsorción de compuestos
aromáticos a las mismas. En consecuencia, estos compuestos son liberados al medio
(Havkin-Frenkel y Belanger, 2008).
Hoy en día existe una tendencia generalizada a la reducción del contenido alcohólico
(etanol) en los vinos. Esto es debido a dos factores fundamentales: en primer lugar, por la
creciente concienciación de la población en temas de salud, además del incremento en la
severidad de las leyes de estatales en cuestiones de consumo de alcohol (Nevoigt, 2008). En
segundo lugar, y más relacionado con la preferencia de los consumidores, porque ha sido

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demostrado el impacto de niveles elevados de etanol en los vinos con el enmascaramiento de

la complejidad aromática de los mismos, siendo posible la percepción de un mayor número de
determinantes sensoriales a concentraciones menores de etanol (Frost et al., 2015). La
reducción del etanol en vinos puede llevarse a cabo mediante diferentes métodos físicos como
procesos de ósmosis inversa, adsorción, evaporación, filtración a través de membranas o
procesos de fermentación parcial. Sin embargo, estos métodos traen consigo altos costes de
maquinaria y procesamiento, así como un impacto considerable en el aroma y el sabor final
del vino. Una alternativa a estos métodos es el uso de cepas de levadura que produzcan
cantidades menores de etanol durante la fermentación completa de los azúcares del mosto. La
estricta legislación en lo que respecta al uso de organismos modificados genéticamente en
productos alimentarios obliga a la búsqueda y selección de cepas con un rendimiento
alcohólico bajo que, de forma natural o mediante técnicas de evolución dirigida, presenten
estos bajos niveles de producción de etanol en las fermentaciones (Tilloy et al., 2014). Sin
embargo, la inmensa mayoría de cepas de S. cerevisiae comerciales disponibles en la
actualidad generan vinos con diferencias que apenas superan el 0,5 % v/v de etanol finalizado
el proceso (Palacios et al., 2007; Varela et al., 2008) en función de su rendimiento alcohólico.
Así, surge en los últimos años el estudio del potencial de las levaduras no-Saccharomyces en
la reducción de alcohol en los vinos mediante el control de su metabolismo, menos
fermentativo y mas respiratorio que el de S. cerevisiae (Contreras et al., 2014; Gobbi et al.,
2014; Quirós et al., 2014). Los resultados más prometedores a este respecto están siendo
obtenidos en el empleo de cepas seleccionadas de M. pulcherrima en condiciones de
oxigenación controladas (Morales et al., 2015), si bien cabe destacar la reducción en los
niveles de etanol obtenidos mediante inoculación secuencial de T. delbrueckii (Bely et al.,
2008; Belda et al., 2015b). Por ello, y como se comentara ya con anterioridad, el estudio de
las levaduras no-Saccharomyces como herramienta para la reducción del contenido en etanol
en los vinos constituye uno de los pilares que sustenta su interés tanto para investigadores
como para la industria.
1.4.2. Glicerol
El glicerol es, cuantitativamente, el producto de fermentación más importante tras el

alcohol etílico. Es un polialcohol incoloro e inodoro con una elevada viscosidad. Su presencia

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Introducción

en vinos tiene un efecto positivo, aportando suavidad, dulzor y densidad en boca (Nieuwoudt
et al., 2002).
El glicerol deriva de la degradación glicolítica de los azúcares en las etapas iniciales

de la fermentación (Figura 4). Aproximadamente un 8% de los azúcares del mosto van a ser
degradados por fermentación gliceropirúvica, generando glicerol y ácido pirúvico. El
contenido final de glicerol en los vinos depende de diversos factores, que fundamentalmente
son el contenido inicial en azúcares del mosto, la temperatura de fermentación y la cepa de
levadura utilizada como inóculo.
La concentración de glicerol suele ser mayor en vinos tintos que en rosados o blancos,
oscilando entre los 4 y 15 g/L (Nurgel y Pickering, 2005). La obtención de concentraciones
que superiores a 10 g/L presenta un efecto muy significativo en la apreciación sensorial de las
propiedades de redondez en boca antes comentadas (Gawel et al. 2007; Jones et al. 2008). Si
bien existen diferencias significativas en la producción de glicerol por distintas cepas de S.
cerevisiae, la obtención de concentraciones mayores en el desarrollo de fermentaciones
espontáneas hizo vislumbrar el uso de especies no-Saccharomyces para el incremento del
contenido en glicerol en los vinos (Ciani y Ferraro, 1996). A este respecto, estudios sucesivos
han demostrado que ciertas especies como L. thermotolerans y C. zemplinina son capaces de
producir grandes concentraciones de glicerol durante la fermentación (Ciani y Ferraro, 1998;
Soden et al., 2000; Comitini et al., 2011; Englezos et al., 2015).
1.4.3. Ácidos
En el vino se pueden encontrar distintos tipos de compuestos ácidos con propiedades

diferentes. Estos compuestos han sido divididos en dos grandes grupos; son los ácidos
volátiles y no volátiles que se describen a continuación.
1.4.3.1. Ácidos volátiles
La acidez volátil de un vino está compuesta por un conjunto de ácidos orgánicos de

cadena corta. El ácido acético constituye aproximadamente el 90% de los estos ácidos
volátiles jugando, por tanto, un papel fundamental en la calidad del vino (Eglinton y
Henschke, 1999). A altas concentraciones, otorga sabores avinagrados que comienzan a ser

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Introducción

muy patentes a concentraciones superiores a 0,7 g/L (Fowles, 1992). Las diferentes cepas de
S. cerevisiae pueden producir cantidades muy variables de ácido acético (de 0,03 g/L a 1 g/L)
dependiendo de su metabolismo y las condiciones de fermentación por lo que la selección de
cepas adecuadas para su uso industrial constituye una de las mayores herramientas para el
control de la acidez volátil durante la fermentación alcohólica (Reynolds et al., 2001;
Paraggio y Fiore, 2004). Se ha descrito que el uso en fermentación de especies alternativas del
género Saccharomyces, S. bayanus y S. uvarum, suelen producir niveles inferiores de ácido
acético que la inmensa mayoría de cepas de S. cerevisiae (Giuduci et al., 1995; Tosi et al.,
2009). Asimismo, el uso de ciertas especies no-Saccharomyces, como T. delbrueckii, como
inóculo en fermentaciones secuenciales ha demostrado tener una influencia significativa en el
descenso de la acidez volátil y el contenido en ácido acético de los vinos fermentados
exclusivamente con S. cerevisiae (Bely et al., 2008; Azzolini et al., 2012; Belda et al.,
2015b).
1.4.3.2. Ácidos no volátiles
La acidez de un mosto o de un vino tiene una influencia directa en sus características

sensoriales, así como en su estabilidad bioquímica y microbiológica (Fowles, 1992). Los
ácidos no volátiles más abundantes en los mostos de uva son el ácido tartárico y el ácido
málico, que constituyen el 90% de la acidez detectable en los mostos. El ácido tartárico es
resistente a la actividad microbiana, permaneciendo casi inalterable durante la fermentación y
siendo, por tanto, independiente de las variables microbiológicas. Los ácidos láctico y cítrico,
aunque son menos abundantes, también contribuyen a la acidez del mosto (Havkin-Frenkel y
Belanger, 2008). Cabe destacar también el ácido pirúvico por como precursor clave de ciertas
rutas anabólicas como la de síntesis de determinados pigmentos estables (Benito et al., 2011).
1.4.3.2.1. Ácido málico
El ácido málico constituye cerca de la mitad de la acidez total de los vinos. Su

concentración en la uva disminuye conforme avanza la maduración, especialmente en
periodos cálidos en las fases finales de la maduración. El ácido málico en los vinos aporta un
carácter acídulo que en vinos tintos de crianza es deseable eliminar. Aunque la reducción de
la concentración de ácido málico en los vinos generalmente se lleva a cabo mediante el
proceso bacteriano de fermentación maloláctica, el uso de levaduras capaces de degradarlo

22

Introducción

constituye una alternativa, aún por valorar a nivel industrial, cuando quiera prescindirse de la
incidencia subyacente de dicho proceso sobre las características sensoriales de los vinos
(Suárez-Lepe et al. 2012; Su et al. 2014). Siendo la mayoría de cepas de S. cerevisiae muy
poco eficaces para la degradación de ácido málico (Su et al., 2014), los resultados mas
exitosos han sido obtenidos mediante el uso de cepas seleccionadas de Schizosaccharomyces
pombe (Benito et al. 2013, 2014, 2015) y Pichia kudriavzevii (Del Mónaco et al., 2014),
aunque cabe también mencionar la acción, menos patente aunque significativa, de T.
delbrueckii en la reducción del contenido en ácido málico en los vinos (Belda et al., 2015b).
1.4.3.2.2. Ácido láctico
La presencia de ácido láctico en el mosto es nula y tampoco se aprecia incremento de

este ácido en fermentación alcohólica. Este ácido es un indicativo de actividad bacteriana en
vinos o mostos, generándose a partir de azucares (picado láctico) o a partir de ácido málico.
Como bacteria responsable del proceso enológico de fermentación maloláctica, O. oeni es
capaz de descarboxilar el ácido málico transformándolo en ácido láctico. El principal
beneficio de este proceso es la conversión del sabor áspero de los vinos, derivado de la
elevada presencia de ácido málico (dicarboxílico), en sabores más suaves propios de ácidos
monocarboxílicos como el ácido láctico.
A pesar de la incapacidad de S. cerevisiae en para la producción de ácido láctico, L.

thermotolerans ha demostrado ser útil como levadura productora de elevadas concentraciones
de ácido láctico (superiores a 3 g/L) en fermentaciones combinadas con S. cerevisiae, lo que
la convierte en una herramienta útil para la corrección de la acidez en mostos poco ácidos (Su
et al., 2014; Kapsopoulou et al., 2007; Gobbi et al., 2013). Recientemente ha sido propuesto
el uso combinado de S. pombe como levadura degradadora de ácido málico y L.
thermotolerans como productora de ácido láctico como alternativa al desarrollo de la
fermentación maloláctica bacteriana en condiciones que dificulten su desarrollo como en
vinos con elevado pH o elevado contenido en etanol (Benito et al., 2015).
1.4.4. Compuestos nitrogenados
Las uvas contienen una gran variedad de compuestos nitrogenados entre los que
podemos destacar los α-aminoácidos, el amonio y pequeños péptidos. Sin embargo, la

23

Introducción

concentración de estos compuestos es relativamente pequeña por lo que la tasa de consumo de

éstos por parte de las levaduras puede ser crítica en ciertos casos (Kunkee, 1991). Bajas
concentraciones de Nitrógeno Fácilmente Asimilable (NFA) a lo largo del proceso de
fermentación pueden dar como resultado fermentaciones lentas e incluso detenidas (Beltrán et
al., 2005). Por ello, las bodegas incorporan a las fermentaciones suplementos nutricionales en
forma de nitrógeno orgánico (hidrolizados de levaduras) o inorgánico (sales de amonio). Por
ello, es importante el conocimiento del metabolismo del nitrógeno en las especies y cepas de
levadura presentes en las fermentaciones. El nitrógeno en forma de amonio se consume en
primer lugar agotándose, en condiciones normales, en un periodo aproximado de 48 horas. A
partir de ese momento la fuente de nitrógeno principal son los aminoácidos. Todos los
aminoácidos presentes en el mosto son metabolizados por las levaduras en condiciones de
anaerobiosis a excepción de la prolina (Zamora, 2009) que sólo puede ser asimilada por S.
cerevisiae en condiciones aerobias, y que precisamente es el aminoácido más abundante en el
mosto.
Figura 4. Esquema representativo del metabolismo básico de las levaduras en fermentación haciendo referencia a
algunos de los metabolitos de mayor interés enológico.

24

Introducción

La importancia del control del nitrógeno en fermentación radica tanto en la

consecución correcta del proceso por parte de S. cerevisiae por motivos nutricionales
(Navascués, 2015) como en la influencia de ciertas formas de nitrógeno sobre la represión de
rutas de síntesis de aromas mediante Represión Catabólica por Nitrógeno (NCR) siendo
determinante en el perfil sensorial de los vinos (Cooper y Sumrada, 1983; Thibon et al.,
2008). Esta importancia ha provocado que, a día de hoy la nutrición nitrogenada en bodega
sea uno de las intervenciones enológicas más consideradas y controladas en el desarrollo de
fermentaciones inoculadas con S. cerevisiae (Ugliano et al., 2007), aunque el metabolismo y
los requerimientos nutricionales de las distintas especies de levadura no-Saccharomyces,
incluso de aquellas ya disponibles como inóculos, apenas han sido estudiados (Kemsawasd et
al., 2015). Un desarrollo excesivo de las poblaciones de levaduras no-Saccharomyces durante
las etapas iniciales de la fermentación puede desencadenar un elevado consumo de nutrientes
que genere a su vez problemas en el posterior desarrollo e implantación de S. cerevisiae. Sin
embargo, el estudio de los mecanismos de NCR en especies no-Saccharomyces, que en S.
cerevisiae provocan una liberación muy limitada de compuestos aromáticos como los tioles,
puede dar lugar al desarrollo de protocolos de fermentación secuencial que mejoren la
intensidad aromática de los vinos.
1.4.5. Compuestos aromáticos
1.4.5.1. Ésteres
Existen dos clases de ésteres en la composición aromática de los vinos, los ésteres
etílicos y los ésteres de acetato. En éstos últimos el grupo acilo deriva del acetato (en forma
de acetil-coA) y el grupo alcohol es el etanol o un alcohol complejo derivado del metabolismo
de los aminoácidos. Los ésteres de acetato más abundantes son: el acetato de etilo (aroma
desagradable a disolvente, especialmente en concentraciones elevadas, aunque aporta cierto
aroma frutal en concentraciones muy bajas), el acetato de isoamilo (olor a plátano, que tiende
a monopolizar el aroma de los vinos en concentraciones elevadas) y el 2-fenil acetato (aromas
florales). Por otro lado, los ésteres etílicos se componen de etanol como grupo alcohólico y un
grupo acilo que deriva de un ácido graso de cadena mediana (Saerens et al., 2010). Éstos
ésteres, como por ejemplo el hexanoato y octanoato de etilo aportan aromas a fruta,
descriptores de manzana y otras frutas blancas (Cordente et al., 2012). La síntesis de esteres
se produce a lo largo del proceso de fermentación y su producción dependerá tanto de la

25

Introducción
presencia de los citados sustratos requeridos para la síntesis de ambos grupos de ésteres, como
de la actividad de las enzimas responsables de su síntesis e hidrólisis (acil transferasas y
esterasas). S. cerevisiae es responsable de gran parte de los ésteres presentes en un vino
puesto que su metabolismo es el responsable de la mayor parte del proceso fermentativo. De
los 5 genes que codifican las enzimas descritas como responsables de su síntesis en S.
cerevisiae (ATF1, ATF2, EHT1, EEB1 e IAH1) es la alcohol acetiltransferasa Atf1p la que ha
presentado la mayor actividad (Sumby et al. 2010). Si bien las técnicas de modificación
genética, como pueda ser la estrategia de sobreexpresión de ATF1 en S. cerevisiae, han
demostrado un incremento en la síntesis de ésteres de acetato en general (Lilly et al. 2000,
2006; Verstrepen et al. 2003), la liberación excesiva de acetato de etilo contribuye de forma
muy negativa a las propiedades sensoriales de esos vinos, por lo que, al margen de la
imposibilidad en el uso de organismos modificados genéticamente, el complejo metabolismo
de estos compuestos dificulta la optimización de esta estrategia (Cordente et al., 2012). En
este contexto, el uso de M. pulcherrima como inóculo combinado con S. cerevisiae presenta
como una de sus principales contribuciones aromáticas la liberación de altas concentraciones
de ésteres (Bisson y Kunkee, 1991; Rodríguez et al., 2010; Sadoudi et al., 2012)
especialmente de ésteres característicos de frutas blancas como el octanoato de etilo
(Lambrechts y Pretorius, 2000; Clemente-Jimenez et al., 2004). La concentración final de
ésteres en un vino dependerá del balance entre la actividad de enzimas de síntesis de ésteres y
enzimas esterasas. Si bien es conocida la existencia de esterasas extracelulares en S.
cerevisiae que contribuyen a la disminución de la concentración final de ésteres en los vinos
(Ubeda-Iranzo et al., 1998), su existencia en especies no-Saccharomyces como M.
pulcherrima debe ser investigada, pero una menor actividad de dichas esterasas podría
contribuir a explicar el aumento en la concentración de estos compuestos (Jolly et al., 2014).
1.4.5.2. Alcoholes superiores y ácidos grasos volátiles
Además de la formación de esteres, la fermentación alcohólica va acompañada de la

síntesis de alcoholes alifáticos y aromáticos conocidos como alcoholes superiores o alcoholes
de fusel. Altas concentraciones de estos compuestos contribuyen negativamente al aroma de
los vinos, por el contrario concentraciones moderadas de estos alcoholes y sus ésteres son
parte fundamental de la complejidad aromática de los mismos (Lambrechts y Pretorius 2000;
Nykanen et al., 1977). Concretamente, el 2-feniletanol es considerado como uno de los

26

Introducción
alcoholes aromáticos más importantes en el aroma de los vinos. Nuevamente, la producción

de alcoholes superiores durante la fermentación es un proceso complejo, que se produce a
partir de α-cetoácidos mediante la degradación de aminoácidos por la vía de Erlich en
levaduras (Hazelwood et al., 2008) cuyo funcionamiento en S. cerevisiae se ha relacionado
con 20 genes distintos; 4 que regulan la fase inicial de transaminación que resulta en la
síntesis del α-cetoácido (BAT1, BAT2, ARO8 y ARO9), 5 genes que codifican enzimas que
regulan su posterior descarboxilación hasta formar un aldehído de fusel (PDC1, PDC5,
PDC6, ARO10 y THI3) y finalmente otros 11 genes que codifican las oxidoreductasas
responsables de la formación final del alcohol de fusel (ADH1-ADH7, SFA1, GRE2, YPR1 y
AAD6) (Bisson y Karpel, 2010). Nuevamente, la complejidad de estas rutas y su conexión con
otras determinantes del metabolismo de las levaduras hace compleja la optimización de
protocolos de modificación genética para la optimización de la liberación, por incremento o
disminución, de alcoholes superiores en el vino. Como ya se ha indicado, en general,
concentraciones elevadas de alcoholes superiores en el vino no son deseables por monopolizar
el aroma de los mismos disminuyendo así su complejidad. En este sentido, la formación de
alcoholes superiores por la mayoría de especies no-Saccharomyces es, generalmente, menor
que en S. cerevisiae (Romano et al., 1992, 1993; Romano y Suzzi, 1993; Zironi et al., 1993)
quedando probado que parte del efecto de T. delbrueckii sobre el perfil sensorial de los vinos
es debido a la disminución de la concentración de estos alcoholes en los mismos (Belda et al.,
2015b).
1.4.5.3. Terpenos
Los terpenos forman parte del aroma de los vinos siendo los compuestos
determinantes del carácter varietal de ciertas variedades de uva blanca definidas por notas
herbáceas y frescas (Albariño, Moscatel, Riesling). Los compuestos terpénicos mas
abundantes en el vino son el linalol, nerol, geraniol, citronelol, y α-terpineol. Parte de los
terpenos se encuentran en el mosto en forma libre, aunque en mayor medida lo están,
conjugados a azúcares. Estos compuestos glicosilados no son volátiles y, por tanto, carecen de
carácter odorante por lo que la liberación de los terpenos bloqueados será necesaria para el
incremento de la concentración de aromas terpénicos en los vinos. La hidrólisis de estos
conjugados por parte de enzimas glicosidasas de las levaduras es la responsable de esta
liberación. Este proceso se produce en dos fases requeridas para la hidrólisis del diglucósido

27

Introducción

que retiene el terpeno aromático (Belda et al., 2015a). En primer lugar, una de las siguientes
enzimas: α-L-ramnosidasa, α-L-arabinofuranosidasa, β-D-xilosidasa o β-D-apiosidasa actúa
sobre la molécula y posteriormente una enzima β-D-glucosidasa libera el terpeno bloqueado
(Figura 5) (Flipphi et al., 1993; LeClinche et al., 1997; Zietsman et al., 2011). Aunque ciertas
actividades como la β-D-xilosidasa se encuentran presentes en S. cerevisiae, la ausencia en la
mayoría de cepas de actividad β-D-glucosidasa en condiciones enológicas hace que esta
especie sea incapaz de completar el proceso de liberación de terpenos de forma natural. En
este caso la ingeniería genética si ha permitido el diseño de cepas de S. cerevisiae capaces de
incrementar la liberación de terpenos en fermentación (Pretorius y Bauer 2002; Manzanares et
al., 2003; Schuller y Casal 2005), aunque nuevamente su aplicación industrial carece de
perspectiva.
Figura 5. Esquema representativo del mecanismo de liberación de terpenos volátiles a partir de sus precursores
glicosídicos no odorantes. Adaptado de Belda et al. (2016a).
El incremento del carácter varietal de los vinos por liberación de compuestos

aromáticos como los terpenos es uno de los principales objetivos que persigue la
investigación en levaduras no-Saccharomyces (Belda et al., 2015c). La inmensa mayoría de
especies no-Saccharomyces presentan actividad β-D-glucosidasa por lo que, a priori, parecen
una herramienta útil para este fin (Belda et al., 2016a). Algunas cepas de C. zemplinina, T.
delbrueckii y M. pulcherrima han sido relacionadas con el incremento de la concentración de
terpenos en el vino (Jolly et al., 2014), sin embargo, su actividad glicolítica en fermentación y
su represión catabólica por glucosa deben ser estudiados en profundidad para confirmar su
aplicación industrial.

28

Introducción
1.4.5.4. Compuestos azufrados
El compuesto azufrado más estudiado en enología, por su implicación negativa sobre el

aroma de los vinos es el ácido sulfhídrico (H2S), descriptor del olor a huevos podridos. Su
metabolismo en S. cerevisiae ha sido profundamente estudiado identificándose los
determinantes genéticos y nutricionales de su producción (Swiegers y Pretorius, 2007). Las
carencias en nitrógeno son la condición fermentativa que más contribuye a la liberación de
este compuesto por parte de las levaduras (Figura 4). Sin embargo, existe un grupo reducido
de compuestos tiólicos cuya presencia en el vino confiere a este aromas característicos a
frutas tropicales, pomelo y boj. Estos compuestos son la 4-mercapto-4-metil-pentan-2-ona (4-
MMP), el 3-mercaptohexan-1-ol (3-MH) y su derivado acetilado el acetato de 3-
mercaptohexilo (3-MHA). Su presencia en el mosto, de igual forma que los terpenos, se
encuentra en bajas concentraciones en forma libre aromática y en mayor medida bloqueados
mediane su unión a cisteína o glutatión (Tominaga et al., 1998; Peyrot Des Gachons et al.,
2002; Fedrizzi et al., 2009; Rolyet al., 2010). La actividad responsable de la hidrólisis de este
complejo es la actividad β-liasa de las levaduras que, por ser intracelular, su actividad
dependerá de la internalización de los precursores a través de transportadores específicos o
generales de aminoácidos (Figura 6) (Darriet et al., 1995). A pesar de que el umbral de
percepción de estos compuestos está en el orden de los 3-60 ng/L (Dubourdieu et al., 2006),
los recursos microbiológicos actuales mediante el uso de las cepas de S. cerevisiae
disponibles comercialmente apenas permiten la liberación de una pequeña fracción de estos
precursores. Esto es debido tanto a la ineficacia de los transportadores y las enzimas con
actividad β-liasa responsables de la internalización e hidrólisis del compuesto conjugado, así
como a la represión que distintas fuentes de nitrógeno (orgánicas e inorgánicas) ejercen sobre
ellas mediante NCR. Los genes CYS3, STR3, BNA5 e IRC7 codifican enzimas responsables de
la hidrólisis de precursores cisteinilados naturales o sintéticos de 3-MH y 4-MMP.
IRC7 ha podido ser confirmado como responsable de la liberación de la práctica

totalidad de 4-MMP (Thibon et al., 2008; Roncoroni et al., 2011) aunque contribuye también
a la de 3-MH, mientras que STR3 parece mostrar mayor repercusión sobre la liberación de 3-
MH (Holt et al., 2011). Los determinantes genéticos de la liberación de 3-MH y 4-MMP están
caracterizados en S. cerevisiae y su sobreexpresión por técnicas de ingeniería genética permite
el incremento significativo de la liberación de tioles en fermentación (Howell et al., 2005;

29

Introducción

Subileau et al., 2008; Thibon et al., 2008; Holt et al., 2011; Roncoroni et al., 2011; Belda et
al., 2016c). Debido a la limitación de las cepas naturales disponibles actualmente en la
liberación de tioles, recientemente ha sido investigado el potencial uso de levaduras no-
Saccharomyces como herramienta para la mejora de este proceso, concluyendo que el uso de
cepas seleccionadas de T. delbrueckii incrementa notablemente la liberación de tioles en
fermentación (Belda et al., 2016c; Renault et al., 2016).
Figura 6. Esquema representativo de los genes y metabolitos implicados en la internalización e hidrólisis de

los precursores cisteinilados y glutationilados de tioles volátiles en Saccharomyces cerevisiae.

30

2. OBJETIVOS

31

32

Objetivos
2. OBJETIVOS
El objetivo general de la tesis consistió en sentar las bases de la variabilidad fenotífica,

en lo que a producción de enzimas de interés enológico se refiere, de una amplia colección de
levaduras no-Saccharomyces para su futura aplicación en la mejora de parámetros de calidad
tecnológica y sensorial de los vinos. Así mismo, se planteó el estudio de la contribución de
ciertas especies no-Saccharomyces a la composicón en manoproteinas de los vinos.
1. Establecimiento de una colección de levaduras fermentativas y no fermentativas

asociadas a distintas regiones vitivinícolas.
2. Desarrollo de métodos rápidos para la evaluación de las propiedades enzimáticas de

las levaduras con interés en enología. Estudio inter- e intraespecífico de la producción
de enzimas de interés enológico.
3. Selección y aplicación de levaduras no-Saccharomyces pectinolíticas en maceración

prefermentativa para la mejora tecnológica de vinos tintos.
4. Selección y aplicación de Saccharomyces cerevisiae y levaduras no-Saccharomyces

para la mejora de las propiedades sensoriales de los vinos: manoproteinas y tioles
varietales.
5. Estudio de la incidencia en la calidad de vinos tintos de la crianza sobre lías de

levaduras no convencionales.

33

34
3. CAPÍTULO 1

3. CAPÍTULO 1

35

36

Capítulo 1
3.1. Estudio de la diversidad de especies de levaduras asociadas a distintas

regiones vitivinícolas y estudio inter- e intraespecífico de la producción de
enzimas de interés enológico.

37
Capítulo 1

38
Capítulo 1
ORIGINAL RESEARCH
published: 20 January 2016
doi: 10.3389/fmicb.2016.00012
Unraveling the Enzymatic Basis of

Wine “Flavorome”: A
Phylo-Functional Study of Wine
Related Yeast Species
Ignacio Belda 1 , Javier Ruiz 1 , Ana Alastruey-Izquierdo 2 , Eva Navascués 1, 3 ,
Domingo Marquina 1 and Antonio Santos 1*
1
Department of Microbiology, Biology Faculty, Complutense University of Madrid, Madrid, Spain, 2 Mycology Reference
Laboratory, National Center for Microbiology, Instituto de Salud Carlos III, Madrid, Spain, 3 Agrovin, S.A., Ciudad Real, Spain
Non-Saccharomyces yeasts are a heterogeneous microbial group involved in the early

stages of wine fermentation. The high enzymatic potential of these yeasts makes them
a useful tool for increasing the final organoleptic characteristics of wines in spite of
their low fermentative power. Their physiology and contribution to wine quality are still
poorly understood, with most current knowledge being acquired empirically and in most
cases based in single species and strains. This work analyzed the metabolic potential
of 770 yeast isolates from different enological origins and representing 15 different
species, by studying their production of enzymes of enological interest and linking
Edited by: phylogenetic and enzymatic data. The isolates were screened for glycosidase enzymes
Alberto Mas,
University Rovira i Virgili, Spain
related to terpene aroma release, the β-lyase activity responsible for the release of
Reviewed by:
volatile thiols, and sulfite reductase. Apart from these aroma-related activities, protease,
Mathabatha Evodia Setati, polygalacturonase and cellulase activities were also studied in the entire yeast collection,
Stellenbosch University, South Africa being related to the improvement of different technological and sensorial features of
María Gabriela Merín,
National University of wines. In this context, and in terms of abundance, two different groups were established,
Cuyo - CONICET, Argentina with α-L-arabinofuranosidase, polygalacturonase and cellulase being the less abundant
*Correspondence: activities. By contrast, β-glucosidase and protease activities were widespread in the
Antonio Santos
yeast collection studied. A classical phylogenetic study involving the partial sequencing
ansantos@ucm.es
of 26S rDNA was conducted in conjunction with the enzymatic profiles of the 770 yeast
Specialty section: isolates for further typing, complementing the phylogenetic relationships established by
This article was submitted to
using 26S rDNA. This has rendered it possible to foresee the contribution different yeast
Food Microbiology,
a section of the journal species make to wine quality and their potential applicability as pure inocula, establishing
Frontiers in Microbiology species-specific behavior. These consistent results allowed us to design future targeted
Received: 05 November 2015 studies on the impact different non-Saccharomyces yeast species have on wine quality,
Accepted: 08 January 2016
Published: 20 January 2016
understanding intra and interspecific enzymatic odds and, therefore, aiming to predict
Citation:
the most suitable application for the current non-Saccharomyces strains, as well as the
Belda I, Ruiz J, Alastruey-Izquierdo A, potential future applications of new strains. This work therefore contributes to a better
Navascués E, Marquina D and
understanding of the concept of wine microbiome and its potential consequences for
Santos A (2016) Unraveling the
Enzymatic Basis of Wine “Flavorome”: wine quality, as well as to the knowledge of non-Saccharomyces yeasts for their use in
A Phylo-Functional Study of Wine the wine industry.
Related Yeast Species.
Front. Microbiol. 7:12. Keywords: microbial terroir, enological enzymes, non-Saccharomyces, phylo-functional study, targeted yeast
doi: 10.3389/fmicb.2016.00012 selection
Frontiers in Microbiology | www.frontiersin.org 1 January 2016 | Volume 7 | Article 12

39
Capítulo 1
Belda et al. Enzymatic Basis of Wine Yeast “Flavorome”
INTRODUCTION release (Belda et al., 2015), and the modulation of wine aroma
profiles and other microbial products (reviewed by Jolly et al.,
Microorganisms coexist and interact in many environments 2014). In addition to fermentative aromas, mainly dependent
and processes, and this fact is of practical relevance for both on S. cerevisiae metabolism, non-Saccharomyces yeasts have
the environmental and industrial fields (Ivey et al., 2013). long been described as a useful tool for revealing the varietal
Grape musts naturally contain a mixture of yeast species, profile of certain grape varieties, whose aroma-determinant
and wine fermentation is not a “single-species” process (Fleet, components are usually found as odorless conjugated precursors
1990). Despite the dominance of Saccharomyces cerevisiae in (Gunata et al., 1990; Tominaga et al., 1998). Trace amounts
fermentation, which is expected and welcomed to avoid stuck of terpenes and thiols could be present in grapes in a free
and sluggish fermentations, the indigenous non-Saccharomyces form, although during fermentation yeasts may also release them
yeasts, already present in the musts, play a critical role during from their corresponding odorless precursors. The cleavage of
the early stages of fermentation. While these yeast species are terpenic glycosides is dependent on the hydrolytic activity of
not the ones mainly responsible for alcoholic fermentation, they glycosidases (Mateo and Di Stefano, 1997) and β-lyases for
can release a wide variety of hydrolytic enzymes depending cysteine-conjugated thiols (Swiegers et al., 2009).
on their diversity (Jolly et al., 2014). Non-Saccharomyces yeasts However, the improvement of the aromatic properties of wine
were originally held responsible for microbe-related problems is not the only aspect dependent on the enzymatic properties
in wine production due to their isolation from spoiled wines. of yeasts, as other sensorial and technological features can be
However, in recent years both empiric and scientific knowledge enhanced by other hydrolytic activities. Pectinolytic enzymes
has emerged concluding that, in some cases, higher microbial (mainly polygalacturonase) are widely used in enology to help
diversity improves wine complexity. degrade the plant cell wall polysaccharides of the grape skin and
The concept of vineyard and wine microbiome has been pulp. They can also help to improve clarification and filterability
addressed in recent years, obtaining extensive and meaningful processes, releasing more color and flavor compounds entrapped
results on the microbial complexity of the fermentation process in the grape skin, and facilitating the release of phenolic
(Liu et al., 2015). These population studies, carried out by both compounds (Lang and Dornenburg, 2000; Van Rensburg and
classical molecular methods and metagenomics, are currently Pretorius, 2000). Finally, the use of proteases in winemaking is
ongoing to better understand and establish the concept of not a widely extended practice at the present time, with bentonite
“microbial terroir” (Bokulich et al., 2013, 2014; Gilbert et al., being used more frequently to solve protein haze problems. The
2014). Considering that a wide variety of yeast species have use of bentonite usually impairs the sensorial properties of wines,
been identified in different scientific studies (Bisson and Joseph, so the use of proteases for this purpose may be a potential
2009; Barata et al., 2012), the role of all these yeast species solution (Marangon et al., 2012).
and their intraspecific variations need to be known. There is an On the other hand, the presence of sulfite reductase in wine
intense debate over the pertinence of the concept of microbial yeast strains is responsible for the production of hydrogen sulfide
terroir in vineyards and wine fermentation. Several factors in wine fermentations, with the consequent appearance of the
have been described as determinants of microbial diversity in characteristic rotten egg off-flavor (Swiegers and Pretorius, 2007).
enological environments. Robust results reported by Bokulich This paper explores the knowledge established between the
et al. (2014) and Wang et al. (2015) have concluded that grape- concepts of wine microbiome and microbial terroir, linking the
associated microbial biogeography is non-randomly associated phylogenetic data provided with the enzymatic characteristics
with regional, varietal and climatic factors across multi-scale determined in a wide yeast collection. These results have allowed
viticultural areas. However, this concept should be studied in us to establish a general enzymatic phenotypical characterization
depth, encompassing a strain-typing level and its final influence of several wine-related yeast species and their intraspecific
on wine quality. variability, predicting the impact of yeast microbiome on wine
A non-Saccharomyces strain was first used intentionally flavor. Thus, since the wine microbial terroir has been defined as
in wine fermentation in the 1960s, when Cantarelli (1955) the distinctive autochthonous microbiome of a wine region and
significantly reduced the volatile acidity of wines by using it has been experimentally demonstrated as a determining feature
selected Torulaspora delbrueckii strains. Nowadays, there is a of wine qualities (Bokulich et al., 2014), this work provides a
wide variety of current and expected applications of non- compelling basis to understand the influence of these microbial
Saccharomyces yeasts whose metabolic heterogeneity not only differences on the wine flavor identity, developing the new
allows overcoming certain shortcomings detected in most S. concept of wine yeast flavorome and also providing some of its
cerevisiae, but also enables the development of innovative enzymatic basis.
fermentation processes to obtain wines with new properties in
sensorial, technological and safety aspects.
Apart from reducing volatile acidity in wines (Moreno et al., MATERIALS AND METHODS
1991; Renault et al., 2009), other specific applications have
been attributed to certain wine yeast species, such as alcohol Grape Samples and Yeast Isolation
reduction (Contreras et al., 2014), modulation of acidity (Gobbi Grape samples were collected from three different Spanish wine
et al., 2013; Benito et al., 2015), increased glycerol content appellations: Tierra de León (vineyard named in this study as
(Ciani and Ferraro, 1998; Soden et al., 2000), mannoprotein G), Ribera del Duero (vineyards named as PDC and EM) and

40
Capítulo 1
Rueda (vineyard named as O). G is a young (20–40 years old) then filtered (0.22 µm). Both fractions were subsequently mixed
vineyard with vines of the Prieto Picudo variety; the PDC and when the agar solution was around 60◦ C. A loop full of each
EM vineyards are between 25 and 91 years old, with vines of yeast strain was spread onto the medium surface and incubated
the Tempranillo variety; and O is an ancient vineyard with pre- at 28◦ C for 3 days. Any significant growth of the colonies
Phylloxera vines between 100 and 200 years old of the Verdejo indicated the presence of β-glucosidase activity. A positive
variety, and also involves biodynamic agricultural practices. control (Rhodotorula glutinis CECT 10143) and a negative one
Representative samples were taken by analyzing a variety of (Torulaspora delbrueckii CECT 10676) were used as reference for
different sample points depending on the particular agronomical growth determinations.
heterogeneity of each vineyard. Three samples points were Additionally, β-D-xylosidase and α-L-arabinofuranosidase
selected in vineyard G, 10 in vineyard PDC, 5 in vineyard EM activities were evaluated using the corresponding
and 9 in vineyard O. methylumbelliferyl-conjugated substrates (methylumbelliferyl-
Seventy-three yeasts were isolated from vineyard G during the β-D-xylopyranoside (MUX) and methylumbelliferyl-α-L-
2012 harvest; 450 yeasts were isolated from vineyards PDC and arabinofuranosidase (MUA), respectively; Sigma-Aldrich),
EM during the 2013 and 2014 harvests; and finally, 247 yeasts according to the method described by Manzanares et al. (1999),
were isolated from vineyard O during the 2013 and 2014 harvests with slight modifications for their development in 96-well
(Table S1). microplates. Methylumbelliferone release was measured by
For the isolation of non-Saccharomyces yeasts, grape samples detecting fluorescence using a Varioskan Flash Mutimode
weighing about 0.5 kg were taken from healthy grape bunches. Reader (Thermo Scientific) with an excitation wavelength at
After pressing, to reduce the number of ubiquitous A. pullulans 355 nm and emission at 460 nm. Once again, R. glutinis CECT
and basidiomycetous species of no interest to the enological 10143 and T. delbrueckii CECT 10676 were used as positive and
objectives of this work, grape musts were incubated overnight negative controls, respectively.
at 20◦ C. A suitable diluted aliquot of grape must was then
spread onto a lysine agar medium (Oxoid) plates at 28◦ C β-Lyase Activity
for 48 h. As stated above, 770 discrete colonies were isolated, β-Lyase activity was evaluated on a medium containing the
and then restreaked on the same medium to obtain pure following: 0.1% S-methyl-L-cysteine (Sigma-Aldrich), 0.01%
cultures that were cryopreserved and included in a yeast pyridoxal-5′ -phosphate (Sigma-Aldrich), 1.2% Yeast Carbon
collection. Base (Difco, Detroit, MI, USA) and 2% agar. This medium was
These yeast isolates were identified by partial sequencing adjusted to pH 3.5 and sterilized as described above to avoid
of the 26S large subunit rRNA gene. Total genomic DNA agar hydrolysis. The agar solution was autoclaved, and all the
was extracted using the isopropanol method (Querol et al., other components were adjusted to pH 3.5 with HCl and filtered
1992), and the DNA for sequencing was amplified by using (0.22 µm), then both fractions were mixed when the agar solution
an Eppendorf Mastercycler, with forward NL-1 primer (5′ - was around 60◦ C. Any significant growth of the colonies after 48–
GCA TAT CAA TAA GCG GAG GAA AAG-3′ ) and reverse 72 h indicated the presence of β-lyase activity (Patent pending). T.
NL-4 primer (5′ -GGT CCG TGT TTC AAG ACG G-3′ ) delbrueckii CECT 10676 and R. glutinis CECT 10143 were used as
(Kurtzman and Robnett, 1997). The sequences obtained to positive and negative controls, respectively.
identify yeasts were analyzed and compared by BLAST-search
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Finally, sequences Pectinase Activities
were deposited in the GenBank database (http://www.ncbi. Yeast isolates were screened for polygalacturonase activity
nlm.nih.gov/genbank/) with the accession numbers listed in in a polygalacturonate agar medium containing 1.25%
Table S1. polygalacturonic acid (Sigma), 0.67% yeast nitrogen base
(YNB, Difco), 1% glucose and 2% agar, adjusted to a final pH
Phylogenetic Tree Analysis 3.5, as previously described (Strauss et al., 2001), with slight
Phylogenetic analyses were conducted with InfoQuest FP modifications. Agar was sterilized separately by autoclaving, and
Software (version 4.5 Bio-Rad Laboratories, Madrid, Spain). The all the other components were adjusted to pH 3.5 and boiled.
clustering was performed following the Neighbor joining (NJ) Both solutions were mixed when agar reached a temperature
method, with Kimura two-parameter correction. of around 60◦ C. Metschnikowia pulcherrima CECT 11202 and
Lachancea thermotolerans CECT 1951 were used as positive and
Culture Media and Enzymatic Screening negative controls, respectively.
Procedures
Glycosidase Activities Protease Activities
β-Glucosidase activity was evaluated as reported by Villena et al. Protease activity was evaluated on YPD plates (containing 1%
(2005) on a medium containing 0.5% cellobiose (4- O-β-D- yeast extract, 2% peptone, 2% glucose, and 2% agar) with 2% skim
glucopyranosyl-D-glucose), 0.67% yeast nitrogen base (Difco) milk powder (Sigma-Aldrich). The plates were incubated for 5
and 2% agar. This medium was adjusted to pH 3.5 as follows. days at 30◦ C. A clear zone around the colony identified protease
The components of the medium were sterilized separately to activity (Strauss et al., 2001). Wickerhamomyces anomalus PYCC
avoid agar hydrolysis. Agar and cellobiose were autoclaved, and 2495 and T. delbrueckii CECT 10676 were used as positive and
the yeast nitrogen base was adjusted to pH 3.5 with HCl and negative controls, respectively.

41
Capítulo 1
Cellulase Activities
Cellulase production was determined on YPGE plates
(containing 1% yeast extract, 2% peptone, 3% glycerol, and
2% ethanol) with 0.4% carboxymethylcellulose, as previously
described (Teather and Wood, 1982). Aureobasidium pullulans
CECT 2660 and T. delbrueckii CECT 10676 were used as positive
and negative controls, respectively.
Sulfite Reductase Activity

Hydrogen sulfide production was evaluated by using a
modification of the lead acetate method (Linderholm et al.,
2008) described by Belda et al. (2015) for its use in 96-well
microplates. Briefly, this method detects volatile H2 S in the
headspace of a culture medium containing 1.17% yeast carbon
base (Difco), 4% glucose anhydrous, and 0.5% ammonium FIGURE 1 | Population distribution across the 770 yeast isolates.
sulfate. Yeasts were grown at 28◦ C for 3 days in 96-well
microplates containing 200 µl of medium with orbital agitation
(200 rpm). Hydrogen sulfide formation was initially detected yeast species only present at levels of less than 4% (Figure 1).
by using paper strips (Whatman filter paper) that had been In spite of this small diversity of species, the high sample size
previously embedded with a 0.1 M lead acetate solution and (770 isolates) allowed us to conduct a functional analysis of
allowed to dry at 65◦ C for 10 min and deposited over microplate the yeast collection in question. Considering the complete yeast
wells. Hydrogen sulfide formation was qualitatively measured collection used here, a phylogenetic analysis of the 770 isolates,
based on the degree of blackening of the lead acetate strip, belonging to 15 yeast species identified on the basis of rDNA
and quantitatively estimated by densitometric measurement 26S sequences, was carried out in order to confirm the success
of the color intensity (Software “My Image Analysis v1.1” of the molecular identification process (Figure S1). It should be
Thermo Scientific). R. glutinis CECT 10143 and T. delbrueckii noted that M. fructicola and M. pulcherrima could not be properly
CECT 10676 were used as positive and negative controls, differentiated by 26S sequence analysis (Guzmán et al., 2013), and
respectively. are henceforth referred to here as Metschnikowia sp.
Notable differences between the diversity and richness of
Statistical Analysis of Enzymatic Data
yeast species in the different vineyards sampled were observed
Enzymatic activity was coded on a scale from 1 (no activity)
(Figure 2, Table S3). Furthermore, some differences could be
to 5 (highest activity) and loaded into InfoQuest FP Software
perceived between yeast populations of different vintages from
(version 4.5 Bio-Rad Laboratories, Madrid, Spain) as a character
the same vineyard. Particular note should be taken of the low
type. A similarity matrix was calculated using the Unweighted
diversity of yeast species in the EM vineyard, which had only
Pair Group Method with Arithmetic Mean (UPGMA). Groups
three yeast species, all of which were identified in both the
were assigned according to the identification of the strains by
2013 and 2014 vintages, with H. uvarum accounting for more
26S analysis. Group separation was calculated with the Jackknife
than three quarters of the total of 196 isolates, followed by L.
method. Principal Components Analysis (PCA) was performed
thermotolerans and Metschnikowia sp. (Figure 2A).
with InfoQuest FP Software.
In the case of the PDC vineyard (Figure 2B), a total of
The species distribution per sample site was introduced into
254 yeast isolates, comprising eight species, were obtained.
R program (R Core Team, 2013). The function vegdist from the
H. uvarum, Metschnikowia sp. and L. thermotolerans were
package vegan version 2.2-1 (Oksanen et al., 2015) was used to
once again the most dominant species (39, 24.8, and 19.7%
calculate a dissimilarity matrix between samples.
of the total population, respectively). However, in this case,
significant differences could be observed between vintages.
RESULTS There was a significant decrease in L. thermotolerans isolates in
the 2014 vintage, and there was a higher diversity. The other
Description of Yeast Populations species identified were Aureobasidium pullulans, Cryptococcus
In this work, 770 yeast isolates from grape musts of different amylolentus, Wickerhamomyces anomalus, Kluyveromyces
origins were identified by partial sequencing of the 26S rRNA marxianus, and Torulaspora delbrueckii, jointly accounting for
gene (Table S1). Fifteen different species were found among less than 16.6% of the PDC population and 5.4% of the total
the yeast collection studied here (Figure 1), which consisted of population.
a wide range of yeast species usually found in vineyards, and Similar diversity was observed in the O vineyard, with six
mostly having been reported to be of enological interest (Fleet, yeast species being identified among the 247 isolates (Figure 2C).
2008; Jolly et al., 2014). Hanseniaspora uvarum was the most H. uvarum was again the most abundant, accounting for 64.4%
abundant species, making up more than half of the total isolates, of the total, with the key observation being the low abundance
followed by Metschnikowia sp. (comprising M. pulcherrima and of L. thermotolerans (one of 247 isolates). It should be noted
M. fructicola) and Lachancea thermotolerans, with the other 12 that in this vineyard M. viticola was identified as an additional

42
Capítulo 1
FIGURE 2 | Total and vintage-specific population distribution from the four sampled vineyards. (A) Population distribution of EM vineyard. (B) Population
distribution of PDC vineyard. (C) Population distribution of O vineyard. (D) Population distribution of G vineyard.
Metschnikowia species. Contrary to what was observed in the aim of this work was to robustly establish the wine-related
PDC vineyard, a higher diversity was found in the 2013 enzymatic profile of a large collection of wine yeasts.
vintage, when compared to 2014, when only H. uvarum and A combined analysis of phylogenetic and enzymatic data (β-
Metschnikowia sp. were isolated. glucosidase, α-L-arabinofuranosidase, β-D-xylosidase, β-lyase,
The G vineyard comprised 10 yeast species (nine non- protease, polygalacturonase (pectinase), cellulase, and sulfite
Saccharomyces species along with some Saccharomyces cerevisiae reductase) was performed to observe whether there were any
isolates). Hanseniaspora genus was distributed among isolates of overall differences in enzyme abundances and their presence
three species: H. uvarum (28.8%), H. osmophila (19.2%), and H. among different phylogenetic groups, inferring species-specific
opuntiae (11%) (Figure 2D). Apart from Hanseniaspora species behaviors (Figure 3, Figure S1). In this context, two different
and L. thermotolerans, in the other vineyards the other five groups of highly and less abundant enzymes could be established,
non-Saccharomyces species were either not isolated (Meyerozyma highlighting α-L-arabinofuranosidase, polygalacturonase and
guilliermondii, Zygosaccharomyces bailii, and Rhodosporidium cellulase as the least abundant activities and, on the other hand,
toruloides) or rarely isolated (W. anomalus and T. delbrueckii). β-glucosidase and protease as the most widespread activities
In this case, the absence of isolates from different vintages made throughout the yeast collection studied.
it impossible to establish any population trends. Finally, contrary Figure 3 shows the overall abundance and activity level of
to what was expected due to the use of a lysine medium, 11 yeast the different enzymes studied in the 770 yeast isolates, and
isolates were identified as S. cerevisiae; nevertheless, they were not their distribution among the 15 species identified. β-Glucosidase
removed from the collection, but instead used as a comparative was widespread among wine yeast species. All the strains of Z.
control for the enzymatic study. bailii and L. thermotolerans were observed to be β-glucosidase
negative, whereas most of the strains belonging to A. pullulans, T.
Phylo-Functional Study delbrueckii and S. cerevisiae were also found to be β-glucosidase
To address a targeted use of non-Saccharomyces species in the negative, without any species-specific behavior. On the other
wine industry, it is required a better understanding of their hand, note should be taken of the activity of H. osmophila, H.
specific metabolic properties and their strain-dependent features. opuntiae, M. guilliermondii, and R. toruloides (Figure 3, Figure
Different yeast species have been reported to modulate wine S1). Regarding the other two glycosidases, the abundance of
flavor and aroma, in part because of their enzymatic properties β-D-xylosidase and α-L-arabinofuranosidase was found to be
(Hernández-Orte et al., 2008; Maturano et al., 2015). The main of medium and low, respectively. Special mention should be

43
Capítulo 1
FIGURE 3 | Abundance and distribution of enzymatic activities among the total yeast collection, individually considering the 15 yeast species
identified. The eight enzymatic activities evaluated were: (A) β-glucosidase; (B) β-D-xylosidase; (C) α-L-arabinofuranosidase; (D) β-lyase; (E) Protease; (F)
Polygalacturonase; (G) Cellulase; (H) Hydrogen sulfide production. Enzymatic activity was determined on a scale from 1 (no activity) to 5 (highest activity)
corresponding to a progressive color code from green to red.

44
Capítulo 1
made of the production of β-D-xylosidase in S. cerevisiae, T. phenotypic differences between M. viticola and the other two
delbrueckii, M. guilliermondii, W. anomalus, R. toruloides, and Metschnikowia species isolates.
A. pullulans, with the production of α-L-arabinofuranosidase Finally, hydrogen sulfide production due to the activity of
being only noteworthy in the three latter species, as well as sulfite reductase was remarkably high in some H. uvarum and
in C. amylolentus. Overall, a glycosidase-active cluster could be in most H. osmophila and H. opuntiae isolates, confirming a
observed in the basidiomycetous group (C. amylolentus and R. genus-related behavior. Regarding the other yeast species, only
toruloides), together with the yeast-like fungus A. pullulans, all of S. cerevisiae and T. delbrueckii had certain H2 S-producer strains.
them located at the bottom of the phylogenetic tree (Figure S1). Thus, Figure S1 shows an active cluster at the lower region of
β-Lyase activity was widespread, albeit in most cases with the phylogenetic tree composed by basidiomycetous species (C.
moderate activity throughout the isolates. Only T. delbrueckii, M. amylolentus and R. toruloides) and by Metschnikowia sp. and A.
guilliermondii, and K. marxianus had a wholly positive specific pullulans isolates. A highly inactive cluster in enzymatic terms
behavior. could also be observed in the lower-middle zone.
Protease activity was, together with β-glucosidase, the most
abundant activity in the yeast population studied. However, 40% Origin-Dependent Intraspecific Study
of the yeast species (six out of 15) had no protease activity. In order to study the concept of microbial terroir in depth, an
This apparent contradiction can be explained by the small intraspecific analysis was conducted on the enzymatic properties
representation these species have in the total number of yeast associated to every strain. Figure 4 shows the intraspecific
isolates. It should be mentioned that protease activity was fully clustering of the isolates of different species (five species isolated
absent in the phylogenetically related species S. cerevisiae, Z. from more than one origin) by carrying out a PCA analysis using
bailii, and T. delbrueckii, as well as in L. thermotolerans, M. enzymatic data.
guilliermondii, and C. amylolentus (Figure 3). Considering the three less abundant species analyzed (T.
On the other hand, pectinase and cellulase activities had a delbrueckii, A. pullulans, and W. anomalus), it was possible
restricted distribution, with pectinase having only a significant to clearly establish origin-dependent strain clusters composed
presence in Metschnikowia sp. and A. pullulans, and cellulase of homogeneous populations that could be distinguished by
only in A. pullulans. Apart from that, almost half of S. cerevisiae their enzymatic profiles. T. delbrueckii was isolated from the G
and a few T. delbrueckii isolates had pectinase activity. It should (seven isolates) and PDC (one isolate) vineyards in the 2012
be mentioned that protease and pectinase activities are the main and 2014 vintages, respectively. Two different groups could be
FIGURE 4 | Intraspecific distribution of isolates from the four origins and their corresponding vintages sampled. Tridimensional plots correspond to the
PCA analysis of specific populations considering their enzymatic activities, and group separation was calculated with the Jackknife method. Color legends: red (EM
2013), pink (EM 2014), blue (PDC 2013), cyan (PDC 2014), dark green (O 2013), pale green (O 2014), and yellow (G 2012). Tridimensional visualization was captured
in order to optimize group distinction.

45
Capítulo 1
statistically identified (with two Principal Components (PCs) and G (2012). On the other hand, H. uvarum isolates from O
explaining 85.2% of the differences, and three PCs explaining (2013 and 2014) did not establish a consistent enzymatic profile
100%), showing a clear origin-dependent differentiation with of their own, with most of the isolates being statistically attributed
β-glucosidase and pectinase mostly affecting this clustering to other origin profiles. Finally, in an intermediate situation,
(Figure 4, Table S2a). A. pullulans was also isolated from EM (2014) and PDC (2013) originated not-fully consistent
two vineyards: PDC (2014) and O (2013), with 20 and 5 groups, with their enzymatic profile overlapping with the profile
isolates, respectively. In this case, two different groups were described by other vineyards from the same appellation (EM
established depending on the isolation origin, composing 100% 2014 with PDC 2014; PDC 2013 with EM 2013) (Figure 4),
homogeneous population groups (Figure 4). The PCA analysis describing a wider origin-specific profile. The PCA analysis of
allowed us to statistically support this clustering, with the first these data gives us statistical evidence of the significance of these
two PCs explaining 94.55% of these differences, and three clustering results. Sulfite reductase and β-D-xylosidase activities
PCs explaining 98.51%. In this case, β-glucosidase and β-D- contributed notably to these differences, significantly affecting
xylosidase were the factors mostly responsible for affecting PC1, which could alone explain 62.62% of the differences between
this clustering, by greatly contributing to the first PC, which groups, and also PC2, which accumulates an explanation of
alone explains 81.84% of the established differences (Table 79.48% of the differences (Figure 4, Table S2e).
S2b). W. anomalus was isolated from three different vineyards:
G (2012), PDC (2014), and O (2013), with 1, 2 and 2
isolates, respectively, and these five isolates again described a DISCUSSION
phenotypic cluster according to their origin, composing three
different phylo-functional groups (Figure 4). This clustering was Diversity and Richness of Yeast Species
again statistically significant in the PCA analysis, explaining The main aim of this work was to establish a large collection
96.8% of the differences with the first two PCs, and 97.5% of non-Saccharomyces yeasts isolated from different Spanish
with three PCs. Protease activity was the most responsible wine appellations in order to perform a joint phylo-functional
factor, explaining the origin-dependent cluster separation, and analysis, linking phylogenetic and phenotypic data on the
contributing significantly to the first PC, which could explain enzymatic properties of the yeast species identified. Furthermore,
63.88% of the differences detected (Table S2c). an attempt has been made to relate certain enzymatic activities,
Due to their large sample size, the other two species evaluated which are usually associated with certain yeasts, to the potential
(L. thermotolerans and H. uvarum) generate more complex role they could play in enology.
clustering but, in most cases, some statistically homogeneous The experimental approach developed for this study was
groups could be composed depending on the origin-dependent based on culture-dependent techniques in order to obtain a yeast
strain phenotype. Regarding L. thermotolerans, a total of 88 collection of enological origin that may have a use in winemaking.
isolates were analyzed from G (2012), PDC (2013, 2014), EM From a general point of view, our population data (Figure 1)
(2013, 2014), and O (2013), with 6, 50, 31 and 1 isolates, were in line with other studies reporting that, apart from the
respectively. Aureobasidium and Rhodotorula species that were intentionally
Clusters were established for the isolates from the four avoided in this study as described in the yeast isolation procedure,
different vineyards, although a less precise separation could be Hanseniaspora spp., Metschnikowia spp., and L. thermotolerans
established between the isolates of different years from the same dominate yeast communities in fresh musts (Prakitchaiwattana
vineyard. Figure 4 shows that L. thermotolerans isolates from et al., 2004; Pinto et al., 2015), with H. uvarum accounting for
EM (2013), PDC (2014), O (2013), and G (2012) established more than half of the total yeast population isolated (Beltran
statistically homogeneous groups, defining their own enzymatic et al., 2002; Wang et al., 2015).
profile. Isolates from EM (2014) did not form a homogeneous There has recently been confirmation of the major differences
group, but 50% of these isolates could be assigned to the EM in population richness values between culture-dependent and
(2013) enzymatic profile. Regarding PDC (2013) isolates, it was independent approaches in enological environments (Wang
not possible to establish a uniform profile, with most of its et al., 2015). Our overall results of yeast diversity using a culture-
isolates being similar to the enzymatic profiles from other origins. dependent approach are wider than those obtained in other
Apart from that, the PCA of the enzymatic properties of the similar studies. Wang et al. (2015) have managed to identify
total L. thermotolerans population could explain 79.28% of the a total of three species (H. uvarum, Issatchenkia terricola, and
differences between origins, considering the first two PCs, and Starmerella bacillaris) from a collection of 179 yeasts isolated
91.87% considering the first three PCs. These differences could from nine different origins by using a lysine medium, and
be attributed mostly to β-D-xylosidase activity, H2 S production, five species (the three previously mentioned, together with S.
and β-glucosidase activity (Table S2d). Finally, regarding the cerevisiae and Hanseniaspora valbyensis) in 183 isolates from
largest species population in this study, the analysis of H. the same nine samples using YPD plates. The higher diversity
uvarum enzymatic profile generated the most complex clustering, obtained in our work (15 vs. 5 species) could be explained by
although in some cases an origin-dependent enzymatic profile both the larger sample size (770 vs. 362 isolates) and the greater
could be defined. H. uvarum was isolated from all the vineyards, heterogeneity in sampling areas (Figure 1). According to data
reaching a total of 431 isolates from all sampled origins. Three reported by Beltran et al. (2002), several differences in yeast
origins established consistent groups: EM (2013), PDC (2014), diversity were observed between years, as shown in Figure 2.

46
Capítulo 1
Differences in the microbial composition among vintages, grape what was observed in β-glucosidase activity, Hanseniaspora spp.
varieties, climate and location have been widely reported by had neither β-D-xylosidase (with the exception of H. osmophila
Bokulich et al. (2014), and could account for the differences and a few H. uvarum strains) nor α-L-arabinofuranosidase
observed for yeast diversity found in the G vineyard compared to activities, which was in complete agreement with previous
the diversity found in the other three vineyards studied (Figure observations reported by Manzanares et al. (1999). However,
S2, Table S3). The different microclimatic conditions, vineyard they also highlighted a remarkable β-D-xylosidase activity for
location and vine variety of this vineyard, with only the 2012 some W. anomalus and H. uvarum strains at the usual enological
vintage sampled, could account for such a difference. The 2012 pH values of 3–3.8, with their use also being recommended
vintage in most Spanish wine appellations was characterized by for terpene release in wine fermentation. Furthermore, lower
low rainfall (Figure S2), which could restrict the filamentous repression levels by glucose and ethanol have been reported
fungi overgrowth that could displace some of the yeast species for W. anomalus glycosidase activities (Mateo et al., 2011).
present in the grape microbial consortia (Liu et al., 2015). Regarding the other yeast isolates, a β-D-xylosidase-active
Additionally, as we show in this work, not only were the diversity cluster was observed in the phylogenetically related species
and richness of yeast species affected by location, but also the T. delbrueckii, Z. bailii, and S. cerevisiae. However, a high
phenotypic profile of the same yeast species differed across glucose-dependent repression has been observed in these species
vineyards, and even in consecutive vintages (Figure 4). (Gueguen et al., 1995; Mateo and Di Stefano, 1997; Mateo
Although most of the current population studies using et al., 2011), restricting their use in terpene release in wine
culture-independent molecular methods report higher diversity fermentation.
values for fresh must than those reported here (Bokulich and Finally, α-L-arabinofuranosidase, as the least distributed
Mills, 2013; David et al., 2014; Pinto et al., 2015), a wide variety glycosidase, was observed in M. guilliermondii, W. anomalus,
of yeast species of enological interest (Jolly et al., 2014) were A. pullulans, R. toruloides, and C. amylolentus. McMahon
represented in the yeast collection established for their enzymatic et al. (1999) have reported the major ability A. pullulans
characterization. glycosidases have to release wine terpene glycosides. According
to Mateo et al. (2011), α-L-arabinofuranosidase, together with
Enzyme Abundance and Species-Specific α-L-rhamnosidase, is the least glucose-repressed glycosidase
Distribution in wine yeasts, so both are of enological interest. Regarding
Regarding enzymatic screening, eight enzymatic activities were Metschnikowia spp., most of them had remarkable β-glucosidase
evaluated to establish an enzymatic profile of enological and β-D-xylosidase activities, although a considerable number
interest for the 15 yeast species studied (Figure 3). A group of Metschnikowia sp. (not considering M. viticola isolates) had
of three glycosidases (β-glucosidase, β-D-xylosidase, and α- also α-L-arabinofuranosidase activity. Along these lines, it has
L-arabinofuranosidase) were determined, recording different been reported that a commercial strain of M. pulcherrima
performances in terms of activity, distribution and abundance. could increase volatile terpenes in wine due to its α-L-
According to other works (Fia et al., 2005), β-glucosidase arabinofuranosidase activity (Lallemand, 2013).
was a widespread activity among wine yeasts. Our results Overall, our results are in agreement with other works
have highlighted the β-glucosidase production of Hanseniaspora reporting that Pichia, Wickerhamomyces, and Hanseniaspora
species, as well as of M. guilliermondii and W. anomalus. These genera are major producers of glycosidase enzymes (Manzanares
results are also consistent with other enzymatic screenings that et al., 1999) and, furthermore, we report the remarkable
additionally reported the ability of some H. uvarum strains glycosidase activity of wine-related basidiomycetes, such as R.
to produce versatile β-glucosidase enzymes with no repression toruloides and C. amylolentus.
by glucose and with no significant activity decrease in a wide β-Lyase activity, which is also directly related to varietal
range of pH values (López et al., 2015). Delcroix et al. (1994) aroma enhancement, recorded a moderate distribution in the
and Hernández et al. (2002) evidenced a loss of stability of overall yeast collection studied. Figure 3 shows moderate β-lyase
β-glucosidase in S. cerevisiae, with a strong reduction in its activity in the majority of yeast species, with its production
enzymatic activity (about 80%) when changing from pH 5 to being remarkable in T. delbrueckii, K. marxianus, and M.
pH 3, while other authors have reported a notable decrease in guilliermondii. Although this activity has been studied in depth
most non-Saccharomyces species at pH values below 4 (Rosi in S. cerevisiae wine strains (Howell et al., 2005; Thibon et al.,
et al., 1994). However, Mateo et al. (2011) have reported that 2008; Roncoroni et al., 2011), actual information on the ability
W. anomalus reached its maximum β-glucosidase activity at pH of non-Saccharomyces to release volatile thiols in wine is very
3.2, also recording lower rates of catabolic repression by glucose. scarce. Zott et al. (2011) have reported that β-lyase activity is
Thus, with β-glucosidase being the final activity responsible for a strain-dependent characteristic in non-Saccharomyces yeasts,
the release of wine terpenes from their glycosylated precursors, as described in S. cerevisiae (Roncoroni et al., 2011; Santiago
both Hanseniaspora species and W. anomalus seem to be a useful and Gardner, 2015). Accordingly, Figure 3 shows that β-lyase
tool to increase wine terpenics, as suggested by Mendes-Ferreira activity has great intraspecific variability. Zott et al. (2011) have
et al. (2001) and Mateo et al. (2011), respectively. reported that, apart from T. delbrueckii, some M. pulcherrima and
Regarding the other two glycosidases analyzed (β-D- L. thermotolerans strains have the ability to release volatile thiols
xylosidase and α-L-arabinofuranosidase), different abundances in Sauvignon Blanc wines, but only a few strains of these species
were observed among the yeast population studied. Contrary to have recorded β-lyase activity in our in vitro assays. Regarding

47
Capítulo 1
the Hanseniaspora genus, and as occurred with β-D-xylosidase, improving phenolic extraction and clarification processes in
H. osmophila recorded higher β-lyase activity compared to H. some pectinase-dependent wine properties, has recently been
opuntiae and H. uvarum species. These phenotypical differences confirmed (Belda et al., unpublished). Furthermore, in light of
were consistent with the observations made in the phylogenetic the behavior of A. pullulans, this was the only cellulase-active
tree (Figure S1), in which H. osmophila was distant from the species in the collection studied, in contrast with data reported
Hanseniaspora genus cluster. Due to the high nitrogen catabolic by Strauss et al. (2001) and Merín et al. (2015) which describe
repression affecting β-lyase activity in S. cerevisiae, which restricts the presence of cellulase activity in some ascomycetous yeasts
its contribution to thiol release in wine fermentation (Thibon (Candida stellata, M. pulcherrima, and H. uvarum) and in the
et al., 2008), these alternative yeasts should be studied in depth as basidiomycetous yeast Rhodotorula dairenensis, respectively.
a way to improve volatile thiol release in enological conditions. It has been reported that at least 75% of the S. cerevisiae
H2 S production, as a result of sulfite reductase activity, is a enological strains have limited pectinolytic activity (Blanco et al.,
rare feature among the majority of non-Saccharomyces species. 1994). However, Merín et al. (2011) and Merín and Morata de
Furthermore, as occurred with β-lyase (the other sulfur-related Ambrosini (2015) have confirmed the existence of a constitutive
activity), major intraspecific variability could be observed in pectinase activity not repressed by glucose in non-Saccharomyces
species such as H. uvarum and L. thermotolerans, as well as in species, in contrast with what occurred in S. cerevisiae (Radoi
S. cerevisiae, as previously reported by Linderholm et al. (2008). et al., 2005). In this context, our results confirm that the vast
Given that the nitrogen composition of musts has been described majority of Metschnikowia sp. and A. pullulans strains are of
to affect H2 S production by yeasts (Linderholm et al., 2008), and interest for their use as pectinase sources in enology, opening a
since non-Saccharomyces yeasts have high nutritional demands new research line for their industrial application.
(Jolly et al., 2014), the lack of sulfite reductase activity in most of
them is a positive characteristic for their application without the Origin-Dependent Intraspecific Phenotypic
risk of wine reduction. Profiles
Protease, pectinase and cellulase have been studied for their Metagenomic approaches have allowed researchers to definitively
involvement in several technological processes in winemaking. establish the concept of microbial terroir, relating location and
Figure 3 shows that protease is a widespread activity when climatic factors to specific microbial populations in vineyards
the total population of yeasts is considered, in agreement with (Bokulich et al., 2014). This finding has been put forward as a
previous works (Lagace and Bisson, 1990; Chomsri, 2008). determinant in the differential flavor and aroma profiles of wines
This is caused by the protease activity of the most abundant from different origins (Gilbert et al., 2014). Additionally, our
species (Hanseniaspora species and Metschnikowia sp.), although results confirm that significant phenotypical differences could
other species of enological interest with a lower relative be observed between strains of the same species from different
abundance recorded no activity (S. cerevisiae, T. delbrueckii, origins, delving further into the concept of microbial terroir, for
and L. thermotolerans, among others). In addition, protease and the first time at strain level.
pectinase seem to be the main differential activities between M. The results shown in Figure 4 allow us to confirm the
viticola and the other Metschnikowia species isolated. The use of possibility of separating single species populations based on their
proteases in winemaking is not a widely extended practice at the enzymatic properties establishing origin-dependent clusters. It
moment, with bentonite being used more often to solve protein has been suggested that high-throughput screening (HTS) assays
haze problems. The use of bentonite usually impairs the sensorial are crucial for discovering interesting enzymes and new sources
properties of wines, so the use of proteases for this purpose (Goddard and Reymond, 2004). Here, we also report the potential
seems to be a potential future application (Marangon et al., 2012). these techniques have to develop phylo-functional analyses of
Special note should be taken of the high protease activity of yeast communities to perform innovative ecological studies. A
W. anomalus, especially in the NS-PDC-167 strain (Figure 3, similar approach has recently been adopted by Zhang et al.
Figure S1), which should be studied in depth for its application (2015) to establish phylo-functional differences among the gut
in protein haze prevention. In fact, an aspartate-protease from M. microbiota of different human populations.
pulcherrima has been characterized and expressed in S. cerevisiae The connecting lines shown in Figure 4 have allowed
by Reid et al. (2012) for its potential wine application, but the role us to decipher the phylogenetic relationships among groups
of proteases from yeasts in winemaking is still poorly understood. of isolates according to their phenotypical similarities. The
Regarding pectinolytic activity, different studies have tridimensional plot for T. delbrueckii, A. pullulans, and W.
confirmed that most yeast species are unable to produce pectic anomalus shows highly defined origin-dependent clusters with
enzymes. It should be mentioned that polygalacturonase activity significant percentages of statistical differences among groups,
has been reported in a few wine yeast isolates without establishing bearing in mind that they were scarcely isolated. The population
a species-specific behavior (Strauss et al., 2001; Merín et al., distribution of L. thermotolerans and H. uvarum isolates shown
2011). In this context, our results suggest that M. pulcherrima, in the tridimensional plot could be better interpreted considering
M. fructicola (jointly identified here as Metschnikowia sp.), and numerical data for group homogeneity (Figure 4) because of
A. pullulans are leading candidates for their use as a source of the high number of isolates considered. The results for both
pectinase in winemaking. Following the confirmed usefulness of species isolated from Ribera del Duero vineyards (EM and PDC)
pectinases from A. pullulans in winemaking conditions (Merín suggest that the EM population isolated in 2014 was more
and Morata de Ambrosini, 2015), the impact of M. pulcherrima, heterogeneous when compared with data for 2013. In contrast,

48
Capítulo 1
yeast populations from the PDC vineyard followed the opposite throughput genomic techniques, together with data on the
trend, with the populations being more homogeneous in 2014 for species-specific enzymatic profiles reported in this work, open
both species, as compared to 2013. These differences, together new possibilities for future comparative genomic works that will
with the different behavior of EM and PDC populations shown in allow for the targeted development of high throughput metabolic
Figure 2, could be related to microclimatic determinants and to screenings.
viticulture practices conditioning the health status of grapes that
could determine microbial populations in them (Sipiczki, 2006;
Barata et al., 2008). In the case of H. uvarum isolates from the AUTHOR CONTRIBUTIONS
O vineyard (Rueda wine appellation), the populations obtained
AS, EN, and DM conceived the project; IB, AS, EN, and
in both the 2013 and 2014 vintages were very heterogeneous. As
JR designed and performed the experiments; IB, AA, and
they were the only species analyzed for consecutive vintages in
AS analyzed the data, and IB and AS wrote and edited the
this vineyard, it is not possible to draw a wider conclusion about
manuscript.
the intraspecific consistency in the O vineyard. It may be the case
that the biodynamic practices applied in this vineyard contribute
to a great microbial diversity, as suggested by Setati et al. (2012). ACKNOWLEDGMENTS
The wide gap between the G population and the other population
groups could be explained by geographic and climatic reasons, as The funding for the research described in this paper was provided
it has been isolated in a wine appellation (Tierra de León) with by Agrovin S.A, within the framework of the project IDI-
several climatic and orographic differences with respect to its 20130192 (Centre for Industrial Technological Development-
Ribera del Duero and Rueda counterparts, as well as in a different CDTI, Spain) and by Pago de Carraovejas, within the framework
vintage (2012) with certain weather peculiarities (remarkably low of the project IDI-20140448 (Centre for Industrial Technological
rainfall). Development-CDTI, Spain). We thank Dr. Cristina Gutiérrez for
In summary, the phenotypical characterization of our yeast her technical support, and Rocío Ramírez for reading the paper
population goes deep into the concept of microbial terroir, and contributing to its final version.
considering the yeast diversity at strain level as an important
factor for determining the microbial influence on the flavor SUPPLEMENTARY MATERIAL
properties of wines. This intraspecific phenotypical clustering
could not have been explored with current metagenomic The Supplementary Material for this article can be found
approaches. However, the exponential growth of genomic data online at: http://journal.frontiersin.org/article/10.3389/fmicb.
for non-Saccharomyces species and the versatility of high 2016.00012
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Renault, P., Miot-Sertier, C., Marullo, P., Hernández-Orte, P., Lagarrigue, Thibon, C., Marullo, P., Claisse, O., Cullin, C., Dubourdieu, D., and Tominaga,
L., Lonvaud-Funel, A., et al. (2009). Genetic characterization and T. (2008). Nitrogen catabolic repression controls the release of volatile thiols
phenotypic variability in Torulaspora delbrueckii species: potential by Saccharomyces cerevisiae during wine fermentation. FEMS Yeast Res. 8,
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doi: 10.1016/j.ijfoodmicro.2009.06.008 Tominaga, T., Peyrot des Gachons, C., and Dubourdieu, D. (1998). A new type of
Roncoroni, M., Santiago, M., Hooks, D. O., Moroney, S., Harsch, M. J., Lee, S. A., flavor precursors in Vitis vinifera L. cv. Sauvignon Blanc: S-cysteine conjugates.
et al. (2011). The yeast IRC7 gene encodes a β-lyase responsible for production J. Agric. Food Chem. 46, 5215-5219.
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28, 926–935. doi: 10.1016/j.fm.2011.01.002 natural catalysts for efficient biotransformations – a review. S. Afr. J. Enol.
Rosi, I., Vinella, M., and Domizio, P. (1994). Characteristion of β-glycosidase Vitic. 21, 52–73. Available online at: http://www.sawislibrary.co.za/dbtw-wpd/
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yeast microbiome, a mixed model microbial map. PLoS ONE 7:e52609. doi: Fungal diversity in grape must and wine fermentation assessed by massive
10.1371/journal.pone.0052609 sequencing, quantitative PCR and DGGE. Front. Microbiol. 6:1156. doi:
Sipiczki, M. (2006). Metschnikowia strains Isolated from botrytized grapes 10.3389/fmicb.2015.01156
antagonize fungal and bacterial growth by iron depletion. Appl. Environ. Zhang, J., Guo, Z., Xue, Z., Sun, Z., Zhang, M., Wang, L., et al. (2015). A
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and composition of Chardonnay wine. Aust. J. Grape Wine Res. 6, 21–30. doi: Zott, K., Thibon, C., Bely, M., Lonvaud-Funel, A., Dubourdieu, D., and
10.1111/j.1755-0238.2000.tb00158.x Masneuf-Pomarede, I. (2011). The grape must non-Saccharomyces microbial
Strauss, M. L. A., Jolly, N. P., Lambrechts, M. G., and Van Rensburg, P. (2001). community: impact on volatile thiol release. Int. J. Food Microbiol. 151,
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Saccharomyces wine yeasts. J. Appl. Microbiol. 91, 182–190. doi: 10.1046/j.1365-
2672.2001.01379.x Conflict of Interest Statement: The authors declare that the research was
Swiegers, J. H., Kievit, R. L., Siebert, T., Lattey, K. A., Bramley, B. R., conducted in the absence of any commercial or financial relationships that could
Francis, I. L., et al. (2009). The influence of yeast on the aroma of be construed as a potential conflict of interest.
Sauvignon blanc wine. Food Microbiol. 26, 204–211. doi: 10.1016/j.fm.2008.
08.004 Copyright © 2016 Belda, Ruiz, Alastruey-Izquierdo, Navascués, Marquina and
Swiegers, J. H., and Pretorius, I. S. (2007). Modulation of volatile sulfur compounds Santos. This is an open-access article distributed under the terms of the Creative
by wine yeasts. Appl. Microbiol. Biotechnol. 74, 954–960. doi: 10.1007/s00253- Commons Attribution License (CC BY). The use, distribution or reproduction in
006-0828-1 other forums is permitted, provided the original author(s) or licensor are credited
Teather, R. M., and Wood, P. J. (1982). Use of Congo red-polysaccharide and that the original publication in this journal is cited, in accordance with accepted
interactions in enumeration and characterization of cellulolytic bacteria from academic practice. No use, distribution or reproduction is permitted which does not
the bovine rumen. Appl. Environ. Microbiol. 53, 41–46. comply with these terms.

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Supplementary material. Belda et al.

Unraveling the enzymatic basis of wine “flavorome”: a phylo-functional study of wine related
yeast species.
Figure S1. Phylogenetic tree for the sequences obtained of the 26S rRNA gene for the 770 isolates
described in this work. In addition,
the activity of these isolates for seven enzymatic activities with
enological relevance was also included. The 8 enzymatic activities evaluated were: A) β-glucosidase;
B) β-D-xylosidase; C) α-L-arabinofuranosidase; D) β-lyase; E) Protease; F) Polygalacturonase; G)
Cellulase; H) Hydrogen sulfide production.
Enzymatic
L-arabinofuranosidase
Phylogenetic tree (26S rRNA gene analysis) activities
Hydrogen sulphide
Global (Gapcost:0%) (Kimura2P)
26s Enzymatic activities
D-glucosidase
D-xilosidase
Pectinase
Cellulase
Protease
A B C D E F G H Strain code
lyase
Species
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
0
NS-EM-198 Hanseniaspora uvarum
NS-PDC-39 Hanseniaspora uvarum
NS-O-49 Hanseniaspora uvarum
NS-G-68 Hanseniaspora uvarum

52
Capítulo 1
NS-EM-192
NS-EM-20
Hanseniaspora uvarum
Hanseniaspora uvarum
53
Capítulo 1
54
Capítulo 1

55
Capítulo 1
NS-G-23 Hanseniaspora opuntiae
NS-G-58 Zygosaccharo. bailii
NS-G-63 Zygosaccharo. bailii
NS-G-48 Saccharomyces cerevisiae
NS-PDC-169 Torulaspora delbrueckii
NS-G-62 Torulaspora delbrueckii
NS-O-24 Hanseniaspora osmophila
NS-G-39 Hanseniaspora osmophila
NS-PDC-100 Kluyveromyces marxianus
NS-PDC-99 Kluyveromyces marxianus
NS-O-46 Lachancea thermotolerans
NS-EM-136 Lachancea thermotolerans
NS-G-32 Lachancea thermotolerans
NS-PDC-41 Lachancea thermotolerans
NS-PDC-96
NS-PDC-97
Lachancea
Lachancea
thermotolerans
thermotolerans
56
Capítulo 1
NS-G-57 Meyerozyma guilliermondii
NS-PDC-171 Wickerhamom. anomalus
NS-PDC-167 Wickerhamom. anomalus
NS-G-34 Wickerhamom. anomalus
NS-O-14 Wickerhamom. anomalus
NS-O-11 Wickerhamom. anomalus
NS-O-94 Metschnikowia viticola
NS-O-92 Metschnikowia sp.
NS-EM-197 Metschnikowia sp.
NS-PDC-192 Metschnikowia sp.
NS-O-236
NS-O-223
Metschnikowia sp.
Metschnikowia sp.
57
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NS-G-61 Rhodosporidium toruloides
NS-G-70 Rhodosporidium toruloides
NS-PDC-249 Cryptococcus amylolentus

58
Capítulo 1
NS-PDC-128 Aureobasidium pullulans
NS-O-82 Aureobasidium pullulans

59

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Unraveling the enzymatic basis of wine “flavorome”: a phylo-functional study of wine related yeast species
Figure S2. Wine appellations sampled in the study indicating geographical and climatic data.
G vineyard

Wine apellation: Tierra de León

Grape variety: Prieto Picudo
Altitude: 747 m
G (42°08′79″N, 05°24′89″W)
Rainfall (total annual data): 224,9 mm (2012)
Mean maximum temperature: 18,5 ºC (2012)
EM (41°39′29″N, 04°12′41″W) Mean minimum temperature: 5,2 ºC (2012)
PDC (41°35′54″N, 04°07′28″W)
EM & PDC vineyards

O (41°04′52″, 04°25′35″W) Wine apellation: Ribera del Duero
Grape variety: Tempranillo
60
Altitude: 746 m (EM) & 754 m (PDC)
Rainfall (total annual data): 258,2 mm (2012) 444,8 mm (2013); 378,2 mm (2014)
Mean maximum temperature: 18,7 ºC (2012); 18 ºC (2013); 19,2 ºC (2014)
Mean minimum temperature: 5,4 ºC (2012); 5,7 ºC (2013); 7,1 ºC (2014)
O vineyards
Wine apellation: Rueda
Grape variety: Verdejo
Altitude: 842 m
Rainfall (total annual data): 280 mm (2012) 416,98 mm (2013); 350,7 mm (2014)
Mean maximum temperature: 19 ºC (2012); 18,6 ºC (2013); 19,1 ºC (2014)
Mean minimum temperature: 5,6 ºC (2012); 5,6 ºC (2013); 6,6 ºC (2014)
Capítulo 1

Unraveling the enzymatic basis of wine “flavorome”: a phylo-functional study of
wine related yeast species
Table S1: Yeast collection with genbank accession numbers

ISOLATES IDENTIFICATION
Origin Strain code Genbank accession number Identification (species)
NS-O-1 KT922724 Hanseniaspora uvarum
NS-O-11 KT922734 Wickerhamomyces anomalus
NS-O-14 KT922737 Wickerhamomyces anomalus
NS-O-24 KT922747 Hanseniaspora osmophila
NS-O-29 KT922752 Metschnikowia sp.
NS-O-32 KT922755 Metschnikowia viticola

61
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NS-O-46 KT922769 Lachancea thermotolerans
O (2013)

NS-O-70 KT922793 Aureobasidium pullulans

62
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63
Capítulo 1

O (2014)

64
Capítulo 1

O (2014)


65
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NS-PDC-1 KT922471 Hanseniaspora uvarum
NS-PDC-14 KT922484 Metschnikowia sp.

66
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NS-PDC-41 KT922510 Lachancea thermotolerans
PDC (2013)


67
PDC (2
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NS-PDC-99 KT922568 Kluyveromyces marxianus
NS-PDC-100 KT922569 Kluyveromyces marxianus

68
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NS-PDC-121 KT922588 Aureobasidium pullulans
NS-PDC-132 KT922597 Cryptococcus amylolentus

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NS-PDC-167 KT922631 Wickerhamomyces anomalus
NS-PDC-169 KT922633 Torulaspora delbrueckii
NS-PDC-171 KT922635 Wickerhamomyces anomalus
PDC (2014)


70
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71
Capítulo 1

NS-EM-1 KT922276 Hanseniaspora uvarum
NS-EM-15 KT922289 Metschnikowia sp.
EM (2013)

72
Capítulo 1

EM (2013)
NS-EM-51 KT922323 Lachancea thermotolerans

73
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74
014)
Capítulo 1

EM (2014)


75
Capítulo 1

NS-G-1 KT922970 Hanseniaspora uvarum
NS-G-2 KT922971 Lachancea thermotolerans
NS-G-4 KT922973 Hanseniaspora opuntiae
NS-G-9 KT922978 Torulaspora delbrueckii
NS-G-24 KT922993 Saccharomyces cerevisiae
G (2012)

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NS-G-33 KT923001 Hanseniaspora osmophila

NS-G-34 KT923002 Wickerhamomyces anomalus
G (2012)

NS-G-57 KT923025 Meyerozyma guilliermondii
NS-G-58 KT923026 Zygosaccharomyces baillii
NS-G-61 KT923029 Rhodosporidium toruloides
NS-G-63 KT923031 Zygosaccharomyces baillii
NS-G-70 KT923038 Rhodosporidium toruloides

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Unraveling the enzymatic basis of wine “flavorome”: a phylo-functional study of

wine related yeast species
Table S2a. Component scores of the PCA analysis of Torulaspora delbrueckii isolates.
PC1 PC2 PC3 PC4

PCA 65,39% 19,84% 14,77% 0,00%
β-glucosidase -2,7525972 -0,4350818 0,4836448 0
β-D-xylosidase -0,454887 -0,2758188 -0,7694166 0
α-L-arabinofuranosidase 0,454887 0,2758188 0,7694166 0
β-lyase 0 0 0 0
Protease 0 0 0 0
Pectinase -1,0191866 1,5443728 -0,2759914 0
Cellulase 0 0 0 0
Hydrogen sulfide 0,454887 0,2758188 0,7694166 0
Table S2b. Component scores of the PCA analysis of Aureobasidium pullulans isolates.
PC1 PC2 PC3 PC4 PC5

PCA 81,84% 12,71% 3,96% 1,49% 0,00%
β-glucosidase -3,9674513 -0,0657378 0,3954627 -0,3140354 0
β-D-xylosidase 2,9145225 0,0674117 0,6352186 -0,3708255 0
α-L-arabinofuranosidase 0,3608266 -0,0006769 -0,7884007 -0,45632 0
β-lyase 0 0 0 0 0
Protease 0 0 0 0 0
Pectinase 0 0 0 0 0
Cellulase 0 0 0 0 0
Hydrogen sulfide 0,2349292 -1,9454368 0,0089224 -0,0020793 0
Table S2c. Component scores of the PCA analysis of Wickerhamomyces anomalus

isolates.
PC1 PC2 PC3 PC4 PC5
PCA 63,88% 32,87% 1,67% 1,57% 0,00%
β-glucosidase 0,3119884 0,8279487 0,1125434 0,0670679 0
β-D-xylosidase 0,1701489 -1,3376974 0,4051581 0,1321433 0
α-L-arabinofuranosidase -0,6239768 -1,6558975 -0,2250869 -0,1341357 0
β-lyase -0,9734537 -0,1301428 -0,1692056 0,4547748 0
Protease 3,0021804 -0,3965892 -0,136305 0,1051224 0
Pectinase 0 0 0 0 0
Cellulase 0 0 0 0 0
Hydrogen sulfide 0 0 0 0 0

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Capítulo 1
Table S2d. Component scores of the PCA analysis of Lachancea thermotolerans

isolates.
PC1 PC2 PC3 PC4 PC5 PC6
PCA 47,17% 32,11% 12,59% 7,54% 0,58% 0,00%
β-glucosidase -2,7019002 -2,2142606 0,1526593 3,1838648 0,0110519 0
β-D-xylosidase -6,0620138 -3,9149433 -1,269482 -1,4245284 -0,0158298 0
α-L-arabinofuranosidase 0,1343755 -0,0228551 0,0113386 0,0581786 -0,9831297 0
β-lyase -3,4338728 0,8871775 4,1287642 -0,4391873 -0,0059975 0
Protease 0 0 0 0 0 0
Pectinase 0 0 0 0 0 0
Cellulase 0 0 0 0 0 0
Hydrogen sulfide 4,7074035 -5,6646144 1,464285 -0,3290471 0,0096475 0
Table S2e. Component scores of the PCA analysis of Hanseniaspora uvarum isolates.
PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8

PCA 62,62% 16,86% 8,30% 6,90% 2,58% 2,43% 0,32% 0,00%
β-glucosidase 1,6052499 -1,2517011 -0,2888223 1,890322 -5,3872489 0,9172722 -0,0133567 0
β-D-xylosidase 10,202646 12,8537732 1,0154342 2,1261501 -0,0610447 0,044465 -0,0316283 0
α-L-arabinofuranosidase -0,0271649 0,6111734 -0,4786401 -1,0740953 -1,1139738 -5,2980603 0,0130678 0
β-lyase 1,6205529 -2,3458289 -5,5246202 7,2533963 1,0370313 -0,6165931 -0,0059899 0
Protease -4,0082486 -2,3418536 8,2548803 4,7039366 0,3929968 -0,6703662 0,0010808 0
Pectinase -0,0563053 0,2368151 -0,0156737 0,0796928 -0,0304144 0,0417455 1,9804444 0
Cellulase 0 0 0 0 0 0 0 0
Hydrogen sulfide 25,3287538 -5,317611 1,2685261 -0,6968938 0,3605937 -0,1482685 0,0185575 0

79

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Unraveling the enzymatic basis of wine “flavorome”: a phylo-functional study of wine related yeast species
Table S3. Dissimilarity matrix for the similarity found during vintages and wine appellations.
EM 2013 EM 2014 PDC 2013 PDC 2014 O 2013 O 2014 G 2012

EM 2013 0.00
EM 2014 0.20 0.00
PDC 2013 0.24 0.36 0.00
PDC 2014 0.66 0.58 0.58 0.00
O 2013 0.53 0.45 0.45 0.34 0.00
O 2014 0.27 0.11 0.45 0.51 0.37 0.00
G 2012 0.63 0.65 0.63 0.68 0.69 0.71 0.00
80
Values in the matrix range from 0 to 1, with 0 representing no differentiation between samples and 1
meaning no similarity.

4. CAPÍTULO 2
4. CAPÍTULO 2

81

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4.1. Aplicación de levaduras pectinolíticas en maceración prefermentativa

para la mejora tecnológica de vinos tintos

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International Journal of Food Microbiology 223 (2016) 1–8
Contents lists available at ScienceDirect
International Journal of Food Microbiology
journal homepage: www.elsevier.com/locate/ijfoodmicro
Selection and use of pectinolytic yeasts for improving clarification and

phenolic extraction in winemaking
Ignacio Belda a, Lorena B. Conchillo a, Javier Ruiz a, Eva Navascués a,b, Domingo Marquina a, Antonio Santos a,⁎
a
Department of Microbiology, Biology Faculty, Complutense University of Madrid, 28040 Madrid, Spain
b
Agrovin, S.A., Alcázar de San Juan, 13600 Ciudad Real, Spain
a r t i c l e i n f o a b s t r a c t
Article history: Pectinase enzymes have shown a considerable influence in both, sensitive and technological properties of wines.
Received 26 October 2015 They can help to improve clarification process, releasing more color and flavor compounds entrapped in grape
Received in revised form 15 January 2016 skin, facilitating the liberation of phenolic compounds. This work aims to find yeasts that, because of their native
Accepted 3 February 2016
pectinases, can be applied on combined fermentations with Saccharomyces cerevisiae obtaining significant bene-
Available online 4 February 2016
fits over single-inoculated traditional fermentations. 462 yeast strains isolated from wineries were identified and
Keywords:
tested for several enzymatic activities of recognized interest for enology industry. Considering the 7 identified
Metschnikowia pulcherrima species, only Aureobasidium pullulans, Metschnikowia pulcherrima and Metschnikowia fructicola showed
Non-Saccharomyces polygalacturonase activity. Because of its interest in winemaking, due to its reported incidence in wine flavor,
Pectinase the impact of M. pulcherrima as a source of pectinolytic enzymes was analyzed by measuring its influence in fil-
Wine clarification terability, turbidity and the increase on color, anthocyanin and polyphenol content of wines fermented in com-
Phenolic extraction bination with S. cerevisiae. Among the strains screened, M. pulcherrima NS-EM-34 was selected, due to its
polygalacturonase activity, for further characterization in both, laboratory and semi-industrial scale assays. The
kinetics concerning several metabolites of enological concern were followed during the entire fermentation pro-
cess at microvinification scale. Improved results were obtained in the expected parameters when M. pulcherrima
NS-EM-34 was used, in comparison to wines fermented with S. cerevisiae alone and combined with other
pectinolytic and non-pectinolytic yeasts (A. pullulans and Lachancea thermotolerans, respectively), even working
better than commercial enzymes preparations in most parameters. Additionally, M. pulcherrima NS-EM-34 was
used at a semi-industrial scale combined with three different S. cerevisiae strains, confirming its potential appli-
cation for red wine improvement on the mentioned sensorial and technological properties.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction the native pectinases of yeasts (Alimardani-Theuil et al., 2011; Merín

et al., 2011, 2015; Pretorius, 2000). It has been reported that at least
Current research in the wine industry pursues different objectives in 50% of the Saccharomyces cerevisiae enological strains tested had limited
agronomic, biochemical and microbiological aspects. The hallmarks of pectinolytic activity (Fernández-González et al., 2004) There has recently
enological microbiology are informed by three different targets: the been increasing interest in the application of non-Saccharomyces wine
sensory, technological and fermentative properties of microbial strains. yeasts, but the ability these yeasts have to secrete efficient pectinases
The enzymatic properties of the different microorganisms involved needs to be studied in depth.
in the winemaking process have been studied for a long time (Van Traditionally, the commercial pectinases used in winemaking com-
Rensburg and Pretorius, 2000; Belda et al., 2016). prise the mixtures of polygalacturonase, pectate lyase and pectin
Pectinase enzymes have a considerable influence on both the sensory methylesterase enzymes (Lang and Dornenburg, 2000). Of these, two
and technological properties of wines (Merín and Morata de Ambrosini, types of polygalacturonases, endo- and exo-polygalacturonase, are
2015). They can help to improve the clarification and filtration process, mainly responsible for pectinolytic activity, and hence are enzymes of
releasing more of the color and flavor compounds contained in the particular importance to industry. Furthermore, cold-active pectinolytic
grape skin, and facilitating the liberation of phenolic compounds (Van enzymes have a number of potential advantages such as their function-
Rensburg and Pretorius, 2000). The addition of commercial enzyme prep- ality during the prefermentative cold soak process that contributes
arations, with filamentous fungi as the main source, can be costly for in- to the color and flavor stability of wines (Merín and Morata de
dustry. Within this context, researchers have focused their attention on Ambrosini, 2015).
Combined fermentations using non-Saccharomyces and S. cerevisiae
⁎ Corresponding author. strains, as sequential inocula in wine fermentations, have a significant
E-mail address: ansantos@ucm.es (A. Santos). impact on the sensorial properties of wines (Ciani et al., 2010; Fleet,
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.02.003
0168-1605/© 2016 Elsevier B.V. All rights reserved.

85
Capítulo 2
2 I. Belda et al. / International Journal of Food Microbiology 223 (2016) 1–8
2008; Lambrechts and Pretorius, 2000). Most studies have been devel- determined in polygalacturonate agar medium containing 1.25%
oped at laboratory scale but scarcely validated on an industrial or polygalacturonic acid (Sigma), 0.67% yeast nitrogen base (YNB, Difco),
semi-industrial scale, questioning their applicability at cellar (Jolly 1% glucose and 2% agar, adjusted to a final pH 3.5, as previously
et al., 2014). Some enzymatic activities related to aroma enhancement described (Strauss et al., 2001).
(glycosidases and β-lyase for terpene and thiol release, respectively) Protease activity was evaluated on YPD plates containing 2% skim
and the release of some interesting products such as glycerol and milk powder (Sigma-Aldrich). The plates were incubated for five days
mannoproteins, among others, are the highlights that justify the in- at 30 °C. A clear zone around the colony identified protease activity
creasing interest in these mixed fermentations (Ciani et al., 2010; (Strauss et al., 2001).
Rojas et al., 2001). In this context, combined fermentations are a very Cellulase production was determined on YPGE plates (containing 1%
useful tool to improve wine fermentations in which aromatic complex- yeast extract, 2% peptone, 3% glycerol and 2% ethanol) with 0.4%
ity of spontaneous fermentations and the safety of industrial targeted carboxymethylcellulose, as previously described (Teather and Wood,
fermentations are joined (Ciani et al., 2010; Romano et al., 2003). The 1982).
wine industry is currently demanding new yeast strains in order to in- β-glucosidase activity was evaluated as reported by Villena
novate and improve wine quality. Within this context, positive results et al. (2005), on a medium containing 0.5% cellobiose (4-O-β- D -
in industrial assays with selected yeast strains have an added value, glucopyranosyl-D-glucose), 0.67% yeast nitrogen base (Difco) and 2%
and may contribute to the deployment of non-Saccharomyces strains agar.
in the enology industry.
Since the incidence of M. pulcherrima on overall wine quality in com-
bined fermentations has been described (Parapouli et al., 2010), modi- 2.3. Pectinolytic activity on microvinifications
fying wine aroma by releasing high amounts of esters (Sadoudi et al.,
2012) or decreasing ethanol content of wines (Contreras et al., 2015; A microvinification assay was conducted to confirm the pectinolytic
Quirós et al., 2014) and also the potential use of its antimicrobial activity activity of M. pulcherrima, in sequential fermentations combined with
(Oro et al., 2014), the study of its pectinolytic activity to improve clari- the commercial S. cerevisiae Viniferm RVA strain. M. pulcherrima NS-
fication and phenolic extraction has not been carried out yet. EM-34 and A. pullulans NS-O-82 strains were used as polygalacturonase
This work aims to validate the industrial use of a selected active strains, and L. thermotolerans NS-G-32 as a negative control. These
M. pulcherrima strain that improves different aspects of wine quality, non-Saccharomyces strains were selected among the complete yeast
such as polyphenol and anthocyanin content, color intensity, turbidity collection analyzed due to their pectinolytic properties and reported
or filterability. enological usage (Jolly et al., 2014). Initial cellular concentrations in
must were of about 106 cells/ml for every strain in sequential fermenta-
2. Materials and methods tions with an inocula ratio of 1:1. Forty-eight hours after the inoculation
of non-Saccharomyces strains, S. cerevisiae Viniferm RVA was used to de-
2.1. Isolation and molecular identification of yeast strains velop sequential fermentations.
Additionally, in order to compare with usual industrial practices,
Grape samples were collected from different districts in the Spanish two commercial enzyme preparations, Enozym Clar and Enozym Lux
Designation of Origin (DO) Ribera del Duero. Samples were taken from (Agrovin S.A.) with high and medium polygalacturonase activity,
Vitis vinifera L.cv. Tempranillo grapes during the 2013 and 2014 har- respectively, were used as positive controls. The time of action of both
vests, at appropriate ripeness and in good sanitary conditions. After enzymes was four hours prior to inoculation, according to the manufac-
pressing, a suitably diluted aliquot of grape must was spread onto lysine turer instructions. After this time, S. cerevisiae RVA was inoculated. All
agar medium (Oxoid) plates at 28 °C for 48 h. Four hundred and sixty- assays were compared with a control assay inoculated solely with
two yeast colonies were taken and restreaked on the same medium to S. cerevisiae RVA.
obtain pure cultures. All the isolates were conserved at −80 °C and de- Furthermore, two temperature conditions were evaluated in the as-
posited in the Complutense Yeast Collection. These isolates were identi- says; first, applying a controlled prefermentative cold soak (12 °C during
fied by partial sequencing of the 26S large subunit rRNA gene. Total the first 48 h, and 25 °C during the remainder of the fermentation) and,
genomic DNA was extracted using the isopropanol method (Querol second, an assay at a constant temperature of 25 °C from the start with-
et al., 1992), and the DNA for sequencing was amplified by using an out prefermentative cold soak.
Eppendorf Mastercycler apparatus, with forward NL-1 primer (5′-GCA The assays were conducted, in triplicate, by using 50 ml Falcon®
TAT CAA TAA GCG GAG GAA AAG-3′) and reverse NL-4 primer (5′- tubes containing 40 g of Tempranillo crushed and destemmed grapes
GGT CCG TGT TTC AAG ACG G-3′) (Kurtzman and Robnett, 1997). The in their own juice. The cap was immersed daily during vinification to
sequences obtained were analyzed and compared by BLAST-search for simulate winemaking procedures.
yeast identification (BLAST; www.ncbi.nlm.nih.gov). The Color Intensity (CI), Total Polyphenol Index (TPI) and Anthocy-
Six yeast strains, three S. cerevisiae and three non-Saccharomyces, were anin Content (AC) of the wines were determined using a NanoDrop
selected for the conducted trials of this study at a microvinification scale 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA)
and an industrial scale. The S. cerevisiae strains were: S. cerevisiae CVA with a 1 cm path-length quartz cuvette. The CI value was calculated as
(Genbank accession number KT222660) and VRI (Genbank accession the sum of the absorbances at 420, 520, and 620 nm (Glories, 1984).
number KT222662) from CYC (Complutense Yeast Collection, Madrid, TPI was measured spectrophotometrically at 280 nm using wine
Spain) and Viniferm RVA (Genbank accession number KT222661) from 1/100 (v/v) diluted with distilled water (Ribéreau-Gayon et al., 2006).
Agrovin S.A., (Alcázar de San Juan, Spain). The non-Saccharomyces strains AC was analyzed by determining the absorbance at 520 nm of wine
were: Metschnikowia pulcherrima NS-EM-34 (Genbank accession number 1/100 (v/v) diluted with 1% (v/v) of HCl (Ruiz-Hernández, 2004).
KT222665), Aureobasidium pullulans NS-O-82 (Genbank accession num- Wine filterability was measured by filtration through a 0.22 μm filter
ber KT222663) and Lachancea thermotolerans NS-G-32 (Genbank acces- (25 mm diameter) applying a vacuum force of 0.1 bars, as described by
sion number KT222664) from CYC. Haight and Gump (1994) with slight modifications, and expressed as
the seconds needed to filtrate 1 ml of wine. Additionally, the turbidity
2.2. Enzymatic characterization of yeast strains of wines produced in microvinifications was evaluated by measuring
the nephelometric turbidity units on a nephelometer (2100N Turbidim-
The 462 yeast strains were screened for polygalacturonase, protease, eter, Hach, Loveland, USA). All the experiments were conducted in
cellulase and β-glucosidase activities. Polygalacturonase activity was triplicate.

86
Capítulo 2
I. Belda et al. / International Journal of Food Microbiology 223 (2016) 1–8 3
2.4. Semi-industrial assays also four Hanseniaspora uvarum isolates (from a total of 260 isolates)
were negative for this activity. All L. thermotolerans, Cryptococcus
2.4.1. Semi-industrial fermentations amylolentus and T. delbrueckii isolates were negative for protease activity
All semi-industrial fermentations were undertaken using V. vinifera and also Kluyveromyces marxianus showed a moderate activity. It should
L. cv. Tempranillo must. 700 kg of freshly pressed grapes were placed be mentioned that H. uvarum NS-EM-87, one of the β-glucosidase nega-
in 1000 L PVC fermentation tanks, and sulfur dioxide (40 mg/kg) was tive isolates, also showed no protease activity. It should be also outstand-
added. The initial must density was 1104 g/L, the yeast assimilable ni- ing the remarkably high protease activity of the H. uvarum isolates from
trogen was 250 mg/L and pH 3.42. All the fermentations were carried EM-A (2014) vineyard that showed a distinctive behavior when com-
out at a cellar temperature of approximately 20 °C. During the first pared with the most of the other H. uvarum isolates from other origins.
48 h, temperature was set at 17 °C and then fermented at cellar temper- In the same line, it is noteworthy the distinctive protease activity of
ature until the end of the fermentation. some M. pulcherrima isolates from EM-B (2013), EM-B (2014), PDC-C
In order to determine the oenological properties of M. pulcherrima NS- (2013) and PDC-D (2013) vineyards that showed lower, but positive,
EM-34 with independence of the S. cerevisiae strain used to complete the protease activities compared with most of the other M. pulcherrima iso-
wine fermentation process, seven assays, 700 kg each, were conducted. lates. Polygalacturonase activity was present only in M. pulcherrima
First, three fermentations inoculated with three different S. cerevisiae (88.5% positive isolates), M. fructicola (88.9% positive isolates) and
strains: CVA, VRI and Viniferm RVA as sole inocula (Sc fermentations). A. pullulans strains (100% positive isolates), with the highest activity
Second, three fermentations performed by inoculation of M. pulcherrima in this latter species. Cellulase activity was found only in A. pullulans
NS-EM-34 on combined fermentations with the previously mentioned (Table S1).
three different S. cerevisiae strains (Mp + Sc fermentations). Finally, Due to its pectinolytic activity and common usage in winemaking,
one fermentation inoculated solely with M. pulcherrima NS-EM-34 an additional characterization was conducted to analyze the influence
followed by spontaneous fermentation (Mp + Spt fermentation). of M. pulcherrima on red wine fermentations. In order to analyze
Cultures were adjusted in order to reach an initial cellular concentra- the influence of the polygalacturonase activity of M. pulcherrima in
tion in must of about 106 cells/ml for every strain, developing mixed cul- some sensorial and technological characteristics of red wines, in both
tures with an inocula ratio of 1:1. During co-fermentations, aliquots microvinifications and semi-industrial fermentations, we decide to use
were taken periodically, and further tenfold dilutions were made serial- M. pulcherrima NS-EM-34 as the strain with the lowest acetic acid pro-
ly. Growth kinetics were followed by plating 50 μL of the appropriate duction and the highest sugar consumption and ethanol production
dilution on Sabouraud glucose agar with chloramphenicol (total yeast rates (data not shown) among the studied M. pulcherrima strains,
counts) and lysine media (non-Saccharomyces counts). Colonies were being the most suitable for winemaking. The results were compared
counted after growth at 30 °C for 48–72 h. M. pulcherrima colonies with those obtained by using A. pullulans as a pectinolytic, but not rec-
were differentiated because of the reddish-brown halo developed sur- ommended for winemaking, and L. thermotolerans as non-pectinolytic
rounding them in lysine agar. but of enological interest. A. pullulans NS-O-82 and L. thermotolerans
NS-G-32 were selected in representation of the isolates of their own
2.4.2. Analytical determinations of wines species.
Glucose, fructose, malic acid, lactic acid, acetic acid, glycerol,
ammonium, primary amino nitrogen (PAN), yeast assimilable nitrogen
(YAN), SO2, TPI and CI were all determined using the Y15 Enzymatic 3.2. Microvinifications
Autoanalyzer (Biosystems S.A, Barcelona, Spain). These analyses were
performed using the appropriate kits supplied by the manufacturer Fermentations were carried out at laboratory scale to evaluate
(BioSystems, Barcelona, Spain). the influence of different microbial or enzymatic pectinase sources on
Total acidity, pH, ethanol, turbidity and density of wines were deter- different enological parameters of enological interest (filterability,
mined following the methods described in the Compendium of Interna- turbidity, TPI, AC and CI). The evolution of TPI (Fig. S1), AC (Fig. S2)
tional Methods of Analysis of Musts and Wines (OIV, 2014). and CI (Fig. S3) during the entire fermentative process was evaluated.
As indicated in Table 1, only the prefermentative cold soak conduct-
2.5. Statistical analysis ed at 12 °C during 48 h with M. pulcherrima NS-EM-34 generated statis-
tically significant differences on CI and TPI compared with S. cerevisiae
All the statistical analyses were performed using PC Statgraphics v.5 RVA alone. A. pullulans has a similar effect on the same features, but
software (Graphics Software Systems, Rockville, MD, USA). The signifi- no significant differences can be established. The final AC data of
cance was set to p b 0.05 for the ANOVA matrix F value. Furthermore, wines showed no significant differences between microvinifications,
the multiple-range test was used to compare the means. Hypothesis but a noticeable increment in AC was seen when non-Saccharomyces
contrast was used to compare means on the industrial scale assays, set- were used. However, the AC extraction rate at the start of the fermenta-
ting the significance to p b 0.05 and remarked significance values to tion process is related to the efficiency of the cold soak process and also
p b 0.01. A principal component analysis (PCA) of the analytic features critical in the final CI of wines, being precursors of stable color pigments
determined in wines was also performed. (Panprivech et al., 2015). In this sense, it should be highlighted the max-
imum AC value obtained during the fermentation in the different assays
3. Results (Fig. S2). When the prefermentative cold soak was applied, both
pectinolytic yeasts, A. pullulans and M. pulcherrima, reached mean
3.1. Yeast population and screening of enzymatic properties values of 46.6 ± 3.93 and 45.1 ± 5.17 at the fifth day of fermentation,
respectively, whereas wines inoculated with L. thermotolerans reached
A total of 462 yeast isolates, pertaining to 9 different species, maximum AC values of 42.1 ± 5.03 (day 13 of fermentation), wines
were tested for different enzymatic activities of enological interest. fermented solely with S. cerevisiae Viniferm RVA reached values of
β-glucosidase, pectinase (polygalacturonase), protease and cellulase 38.4 ± 1.84 (day 9 of fermentation) and finally, those treated with
activities were analyzed because of their influence on certain technolog- Enovin Clar enzymes reached 41.4 ± 2.42 (day 5 of fermentation) or
ical properties, such as turbidity and filterability (Table S1). Enozym Lux enzymes reached AC values of 41.3 ± 4.58 (day 2 of fer-
β-glucosidase and protease activities were widely distributed among mentation) (Fig. S2a). These results indicated that the prefermentative
the yeast collection. Three species, L. thermotolerans, A. pullulans and cold soak conducted by certain non-Saccharomyces are of interest
Torulaspora delbrueckii showed full negative β-glucosidase activity and in winemaking for color increment purposes, being their effect

87
Capítulo 2
Lt: L. thermotolerans followed by S. cerevisiae Viniferm RVA; Sc: S. cerevisiae Viniferm RVA alone; Clar: S. cerevisiae Viniferm RVA previously treated with Enovin Clar; Lux: S. cerevisiae Viniferm RVA previously treated with Enozym Lux. CI, Color
Results represent the mean SD for three replicates. Means in the same row with the same letter are not significantly different (s b 0.05). Ap: A. pullulans followed by S. cerevisiae Viniferm RVA; Mp: M. pulcherrima followed by S. cerevisiae Viniferm RVA;
quantitatively similar to the use of commercial enzyme preparations of
27.17 ± 10.20b
45.45 ± 1.34a
2.60 ± 1.68c
8.44 ± 1.85c
1.55 ± 0.25c
3.98 ± 2.39c
fungal origin.
Enzymatic treatment with industrial preparations do not increase CI,
25 °C TPI and AC values of wines, but both enzymatic treatments have a very
significant effect on the filterability and turbidity of wines, highlighting
the effect of Enozym Lux on wine filterability, accordingly to its high
polygalacturonase activity. The influence of non-Saccharomyces on
Turbidity (NTU)
2.23 ± 0.81cd
2.83 ± 0.70d
24.00 ± 2.69b
42.97 ± 5.33a
11.28 ± 7.76c
3.79 ± 2.8cd
wine filterability was also directly related to their pectinase activity,
showing that the highest polygalacturonase activity observed (in plate
assays) corresponded to the lowest filtration time reached in wines. Re-
12 °C
garding turbidity data, all treatments had a statistically significant effect

compared with fermentations with S. cerevisiae Viniferm RVA as sole in-
oculum, standing out the unexpected effect of L. thermotolerans NS-G-
104.24 ± 17.08bc
119.00 ± 12.43bc
130.97 ± 11.93b
158.79 ± 28.57a
32 on this parameter applying cold soak (Table 1).

31.51 ± 0.86d
96.88 ± 5.04c
The maintenance of a constant temperature of 25 °C (no

prefermentative cold soak) during the entire fermentation process no-
tably reduced the differences between treatments (Table 1). No signifi-
25 °C
cant differences were observed between S. cerevisiae Viniferm RVA and

non-Saccharomyces treatment for CI and TPI, and only A. pullulans was
able to significantly increase the final value of AC. The initial extraction
Filterability (s/mL)
79.74 ± 11.61bc
89.35 ± 18.43b
88.64 ± 19.00b
of anthocyanins, as occurred in the other studied parameters when

26.99 ± 0.88d
125.92 ± 8.56a
65.53 ± 2.79c
prefermentative soak was developed at 25 °C, the pectinolytic effect of

both, non-Saccharomyces and enzyme preparations, was not clear
12 °C
being less dependent of those pectinase sources. Only the delay in

the start of the fermentation process contributed to the differences
observed between the presence or absence of non-Saccharomyces
yeasts at the early stages. In this case A. pullulans, M. pulcherrima
40.73 ± 1.01bcd
44.80 ± 1.01ab
42.17 ± 2.11bc
35.50 ± 2.00d
48.47 ± 6.91a
37.30 ± 1.45c
and L. thermotolerans reached similar maximum mean AC values of

51.3 ± 3.90, 51.4 ± 3.18 and 50.9 ± 0.70 at the fifth day of fermentation,
respectively. Nevertheless, wines fermented with S. cerevisiae Viniferm
25 °C
Analytical results for the studied microvinifications applying prefermentative cold soak at 12 °C or at a constant temperature of 25 °C.
RVA and treated with pectinolytic enzymes reached AC values of

44.0 ± 4.03, 39.4 ± 1.64 and 42.5 ± 1.92, respectively, at the fifth day
of fermentation (Fig. S2b).
38.40 ± 1.84a
41.20 ± 6.34a
41.57 ± 2.22a
42.13 ± 5.03a
39.17 ± 2.99a
37.90 ± 1.40a
Furthermore, the effect observed at 25 °C on filterability was also

slighter in both S. cerevisiae Viniferm RVA and non-Saccharomyces strains.
According to the results observed at 12 °C, the effect on filterability of the
12 °C
AC
enzyme preparation Enozym Lux was higher than the effect of Enovin
Clar; however, in both cases their effect was higher than any other micro-
bial treatment. The turbidity data were similar to those observed at a low
74.77 ± 3.46ab
74.90 ± 3.08ab
74.07 ± 0.51ab
70.40 ± 3.81bc
77.73 ± 2.25a
68.77 ± 3.10c
temperature, but in this case, L. thermotolerans NS-G-32 recorded the

highest turbidity value, contrary to that observed with the same strain
at a low temperature (Table 1).
25 °C
3.3. Semi-industrial fermentations

72.57 ± 4.50ab
72.83 ± 3.43ab
73.97 ± 3.63ab
In order to confirm the incidence of M. pulcherrima NS-EM-34 on

70.13 ± 2.76b
69.23 ± 0.50b
75.53 ± 0.59a
wine properties, due to its remarkable pectinase activity, seven industri-

al trials were performed. Three fermentations were inoculated solely
12 °C
with one of the following three S. cerevisiae strains: RVA, CVA and
TPI
Intensity; TPI, Total Polyphenol Index; AC, Anthocyanin Content.
VRI (Sc fermentations). Three sequential fermentations (Mp + Sc)

of M. pulcherrima NS-EM-34 were carried out in combination with
every one of the mentioned three S. cerevisiae strains (Mp + RVA,
10.60 ± 0.92ab
10.70 ± 0.70ab
9.97 ± 0.40b
10.13 ± 0.93b
11.57 ± 0.40a
11.50 ± 0.36a
Mp + CVA and Mp + VRI). Finally, another fermentation was inoculat-

ed solely with M. pulcherrima NS-EM-34 followed by spontaneous
25 °C
fermentation (Mp + Spt).
3.3.1. Fermentation kinetics

Fermentations were carried out at a cellar temperature (20 °C ap-
10.57 ± 0.40ab
9.97 ± 0.42b
10.43 ± 0.38b
10.33 ± 0.35b
9.97 ± 0.25b
11.23 ± 0.71a
proximately) requiring between six (Sc fermentations) and eight days

(Mp + Spt fermentations) to finalize (Fig. 1). Mp + Spt fermentations
12 °C
recorded the slowest fermentation kinetics due to the absence of a

CI
S. cerevisiae inoculum, with S. cerevisiae wild yeasts being responsible

for completing the fermentation process. The other three combined fer-
mentations (Mp + RVA, Mp + CVA and Mp + VRI) showed no notice-
Assays
Table 1
able differences in the fermentation kinetics compared with their

Clar
Lux
Mp
Ap
Sc
Lt
respective Sc fermentations, completing the process after seven days.

88
Capítulo 2
Fig. 1. Fermentation kinetics of semi-industrial trials. A) Sc fermentations. Total yeast cell counts (blue) and must density evolution (red) of Sc fermentations. B) Mp + Sc fermentations.
Total yeast cell counts (blue), M. pulcherrima NS-EM-34 cell counts (green) and must density (red) of Mp + Sc fermentations. C) Mp + Spt fermentations. Total yeast cell count (blue),
M. pulcherrima NS-EM-34 cell count (green) and must density decrease (red) of Mp + Spt fermentations. D) Temperature evolution in Sc (black), Sc + Mp (gray) and Mp + Spt (white)
fermentations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
S. cerevisiae maintained high cell viability until the end of fermentation Fig. 2a. Fig. 2b represents the two-dimensional projection of the data
in both, as the sole inoculum (Fig. 1a) or sequentially co-inoculated with according to the parameters used, explaining 77.8% of the variability
M. pulcherrima (Fig. 1b). The total cell counts from Sc fermentations re- in the first two dimensions. PC1 accounted for 52.9%, and PC2 accounted
corded a higher growth rate at the early stage of fermentation. However, for an additional 24.8% of the total variability. PC1, which accounts
the final yeast population was higher in combined fermentations for almost a half of the total variability, was positively loaded by
(Mp + Sc). Besides the slower fermentation kinetics, Mp + Spt record- pectinase-dependent parameters such as CI and TPI, detected in higher
ed lower biomass counts compared with, Sc and Mp + Sc (Fig. 1c). values in fermentations where M. pulcherrima were involved. It should
be mentioned that PC1 is also loaded in the other direction by turbidity
3.3.2. Wine composition data, detected in lower values in Mp + Spt and Mp + Sc fermentations.
In order to detect and highlight differences between wines Thus, PCA analysis showed that the global characteristics of the wines
fermented with single or mixed inocula, principal component analysis can be used to separate them into two defined groups depending on
(PCA) was applied to all the analytical data obtained for the final com- the presence of M. pulcherrima as inoculum, and notably influenced by
position of wines (Fig. 2). Wines positioned in the right quadrants of pectinase-dependent parameters.
Fig. 2a correspond to combined fermentations, forming a homogeneous Table 2 shows the final chemical composition of wines that, accord-
group. The fermentations inoculated solely with S. cerevisiae formed ing to PCA results, only showed significant differences in a few parame-
a heterogeneous group, and are positioned in the left quadrants of ters. Apart from CI, TPI and turbidity, only malic and lactic acid recorded
Fig. 2. Principal Component Analysis (PCA) of the analytical composition of wines. A) Scores for the seven wine samples for the two first principal components. Wines were fermented by
using S. cerevisiae CVA, RVA and VRI strains and their combinations in sequential fermentations with M. pulcherrima NS-EM-34 (Mp + CVA, Mp + RVA, Mp + VRI). Finally, a fermentation
with M. pulcherrima NS-EM-34 followed by a spontaneous fermentation (Mp + Spt) was conducted. B) Loadings of the variables on the two first principal components. The compounds
considered, listed by PC1 loading value, were: TPI, lactic acid, CI, YAN, malic acid, acetic acid, pH, sugars, alcoholic grade, free SO2, glycerol, total SO2, turbidity, and molecular SO2.

89
Capítulo 2
Table 2
Analytical results of the semi-industrial assays developed with S. cerevisiae as sole inoculum (Sc) or combined with M. pulcherrima NSEM 34 (Mp + Sc).
Parameter S. cerevisiae (Sc) Combined fermentations (Mp + Sc) Mean values
RVA CVA VRI Mp + RVA Mp + CVA Mp + VRI Mp + Spt X Sc X Mp + Sc
Glucose + Fructose (g/L) 0.21 0.25 0.3 0.33 0.3 0.26 0.29 0.25 ± 0.05 0.30 ± 0.03
Acetic acid (g/L) 0.21 0.18 0.27 0.26 0.3 0.24 0.27 0.22 ± 0.05 0.27 ± 0.03
Malic acid (g/L) 3.13 3.18 3.22 3.5 3.42 3.44 3.45 3.18 ± 0.05 3.45 ± 0.03⁎⁎
Lactic acid (g/L) 0.21 0.2 0.24 0.28 0.31 0.3 0.27 0.22 ± 0.02 0.29 ± 0.02⁎⁎
Glycerol (g/L) 7.7 7.8 8.4 7.7 7.9 7.5 7.7 7.97 ± 0.38 7.70 ± 0.16
Ammonium (mg/L) 34 31 39 41 39 37 38 34.7 ± 4.0 38.8 ± 1.7
PAN (mg/L) 59 54 81 93 91.2 88 106 64.7 ± 14.4 94.6 ± 7.9⁎
YAN (mg/L) 86 78 111 125 121.6 107. 0 136 91.7 ± 17.2 122.4 ± 12.0⁎
Alcohol (% v/v) 14.09 14.05 13.98 14.03 14.12 14.08 14.02 14.04 ± 0.06 14.06 ± 0.05
pH 3.66 3.58 3.8 3.73 3.73 3.72 3.71 3.68 ± 0.11 3.72 ± 0.01
Free SO2 (mg/L) 99 99 121 111 93 104 94 106.3 ± 12.7 100.5 ± 8.6
Molecular SO2 (ppm) 1.38 1.65 1.23 1.32 1.1 1.26 1.17 1.42 ± 0.21 1.21 ± 0.10
Total SO2 (mg/L) 168 193 164 199 162 152 187 175 ± 15.7 175 ± 21.7
CI 7.16 7.05 7.54 11.81 13.62 10.74 12.6 7.25 ± 0.26 12.20 ± 1.22⁎⁎
TPI 65.8 63.6 73.8 85.8 84.6 83.1 83.5 67.7 ± 5.4 84.3 ± 1.2⁎⁎
Turbidity (NTU) 587 441 614 451 235 255 324a 547 ± 93 316 ± 98⁎
Results in the seven left columns show the values for the seven individual assays. The two right columns represent the mean SD for the three single (Sc) fermentations (Viniferm RVA, CVA
and VRI) and the four combined (Mp + Sc) fermentations (Mp + RVA, Mp + CVA, Mp + VRI and Mp + Spt). Means in the same row with single asterisk (*) indicate significantly different
(p b 0.05) and with double asterisk (**) indicate significantly different (p b 0.01). RVA: S. cerevisiae Viniferm RVA alone; CVA: S. cerevisiae CVA alone; VRI: S. cerevisiae VRI alone; Mp +
RVA: M. pulcherrima NSEM-34 followed by S. cerevisiae Viniferm RVA; Mp + CVA: M. pulcherrima followed by S. cerevisiae CVA; Mp + VRI: M. pulcherrima NSEM-34 followed by
S. cerevisiae VRI; Mp + Spt: M. pulcherrima NSEM-34 followed by spontaneous fermentation.
significant differences between wines fermented with or without therefore, on the final composition of the wine. Differences between as-
M. pulcherrima. says were observed depending on the pectinolytic activity of the strains
when prefermentative cold soak was applied. Nevertheless, these differ-
4. Discussion ences decreased significantly when a constant temperature (25 °C) was
applied from the start of the process, obtaining more homogeneous
The present work has afforded the study of the enzymatic properties results.
of a wide collection of yeasts isolated from the winemaking environ- Most winemakers usually apply a prefermentative cold soak to im-
ment that comprised eight species of enological interest. The studied prove certain aspects of wine quality, especially those related with
enzymatic properties were those related with the implementation of color intensity and stability. Apart from the fact that the extraction of
clarification and color extraction processes in winemaking and the some color compounds increases when a cold soak is applied, due to
study was reinforced by the application of these strains in enological the increased permeabilization of the grape's cellular membranes as a
conditions. result of longer contact time, our results suggest that the presence of
certain yeast species contributes significantly to the increase in phenolic
4.1. Population and enzymatic distribution and color extraction. Those results were strengthened by the fact that
L. thermotolerans inoculation (with no pectinolytic activity) did not in-
Two different groups of high and low distributed enzymatic activi- crease color extraction rates, if compared with S. cerevisiae fermenta-
ties, among the yeast collection studied, were established. According tions, in spite of the delay caused by the prefermentative cold soak
to several studies, the presence of β-glucosidase activity is widespread (Table 1). We may therefore posit that a prefermentative cold soak
in most wine-related yeast species (Fia et al., 2005), although it is scarce not only contributes to wine composition by chemical means, but also
in S. cerevisiae strains. Moreover, the proteolytic activity is also abun- that microbiological aspects are involved through the intervention of
dant among yeasts (Chomsri, 2008), being the most extended activity certain non-Saccharomyces yeasts. It has been recently reported that a
across the 462 yeast isolates studied in this work. On the other hand, longer prefermentative cold soak leads to higher color intensity values
pectinase and cellulase activities are the most restrictively distributed at the end of this process, but such differences usually disappear at the
activities. Contrary to other authors that reported the presence of cellu- end of alcoholic fermentation (Panprivech et al., 2015). It should be
lase activity in some yeast species (Candida stellata, M. pulcherrima and mentioned that, in this study, the higher values reached for CI, TPI and
Kloeckera apiculata) (Strauss et al., 2001), in this work cellulase activity AC in wines fermented without a prefermentative cold soak (Table 1;
was detected only in A. pullulans strains (Table S1). 25 °C vs. 12 °C) could be explained by the faster extraction of phenolic
It should be mentioned that polygalacturonase activity has been re- compounds due to the effect of temperature and of ethanol as solvent
ported in a few wine yeast isolates without establishing a species- (Sacchi et al., 2005), so only an internal comparison of the effect of
specific behavior (Merín et al., 2011; Strauss et al., 2001). Within this yeast strains in assays with or without a cold soak can be made with
context, the selection of pectinolytic yeast strains for their use as inocu- any certainty. Thus, the results shown in Table 1 prove that there are
lum in industrial fermentations seems to be a useful tool to produce significant increases in CI and TPI when M. pulcherrima was inoculated
higher quality wines without the addition of expensive commercial en- during the prefermentative cold soak. These increases were not very
zyme preparations. remarkable, although statistically significant. It could be explained be-
cause of the experimental conditions at a laboratory scale where fer-
4.2. Incidence of different polygalacturonase sources on wine composition mentations were performed in 50 mL Falcon® tubes that increase the
grape-juice contact surface in comparison to semi-industrial assays.
The results obtained in this work not only contributes to the Confirming these facts in real winemaking conditions, Table 2 shows
knowledge about the usefulness of M. pulcherrima, but also open a that the mean increments observed in TPI and CI values for semi-
new research line on the influence of different variables, such as tem- industrial assays when M. pulcherrima was inoculated were 19.7%
perature, on its metabolic efficiency. Data shown in Table 1 reveals the and 40.6%, respectively, compared with wines only inoculated with
effect of low temperature on non-Saccharomyces metabolism and, S. cerevisiae (Table 2). These increases were remarkably higher than

90
Capítulo 2
those obtained by applying a conventional cold soak (Panprivech et al., L. thermotolerans at temperatures close to 12 °C has not yet been report-
2015) and also higher compared with other studies using genetic engi- ed, as far as we know. An increased metabolic rate of L. thermotolerans at
neering approaches (Radoi et al., 2005; Fernández-González et al., low temperatures could explain the decrease in wine turbidity
2005) and other innovative treatments for wine phenolic extraction, through the release of higher amounts of organic compounds, such
such as the pulsed electric fields technology (Puértolas et al., 2009). as proteins, which contribute to the precipitation of suspended par-
Additionally, as reported by Panprivech et al. (2015), and in agree- ticles (Deckwart et al., 2014) as observed by Fernandes et al. (2015)
ment with our results with pectinolytic yeasts (Fig. S2a), greater rates by applying yeast protein extracts as fining agent, but this fact should
of anthocyanin extraction during the early stages of fermentation are be studied in depth.
observed when a cold soak was used. Furthermore, increased maximum The industrial use of M. pulcherrima NS-EM-34 on a semi-industrial
values of anthocyanin along the process have been reached (Fig. S2a) scale combined with three different S. cerevisiae strains (two autochtho-
that could contribute to the final CI values because of the formation of nous ones and a commercial one) allows us to robustly confirm its glob-
stable pigments by copigmentation (Casassa et al., 2013). However, al application, independently of the S. cerevisiae strain used for alcoholic
our results show that, during the prefermentative cold soak, the increase fermentation. The fermentation kinetics and population dynamics
in the anthocyanin extraction rate only occurs when a source of (Fig. 1) recorded similar results to those reported in studies of sequen-
pectinases is applied, contrary to observed with a prefermentative soak tial fermentations with M. pulcherrima (Sadoudi et al., 2012), where
at 25 °C, where the lack of action of non-Saccharomyces yeasts eliminates the presence of M. pulcherrima is limited to the first half of the fermen-
the differences between treatments with pectinolytic (A. pullulans and tation process. This moderate implantation allowed S. cerevisiae to eas-
M. pulcherrima) and non-pectinolytic (L. thermotolerans) yeasts. It may ily govern the alcoholic fermentation, achieving the completion of the
be concluded that a prefermentative cold soak contributes positively to process without significant delays.
the extraction rates of color compounds when a source pectinase (micro- According to other studies (Sun et al., 2014), M. pulcherrima does not
bial or enzymatic) was added. Due to the difficulty of analytically- modify enological analytical parameters such as ethanol or glycerol con-
monitoring semi-industrial fermentations, AC extraction rates were not centrations (Table 2). However, it should be mentioned the differences
quantified, but the notable increase in CI for Mp + Sc compared with observed in the malic acid concentrations between assays, that was
Sc wines (Table 2) could be related with both, the higher TPI values ob- higher using M. pulcherrima (Table 2). It has been reported the capabil-
served and, possibly, with greater AC extraction rates. ity of other non-Saccharomyces species such as Schizosaccharomyces
Filterability values were observed to be directly related with pombe to consume malic acid during wine fermentation modifying the
the pectinolytic activity of yeast strains, diminishing when the final sensorial properties of wines that has been described to be less bit-
polygalacturonase activity from yeasts or enzyme preparations increased ter (Benito et al., 2015). It could be also mentioned the repercussion of
(Table 1). The reduction of filtration time obtained with pectinolytic non- M. pulcherrima NS-EM-34 in acetic acid concentrations that are slightly
Saccharomyces yeasts (A. pullulans and M. pulcherrima) was slightly higher compared with fermentations inoculated only with S. cerevisiae,
higher than the obtained with the less active pectinolytic enzyme prepa- however this increase cause no significant differences. This fact is of im-
ration (Enovin Clar) when cold soak was applied. Furthermore, the re- portance to winemaking industry, since an excessive increase of acetic
duction time was slightly lower when cold soak was not applied; and in acid in wines has been traditionally associated with the presence of
all cases it was notably lower than that obtained with the high efficient non-Saccharomyces yeasts during the fermentation process (Jolly et al.,
pectinolytic enzyme preparation (Enozyn Lux) (Table 1). 2014). On the same line, other non-Saccharomyces species such as
Some industrial methods have been developed to reduce turbidity in T. delbrueckii has been described to keep or slightly reduce the acetic
winemaking; their effects have been recently evaluated by Fernandes acid content of wines (Bely et al., 2008; Belda et al., 2015). This work
et al. (2015) showing great turbidity reductions, especially when yeast has reported a novel usage of M. pulcherrima through the exploitation
protein extracts were applied as fining agents. All of these techniques of its polygalacturonase activity. Tables 1 and 2 indicates that CI, TPI
were applied in postfermentative stages, with the time and economic and turbidity data show significant differences that could be related to
costs that involved. Our results show that there was a clear improve- pectinolytic activity, Furthermore, PCA analysis of semi-industrial
ment in this parameter with both sources of pectinases, from enzymatic wines confirm that wines inoculated with M. pulcherrima NS-EM-34
or microbial origin, as well as with or without cold soak. A. pullulans can be clearly distinguished from wines fermented solely with
showed a remarkable effect at higher prefermentative temperatures re- S. cerevisiae, with these differences mostly affecting CI, TPI and turbidity
ducing turbidity values in a 90.4% but only a 44.2% when cold soak was (Fig. 2).
applied. This fact are partially in agreement with Merín et al. (2011) that This study broadly contributes to the knowledge on the enzymatic
reported pectinolytic activity in A. pullulans at 12 °C, however our re- properties of non-Saccharomyces yeasts and their applicability in
sults show that this activity are lower than the activity found at 25 °C. winemaking, and specifically to the understanding of the behavior of
On the contrary, M. pulcherrima showed a noticeable effect at both M. pulcherrima in wine fermentations. In addition to the previously re-
prefermentative temperatures, reducing 73.8% and 68.9% by applying ported impact on sensorial aspects of wines such as their aromatic com-
cold and conventional soak, respectively (Table 1). These turbidity re- plexity and alcoholic content (Morales et al., 2015), this work confirms
duction rates are not really far from those obtained by Fernandes et al. the usefulness of M. pulcherrima NS-EM-34 to improve some aforemen-
(2015) (turbidity reduction of 81.3%) using their best fining agent tioned technological aspects of wines like clarification and phenolic ex-
(yeast protein extracts) as an additional post-fermentative treatment traction processes. In this sense, the increase in the knowledge about
and that in all cases are less efficient than the commercial enzyme prep- the physiological properties and the metabolic determinants of non-
arations evaluated in this study (Table 1). Additionally, as occurred with Saccharomyces yeasts will be the only way to achieve their deployment
CI and TPI values, the effect of M. pulcherrima in wine turbidity at semi- in the enology industry.
industrial scale confirmed its usefulness for this objective with a mean
value of turbidity reduction of 42.23% with its greatest effect (58.47%)
combined with S. cerevisiae VRI (Table 2). Acknowledgments
Special note should be taken on the effect of L. thermotolerans on
wine turbidity when it was inoculated during the prefermentative The funding for the research in this paper was provided by Agrovin
cold soak. There was a sharp decrease in wine turbidity, recording the S.A, under the framework of the project IDI-20130192-ENZIOXIVIN
highest microbial yield for this parameter. As previously reported, (Centre for Industrial Technological Development—CDTI, Spain). We
L. thermotolerans is positively promoted at lower temperatures (20 °C thank Dra. María de los Ángeles Gómez-Flechoso for helpful discussion
vs. 30 °C) (Gobbi et al., 2013); however, the cryophilic nature of on statistical analysis.

91
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Appendix A. Supplementary data Lambrechts, M.G., Pretorius, I.S., 2000. Yeast and its importance to wine aroma—a review.
S. Afr. J. Enol. Vitic. 21, 97–129.
Lang, C., Dornenburg, H., 2000. Perspectives in the biological function and the technolog-
Supplementary data to this article can be found online at http://dx. ical application of polygalacturonases. Appl. Microbiol. Biotechnol. 53, 366–375.
doi.org/10.1016/j.ijfoodmicro.2016.02.003. Merín, M.G., Morata de Ambrosini, V.I., 2015. Highly cold-active pectinases under wine-
like conditions from non-Saccharomyces yeasts for enzymatic production during
winemaking. Lett. Appl. Microbiol. 60, 467–474.
Merín, M.G., Mendoza, L.M., Farías, M.E., Morata de Ambrosini, V.I., 2011. Isolation and se-
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Supplementary material – International Journal of Food Microbiology
Selection and use of pectinolytic yeasts for improving clarification and phenolic
extraction in winemaking
Ignacio Beldaa, Lorena B. Conchilloa, Javier Ruiza, Eva Navascuésa,b, Domingo

Marquinaa, Antonio Santosa*
a
Department of Microbiology, Biology Faculty, Complutense University of Madrid,
28040 Madrid, Spain
b
*Corresponding author. Dr. Antonio Santos

Tel.: +34 913 944 962
E-mail address: ansantos@ucm.es

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Figure S1. Evolution of Total Polyphenol Index (TPI) during microvinification

assays. A) Fermentations carried out applying prefermentative cold soak; B)
Fermentations carried out at a constant temperature. Ap (orange line): A. pullulans
followed by S. cerevisiae RVA; Mp (grey line): M. pulcherrima followed by S.
cerevisiae RVA; Lt (yellow line): L. thermotolerans followed by S. cerevisiae RVA; Sc
(light blue line): S. cerevisiae RVA alone; Clar (dark blue line): S. cerevisiae RVA
previously treated with Enovin Clar; Lux (green line): S. cerevisiae RVA previously
treated with Enozym Lux.
Figure S2. Evolution of Anthocyanin Content (AC) during microvinification

assays. A) Fermentations carried out applying prefermentative cold soak; B)
Fermentations carried out at a constant temperature. Ap (orange line): A. pullulans
followed by S. cerevisiae RVA; Mp (grey line): M. pulcherrima followed by S.
cerevisiae RVA; Lt (yellow line): L. thermotolerans followed by S. cerevisiae RVA; Sc
(light blue line): S. cerevisiae RVA alone; Clar (dark blue line): S. cerevisiae RVA
previously treated with Enovin Clar; Lux (green line): S. cerevisiae RVA previously
treated with Enozym Lux.

95
Capítulo 2
Figure S3. Evolution of Color Intenstity (CI) during microvinification assays. A)

Fermentations carried out applying prefermentative cold soak; B) Fermentations
carried out at a constant temperature. Ap (orange line): A. pullulans followed by S.
cerevisiae RVA; Mp (grey line): M. pulcherrima followed by S. cerevisiae RVA; Lt
(yellow line): L. thermotolerans followed by S. cerevisiae RVA; Sc (light blue line): S.
cerevisiae RVA alone; Clar (dark blue line): S. cerevisiae RVA previously treated
with Enovin Clar; Lux (green line): S. cerevisiae RVA previously treated with Enozym
Lux.

96

Table S1. Identification of the yeast collection analyzed (species and isolation source) and enzymatic characterization.
Collection number Identification Isolation source β-Glucosidase Protease Polygalacturonase Cellulase

(vineyard code and year)
Aureobasidium pullulans (n=22)
NS-PDC-121 A. pullulans PDC-H (2014) - + ++ +


97
NS-PDC-161 A. pullulans PDC-J (2014) - + ++ +
Cryptococcus amylolentus (n=17)
NS-PDC-132 C. amylolentus PDC-H (2014) + - - -
NS-PDC-133 C. amylolentus PDC-H (2014) + - - -
Capítulo 2

NS-PDC-178 C. amylolentus PDC-K (2014) + - - -
NS-PDC-242 C. amylolentus PDC-O (2014) + - - -
Capítulo 2


NS-PDC-261 C. amylolentus PDC-P (2014) + - - -
NS-PDC-262 C. amylolentus PDC-P (2014) + - - -
Hanseniaspora uvarum (n=260)
NS-EM-1 Hanseniaspora uvarum EM-A (2013) + +- - -
98
NS-EM-2 Hanseniaspora uvarum EM-A (2013) + + - -


NS-EM-26 Hanseniaspora uvarum EM-B (2013) + + - -

99
NS-EM-41 Hanseniaspora uvarum EM-B (2013) + +- - -
Capítulo 2

Capítulo 2

NS-EM-76 Hanseniaspora uvarum EM-D (2013) + + - -

NS-EM-81 Hanseniaspora uvarum EM-D (2013) + +- - -
NS-EM-87 Hanseniaspora uvarum EM-D (2013) - - - -
100


NS-EM-117 Hanseniaspora uvarum EM-B (2014) + ++ - -
NS-EM-122 Hanseniaspora uvarum EM-B (2014) + ++ - -

101
Capítulo 2

Capítulo 2


NS-EM-151 Hanseniaspora uvarum EM-A (2014) + ++ - -
102

NS-EM-176 Hanseniaspora uvarum EM-E (2014) + + - -


103
NS-PDC-1 Hanseniaspora uvarum PDC-A (2013) + +- - -
NS-PDC-3 Hanseniaspora uvarum PDC-A (2013) + + - -
Capítulo 2

Capítulo 2


NS-PDC-21 Hanseniaspora uvarum PDC-B (2013) + + - -
104
NS-PDC-101 Hanseniaspora uvarum PDC-G (2013) + + - -

105
NS-PDC-117 Hanseniaspora uvarum PDC-G (2013) + +- - -
NS-PDC-119 Hanseniaspora uvarum PDC-G (2013) + +- - -
NS-PDC-162 Hanseniaspora uvarum PDC-J (2014) - ++ - -
NS-PDC-163 Hanseniaspora uvarum PDC-J (2014) - ++ - -
NS-PDC-164 Hanseniaspora uvarum PDC-J (2014) - + - -
NS-PDC-166 Hanseniaspora uvarum PDC-J (2014) + + - -
NS-PDC-181 Hanseniaspora uvarum PDC-C (2014) + + - -
Capítulo 2

Capítulo 2


NS-PDC-203 Hanseniaspora uvarum PDC-M (2014) + ++ - -
NS-PDC-209 Hanseniaspora uvarum PDC-M (2014) + + - -
NS-PDC-218 Hanseniaspora uvarum PDC-M (2014) + ++ - -
NS-PDC-221 Hanseniaspora uvarum PDC-N (2014) + + - -
106
Kluyveromyces marxianus (n=2)
NS-PDC-99 Kluyveromyces marxianus PDC-F (2013) + +- - -
NS-PDC-100 Kluyveromyces marxianus PDC-F (2013) + +- - -
Lachancea thermotolerans (n=81)
NS-EM-51 Lachancea thermotolerans EM-C (2013) - - - -

107
Capítulo 2

Capítulo 2

NS-EM-104 Lachancea thermotolerans EM-B (2014) - - - -
NS-EM-119 Lachancea thermotolerans EM-B (2014) - - - -
NS-EM-130 Lachancea thermotolerans EM-D (2014) - - - -

NS-PDC-41 Lachancea thermotolerans PDC-C (2013) - - - -
108
NS-PDC-58 Lachancea thermotolerans PDC-E (2013) - - - -


NS-PDC-82 Lachancea thermotolerans PDC-F (2013) - - - -

109
NS-PDC-174 Lachancea thermotolerans PDC-J (2014) - - - -
NS-PDC-205 Lachancea thermotolerans PDC-M (2014) - - - -
Capítulo 2

Metschnikowia fructicola (n=18)
NS-EM-15 Metschnikowia fructicola EM-A (2013) + + + -
Capítulo 2
NS-EM-113 Metschnikowia fructicola EM-B (2014) + + + -

NS-EM-172 Metschnikowia fructicola EM-A (2014) + ++ + -
NS-EM-194 Metschnikowia fructicola EM-E (2014) + ++ + -
NS-PDC-14 Metschnikowia fructicola PDC-A (2013) + + + -
NS-PDC-54 Metschnikowia fructicola PDC-D (2013) + ++ - -
NS-PDC-57 Metschnikowia fructicola PDC-D (2013) + + - -
NS-PDC-177 Metschnikowia fructicola PDC-K (2014) + ++ + -

NS-PDC-191 Metschnikowia fructicola PDC-L (2014) + ++ + -
NS-PDC-207 Metschnikowia fructicola PDC-M (2014) + ++ + -
110
Metschnikowia pulcherrima (n=61)
NS-EM-34 Metschnikowia pulcherrima EM-B (2013) + + + -
NS-EM-167 Metschnikowia pulcherrima EM-A (2014) + ++ + -
NS-EM-184 Metschnikowia pulcherrima EM-E (2014) + ++ + -
NS-PDC-48 Metschnikowia pulcherrima PDC-C (2013) + + + -
NS-PDC-50 Metschnikowia pulcherrima PDC-C (2013) + + + -
NS-PDC-51 Metschnikowia pulcherrima PDC-D (2013) + + - -
NS-PDC-81 Metschnikowia pulcherrima PDC-F (2013) + ++ + -
NS-PDC-141 Metschnikowia pulcherrima PDC-I (2014) + ++ + -

111
NS-PDC-176 Metschnikowia pulcherrima PDC-K (2014) + ++ + -
NS-PDC-197 Metschnikowia pulcherrima PDC-L (2014) + ++ + -
Capítulo 2

Capítulo 2

NS-PDC-201 Metschnikowia pulcherrima PDC-M (2014) + ++ + -
NS-PDC-202 Metschnikowia pulcherrima PDC-M (2014) + ++ - -
NS-PDC-206 Metschnikowia pulcherrima PDC-M (2014) + ++ - -
NS-PDC-241 Metschnikowia pulcherrima PDC-O (2014) + ++ + -
112
NS-PDC-259 Metschnikowia pulcherrima PDC-O (2014) + + + -
Torulaspora delbrueckii (n=1)
NS-PDC-169 Torulaspora delbrueckii PDC-J (2014) - - - -
Wickerhamomyces anomalus (n=2)
NS-PDC-167 Wickerhamomyces anomalus PDC-J (2014) + +++ - -
NS-PDC-171 Wickerhamomyces anomalus PDC-J (2014) + ++ - -

5. CAPÍTULO 3
5. CAPÍTULO 3

113

114
Capítulo 3
5.1. Desarrollo de un métodos rápido para la selección de levaduras con

elevada actividad β-liasa

115
Capítulo 3

116
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International Journal of Food Microbiology 225 (2016) 1–8
Contents lists available at ScienceDirect
International Journal of Food Microbiology
journal homepage: www.elsevier.com/locate/ijfoodmicro
Improvement of aromatic thiol release through the selection of yeasts

with increased β-lyase activity
Ignacio Belda a, Javier Ruiz a, Eva Navascués b, Domingo Marquina a, Antonio Santos a,⁎
a
Department of Microbiology, Biology Faculty, Complutense University of Madrid, 28040 Madrid, Spain
b
a r t i c l e i n f o a b s t r a c t
Article history: The development of a selective medium for the rapid differentiation of yeast species with increased aromatic
Received 1 August 2015 thiol release activity has been achieved. The selective medium was based on the addition of S-methyl-L-
Received in revised form 22 January 2016 cysteine (SMC) as β-lyase substrate. In this study, a panel of 245 strains of Saccharomyces cerevisiae strains was
Accepted 1 March 2016
tested for their ability to grow on YCB-SMC medium. Yeast strains with an increased β-lyase activity grew rapidly
Available online 3 March 2016
because of their ability to release ammonium from SMC in comparison to others, and allowed for the easy isola-
Keywords:
tion and differentiation of yeasts with promising properties in oenology, or another field, for aromatic thiol re-
Wine fermentation lease. The selective medium was also helpful for the discrimination between those S. cerevisiae strains, which
Selective medium present a common 38-bp deletion in the IRC7 sequence (present in around 88% of the wild strains tested and
β-lyase activity are likely to be less functional for 4-mercapto-4-methylpentan-2-one (4MMP) production), and those
Aromatic thiols S. cerevisiae strains homozygous for the full-length IRC7 allele. The medium was also helpful for the selection
of non-Saccharomyces yeasts with increased β-lyase activity. Based on the same medium, a highly sensitive, re-
producible and non-expensive GC–MS method for the evaluation of the potential volatile thiol release by differ-
ent yeast isolates was developed.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction 2008). Recently, IRC7 and STR3 genes have been confirmed to be re-
sponsible for 4MMP and 3MH production due to their encoded
Several cysteine-S-conjugates found in foods and beverages (garlic, carbon-sulfur β-lyase activity (EC 4.4.1.8) (Holt et al., 2011; Roncoroni
onion, grape must, etc.) are β-lyase substrates. Cysteine-S-conjugated et al., 2011). However, only irc7Δ mutant had shown significant reduc-
compounds are precursors of potent aromatic thiols that contribute to tions in the release of both, 4MMP and 3MH, independently of the initial
aroma descriptors such as grapefruit, passion fruit, citrus and boxwood cysteine-S-conjugated precursor concentration indicating its central
of many white wines with a sensory perception threshold range in the role in volatile thiol release. The presence of carbon–sulfur β-lyase ac-
parts per trillion (Bailly et al., 2006; Bouchilloux et al., 1998; Darriet tivity has been determined to be the main responsible for cleavage
et al., 1995). These aromatic thiols are practically absent in grape juice of cysteine-S-conjugated forms of 3MH and 4MMP into free thiols
and develop only during the alcoholic fermentation. This explains the (Harsch and Gardner, 2013; Howell et al., 2005; Swiegers and
commonly held notion that the wine yeast Saccharomyces cerevisiae is Pretorius, 2007; Tominaga et al., 1998). The release of aromatic thiols
responsible for the formation of volatile thiols during fermentation. by other microorganisms has been related to the activity of cystathio-
Darriet et al. (1995) found that volatile thiols occur in the grape in nine γ- and β-lyases (Irmler et al., 2008; Martínez-Cuesta et al., 2006;
the form of aroma-free, non-volatile, and cysteine-bound compounds Troccaz et al., 2008; Wu and Morris, 1973). Furthermore, apart from
and that yeast, is only involved in releasing the aromatic thiols from their role in aromatic thiol release, cystathionine β-lyases catalyze the
the aroma-free grape precursor compounds. For example, the aromatic conversion of cystathionine into homocysteine in an α, β-elimination
thiols 3-mercaptohexan-1-ol (3MH) and 4-mercapto-4-methylpentan- reaction, which generates methionine and the by-products pyruvate
2-one (4MMP) are released from odorless cysteine-S-conjugated pre- and ammonia employing pyridoxal-5′-phosphate as cofactor (Thomas
cursors of the grape must during fermentation (Holt et al., 2012; and Surdin-Kerjan, 1997) (Fig. S1).
Swiegers et al., 2009). It is generally accepted that grape harvesting practices and process-
Some genes (BNA3, CYS3, GLO1, IRC7, STR3) have been suggested to ing can have an important influence on thiol yield (Allen et al., 2011;
be involved in volatile thiol release (Howell et al., 2005; Thibon et al., Capone and Jeffery, 2011). However, conversion of the cysteinylated
precursors into their corresponding thiols is accepted to be very limited,
⁎ Corresponding author. typically less than 5%, taking into account an efficient S. cerevisiae strain
E-mail address: ansantos@ucm.es (A. Santos). (Coetzee and du Toit, 2012; Murat et al., 2001; Peña-Gallego et al., 2012;
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.03.001
0168-1605/© 2016 Elsevier B.V. All rights reserved.

117
Capítulo 3
Roland et al., 2011; Winter et al., 2011). For instance, Str3p showed a (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) and reverse NL-4 primer
modest side activity, being able to release an amount of free 3MH and (5′-GGT CCG TGT TTC AAG ACG G-3′) (Kurtzman and Robnett, 1997).
4MMP corresponding to approximately 0.1% and 0.6%, respectively, of Isolates were compared for aromatic thiol release with Torulaspora
the specific activity against L-cystathionine (Holt et al., 2012). β-lyases delbrueckii Viniferm NSTD, a strain of industrial usage for thiol release
are enzymes involved in amino acid metabolism that do not normally as described below (Fig. 4).
catalyze a β-lyase reaction, but catalyze a non-physiological related β-
lyase side reaction that depends on the electron-withdrawing charac- 2.2. Interdelta analysis for S. cerevisiae strain fingerprinting
teristics of the cysteine S-conjugates (Cooper et al., 2010). Due to the
very low sensory perception threshold range, even a modest increase Eight hundred and eighty S. cerevisiae strains isolated from wineries
in yeast β-lyase activity, due to this side reaction, could alter the compo- in this study were checked for fingerprinting on interdelta polymor-
sition of volatile thiols in wine and improve flavor (Murat et al., 2001). phisms by PCR amplification using delta12 (5′-TCAACAATGGAATCCC
Many types of yeast, including Saccharomyces sp., possess limited capa- AAC-3′) and delta21 (5′- CATCTTAACACCGTATATGA-3′) primers
bilities in terms of enzymatic hydrolysis of precursors and formation of (Legras and Karst, 2003). Agarose gels were stained with GelRed®
volatile products. Cleavage of volatile thiols during fermentation ap- and analyzed under an UV transilluminator. The selection of the
pears to be strain dependent and a particular strain ability to release S. cerevisiae strains was achieved according to the different patterns ob-
one thiol does not seem to be linked to the formation of a different served using SigmaGel software.
thiol (Holt et al., 2012; Roncoroni et al., 2011). Research suggests that
by using different strains, differences in the release of these volatile
thiols can be achieved. 2.3. The selective YCB-SMC medium
Due to the demanding nature of modern winemaking practices and
an increasingly consumer quality demand, there is a growing need for The selective medium described in this work was based on the reac-
wine strains possessing a wide range of improved, optimized or novel tion catalyzed by the β-lyase activity over cysteinylated thiol precur-
enological features. One challenge today is the development of screen- sors. The YCB-SMC medium was: 0.1% (wt/vol) S-methyl-L-cysteine
ing methods to identify strains that improve wine quality from the (Sigma-Aldrich, Madrid, Spain), 0.01% (wt/vol) pyridoxal-5′-phosphate
great, unexplored diversity of natural grape yeasts. Based on several (Sigma-Aldrich) and 1.2% (wt/vol) Yeast Carbon Base (Difco, Detroit, MI,
criteria, winemakers often use selected strains to improve flavor, palate USA). For solid medium 2% agar was added. This medium was adjusted
structure and alcohol and phenolic content, among others (Belda et al., to pH 3.5 with HCl. All components, except agar, were sterilized by using
2016; Pretorius, 2000). Commercial wine S. cerevisiae has been selected 0.22 μm filters. The medium was kept under refrigeration at 4 °C before
on the basis of enhanced tropical fruity characters produced during fer- use. The strains were seeded on YCB-SMC medium following the proce-
mentation and similarly several non-Saccharomyces yeasts have been dure for nitrogen assimilation tests used for classical methods on yeast
shown to release significant concentrations of volatile thiols. Indeed, taxonomy. Yeasts grown on a rich medium may carry a reserve of nitro-
Pichia kluyveri was recently commercialized (Frootzen, Chr. Hansen, gen in the form of protein. Possible errors due to this reserve are elimi-
Denmark) for winemaking with the aim of enhancing fruity flavors nated by making two serial transfers in the YCB-SMC medium. When
(Anfang et al., 2009; Zott et al., 2011). Therefore, selection programs the first transfer was three days old, one loopful was transferred to a
of wine yeast starters able to produce more volatile thiols constitute second plate of the YCB-SMC medium containing the same source of ni-
an important goal for the wine industry, which has never conducted trogen. Colony growth was checked after 48–72 h at 20 °C.
rationally. Based on the YCB-SMC medium and with the objective of validating
Selective media are formulated to support the growth of one group it, a Cys-4MMP based medium, instead of SMC, was developed
of organisms, but inhibit the growth of another. The aim of this study as follows: 0.1% (wt/vol) S-4-(4-methylpentan-2-one)-L-cysteine
was to develop a selective medium for the differentiation of yeast spe- (Cys-4MMP), 0.01% (wt/vol) pyridoxal-5′-phosphate (Sigma-Aldrich),
cies according to their β-lyase activity and to discuss their potential ap- and 1.2% (wt/vol) Yeast Carbon Base (Difco). Cys-4MMP was syn-
plication in oenology. Additionally, based on the same medium, a highly thetized according to the procedure of Howell et al. (2004). Purity was
sensitive, reproducible and non-expensive method for the evaluation of determined by 1H-NMR (Bruker DPX 300 MHz) and ESI-MS (HPLC)
the potential volatile thiol release by different yeast isolates has been with Bruker EsquireLC quadrupole ion trap instrument (Bruker Daltonik
developed. GmbH, Bremen, Germany) (Fig. S7).
2. Material and methods 2.4. PCR analysis of IRC7 genotypes
2.1. Strains and general media With the initial aim being to gather new information on the compar-
ison of the IRC7 sequences of the industrial and wild S. cerevisiae strains, a
223 S. cerevisiae strains, originally isolated from wineries from Desig- PCR protocol was conducted with the primers PF6, 5′-AGCTGGTCTGGA
nation of Origin (D.O.) Ribera del Duero, D.O. Rueda and D.O. Tierra de GAAAATGG-3′ and PR7, 5′-TCTTCTGCGAGACGTTCAAA-3′ (Roncoroni
León (deposited in CYC, Complutense Yeast Collection, Complutense et al., 2011). The DreamTaq Green PCR Master Mix (Life Technologies
University of Madrid, Spain) and 22 industrial strains (Agrovin S.A., Al- Ltd., Paisley, UK) was used. The PCR reaction conditions were an initial
cázar de San Juan, Spain). denaturing step of 2 min at 94 °C followed by 35 cycles of 94 °C for
Sabouraud-Chloramphenicol (Oxoid, Hampshire, UK) was routinely 15 s, 56 °C for 30 s and 72 °C for 1 min and then a final extension at
used for S. cerevisiae isolation and Lysine Agar (Oxoid) was used for the 72 °C for 5 min. The PCR products were run on 2% agarose gels with an
isolation of non-Saccharomyces strains. appropriate molecular weight marker and the size of the amplified prod-
In order to determine if the methods described in this work were ucts was checked (Fig. S3 and Fig. S4). Six strains were selected in repre-
also helpful with non-Saccharomyces yeasts, a selection of 13 non-Sac- sentation of the three IRC7 genotypes (Fig. 1). The selected S. cerevisiae
charomyces strains isolated from wineries was used for the determina- strains were: (homozygous for the short IRC7 allele: 1- SEM-73, 2-
tion of their β-lyase activity. These non-Saccharomyces strains were SEM-25, 3- SEM-107, 4- SEM-251, 5- SEM-271 and 6- SO-320); (hetero-
identified by partial sequencing of the 26S large subunit rRNA gene. zygous strains for IRC7 allele: 7-SEM-10, 8- SO-166, 9- SO-353, 10- SO-
Total genomic DNA was extracted using the isopropanol method 10, 11- SEM-113, 12- SEM-294) and (homozygous for the full-length
(Querol et al., 1992), and the DNA for sequencing was amplified by IRC7 allele: 13- SO-213, 14- SO-335, 15- SO-331, 16- SEM-115, 17-
using an Eppendorf Mastercycler apparatus, with forward NL-1 primer SEM-129 and 18- SO-203).

118
Capítulo 3
2.6. Blind test for validation of YCB-SMC medium
Two different S. cerevisiae strains, S-EM-129 and S-EM-251; in repre-

sentation of two IRC7 genotypes (the homozygous for the full-length
IRC7 allele and the homozygous for the short IRC7 allele, respectively)
were grown in YCB-SMC agar medium during 48 h to develop a cellular
suspension of 106 cells/ml in NaCl 0.9%. Both strains were mixed togeth-
er (ratio 1:1) and then serially diluted and spread in YCB-SMC agar
medium. Plates were incubated at 20 °C during 72 h and colony devel-
opment was followed. Plates were photographed with a Nikon Eclipse
50i microscope at 40 × magnification using a ProgRes® CT3 camera
with a ProgRes® CapturePro 2.6 software (Jenoptik Laser, Optik,
Systeme GmbH). Colony area was calculated by using the colony diam-
eter expressed in number of pixels. Based on their size, colonies were
Fig. 1. PCR products of the main three IRC7 genotypes present in S. cerevisiae selected and analyzed by PCR for IRC7 genotypes as described before
amplified with PF6 and PR7 primers. S. cerevisiae strains used as PCR templates were as (Fig. S5).
follows: HS (1- SEM-73, 2- SEM-25, 3- SEM-107, 4- SEM-251, 5- SEM-271, 6- SO-320),
HT (7-SEM-10, 8- SO-166, 9- SO-353, 10- SO-10, 11- SEM-113, 12- SEM-294, 13- SO-
213) and HL (14-SO-335, 15- SO-331, 16- SEM-115, 17- SEM-129 and 18- SO-203). 2.7. Quantification of thiol release by gas chromatography–mass spectrom-
etry in YCB-SMC medium
The liquid YCB-SMC medium was used to prepare inocula in 20 ml

2.5. IRC7 genotype differentiation based on the spot growth assay on YCB- headspace vials with magnetic screw caps (Supelco Inc., Bellefonte,
SMC medium Pennsylvania, USA) at 10 ml per tube. Inocula of the different yeast
strains were standardized at an OD600 nm of 0.1. The strains used are
In order to determine whether there is a correlation between the listed in Fig. 3 (S. cerevisiae strains) and Fig. 4 (non-Saccharomyces
growth observed in the YCB-SMC medium and the different IRC7 alleles, strains). These liquid cultures were incubated with shaking during
the six wild strains belonging to each of the three IRC7 genotypes re- 24 h at 28 °C. Then, the production of methanethiol (MTL), and its
ferred in this work were grown on solid YCB-SMC and YCB-DAP dimer (dimethyl disulfide, SMDS), were determined in the headspace
media. Every strain was seeded onto YCB-SMC medium and incubated by GC–MS. A Varian gas chromatograph CP-3800 coupled to a Saturn
at 20 °C for 48–72 h and restreaked on the same medium to avoid nutri- 2200 ion trap mass spectrometer was used to analyze 200 μl of the sam-
ent carryover. Then the strains were suspended in saline buffer to reach ple headspace. The properties and basic settings of the GC–MS were as
an optical density at λ600 nm of 0.5. The indicated strains were tested for follows. The GC column used in this instrument was a capillary column
growth by applying 5 μl of serial dilutions from 10−1 to 10−6 onto YCB- Phenomenex ZB-5MS (30 m × 0.25 mm i.d. × 0.25 μm film thickness).
SMC and YCB-DAP media. YCB-DAP medium (containing the same com- The volatilized compound was carried in a helium flow at 1 ml/min.
ponents and concentrations of YCB-SMC medium but containing 0.1% The spectrometer operated in full-scan mode in a mass interval be-
diammonium phosphate instead of SMC) was used as positive control tween 30 and 400 amu. The injection-port temperature was set at
for growth (Fig. 2). 260 °C. The temperature program was initially set at 30 °C for 2 min
Fig. 2. Plot test study developed in order to determine the growth ability in YCB-SMC and YCB-DAP media of the six strains selected belonging to the three IRC7 allele groups present
in S. cerevisiae (A). The first group (HS) was formed by the strains homozygous for the short IRC7 allele (1- SEM-73, 2- SEM-25, 3- SEM-107, 4- SEM-251, 5- SEM-271 and 6- SO-320),
that showed a slow growth in YCB-SMC. The second group (HT) was composed by the heterozygous strains for IRC7 allele (7-SEM-10, 8- SO-166, 9- SO-353, 10- SO-10, 11- SEM-113,
12- SEM-294). (HL) Group of strains homozygous for the full-length IRC7 allele (13- SO-213, 14-SO-335, 15- SO-331, 16- SEM-115, 17- SEM-129 and 18- SO-203) that showed the
highest growth in YCB-SMC, but very similar to the heterozygous strains for IRC7. The same medium supplemented with diammonium phosphate (DAP), instead of SMC, was used as
control. Box plots (B) represent data for the six strains analyzed of the three different genotypes (HS, HT, HL). Different letters among boxes represent different statistical groups (p b 0.05).

119
Capítulo 3
In the conditions tested, S-methyl-L-cysteine was transformed to

MTL, pyruvate and ammonium due to the β-lyase activity of yeasts. In
these conditions, MTL dimerized in part through reoxidation to DMDS,
and so, both compounds were detected simultaneously by GC–MS
(Fig. S6). Sodium methanethiolate and dimethyl disulfide (Sigma)
were used as standards for quantification. Each determination was
done by triplicate in three independent assays. The results obtained
were expressed as relative β-lyase activity.
2.8. Quantification of thiol release by gas chromatography–mass spectrom-

etry in YCB-Cys-4MMP medium
With the aim of validating the results obtained with the new de-
scribed methodology and also to compare them with the results
obtained with naturally occurring substrates, the β-lyase activity of
the same S. cerevisiae strains was analyzed (Fig. 3b) in a medium
supplemented with Cys-4MMP instead of SMC. The method for
the determination of 4MMP production was based in the method de-
scribed by Howell et al., 2004, but detecting 4MMP directly from the
vial headspace without the use of SPME fiber. The detailed method
was exactly the same described above for SMC products (MTL/DMDS)
but with the injection of 1000 μl of the sample headspace instead of
200 μl.
2.9. Statistical analysis
Fig. 3. β-lyase activity of 18 S. cerevisiae wild strains determined by GC–MS using YCB-SMC All the statistical analyses were performed using PC Statgraphics
(A) and YCB-Cys-4MMP (B) as substrates for β-lyase activity. The strains homozygous for v.5 software (Graphics Software Systems, Rockville, MD, USA). The sig-
the short IRC7 allele (SO-320, SEM-271, SEM-251, SEM-107, SEM-25 and SEM-73) had a
nificance was set to p b 0.05 for the ANOVA matrix F value. Furthermore,
residual activity (HS). The heterozygous strains for IRC7 allele (SO-353, SO-166, SO-10,
SEM-294, SEM-113 and SEM-10) reached intermediate activity values (HT). The highest the multiple-range test was used to compare the means.
β-lyase activity (HL) was obtained for the homozygous S. cerevisiae strains for the full-
length IRC7 allele (SO-335, SO-331, SO-213, SO-203, SEM-129 and SEM-115). The same
pattern of β-lyase activity was observed for both substrates (SMC or Cys-4MMP). Box plots
3. Results
represent data for the six strains analyzed of the three different genotypes (HS, HT, HL).
Different letters among boxes represent different statistical groups (p b 0.05). 3.1. Interdelta analysis for S. cerevisiae strain fingerprinting
and ramped to 40 °C at 2 °C/min, then to 100 °C at 15 °C/min and finally With the initial aim of checking a significant number of yeast
ramped to 200 °C at 25 °C/min and stated at this temperature during isolates with oenological interest, 880 isolates of S. cerevisiae, from win-
5 min. The total program was 20 min. A TIC (Total Ion Chromatogram) eries of three different D.O., were checked for interdelta polymor-
was created by summing up intensities of all mass spectral peaks be- phisms. The analysis of the polymorphisms in agarose gels obtained
longing to the same scan. for the entire collection of S. cerevisiae isolates revealed the existence
Fig. 4. β-lyase activity of 14 non-Saccharomyces strains determined by GC–MS by using the selective medium developed in this work. Non-Saccharomyces strains had a low β-lyase activity
with the exception of T. delbrueckii Viniferm NSTD and K. marxianus NSPDC-99.

120
Capítulo 3
of 223 different polymorphisms (Fig. S2), named in this study as “wild 3.5. Determination of the β-lyase activity on YCB-SMC medium by GC–MS
strains”.
With the aim of determining if the YCB-SMC medium was useful for
3.2. Growth industrial and wild S. cerevisiae strains on YCB-SMC medium quantification purposes of the β-lyase activity, the YCB-SMC medium
was inoculated with the aforementioned S. cerevisiae strains and the
Two growth phenotypes on YCB-SMC medium were observed β-lyase reaction products (MTL and DMDS) determined by GC–MS. Ac-
among the 245 industrial and wild strain collections. Approximately, cording to the results, it was possible to establish a relationship between
15% of the strains were able to grow more efficiently on the selective the IRC7 genotypes of the S. cerevisiae strains and the release of thiols
SMC medium, generating bigger colonies. The percentage of this pheno- (Fig. 3). The homozygous strains for the full-length IRC7 allele (SEM-
type varied between the industrial (10 of 22; 45.5%) and the wild strains 115, SEM-129, SO-203, SO-213, SO-331 and SO-335) had, approximate-
(27 of 223; 12%) isolated in this study, showing the influence of the se- ly, a 50% more β-lyase activity than the heterozygous strains for IRC7 al-
lection processes of industrial strains. lele (SEM-10, SEM-113, SEM-294, SO-10, SO-166 and SO-353). It was
also noticeable that the activity showed by the strain SEM-129, being
30–40% higher than the rest of the strains belonging to the same
3.3. Determination of IRC7 genotypes in wild and industrial strains group. The β-lyase activity showed for the strains SEM-25, SEM-73,
SEM-107, SEM-251, SEM-271 and SO-320 bearing a deletion in the
With the aim of determining the presence of different genotypes in IRC7 sequence, was comprised between 5 and 10% in comparison to
IRC7, according to the findings by Roncoroni et al. (2011), 223 wild iso- the average value of β-lyase activity obtained for the homozygous
lates were checked for the determination of the presence of a 38-bp de- strains for the full-length IRC7 allele (Fig. 3a).
letion commonly found between yeasts isolates (Liti et al., 2009). As Among the selection of non-Saccharomyces strains used in the pres-
expected, two sizes of PCR products were detected, 219 bp for the full- ent study, only Kluyveromyces marxianus NSPDC-99 was observed to
length copy and 181 bp for the 38-bp deletion of IRC7. The percentage cleave an important amount of SMC, in comparison to T. delbrueckii
of wild yeast strains with the reported 38-bp deletion in IRC7 was 88% Viniferm NSTD, a commercial strain indicated for volatile thiol release
(196 strains of 223). Additionally, there were 2.7% (6 strains of 223) of (Fig. 4).
yeast strains homozygous for the full-length IRC7 allele and 9.4%
(21 strains of 223) that were found to be heterozygous for the full- 3.6. β-lyase activity on YCB-Cys-4MMP medium
length allele (Fig. 1; Fig. S3).
In addition, the industrial collection (Agrovin, S.A.) of S. cerevisiae Six strains belonging to each of the three IRC7 genotypes described
strains was tested in the same way (Fig. S4). In this collection, 23% were inoculated in a liquid medium in which SMC was substituted by
(5 strains of 22) of the strains were found to be homozygous for the Cys-4MMP as the natural thiol-cysteine precursor present in grapes.
full-length IRC7 allele and an additional 23% was found to be heterozy- The products were detected by GC–MS. The strains bearing a full-
gous for the full-length IRC7 allele. length copy of IRC7 (SEM-115, SEM-129, SO-203, SO-213, SO-331 and
SO-335) had the best production of 4-MMP when growing in YCB-
3.4. Relationship between ICR7 genotypes and phenotypes detected in YCB- Cys-4MMP medium, followed by the heterozygous strains for IRC7 allele
SMC medium (SEM-10, SEM-113, SEM-294, SO-10, SO-166 and SO-353). The strains
found to be homozygous for the deleted IRC7 allele (SEM-25, SEM-73,
Comparison of the IRC7 genotypes of S. cerevisiae and the growth SEM-107, SEM-251, SEM-271 and SO-320) showed no production of
ability in YCB-SMC medium indicated that there is a clear relationship 4-MMP or a production under the detection limits.
between those features. The strains homozygous and heterozygous for
the full-length IRC7 allele were able to grow faster in YCB-SMC medium 4. Discussion
than those strains homozygous for the short-length IRC7 allele (Fig. 2). It
was also noticeable that, according to colony growth, S. cerevisiae strains 4.1. The selective YCB-SMC medium
described to be homozygous and heterozygous for the full-length IRC7
allele were difficult to differentiate in YCB-SMC medium. The homozy- The final aromatic profile of wine is one of the most important fac-
gous and the heterozygous strains for the full-length IRC7 allele were tors when determining its quality and intrinsic value. Winemakers gen-
able to grow faster in YCB-SMC medium, giving a phenotype clearly dif- erally focus on maximizing aroma intensity and complexity while
ferent than the observed for the strains that were homozygous for the minimizing aromas that may dominate or produce a negative percep-
short-length IRC7 allele (Fig. 2). The average colony area comprised by tion for the production of wines with varietal distinction. The aroma
the group of strains homozygous for the full-length allele was 17.3 of a wine is one of the most important determinants of its quality
times higher than the homozygous for the short-length allele and 1.26 (Selli et al., 2004). Current strategies intended for the increment of
times higher than the heterozygous strains for IRC7 allele, indicating wine aroma comprise the choice of grape variety to optimize production
that the YCB-SMC medium generated a selective advantage over the of thiol precursors, the choice of yeast strain to optimize release of vol-
group of strains that were homozygous for the short IRC7 allele atile thiols in the winery and the coinoculation with yeast blends. Here-
(Fig. S5a). These observations gave the opportunity to easily differenti- tofore, the yeast selection programs do not have a tool for the selection
ate one of the main IRC7 genotypes. of yeasts with an increased capacity of varietal thiol release. In this work
Two strains (S-EM-129 and S-EM-251; in representation of the ho- a selective medium for the isolation of yeasts with an increased capacity
mozygous full- and the homozygous short-length IRC7 alleles, respec- for volatile thiol release has been developed. Cysteine S-conjugate β-
tively) were mixed (ratio 1:1) and used as inocula to develop a blind lyases are pyridoxal 5′-phosphate-dependent enzymes that catalyze
test to differentiate IRC7 genotypes based on the phenotypes showed β-elimination reactions with cysteine S-conjugates that possess an
in YCB-SMC medium. As expected, yeast colonies, isolated from YCB- electron-withdrawing group attached at the sulfur. The end-products
SMC medium, showed different sizes and were ascribed to two colony of the β-lyase reaction are pyruvate, ammonium and a sulfur-
size groups. Ten colonies of each group were subjected to PCR for IRC7 containing fragment. The selective medium was designed by using S-
genotype differentiation (Fig. S5b). The group of smaller colonies was methyl-L-cysteine (SMC) as the only nitrogen source to provide an
observed to have the reported 38-bp deletion in IRC7 whereas the easy and rapid method for isolation of yeast strains for their ability to
group of bigger colonies had the homozygous full-length allele. 100% cleave thiol precursors to release varietal thiol aroma. SMC was chosen
of the strains were correctly assigned to their respective genotype. because it structurally resembled naturally occurring cysteinilated

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precursors in grape must, being commercially available, water-soluble SMC. The GC–MS method described in this work was developed in the
and non-expensive, avoiding the process of synthesis or extraction hope that it could be helpful for the determination of the β-lyase activity
and purification of naturally occurring cysteinilated precursors in avoiding the use of natural substrates such as Cys-4MMP, more difficult
grapes. Additionally, SMC was found to be less toxic than other similar to detect by GC–MS and not widely available as commercial products.
cysteinylated compounds, such as S-ethyl-L-cysteine, and it is metabo- The detection of MTL/DMDS by GC–MS was observed to be simplest,
lized to less-toxic end-products (Maw, 1961). because the method only considered injection of a small headspace vol-
ume without the utilization of a SPME fiber to concentrate the products
4.2. The selective YCB-SMC medium and IRC7 in S. cerevisiae of the reaction, as it was described to be required for the detection of
4MMP (Howell et al., 2004).
As described by Roncoroni et al. (2011) in S. cerevisiae strains the
main β-lyase activity, responsible for the release of the varietal thiol 4.3. Contribution of non-Saccharomyces to thiol release
4MMP, is coded by the IRC7 gene (Howell et al., 2005; Roncoroni
et al., 2011). Two alleles have been described for this gene; the most In addition to the presence of a highly functional IRC7-coded β-lyase,
common allele has a 38-bp deletion, missing a conserved region found it is known that Nitrogen Catabolite Repression (NCR) affects concen-
in other β-lyase genes, which generates a C-terminally truncated pro- trations of varietal thiols in wine through the repression of IRC7, special-
tein of about 340 amino acids instead of the 400 amino acids protein ly by inorganic nitrogen forms such as ammonium, usually added as
present in other yeasts and bacteria (Hall et al., 2005). This deletion im- diammonium phosphate during the middle and final fermentation
plies the production of an enzyme, which is characterized by its low β- stages (Subileau et al., 2008; Thibon et al., 2008), and also low fermen-
lyase activity, and therefore a lower 4MMP production (Roncoroni et al., tation temperatures diminish 4MMP production (Howell et al., 2004;
2011). The observation, also reported by Roncoroni et al. (2011), that Masneuf et al., 2006). That resulted in a small conversion of the
the short allele of IRC7 is present in a higher proportion in commercial cysteinylated thiol precursors into their corresponding free thiols, usu-
wine yeasts helps to explain why certain commercial strains are ally about 5%, or even less, (Murat et al., 2001; Roland et al., 2011;
known as high 4MMP releasers (Lee et al., 2008; Masneuf et al., 2002, Winter et al., 2011; Coetzee and du Toit, 2012; Peña-Gallego et al.,
2006). On the contrary, 88% of the autochthonous S. cerevisiae strains 2012). Although some genetic engineering techniques have been de-
isolated in this study bear the truncated IRC7 allele (Fig. S3), and being veloped to enhance the cleavage of cysteinylated precursors into free
therefore low 4MMP producers. The reasons about why this short allele thiols (Swiegers et al., 2007), these approaches are not acceptable
of IRC7 have been selected by nature remains to be elucidated. The high for consumers and winemakers because of the use of genetically
diversity observed among natural strains reinforces the usefulness of modified organisms. Exploiting the natural genetic variations offered
the screening proposed to be added to the current yeast selection by different S. cerevisiae (Liti et al., 2009) and non-Saccharomyces
criteria used. However, it must be considered that the findings de- strains is a powerful tool to improve wine yeast characters (Zott
scribed in this work could be affected by the existence of additional con- et al., 2011).
siderations such as the intrinsic characteristics of the strains which Non-Saccharomyces species are limited to the early stages of fermen-
could affect metabolism behavior or the variable toxicity of the precur- tation, while Saccharomyces dominated towards the end of the alcoholic
sors that are substrates of β-lyase (Santiago and Gardner, 2015a). fermentation. However, the presence of non-Saccharomyces (autoch-
In our laboratory, in addition to SMC, other potential substrates of β- thonous or commercial strains) generates significant differences in
lyase have been considered (S-benzyl-L-cysteine, S-(2-aminoethyl)-L- wine quality (Belda et al., 2015; Benito et al., 2015). For that reason
cysteine and S-(4-tolyl)-L-cysteine) but without valuable results due YCB-SMC medium was intended for the selection of both, S. cerevisiae
to growth inhibition in the conditions tested (not shown). and non-Saccharomyces yeasts. Due to the cleavage of SMC, yeasts
In addition, the described method for yeast isolation was also useful with an increased β-lyase activity were able to grow rapidly probably
when performed to quantitatively detect β-lyase activity by determin- because of the ammonium released in the β-lyase reaction and, so, gen-
ing the release of MTL/DMDS (Fig. S6). This method uses SMC as sub- erating big colonies (Fig. 2). Furthermore, non-Saccharomyces yeast
strate for β-lyase avoiding the use of natural precursors such as Cys- strains, such as T. delbrueckii Viniferm NSTD, were able to grow faster
4MMP. SMC is commercially available, water soluble and non-toxic for on YCB-SMC medium, being also able to release higher amounts of
yeast cells in the conditions tested. In order to phenotypically differen- MTL/DMDS as detected by GC–MS (Fig. 4b), indicating that the medium
tiate between the three different IRC7 genotypes present in S. cerevisiae was useful for the selection of a wide variety of yeast species.
strains, six S. cerevisiae strains belonging to each genotype were selected In conclusion, there is a good margin to further multiply thiol
and inoculated in the YCB-SMC medium and then subjected to analysis production by non-Saccharomyces yeasts by breeding. In that sense,
for MTL/DMDS production. According to the results presented in the non-Saccharomyces yeasts are dominant during the first stages of wine
Fig. 3a, the strains with the full-length copy of IRC7 were the higher fermentation, such as pre-fermentative cold soak (Zott et al., 2008).
MTL/DMDS producers indicating an increased β-lyase activity. On In accordance with that, their impact on wine quality is mainly
the contrary, those strains with the 38-bp deletion produced small circumscribed to the early stages in winemaking, when the addition of
amounts of MTL/DMDS indicating a less efficient β-lyase activity. inorganic nitrogen sources (as the major catabolic repressor) could be
Furthermore, this is in agreement with recent findings that indicate avoided or substituted by the addition of organic nitrogen complexes
that a full-length copy of IRC7 allele is required for the cleavage of two that has been shown to be less repressive. YCB-SMC medium was useful
thiol precursors (cysteine-4MMP and glutathione-3MH) in a panel of for the determination of the potential of varietal thiol release by both,
S. cerevisiae strains (Santiago and Gardner, 2015b). S. cerevisiae and non-Saccharomyces strains, making possible the effi-
In order to validate the proposed methods for, qualitatively and cient selection of yeasts with increased volatile thiol release. This
quantitatively, determine the β-lyase activity in yeast isolates, the use possibility allows us to contemplate the possibility of using non-
of SMC was compared with the natural precursor Cys-4MMP (Fig. 3). Saccharomyces yeasts as tools to increase the volatile thiol release in a
The results indicate that although the reported data are not exactly time in which multi-starter fermentations are increasingly being used
the same, the conclusions for these findings were similar; confirming for organoleptic and quality improvement (Belda et al., 2015; Ciani
that yeast strains possessing a long size copy of IRC7 are more efficient et al., 2010). According to Zott et al. (2011), T. delbrueckii and
for thiol aroma release and YCB-SMC medium is a suitable simplified K. marxianus had a high capacity to release MTL/DMDS indicating their
method for the isolation of yeasts with good properties for thiol aroma high β-lyase activity. However, the strains of Metschnikowia pulcherrima
production. These results indicate that the same β-lyase activity could tested in our study were found to have a moderate β-lyase activity
be the responsible for the cleavage of both substrates, Cys-4MMP and (Fig. 4). Other yeasts species usually found in wineries released low

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amounts of SMC-related thiols, indicating their poor contribution to Howell, K.S., Swiegers, J.H., Elsey, G.M., Siebert, T.E., Bartowsky, E.J., Fleet, G.H.,
Pretorius, I.S., 2004. Variation in 4-mercapto-4-methyl-pentan-2-one release by
thiol production. Taking into account that NCR has not been studied in Saccharomyces cerevisiae commercial wine strains. FEMS Microbiol. Lett. 240,
depth in several non-Saccharomyces species, and that the relationship 125–129.
between NCR and thiol release has been studied only in S. cerevisiae, Howell, K.S., Klein, M., Swiegers, J.H., Hayasaka, Y., Elsey, G.M., Fleet, G.H., Hoj, P.B.,
Pretorius, I.S., de Barros Lopes, M.A., 2005. Genetic determinants of volatile-thiol re-
our ongoing research is to investigate whether the positive effect of lease by Saccharomyces cerevisiae during wine fermentation. Appl. Environ. Microbiol.
our promising non-Saccharomyces yeasts on thiol release is less affected 71, 5420–5426.
by NCR in wine, giving the opportunity to develop a promising applica- Irmler, S., Raboud, S., Beisert, B., Rauhut, D., Berthoud, H., 2008. Cloning and characteriza-
tion of two Lactobacillus casei genes encoding a cystathionine lyase. Appl. Environ.
tion for exploiting the potential thiol content of grape musts.
Microbiol. 74, 99–106.
Kurtzman, C.P., Robnett, C.J., 1997. Identification of clinically important ascomycetous
yeasts based on nucleotide divergence in the 5′ end of the large-subunit (26S) ribo-
Acknowledgement somal DNA gene. J. Clin. Microbiol. 35, 1216–1223.
Lee, S.A., Rick, F.E., Dobson, J., Reeves, M., Clark, H., Thomson, M., Gardner, R.C., 2008.
Funding for the research in this paper was provided by the Agrovin Grape juice is the major influence on volatile thiol aromas in sauvignon blanc. Aust.
N. Z. Grapegrower Winemaker 533, 78–86.
S.A, under the framework of the project IDI20130192-ENZIOXIVIN Legras, J., Karst, F., 2003. Optimisation of interdelta analysis for Saccharomyces cerevisiae
(Centre for Industrial Technological Development-CDTI, Spain). We strain characterisation. FEMS Microbiol. Lett. 221, 249–255.
are grateful to Dra. Cristina Gutiérrez from the Mass Spectrometry Cen- Liti, G., Carter, D.M., Moses, A.M., Warringer, J., Parts, L., James, S.A., Davey, R.P., Roberts,
I.N., Burt, A., Koufopanou, V., Tsai, I.J., Bergman, C.M., Bensasson, D., O'Kelly, M.J.T.,
tre of the Complutense University of Madrid for technical assistant and van Oudenaarden, A., Barton, D.B.H., Bailes, E., Nguyen, B.A.N., Jones, M., Quail, M.A.,
helpful discussions. We also thank Dr. Luis Sánchez and Julia Buendía Goodhead, I., Sims, S., Smith, F., Blomberg, A., Durbin, R., Louis, E.J., 2009. Population
from Analytical Chemistry Department of the Complutense University genomics of domestic and wild yeasts. Nature 458, 337–341.
Martínez-Cuesta, M.C., Pelárez, C., Eagles, J., Gasson, M.J., Requena, T., Hanniffy, S.B., 2006.
of Madrid for their help with 4MMP and Cys-4MMP synthesis. YtjE from Lactococcus lactis IL1403 is a C-S lyase with α, γ-elimination activity toward
methionine. Appl. Environ. Microbiol. 72, 4878–4884.
Masneuf, I., Murat, M.L., Naumov, G.I., Tominaga, T., Dubourdieu, D., 2002. Hybrids Saccha-
Appendix A. Supplementary data romyces cerevisiae × Saccharomyces bayanus var. uvarum having a high liberating
ability of some sulfur varietal aromas of Vitis vinifera sauvignon blanc wines. J. Int.
Sci. Vigne Vin 36, 205–212.
Supplementary data to this article can be found online at http://dx.
Masneuf, I., Mansour, C., Murat, M.L., Tominaga, T., Dubourdieu, D., 2006. Influence of fer-
doi.org/10.1016/j.ijfoodmicro.2016.03.001. mentation temperature on volatile thiols concentrations in sauvignon blanc wines.
Int. J. Food Microbiol. 108, 385–390.
Maw, G.A., 1961. Ability of S-Methyl-L-cysteine to annul the inhibition of yeast growth by
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thiols 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) dur- Zott, K., Miot-Sertier, C., Claisse, O., Lonvaud-Funel, A., Masneuf, I., 2008. Dynamics and di-
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Belda et al. 2016

Supplementary material
Figure S1. Cystathionine β-lyases catalyze the conversion of cystathionine into

homocysteine and the by-products pyruvate and ammonia, employing pyridoxal-
5’-phosphate as cofactor. They can also release the aromatic thiols 3MH and 4MMP
from their respective cysteine-S-conjugated precursors present in grape must. The
selective medium described in this work is composed by S-methyl-L-cysteine
(SMC). Yeasts with an increased β-lyase activity are able to grow efficiently due to
the assimilation of the released ammonia from SMC.

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Figure S2a. PCR amplification of inter-delta regions. Electrophoretical patterns

obtained with delta1-2 and delta 2-1 primers for the 120 S. cerevisiae isolates obtained
from cellars of the D.O. Tierra de Leon. Highlighted lanes represents every one of the
223 different polymorphisms selected in the whole study.
60

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Figure S2b. PCR amplification of inter-delta regions. Electrophoretical patterns

from cellars of the D.O. Ribera de Duero. Highlighted lanes represents every one of the
223 different polymorphisms selected in the whole study.

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129
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130
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284

131
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132
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Figure S2c. PCR amplification of inter-delta regions. Electrophoretical patterns

from cellars of the D.O. Rueda. Highlighted lanes represents every one of the 223
different polymorphisms selected in the whole study.

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134
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135
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136
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Figure S3. The main three IRC7 genotypes present in 223 wild strains of S.
cerevisiae. PCR products of yeast genomic DNA amplified with PF6 and PR7
primers. Yeast strains used as PCR templates were named as indicated in the
figure.

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Figure S4. PCR amplification of IRC7 for the 22 industrial S. cerevisiae strains
obtained from Agrovin, S.A.
Figure S5. A micrograph taken with a phase-contrast microscopy (X40) after 24 h of

cultivation of the colonies developed in SMC medium (A) in a blind test developed by
mixing two strains of S. cerevisiae (S-EM-129 and S-EM-251; in representation of the
homozygous full- and the homozygous short-length IRC7 alleles, respectively, as
detected by PCR (B).

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Figure S6. Detection by GC-MS of the end-products (MTL/DMDS) of the SMC

metabolism for the determination of the β-lyase activity. Yeast strains with an increased
β-lyase activity released high amounts of MTL in comparison with those strains with
low β-lyase activity. MTL dimerized, in part, to dimethyl disulfide (DMDS) and both
were simultaneously detected by GC-MS.

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Figure S7. Cys-4MMP was synthetized according to the procedure of Howell et al. (2004). Cys-4MMP
purity was determined by (A) 1H-NMR (Bruker DPX 300MHz) and (B) ESI-MS (HPLC) with Bruker
EsquireLC quadrupole ion trap instrument (Bruker Daltonik GmbH, Bremen, Germany).

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5.2. Caracterización de la fisiología en fermentación de Torulaspora

delbrueckii y su contribución a la complejidad de vinos tintos.

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Appl Microbiol Biotechnol (2015) 99:1911–1922

DOI 10.1007/s00253-014-6197-2
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Dynamic analysis of physiological properties of Torulaspora

delbrueckii in wine fermentations and its incidence on wine quality
Ignacio Belda & Eva Navascués & Domingo Marquina &
Antonio Santos & Fernando Calderon & Santiago Benito
Received: 12 August 2014 / Revised: 26 October 2014 / Accepted: 27 October 2014 / Published online: 19 November 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract This work examines the physiology of a new com- Keywords Torulaspora delbrueckii . Manno proteins .
mercial strain of Torulaspora delbrueckii in the production of Glyceropyruvic pathway . Malic acid . Pyruvic acid .
red wine following different combined fermentation strate- Combined fermentation
gies. For a detailed comparison, several yeast metabolites
and the strains implantation were measured over the entire
fermentation period. In all fermentations in which Introduction
T. delbrueckii was involved, the ethanol concentration was
reduced; some malic acid was consumed; more pyruvic acid Many research groups are currently studying non-
was released, and fewer amounts of higher alcohols were Saccharomyces yeasts (Comitini et al. 2011; Contreras et al.
produced. The sensorial properties of final wines varied wide- 2014; Garde-Cerdán and Ancín-Azpilicueta 2006; Jolly et al.
ly, emphasising the structure of wine in sequential fermenta- 2006) due to their unique physiological metabolic properties,
tions with T. delbrueckii. These wines presented the maximum which may be advantageous in winemaking. The presence of
overall impression and were preferred by tasters. Semi- non-Saccharomyces wild yeasts in fermentations has been
industrial assays were carried out confirming these differences associated, traditionally, with high levels of acetic acid and
at a higher scale. No important differences were observed in other off-flavours. Nevertheless, nowadays, researchers and
volatile aroma composition between fermentations. However, winemakers are aware of the influence of non-Saccharomyces
differences in mouthfeel properties were observed in semi- in wine aroma complexity (Egli et al. 1998; Esteve-Zarzoso
industrial fermentations, which were correlated with an in- et al. 1998; Fleet 2003, 2008; Fleet and Heard 1993; Gil et al.
crease in the mannoprotein content of red wines fermented 1996; Henick-Kling et al. 1998; Lambrechts and Pretorius
sequentially with T. delbrueckii. 2000; Romano et al. 2003; Viana et al. 2008). The difficulty
with which non-Saccharomyces wine yeast finishes the alco-
holic fermentation requires the development of combined
fermentation with Saccharomyces cerevisiae as a binding
Electronic supplementary material The online version of this article partner. Some enzymatic activities related to aroma enhance-
(doi:10.1007/s00253-014-6197-2) contains supplementary material, ment (glycosidases and β-lyase for terpene and thiol release,
which is available to authorized users. respectively) and the release of some interesting products such
I. Belda : E. Navascués : D. Marquina : A. Santos as glycerol and mannoproteins, among others, are the high-
Department of Microbiology, Biology Faculty, Complutense lights that justify the interest in these mixed fermentations
University of Madrid, 28040 Madrid, Spain (Ciani et al. 2010; Rojas et al. 2001). In this context, combined
fermentations are a very useful tool to improve wine fermen-
E. Navascués
Agrovin, S.A., Alcázar de San Juan, 13600 Ciudad Real, Spain tations in which aromatic complexity of spontaneous fermen-
tations and the safety of industrial targeted fermentations are
F. Calderon : S. Benito (*) joined (Ciani et al. 2010; Romano et al. 2003).
Department of Chemistry and Food Technology, Polytechnic
University of Madrid, Ciudad Universitaria S/N, 28040
Some studies have analysed the use and influence of dif-
Madrid, Spain ferent non-Saccharomyces species in wine fermentations,
e-mail: santiago.benito@upm.es such as Kloeckera apiculata (Herraiz et al. 1990), other

145
Capítulo 3
1912 Appl Microbiol Biotechnol (2015) 99:1911–1922
apiculated yeasts like Hanseniaspora uvarum (Zironi et al. methods as follows. Yeast isolates were identified by sequence
1 99 3) , To r u l a s po r a d el b r u ec ki i, Kl uy ve romy ce s analysis of the 26S large subunit rRNA gene. Total genomic
thermotolerans, Hansenula anomala, and Metschnikowia DNA was extracted using the isopropanol method (Querol
pulcherrima (Ciani et al. 2006; Izquierdo-Cañas et al. 2011, et al. 1992), and DNA for sequencing was amplified using
2014; Oro et al. 2014). an Eppendorf Mastercycler apparatus as described by
Despite that studies of industrial or semi-industrial use of Kurtzman and Robnett (1997) with forward NL-1 primer
T. delbrueckii and its repercussion on wine quality are scarce, (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) and
most scientific studies report its relationship with wines with reverse NL-4 primer (5′-GGT CCG TGT TTC AAG ACG
low acetic acid content and great mouthfeel properties (Bely G′). Sequences obtained to identify yeasts were analysed and
et al. 2008). Furthermore, the fermentative capacity of compared by BLAST-search (GenBank;www.ncbi.nlm.nih.
T. delbrueckii (Quirós et al. 2014) allows its implantation at gov). The 26S rRNA nucleotide sequences has been
the beginning of fermentation process, contrary to other strict- submitted to Genbank-NCBI under accession numbers
ly oxidising non-Saccharomyces yeasts. At the same time that KM434246 (S. cerevisiae CT007) and KM434245 (T.
several authors are studying the potential use of non- delbrueckii NSA-1).
Saccharomyces yeasts in wine fermentations (De Benedictis Additionally, S. cerevisiae CT007 identification was con-
et al. 2010; Domizio et al. 2011; Viana et al. 2008), the firmed by the polymerase chain reaction amplification of the
enology industry has been able to accept this trend, and most interdelta region of S. cerevisiae (Legras and Karst 2003)
wine yeast distribution companies already have non- using delta12 (5′-TCAACAATGGAATCCCAAC-3′) and
Saccharomyces strains for its use in winery. delta21 (5′- CATCTTAACACCGTATATGA-3′) primers.
The possibility to modulate the flavour and style of wine by
different fermentation strategies forced the study on all possible
combinations of non-Saccharomyces and Saccharomyces yeast Characterisation of yeast strains
strains (Azzolini et al. 2012). In this sense, most of studies
analyse fermentations carried out with non-Saccharomyces β-Glucosidase activity was evaluated as reported by Rosi
strains by itself, with mixed fermentations by simultaneous et al. (1994), on a medium containing 0.5 % cellobiose (4-
and sequential inoculation, comparing all of them with the O-β-D-glucopyranosyl-D-glucose), 0.67 % yeast nitrogen ba-
alcoholic fermentation with S. cerevisiae by itself. se (Difco) and 2 % agar. Yeast strains were inoculated as
This study aims to validate the industrial use of a new above and incubated at 28 °C for 3 days. A significant growth
commercial strain of T. delbrueckii from Agrovin S.A., study- of the colonies indicated the presence of β-glucosidase activ-
ing their physiology throughout fermentation in order to explain ity. Additionally, β-D-xylosidase and α-L-arabinofuranosidase
the chemical composition, aromatic profile and sensorial prop- activities were evaluated using the correspondent
erties of the red Tempranillo wines produced by different mixed methylumbelliferyl-conjugated substrates
cultures of the strain T. delbrueckii NSA-1 with S. cerevisiae. (methylumbelliferyl-β-D-xylopyranoside and
Most of studies reported to analysing the properties and methylumbelliferyl-α-L-arabinofuranosidase, respectively),
that advantages of some non-Saccharomyces yeast are devel- according to the method described by Manzanares et al.
oped following a microvinification trend, but results are rarely (1999). Strains of T. delbrueckii CECT 10676 from the
validated in an industrial or semi-industrial scale, questioning Spanish Type Culture Collection (CECT Valencia, Spain)
its potential applicability (Jolly et al. 2014) due to the influ- and Rhodotorula mucilaginosa NSG-61 from the
ence of scale on yeast gene expression (Rossouw et al. 2012). Complutense Yeast Collection (CYC Madrid, Spain) were
In order to validate microvinification results in this study, used as were used as negative and positive controls,
semi-industrial fermentation was carried out in 100-L stainless respectively.
tanks. Production of hydrogen sulfide was evaluated by using a
modification of the lead acetate method (Linderholm et al.
2008). This method detects volatile H2S in the headspace of
Materials and methods the fermentation in a culture medium containing 1.17 % yeast
carbon base (Difco), 4 % glucose anhydrous, and 0.5 %
Microorganisms ammonium sulfate. Yeasts were grown at 28 °C for 3 days
in 96-well microplates containing 200 μl of medium with
Yeast strains and molecular identification orbital agitation (200 rpm). Hydrogen sulfide formation was
initially detected by using paper strips (Whatman filter paper)
S. cerevisiae CT007 and T. delbrueckii NSA-1 Viniferm NS- that had been previously embedded with a 0.1 M lead acetate
TD were obtained from the Agrovin S.A. (Alcázar de San solution and allowed to dry at 65 °C for 10 min and deposited
Juan, Spain) collection and identified by using molecular over microplate wells. Hydrogen sulfide formation was

146
Capítulo 3
Appl Microbiol Biotechnol (2015) 99:1911–1922 1913
qualitatively measured based on the degree of blackening of followed by plating 50 μL of the appropriate dilution on
the lead acetate strip and quantitatively estimated by densito- Sabouraud glucose agar with chloramphenicol (total yeast
metric measure of the intensity (Software “My Image counts) and lysine media (non-Saccharomyces counts).
Analysis v1.1” Thermo Scientific). Colonies were counted after growth at 30 °C for 48–72 h.
Killer activity was measured by the method described by All fermentation processes were carried out at 20 °C. Once
Santos et al. (2009). Yeast to be tested for killer activity were fermentation of sugars was completed (deemed to be repre-
inoculated in ∼1-cm diameter concentrated zones onto YMA- sented by a remaining glucose+fructose concentration lower
MB plates (1 % glucose, 0.3 % yeast extract, 0.3 % malt than 3 g/L), 50 mg/L of sulphur dioxide was added in potas-
extract and 0.5 % proteose peptone no. 3, supplemented with sium metabisulfite form to the wines, and they were racked
30 mg/L of methylene blue, 3 % NaCl and 2 % agar) previ- and stabilised during 7 days at 4 °C, and the final product was
ously seeded with a lawn (5.0×105 cells/ml) of the sensitive bottled. Bottles were placed horizontally in a climate chamber
yeast (S. cerevisiae Hansen BY4741). The sensitive strain was TR2V120 (La Sommelie`re, Saint-Saturnin, France) at 18 °C
grown on YMA medium (YMA-MB without NaCl and and 70 % relative humidity. These conditions were maintained
methylene blue) and suspended in sterile water just before until the sensory evaluation took place.
inoculation. The plates were incubated for a week at 20 °C.
Killer yeasts were identified by a clear zone of inhibition Semi-industrial fermentations
surrounding them (Llorente et al. 1997).
All semi-industrial fermentations were undertaken using
Biomass production V. vinifera L. cv. Tempranillo must. Eighty kilograms of fresh
crushed grapes were placed in 100 L stainless steel fermenta-
S. cerevisiae CT007 was obtained as active dry yeast and tion tanks, leaving enough space for the emission of carbon
rehydrated following the manufacturer’s instructions. dioxide. Sulphur dioxide (40 mg/kg) was added to each. The
T. delbrueckii cultures were obtained by using an enriched sugar content was 247 g/L, yeast assimilable nitrogen 188 mg/
must medium (12.5 % concentrated must (final concentration, L, pH 3.42.
50 g/L glucose+fructose), 1 % yeast extract, 0.5 % proteose Four assays were performed as described above for
peptone no.3, pH 3.5) at 25 °C. Upon reaching the necessary microvinifications. All fermentation processes were carried
cell concentration, T. delbrueckii was concentrated by decan- out at winery temperature of 20 °C. Once fermentation of
tation and then used as inoculum for vinifications. sugars was complete (deemed to be represented by a remain-
ing glucose fructose concentration lower than 3 g/L), the
wines fermented with T. delbrueckii were racked and stabi-
Microvinifications and growth kinetics
lized during 15 days at 4 °C, and the final product was bottled.
Fifty milligrams per liter of sulphur dioxide were added in
All fermentations were prepared using the must from Vitis
potassium metabisulfite form. Corked bottles were placed as
vinifera L.cv. Tempranillo grapes from El Socorro
described above. These conditions were maintained for
(Experimental Vineyard, Madrid, Spain) and processed accord-
7 weeks until the sensory evaluation took place.
ingly to the methods described previously with slight modifi-
cations (Benito et al. 2012; Sampaio et al. 2007). Fresh must
was bleeding from crushed-grapes (3.5 L) and placed in 4.9-L Analytical determinations of non-volatile compounds
glass fermentation vessels, leaving enough space for carbon
dioxide emission. Sulphur dioxide (40 mg/L) (Panreac, Glucose fructose, malic acid, lactic acid, acetic acid, glycerol,
Barcelona, Spain) was added to each vessel. The sugar content pyruvic acid and colour intensity were all determined using the
was 247 g/L, yeast assimilable nitrogen 188 mg/L, pH 3.42. Y15 Enzymatic Autoanalyzer (Biosystems S.A, Barcelona,
By triplicate, four assays were performed: (1) inoculation Spain). These analyses were performed using the appropriate
with T. delbrueckii (Td), (2) sequential inoculation (SQ) with kits supplied by the manufacturer (www.biosystems.pt).
T. delbrueckii followed by S. cerevisiae CT007 after 15 g/L Total acidity, pH, ethanol and density were determined
sugar consume was detected, (3) simultaneous co-inoculation following the methods in the Compendium of International
(SM) with T. delbrueckii and S. cerevisiae CT007 and (4) Methods of Analysis of Musts and Wines (OIV 2014).
inoculation with S. cerevisiae CT007 (Sc).
Cultures were adjusted in order to reach an initial cellular Analytical determinations of volatile compounds
concentration in must of about 106 cells/ml for every strain,
developing mixed cultures with an inocula ratio of 1:1. During Volatile compounds from microvinifications The concentra-
co-fermentations, aliquots were taken periodically, and further tion of volatile compounds (Tables 2 and S2), all of which
tenfold dilutions were made serially. Growth kinetics were influence wine quality, were measured at the end of alcoholic

147
Capítulo 3
fermentations by gas chromatography using an Agilent end of fermentations, both as only inoculum or as coinoculated
Technologies 6850 gas chromatograph with a flame ionisation with T. delbrueckii (Fig. 1). Figure 1a shows microbial kinetics of
detector (Hewlett Packard, Palo Alto, CA, USA) (Ortega et al. a fermentation carried out with T. delbrueckii by itself (Td), so
2001). The apparatus was calibrated with a 4-methyl-2- total viable cells counted in Sabouraud medium are relating to the
pentanol internal standard. Gas chromatography quality com- wild yeasts in the must. The sequential inoculation (SQ), in
pounds (Fluka, Sigma–Aldrich Corp., Buchs SG, which S. cerevisiae was inoculated at day 4, showed a similar
Switzerland) were used to provide standard patterns. Higher fermentation kinetic compared with Td fermentation, but with
alcohols were separated as described in the Compendium of greater homogeneity in yeast populations between replicates
International Methods of Analysis of Musts and Wines (OIV (Fig. 1b). In these fermentations, non-Saccharomyces can be
2014). The detection limit was 0.1 mg/L. Minor compounds isolated until advanced stages of the process (day 17) contrary
were quantified by gas chromatography–mass spectrometry as to what could be observed in the simultaneous inoculation of
described by Lopez et al. (2002) with the modifications intro- T. delbrueckii and S. cerevisiae (SM), where non-Saccharomyces
duced by Loscos et al. (2007). can be only observed until the day 7 (Fig. 1c). Figure 1d shows
the total cell count corresponding to the fermentation inoculated
Analysis of mannoprotein content of wines Total soluble wine only with S. cerevisiae (Sc).
polysaccharides were evaluated in duplicate by using a HPLC
apparatus (Surveyor Plus chromatograph, Thermo Fisher
Sugar consumption and ethanol production
Scientific, Waltham, MA) equipped with a refraction index
detector (Surveyor RI Plus Detector) as reported (Quirós et al.
Figure 2 shows the different fermentation kinetics of
2012). The column employed was a 300 × 7.7 mm PL Hi-Plex
microvinifications and semi-industrial fermentations by sugar
Pb 8 lm (Varian, Inc., Shropshire, UK). MilliQ water was used
consumption. In the case of the laboratory-scale assays, fermen-
as the mobile phase at a flux of 0.6 mL/min and a column
tations which were started with T. delbrueckii by itself (Td) and
temperature of 70 °C. The retention time valued was between
sequentially (SQ) with S. cerevisiae required 24 and 21 days,
0 to 30 min.
respectively, to complete fermentation, despite fermentations
with S. cerevisiae by itself (Sc), and its simultaneous (SM)
Sensorial analysis The final wines were assessed (blind) by a
inoculation with T. delbrueckii only required 14 days to finish
panel of ten experienced wine tasters, all members of the staff
(Fig. 2a). Regarding semi-industrial fermentations, all of them
of the Food Technology Department of the Polytechnic
required only 12 days to complete fermentation, despite fermen-
University of Madrid. Assessments took place in standard
tations started only with T. delbrueckii (Td and SQ) followed
sensory analysis chambers with separate booths. Following
slower kinetics at the beginning compared with fermentations
the generation of a consistent terminology by consensus, two
started with S. cerevisiae (Sc and SM) (Fig. 2b). The final alcohol
visual descriptors, five aromas and four taste attributes were
content of the wines obtained in fermentations involving
chosen to describe the wines. Formal assessment consisted of
T. delbrueckii NSA-1 was lower than those only fermented by
two sessions held on different days where wine tasters tasted
S. cerevisiae CT007 (Table 1). The semi-industrial fermentations
all fermented triplicates. The panelists used a 10 cm unstruc-
confirmed this reduction, so final alcohol degree produced in
tured scale, from 0 (no character) to 10 (very strong character),
different fermentations was gradually lower, depending on the
to rate the intensity of ten attributes.
higher T. delbrueckii presence (Supplementary material,
Table S1).
Statistical analysis All statistical analyses were performed
using PC Statgraphics v.5 software (Graphics Software
Systems, Rockville, MD, USA). The significance was set to Acetic acid and malic acid production
p<0.05 for the ANOVA matrix F value. The multiple-range
test was used to compare the means. Slight differences in acetic acid production were observed be-
tween assays (Fig. 3). Figure 3a shows the acetic acid release
kinetics in microvinifications, where SQ and SM fermentations
Results produced final acetic acid concentrations ranging from 0.29 to
0.32 g/L, similar to Sc fermentations (0.31 g/L). Similar data
Fermentation kinetics were obtained from semi-industrial fermentations in which SQ
fermentation shows the minimum acetic acid release (0.29 g/L)
Population dynamics (Fig. 3b). SM and Sc fermentations show again similar acetic
acid content (0.35 and 0.33 g/L, respectively) (Fig. 3b).
S. cerevisiae population showed the typical growth kinetic In addition, lower levels in total acidity and higher pH
where, in all cases, it maintained high cell viability until the values in Torulaspora related fermentations were detected

148
Capítulo 3
Fig. 1 Total yeast cell count (black triangle) and T. delbrueckii NSA-1 NSA-1 and S. cerevisiae CT007. c Simultaneous inoculation of
cell count (black circle) during fermentation. a Fermentation inoculated T. delbrueckii NSA-1 and S. cerevisiae CT007. d Fermentation inoculated
only with T. delbrueckii NSA-1. b Sequential inoculation of T. delbrueckii only with S. cerevisiae CT007
(Table 1). It also could be related to the higher malic acid initial malic acid content of 1.77 g/L. The final values in
consumption by T. delbrueckii. fermentations involving T. delbrueckii NSA-1 were lower
than Sc fermentation ones (Table 1), detecting a maximum
Fermentations involving T. delbrueckii NSA-1 consumed malic acid reduction rate of 13.56 % in Td fermentation and a
part of the malic acid present in the must, which showed an 4.52 % of malic acid reduction in Sc fermentation. Table 1
Fig. 2 Change in glucose fructose concentration of the studied T. delbrueckii NSA-1 S. cerevisiae (SM); fermentation with S. cerevisiae
Tempranillo-based wines during fermentation with T. delbrueckii NSA- CT007 alone (Sc). a Laboratory-scale assays; b semi-industrial-scale
1 alone (Td); sequential fermentation with T. delbrueckii NSA-1 followed assays
by S. cerevisiae CT007 (SQ); simultaneous fermentation with

149
Capítulo 3
Td T. delbrueckii NSA-1 alone, SQ sequential fermentation with T. delbrueckii NSA-1 followed by S. cerevisiae CT007, SM simultaneous fermentation with T. delbrueckii NSA-1+S. cerevisiae CT007,
Colour intensity
shows final concentrations of lactic acid; the absence of ma-

2.96±0.02b
2.77±0.02c
3.09±0.03a
3.16±0.05a
lolactic fermentation confirmed that no contamination by

lactic acid bacteria occurred.
Pyruvic acid and glycerol production

.51±0.04c
.53±0.02c
3.54±0.02c
3.56±0.01a
S. cerevisiae CT007 by itself (Sc) and SM fermentation

Assays Glucose fructose (g/l) Acetic acid (g/l) Malic acid (g/l) Lactic acid (g/l) Glycerol (g/l) Free SO2 (mg/L) Total SO2 (mg/l) Total acidity(g/l) Alcohol (%v/v) pH
showed maximum pyruvic acid production at fourth day,

r e a c h i n g 111 a n d 1 4 1 m g / L , r e s p e c t i v e l y, i n
14.53±0.02b
14.46±0.02b
14.38±0.07a
14.39±0.03a
microvinifications (Fig. 4a). Td and SQ fermentations showed

higher values with maximum figures of 156 and 143 mg/L,
respectively, at day 6. Similar values and kinetics can be
observed in the semi-industrial fermentations where
T. delbrueckii contributed to the pyruvic acid production
6.77±0.01b
6.75±0.02b
6.67±0.06a
6.62±0.08a
obtaining its maximum values times depending on the differ-

ent fermentation kinetics (Fig. 4b).
The glycerol content in Td and SQ fermentations was also
Results represent the mean SD for three replicates. Means in the same row with the same letter are not significantly different (s<0.05)
slightly higher than the one observed in Sc and SM fermenta-

77.13±2.21ab
79.31±2.33ab
4.13±2.02b
82.13±3.21a
tions in microvinifications (Table 1) and semi-industrial trials

(Supplementary material, Table S1).
Volatile compounds
24.13±1.76a
26.38±2.46a
23.42±1.21a
25.13±3.21a
Table 2 shows that fermentations involving T. delbrueckii pro-

duced lower concentrations of higher alcohols; nevertheless, all
fermentations produced these compounds in moderate quantities.
A similar effect was observed in the case of esters and fatty acids.
6.63±0.01b
6.63±0.02b
6.70±0.03a
6.71±0.02a
Some compounds such acetaldehyde, diacetyl and ethyl acetate

were detected in higher values when Torulaspora was used
Table 1 Analytical results for the wines produced by the different fermentation systems
alone. These results were confirmed in semi-industrial-scale

vinifications (Supplementary material, Table S2). No differ-
ences of terpenic compounds between assays (Supplementary
0.09±0.01a
0.09±0.01a
0.11±0.02a
0.10±0.02a
material, Table S2) were found in this trial.
Sensorial analysis
1.57±0.04b
1.61±0.02b
1.53±0.03a
1.69±0.02c
Figure 5 shows a “spider web” diagram for the average scores of

some olfactory and taste attributes. Light differences in the
perception of acidity were recorded. Colour intensity perception
was higher in those fermentations in which T. delbrueckii NSA-1
Sc fermentation with S. cerevisiae CT007 alone
took place. Fermentation with S. cerevisiae CT007 alone pro-

0.29±0.02b
0.31±0.01b
0.32±0.02b
0.37±0.02a
duced slightly stronger sensations of oxidation. None of the

wines that involved fermentation with T. delbrueckii had any
perceptible organoleptic problems; indeed, sequential and
mixed fermentations received the best scores from all tasters.
The greatest virtue attributed to SQ fermentation was the
complexity and structure of its mouthfeel properties.
.88±0.13b
2.49±0.47a
1.99±0.37a
2.03±0.22a
Mannoproteins content in semi-industrial fermentations
Final content of mannoproteins in semi-industrial scale fer-

SM
SQ
sss
Td
mentation in tanks containing 80 kg of crushed grapes were

150
Capítulo 3
Fig. 3 Change in acetic acid concentration of the studied Tempranillo- S. cerevisiae CT007 (SQ); simultaneous fermentation with T. delbrueckii
based wines during fermentation with T. delbrueckii NSA-1 alone (Td); NSA-1+S. cerevisiae (SM); fermentation with S. cerevisiae CT007 alone
sequential fermentation with T. delbrueckii NSA-1 followed by (Sc). a Laboratory-scale assays; b semi-industrial scale assays
analysed. In Fig. 6, the increase of mannoproteins can be seen both, S. cerevisiae and non-Saccharomyces strains, limited
in the fermentations in which T. delbrueckii acts for longer, the presence of non-Saccharomyces to the early stages of
especially in controlled sequential fermentation. fermentation (Azzolini et al. 2012; Oro et al. 2014). In this
work, the studied yeast strains were observed to present killer
phenotype and were active against the sensitive strain used as
Discussion control (S. cerevisiae BY4741). However, there was no cross-
activity between them, so killer activity was not considered as
Similar results in fermentation kinetics and population dynam- a relevant feature in the growth kinetics of both strains.
ics (Fig. 1) can be seen in recent analogous studies using non- The sugar consumption results showed in this work (Fig. 2)
Saccharomyces yeasts, where simultaneous inoculation of agree with the lower fermentative power of Torulaspora spp.
Fig. 4 Change in pyruvic acid concentration of the studied Tempranillo- S. cerevisiae CT007 (SQ); simultaneous fermentation with T. delbrueckii
based wines during fermentation with T. delbrueckii NSA-1 alone (Td); NSA-1+S. cerevisiae (SM); fermentation with S. cerevisiae CT007 alone
sequential fermentation with T. delbrueckii NSA-1 followed by (Sc). a Laboratory-scale assays; b semi-industrial scale assays

151
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Table 2 Volatile compounds (micrograms per liter) detected in the different fermentations
Compounds Sc SM SQ Td
1-Hexanol 1,835.33±80.03b 1,983.00±45.90a 1,557.00±72.63c 1,859.00±77.32ab

3-Hexanol 684.67±7.02a 680.67±21.94a 457.67±17.95c 564.33±23.59b
Isoamylalcohol 391,316.33±13,882.09a 403,590.33±4,815.73a 334,420.00±9,634.85b 386,564.67±9,624.89a
Isobutanol 55,502.00±1,213.69b 60,904.33±1,608.61a 54,783.00±1,371.60b 61,803.33±1,533.15a
Alcohols 449,337.99±12,961.9b 467,158.33±4,523.64a 391,217.67±8,922.84c 450,791.33±8,930.99b
Acetaldehyde 332.27±631.43a 7,332.27±631.43a 7,254.34±672.34a 11,342.13±792.23b
Diacetyl 1,243.45±65.13a 1,425.32±88.43b 1,223±77.34a 1,634.43±104.47c
Carbonyl compounds 8,575±584.41b 9,846±687.29b 8,477.34±622.67c 1,2976±741.61a
Ethyl acetate 21,365.23±1,365.45b 23,456.34±1,567.76ab 21,897.65±1,645.54b 5,764.26±1,876.54a
Ethyl butyrate 157.33±4.73b 182.00±5.29a 144.67±7.02c 181.67±6.51a
Ethyl decanoate 86.33±4.52a 95.00±6.00a 74.33±4.06b 80.00±8.73ab
Ethyl hexanoate 613.67±17.16a 652.33±16.56a 339.67±20.60c 462.33±23.29b
Ethyl lactate 3,700.33±121.38a 3,711.33±104.31a 2,832.67±120.02c 2,832.67±120.02c
Ethyl octanoate 372.00±33.15a 425.67±46.74a 297.00±12.53b 377.67±36.74a
Isoamyl acetate 1,381.67±43.25a 1,073.33±47.48b 974.00±12.53c 1,136.00±79.54d
Esters 2,7676.23±1,200.65bc 29,596±1,396.32b 26,559.99±1,494.77c 31,156.93±1,706.91a
hexanoic acid 3,614.33±140.47a 3,521.33±151.64a 3,154.00±157.29b 3,383.33±161.98ab
Isobutyric acid 3,614.33±140.47a 3,521.33±151.64a 3,154.00±157.29b 3,383.33±161.98ab
isovaleric acid 385.67±7.51a 368.00±4.58b 331.33±13.61c 363.67±6.66b
octanoic acid 9,919.33±74.59a 10,125.67±145.29a 6,703.00±220.96c 8,544.00±224.01b
valeric acid 598.67±12.22a 584.33±20.40a 531.33±25.20b 567.67±11.72ab
2-Phenylethanol 54,308.67±1,151.98a 53,194.00±2,022.95ab 52,531.67±1,170.21ab 51,485.33±719.18b
2-phenylethyl acetate 8.00±1.00a 7.67±2.31a 7.67±1.53a 7.00±1.00a
Phenols 8.00±1.00a 53,201.67±2,022.82ab 52,539.34±1,170.13ab 51,492.33±719.13b
Results represent the mean SD for three replicates. Means in the same row with the same letter are not significantly different (s<0.05)
T. delbrueckii NSA-1 alone (Td); Sequential fermentation with T. delbrueckii NSA-1 followed by S. cerevisiae CT007 (SQ); Simultaneous fermentation
with T. delbrueckii NSA-1+S. cerevisiae CT007 (SM); fermentation with S. cerevisiae CT007 alone (Sc)
compared with S. cerevisiae reported by other authors (Bisson 2012). The slower kinetics of T. delbrueckii fermentations
and Kunkee 1991; Jolly et al. 2006), due to the fact that, in the (Fig. 2a) was attributed to a high nutrient demand by these
last stages, only Saccharomyces was detected (Azzolini et al. species that limited the later S. cerevisiae fermentation activity
(Romano et al. 2003). Data obtained for fermentation kinetics
in semi-industrial fermentations confirm this trend. The fact
Fig. 6 Mannoprotein content (milligrams per liter of mannose) of wines

fermented at semi-industrial scale with: S. cerevisiae CT007 (Sc);
T. delbrueckii NSA-1 and S. cerevisiae CT007 by using simultaneous
inoculation (SM); sequential inoculation (SQ); and T. delbrueckii NSA-1
Fig. 5 Taste and olfactory attribute scores for the final wines alone (Td)

152
Capítulo 3
that the semi-industrial fermentation kinetics was faster than of wine colour (Benito et al. 2011, 2014c; Morata et al. 2012).
microvinifications (Fig. 2) can be explained because of the Final OD values in colour intensity of 3.16, 3.09, 2.96 and
different composition of the must. In microvinifications, the 2.77 were returned for Td, SM, SQ and Sc fermentations,
fermentative media contained only fresh must and must with respectively (Table 1). The formation of highly stable pig-
crushed grapes in semi-industrial fermentations, which con- ments such as vitisin A, due to the higher pyruvic acid forma-
tribute to nutrient enrichment of musts. tion, could explain these chromatic differences between
Several authors argue the usefulness of non- wines. Also, colour material absorption could be different
Saccharomyces yeast in the production of lower concentra- between species and strains (Morata et al. 2005).
tions of alcohol in wines (Contreras et al. 2014; Kutyna et al. Furthermore, higher total sulphur dioxide levels (Table 1) in
2010), reporting reductions higher than 1 % in final alcohol fermentations involving T. delbrueckii could also be explained
content. These previous results agree with the lower final by higher combinations of anthocyanins with pyruvic acid
alcohol content of the wines produced in fermentations in- during fermentation (Morata et al. 2003).
volving T. delbrueckii NSA-1 in this work (Table 1); however, The increase of glycerol content in wines is one of the most
in this assay, the ethanol reduction was lower than 0.2 %. recognised contributions of non-Saccharomyces species to the
Sugar consumption could also be used to produce alternative quality of wines (Jolly et al. 2006). However, some authors
compounds to ethanol, such as glycerol or pyruvic acid, or to reported that an increase in glycerol production is usually
increase the yeast biomass by T. delbrueckii due to its reported linked with a rise in acetic acid production (Prior et al.
lower Crabtree effect (Bely et al. 2008; Merico et al. 2007). 2000), which can be detrimental to wine quality. This fact
Higher acetic acid values from Td fermentations (Table 1) was observed in Candida stellata strains that can produce
than the others, both in microvinifications and in semi- elevated concentrations of glycerol (10 to 14 g/L) compared
industrial fermentations, can be attributed to the wild with S. cerevisiae (4 to 10 g/L); on the contrary, our results
Saccharomyces yeasts that finish the fermentation, observing confirm that these facts seem to be irrelevant in the case of
the increase in the release of acetic acid at the beginning of T. delbrueckii.
tumultuous fermentation (Fig. 3) and confirming the useful- The values observed in pyruvic acid and glycerol produc-
ness of selected strains to reduce the acetic acid content in tion could indicate that T. delbrueckii possesses a highly active
wines. One of the questions raised by winemakers is the glyceropyruvic pathway (Ciani and Maccarelli 1998; Renault
excessive increase of acetic acid in wines with high presence et al. 2009). Besides, some authors have been reported that
of non-Saccharomyces yeasts (Jolly et al. 2014). Our results there is a big difference in glycerol production depending on
show that using T. delbrueckii in mixed fermentations does strain level (Loira et al. 2012).
not cause an increase of acetic acid (Table 1), according to the The detected lower production of higher alcohols by
results reported in similar studies (Azzolini et al. 2012). Other T. delbrueckii could have increased the varietal Tempranillo
authors also described T. delbrueckii as a low acetic acid aroma perception. Different non-Saccharomyces yeasts pro-
producer compared with most non-Saccharomyces yeasts duce different levels of higher alcohols (Lambrechts and
(Bely et al. 2008; Moreno et al. 1991; Renault et al. 2009). Pretorius 2000; Romano et al. 1992). This can be important
The higher decrease in malic acid content observed in the because a large concentration of higher alcohols can generally
trials involving T. delbrueckii (Table 1) agrees with the report- not be desired, whereas lower values can contribute to wine
ed by other authors who confirm that malic acid can be complexity (Romano and Suzzi 1993). Non-Saccharomyces
metabolised by several yeast species (Benito et al. 2013, yeasts often form lower levels of these alcohols than
2014a, 2014b; Suárez-Lepe et al. 2012; Su et al. 2014) in S. cerevisiae, but there is great strain variability (Romano
levels lower than 20 %, unless Schizosaccharomyces species et al. 1992; Zironi et al. 1993). The higher values detected in
are used. acetaldehyde, diacetyl and ethyl ethanol when Torulaspora
Previous pyruvic acid-based selection studies on was used alone could be attributed to wild high fermentative
S. cerevisiae strains returned maximum values of 60– non-selected yeasts involved in a spontaneous process. Other
132 mg/L after 4 days of fermentation (Morata 2004), values authors have reported a higher production of terpenic com-
below those obtained in the present study with T. delbrueckii pounds by T. delbrueckii in Muscat variety (King and Dickson
fermentations (Fig. 4a). A strong correlation has been reported 2000). In contrast, our results show no differences between
between the amount of pyruvic acid released into the medium assays in this kind of compounds (Supplementary material,
and the formation of vitisin A (Morata et al. 2003, 2012) Table S2). However, this ability is attributed to specific strains
which is also related to colour stability. Other authors have and T. delbrueckii NSA-1 does not possess the terpenic-
reported before a high production of other acid in yeast related enzymatic properties that were analysed (β-glucosi-
metabolism by T. delbrueckii such as succinic acid (Ciani dase, β-D-xylosidase and α-L-arabinofuranosidase).
and Maccarelli 1998). Different non-Saccharomyces yeasts The recorded differences in acidity perception could be
have been found to have influence in intensity and stability related to the small malic acid consumption detected in

153
Capítulo 3
fermentations in which T. delbrueckii NSA-1 was involved aromatic properties already mentioned, because of the in-
and to the lower total acidity levels obtained in these wines crease in mannoprotein content of these wines. The use of
(Table 1). Differences in colour intensity perception could be non-Saccharomyces yeast in winemaking implies its adapta-
partially explained because of the higher pyruvic acid content tion to a cellar environment, so semi-industrial and industrial
detected and its influence in high stable colour forms (Benito assays should be considered as important and ought to be
et al. 2011). Other authors described that wines fermented by included in scientific reports.
coinoculation with T. delbrueckii and S. cerevisiae are better
than the regular S. cerevisiae control for the varieties
Acknowledgements Funding for the research in this paper was provid-
Sauvignon Blanc, Chenin Blanc and Amarone (Azzolini ed by Agrovin S.A, under the framework of the project IDI20130192-
et al. 2012; Jolly et al. 2003). In this work, a similar effect ENZIOXIVIN (Centre for Industrial Technological Development-CDTI,
was found for the Tempranillo variety. The tasters perceived Spain).
higher aroma quality in the specific cases of SQ and SM
fermentations, but no strong difference in aroma compounds
was observed. This could be explained due to a lower higher-
alcohol content which generally overlays other minor com- References
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Supplementary material - Applied Microbiology and Biotechnology
Dynamic analysis of physiological properties of Torulaspora delbrueckii in wine

fermentations and its incidence on wine quality
Ignacio Beldaa, Eva Navascuésa,b, Domingo Marquinaa, Antonio Santosa, Fernando

Calderónc and Santiago Benitoc*
a
28040 Madrid, Spain
b
c
Departament of Food Technology. Escuela Técnica Superior de Ingenieros
Agrónomos, Polytechnic University of Madrid, Ciudad Universitaria S/N, 28040
Madrid, Spain
*Corresponding author. Santiago Benito Sáez

Tel.: +34913363710
E-mail address: santiago.benito@upm.es

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Table S1. Analytical results for the wines produced by the different fermentation systems on a semiindustrial scale
Glucose + Fructose Acetic Malic Lactic Glycerol Total Acidity Alcohol pH
Assays
(g/l) Acid (g/l) Acid (g/l) Acid (g/l) (g/l) (g/l) (% v/v)
Td 0.83 0.43 1.71 0.11 5.78 6.55 14.34 3.51
SQ 1.39 0.29 1.93 0.13 5.77 7.27 14.38 3.48
SM 1.33 0.35 1.73 0.09 5.59 6.67 14.44 3.54
Sc 0.57 0.33 1.80 0.09 5.44 6.75 14.48 3.53
Results represent the value for a single semiindustrial sample

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Table S2. Volatile compounds (µg/l) detected in the different fermentations on a semiindustrial scale
Compounds Sc SM SQ Td
1-Hexanol 1750 2020 1690 1980

3-Hexanol 330 340 250 290
Isoamylalcohol 322470 363570 323620 386560
Isobutanol 55280 60040 59050 69020
Alcohols 379830 425970 384610 457850
Acetaldehyde 11940 16460 10080 18850

Diacetyl 1080 2970 910 4310
Carbonyl compounds 13020 19430 10990 23160
Ethyl acetate 32900 33070 32190 36030

Ethyl butyrate 140 170 150 170
Ethyl decanoate 70 110 70 100
Ethyl hexanoate 190 620 260 430
Ethyl lactate 3650 2740 3220 2790
Ethyl octanoate 310 410 290 350
Isoamyl acetate 1160 1170 1090 1200
Esters 38420 38290 37270 41070
hexanoic acid 990 2480 1170 2150

Isobutyric acid 1860 1580 1880 2260
isovaleric acid 1740 1760 1620 2430
octanoic acid 950 2200 1210 1790
valeric acid
Fatty acids 5540 8020 5880 8630
2-Phenylethanol 85740 67460 79710 57100

2-phenylethyl acetate N.D N.D. 10 N.D.
Phenols 85740 67460 79720 57100
Linalool (p.p.b) 19.68 18.27 17.89 18.81

linalool acetate (p.p.b) 1.81 1.74 2.06 2.28
α-Terpineol (p.p.b) 1.45 1.26 1.24 1.28

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β-Citronellol (p.p.b) 10.09 12.46 10.34 10.01

Geraniol (p.p.b) 5.86 4.17 4.71 4.6
Terpenes (p.p.b) 38.89 37.9 36.24 36.98
Results represent the value for a single semiindustrial sample

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ANEXO

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162
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Levaduras Ficha técnica
PROPIEDADES ENOLÓGICAS PROPIEDADES MICROBIOLÓGICAS Y FISICOQUÍMICAS

� Poder alcoholígeno 9,5 %vol. Requiere inoculación posterior de Recuento de levaduras (Torulaspora delbrueckii.) [UFC/g] > 1010
cepa de Saccharomyces cerevisiae.
Otras levaduras [UFC/g] < 105
� Temperatura de trabajo 17 oC - 28 oC
� Necesidades de NFA medias. Mohos [UFC/g] < 103
� Producción de sulfhídrico ausente. Bacterias lácticas [UFC/g] < 105
� Producción de acidez volátil, muy baja. Bacterias acéticas [UFC/g] < 104
� Producción de compuestos carbonílicos (acetaldehído, acetoína),
muy baja. Salmonella [UFC/25 g] Ausencia
� Cinética fermentativa, media. E. coli [UFC/g] Ausencia
� Resistencia al sulfuroso, baja. Staphylococcus aureus [UFC/g] Ausencia
� El empleo de fermentaciones secuenciales No Saccharomyces +
Saccharomyces cerevisiae, permite la obtención de vinos con Coliformes totales [UFC/g] < 102
menor graduación alcohólica. Humedad [%] <8
Viniferm NSTD ha sido galardonada con el Premio a la Innovación
Enomaq 2015.
Pb [mg/kg] <2
Hg [mg/kg] <1
DOSIS
As [mg/kg] <3
Vinificación 20-30 g/HL Cd [mg/kg] <1
MODO DE EMPLEO
ASPECTO FÍSICO
Para obtener los mejores resultados es indispensable asegurar la
buena implantación de la cepa en el medio, por lo tanto es importante: Gránulos de color tostado, desprovistos de polvo.
� Mantener una buena higiene en la bodega.
� Añadir la levadura lo antes posible.
� Respetar la dosis prescrita. PRESENTACIÓN
� Rehidratar bien la levadura.
Paquetes de 500 g envasados al vacío en envuelta multilaminar de
Rehidratación:
aluminio en cajas de 10 kg.
1.- Añadir las levaduras secas en 10 veces su peso en agua a
35 oC - 40 oC (10 litros de agua por 1 kg de levadura).
CONSERVACIÓN
2.- Esperar 10 minutos.
3.- Agitar la mezcla. El producto conforme a los estándares cualitativos se conserva en su
envase sellado al vacío durante un periodo de cuatro años en
4.- Esperar 10 minutos e incorporar al mosto, procurando que
cámara refrigerada entre 4 oC y 10 oC.
no haya una diferencia de más de 10 oC entre el medio
rehidratado y el mosto. Eventuales exposiciones prolongadas a temperaturas superiores a
Precauciones de trabajo: 35 oC y/o con humedad reducen su eficacia.
- En cualquier caso, la levadura no deberá estar rehidratándose
más de 30 minutos en ausencia de azúcares.
- El respeto del tiempo, temperatura y modo de empleo descrito
garantizan la máxima viabilidad de la levadura hidratada.
- La siembra secuencial de levadura Saccharomyces debe
realizarse cuando se evidencie una bajada de la densidad
Registro:
(48-72 horas en función de la temperatura). No se recomienda R.G.S.A: 31.00391/CR
la siembra simultanea de ambas cepas. Producto conforme con el Codex Enológico
Internacional y el Reglamento CE 606/2009.
- Protocolo de trabajo: ver ficha adjunta. VINIFERM NSTD EP 871 / Rev.: 1 / Fecha: 14/09/15
2/2

164

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6. CAPÍTULO 4

165

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6.1. Estudio de la incidencia en la calidad de vinos tintos de la crianza sobre

lías de levaduras no convencionales

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Outlining the influence of non-conventional yeasts in wine ageing over-lees
Artículo aceptado para su publicación en la revista “Yeast”
(doi: 10.1002/yea.3165)

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Outlining the influence of non-conventional yeasts in wine ageing over-lees
Ignacio Beldaa, Eva Navascuésa,b,c, Domingo Marquinaa, Antonio Santosa, Fernando
Calderónc and Santiago Benitoc*
a
28040 Madrid, Spain
b
c
Department of Chemistry and Food Technology. Polytechnic University of Madrid, Ciudad
Universitaria S/N, 28040 Madrid, Spain
*Corresponding author. Santiago Benito Sáez
Tel.: +34 913363984
E-mail address: santiago.benito@upm.es
Abstract
During the last decade, the use of innovative yeast cultures of both Saccharomyces cerevisiae
and non-Saccharomyces yeasts as alternative tools to manage the winemaking process have
turned the oenology industry. Although the contribution of different yeast species to wine
quality during fermentation is increasingly understood, the information about their role in
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/yea.3165
This article is protected by copyright. All rights reserved.

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Capítulo 4
wine ageing over-lees is really scarce. This work aims to analyse the incidence of 3 non-
Saccharomyces yeast species of oenological interest (Torulaspora delbrueckii, Lachancea
thermotolerans and Metschnikowia pulcherrima) and of a commercial mannoprotein-
overproducer S. cerevisiae strain compared with a conventional industrial yeast strain during
wine ageing over-lees. To evaluate their incidence in mouthfeel properties of wine after 4
months of ageing, mannoprotein content of wines was evaluated, together with other wine
analytic parameters such as colour and aroma, biogenic amines and amino acids profile.
Some differences among the studied parameters were observed during the study, especially
regarding the mannoprotein concentration of wines. Our results suggest that the use of T.
delbrueckii lees in wine ageing is a useful tool for the improvement of overall wine quality by
notably increasing mannoproteins, reaching values higher than obtained using a S. cerevisiae
overproducer strain.
Keywords: Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia
pulcherrima, ageing over-lees, mannoprotein, amino acids.
Introduction
The incidence of yeasts in winemaking not only works during the alcoholic fermentation
process, but also in both pre- and postfermentative stages. In an industrial context that
demands products with increasingly high quality and safety, the development of new
techniques to improve wine attributes and to avoid a global-market wine homogeneity is
advisable (Moreno-Arribas and Polo, 2005).

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The technique of ageing over-lees is gaining importance in the production of red wine
because of its impact on wine mouthfeel properties. Mannoproteins have been recognized to
have many positive sensorial attributes such as improving wine mouthfeel and roundness
(Vidal et al., 2004), increasing aromatic persistence (Chalier et al., 2007), and decreasing
astringency (Saucier et al., 2002). Additionally, some technological advantages have been
described for mannoproteins in wines, by reducing protein and tartrate instability (González-
Ramos et al., 2008) and also by removing (by absorption) ochratoxin A from wine (Ringot et
al., 2005). On the other hand, the use of wine ageing over-lees can also involve certain risks
such as the formation of biogenic amines or the release of their amino acid precursors
( - and Polo, 2000).
Keeping in contact the wine with the resting dead yeast cells after the alcoholic fermentation
may cause the release of the polysaccharide fraction from the yeast cell wall due to the
autolysis process and to the action of the yeast-derived e ym s β-glucanase and cell wall
mannosidase (Charpentier and Freyssinet, 1989). Nowadays, the influence of different
Saccharomyces cerevisiae strains in wine ageing over-lees is known (Loira et al., 2013). It
also has been reported a strain-dependent behaviour regarding to the mannoprotein release
rate during alcoholic fermentation, nevertheless this performance is not directly correlated
with mannoprotein release during ageing over-lees (del Barrio-Galán et al., 2015).
The yeast cell-wall composition is variable between species and the polysaccharide
composition of some relevant wine yeast species have already been described (Domizio et al.,
2014). There is an increasing interest in non-Saccharomyces yeasts to improve wine quality
(Jolly et al., 2006, 2014) and it should be mentioned that certain yeast species, such as
Torulaspora delbrueckii, have been reported as adequate to increase mannoprotein content of
wines during wine fermentation (Belda et al., 2015; Domizio et al., 2014). However, the
information about their influence during wine ageing over-lees is really scarce. The influence

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of certain non-Saccharomyces yeasts such as Schizosaccharomyces pombe, Saccharomycodes
ludwigii, Wickerhamomyces anomalus and Pichia mambranifaciens has been evaluated for
over-lees ageing (Palomero et al., 2009). Other species such as T. delbrueckii (Azzolini et al.,
2015; Belda et al., 2015; Renault et al., 2015), Lachancea thermotolerans (Benito et al.,
2015a; Benito et al., 2016; Comitini et al., 2011; Gobbi et al., 2013) and Metschnikowia
pulcherrima (Belda et al., 2016; Benito et al., 2015b; Contreras et al., 2014; Jolly et al.,
2003) have been proved to improve wine quality during alcoholic fermentation. However,
they have not been studied in ageing over-lees yet.
This work aims to study the incidence of some of the currently most used non-
Saccharomyces yeasts in wine industry (T. delbrueckii, L. thermotolerans and M.
pulcherrima) and also of a mannoprotein-overproducer S. cerevisiae commercial strain in
wine composition during red wine ageing over-lees.
Materials and Methods
Yeasts used in experimental over-lees ageing
Five different yeast strains, pertaining to 4 different species, were used: Saccharomyces
cerevisiae CT007 (Agrovin S.A., Ciudad Real, Spain) which was used as a control; S.
cerevisiae 3D (Agrovin S.A.), as a mannoprotein overproducing industrial strain.
Torulaspora delbrueckii NS-TD (Agrovin S.A.; also referred in past literature as T.
delbrueckii NSA-1 from Complutense Yeast Collection-CYC, Madrid, Spain) (
KM434245), Lachancea thermotolerans NS-G-32 (CYC) (
and Metschnikowia pulcherrima NS-EM-34 (CYC) (
as non-Saccharomyces yeasts.

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Capítulo 4
The yeast biomass used in the over-lees ageing assay was obtained according to Palomero et
al. (2009) with some modifications. Briefly, yeasts were grown using an enriched must
medium (12.5 % concentrated must [final concentration, 50 g/L glucose+fructose], 1 % yeast
extract, 0.5 % proteose peptone no.3, pH 3.5) at 25 °C with orbital agitation (100 rpm)
(Orbital shaker Infors AG, Bottminger CH-4103, Switzerland) for 48 hours. After that, the
yeast biomass was washed with 10:1 volumes of sterile distilled water, centrifuged at 3000
rpm for 2 min, and the supernatant discarded. This procedure was repeated twice to provide
yeast biomass with no remains of nutrients. Finally, these yeasts were lyophilised using a
Cryodos apparatus (Telstar, Spain) and added, under sterile conditions (laminar flow cabinet
Telstar PV-100, Spain), to wines at a concentration of 0.8 g/L.
Wine ageing over-lees assays were performed, by triplicate, in 1L crystal bottles (Fisherbrand
FB-800-1000, UK) with its proper hermetic seal (Fisherbrand GL45, UK) filling up the entire
bottle to avoid any oxidation problems during ageing. Wine ageing was carried out for 4
months, at a constant temperature of 16ºC, using a young commercial wine (var.
Tempranillo; Bodegas Urbina S.L., Spain) from La Rioja wine appellation.
Analytical determinations of non-volatile compounds
Color Intensity (CI), Total Polyphenol Index (TPI), Anthocyanin Content (AC), urea,
acetaldehyde, acetic acid, lactic acid, malic acid, glucose/fructose citric acid and glycerol
were measured using a Y15 enzymatic autoanalyzer (Biosystems S.A, Barcelona, Spain) and
their corresponding kits (http://www.biosystems.es/products/). Ethanol and pH were
determined following the methods in the Compendium of International Methods of Analysis
of Musts and Wines (http://www.oiv.int/oiv/info/enmethodesinternationalesvin).

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Analysis of mannoprotein content of wines
Mannoprotein concentration of wines were evaluated in duplicate by using a HPLC apparatus
(Surveyor Plus chromatograph, Thermo Fisher Scientific, Waltham, MA) equipped with a
refractive index detector (Surveyor RI Plus Detector) as reported (Quirós et al., 2012). The
column employed was a 300 x 7.7 mm PL Hi-Pl x Pb 8 μm (Varian, Inc., Shropshire, UK).
MilliQ water was used as the mobile phase at a flux of 0.6 ml/min and a column temperature
of 70ºC. The retention time valued was between 0 and 30 minutes.
Analytical determinations of amino acids
Amino acids were analyzed using a Jasco (Tokyo, Japan) UHPLC chromatograph series X-
LCTM, equipped with a fluorescence detector 3120-FP. Gradients of solvent A
(methanol/acetonitrile, 50:50, v/v) and B (sodium acetate /tetrahydrofuran, 99:1, v/v) were
used in a C18 (HALO, USA) column (100 mm × 2.1 mm; particle size 2.7 µm) as follows:
90 % B (0.25 mL/min) from 0 to 6 min, 90–78 % B linear (0.2 mL/min) from 6 to 7.5 min,
78 % B from 7.5 to 8 min, 78–74 % B linear (0.2 mL/min) from 8 to 8.5 min, 74 % B (0.2
mL/min) from 8.5 to 11 min,74–50 % B linear (0.2 mL/min) from 11 to 15 min, 50 % B (0.2
mL/min) from 15 to 17 min, 50–20 % B linear (0.2 mL/min) from 17 to 21 min, 20–90 % B
linear (0.2 mL/min) from 21 to 25 min and re-equilibration of the column from 25 to 26 min.
Detection was performed by scanning in the 340–455 nm range. Quantification was
performed by comparison against external standards of the studied amino acids. The different
amino acids were identified by their retention times.
Analytical determinations of biogenic amines
Biogenic amines were analyzed using a Jasco (Tokyo, Japan) UHPLC chromatograph series
X-LCTM, equipped with a fluorescence detector 3120-FP. Gradients of solvent A

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(methanol/acetonitrile, 50:50, v/v) and B (sodium acetate /tetrahydrofuran, 99:1, v/v) were
used in a C18 (HALO, USA) column (100 mm × 2.1 mm; particle size 2.7 µm) as follows:
60% B (0.25 ml/min) from 0 to 5 min, 60–50% B linear (0.25 ml/min) from 5 to 8 min, 50%
B from 8 to 9 min, 50-20% B linear (0.2 ml/min) from 9 to 12 min, 20% B (0.2 ml/min) from
12 to 13 min, 20–60% B linear (0.2 ml/min) from 13 to 14.5 min, and re-equilibration of the
column from 14.5 to 17 min. Detection was performed by scanning in the 340–420 nm range.
Quantification was performed by comparison against external standards of the studied amines.
The different amines were identified by their retention times.
Analytical determination of volatile compounds
Volatile compounds were quantified by headspace gas chromatography–mass spectrometry
(HS-GC-MS). Analyses were carried out using a Perkin-Elmer Clarus 500 gas chromatograph
with a flame ionization detector, coupled to a mass spectrometer single quadrupole Clarus
560 S, all coupled to an automatic headspace sampler Turbomatrix 110 Trap (Perkin-Elmer,
Massachusetts, USA). The headspace sampler conditions were: temperature of thermostating:
80ºC; time of thermostating: 45 min; type of trap: Tenax TA; cycles of purge and trap: 4;
temperature of trap capture: 45ºC; desorption temperature of the trap: 290ºC; time of dry trap
purge: 10 min; desorption time of trap: 2 min; trap cleaning time: 5 min; needle temperature:
110ºC; temperature of HS-GC transfer line: 150ºC; vial pressure: 30 psi; and constant
pressure column: 28 psi. A Free Fatty Acid Phase (FFAP) capillary column (60 m × 0.25 mm
DI x 0.25 μm film hick ss) w s s . H li m (Ai Liq i , Spain) was used as carrier gas.
Gradient analysis was run using the following temperature program: 40ºC (3 min); 40–80ºC
(2ºC/min); 80-180ºC (3ºC/min); and 210ºC (5 min). Identification of individual compounds
was based on a comparison of the obtained mass spectra of the individual chromatographic
peaks with those valid for the standards and available from the National Institute of Standards
and Technology (Gaithersburg, MD) software library. We also compared the retention times

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Capítulo 4
valid for individual peaks from the wine samples with those of the known volatile
components used as standard patterns. To this effect, we used Gas chromatography quality
compounds as the sets of the volatile standards (Fluka, Sigma–Aldrich Corp., Buchs SG,
Switzerland).
Sensory analysis
The final wines were assessed (blind test) by a panel of 15 experienced wine tasters; all staff
members of the Chemistry and Food Technology Department of Polytechnic University of
Madrid and the Department of Microbiology of the Biology Faculty of the Complutense
University of Madrid. Following the generation of a consistent terminology by consensus,
two aromas and nine taste attributes were chosen to describe the wines. The panellists used an
8-cm unstructured scale, from 0 (no character) to 8 (very strong character), to rate the
intensity of the 11 attributes.
Statistical analysis
All statistical analyses were performed using PC Statgraphics v. 5 software (Graphics
Software Systems, Rockville, MD, USA). The significance was set to p < 0.05 for the
ANOVA matrix F value. The multiple range test was used to compare the means.
Results and Discussion
General chemical analyses
No statistical differences were observed among most of the different studied basic chemical
parameters (Table 1). These results could be related with no deviations during the studied

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ageing over-lees period. Nevertheless, differences in other parameters are explained below as
consequence of the ageing over-lees process.
Mannoproteins
One of the main reasons to use yeast lees for wine ageing is to improve its mouthfeel
properties by releasing most of the mannoproteins contained in the yeast cell wall. Figure 1
shows that the use of different S. cerevisiae strains lees could increase, in a significant way,
the mannoprotein content of wines after a short ageing period. However, several differences
in mannoprotein content of about 240 mg/L were observed between CT007 and 3D assays, as
expected taking into account that S. cerevisiae 3D is a commercial mannoprotein
overproducer strain. Our results show that the use of T. delbrueckii lees was able to greatly
increase the mannoprotein concentration after four months of wine ageing, reaching
significant higher values of about three times when compared with the conventional S.
cerevisiae CT007 strain and being also slightly higher (7,9%) than S. cerevisiae 3D strain,
but without establishing significant statistical differences in this last case. In the same line, M.
pulcherrima showed a significant increase in the final mannoprotein content of wines, being
remarkably higher than CT007 and Lt levels but lower than the concentrations obtained using
T. delbrueckii lees. These results are in agreement with the results of Domizio et al. (2014)
reporting the higher mannoprotein release of both M. pulcherrima and T. delbrueckii, during
alcoholic fermentation, when compared with S. cerevisiae or other non-Saccharomyces such
as L. thermotolerans. Here we confirm, for the first time, the usefulness of these yeast species,
not only during the alcoholic fermentation, but also during wine ageing over-lees by releasing
their mannoproteins. The ability of T. delbrueckii NS-TD to release significant amounts of
mannoproteins during the alcoholic fermentations has been described (Belda et al., 2015) and,
on the other hand, the contrary has been observed using M. pulcherrima NS-EM-34 (Belda et
al., unpublished). However, other authors have reported different results of high

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Capítulo 4
mannoprotein release levels using other M. pulcherrima strain (Domizio et al., 2014). In this
sense, del Barrio-Galán et al. (2015), showed a strain-dependent behaviour in mannoprotein
release for their S. cerevisiae strains used during both alcoholic fermentation and wine ageing
over-lees period. Commercial information of S. cerevisiae 3D strain
(http://www.agrovin.com/agrv/pdf/enologia/levaduras/en/Viniferm_3D_en.pdf) shows that
using this strain during the alcoholic fermentation causes an increase of about 30% compared
with a conventional S. cerevisiae strain. Our results show that the use of their lees could
increase this difference, making it more interesting for winemakers to improve wine
mouthfeel properties. In summary, the use of T. delbrueckii seems to be a useful tool to
increase mannoprotein concentration in wines in both fermentation process and, as we show
here, during wine ageing over-lees.
Colour characteristics
Colour Intensity (CI), Total Polyphenol Index (TPI) and Anthocyanin Content (AC) of wines
were evaluated, since they have been described as influenced by ageing process over-lees
(Loira et al., 2013; Palomero et al., 2009). Table 2 shows different final values of CI, with
certain statistical differences depending on the assay. Wine ageing has been related with both
colour stabilization and colour loss depending on the physicochemical environment of the
ageing process and also the characteristics of yeast cells, such as their adsorption capacity
depending on their porosity, in the case of over-lees ageing processes (Gómez-Cordovés and
Gonzalez-San José, 1995; Morata et al., 2003). Our results showed, in all cases, a CI loss
with slight differences among treatments after 4 months of ageing. Trials Td and Lt showed
higher values in colour measurements at 520 nm than CT007 trial, up to 0.4 absorbance units,
keeping better their red colour. The role of mannoproteins in the stabilization of colour
properties of wines has been previously reported (Feuillat et al., 2001; Fuster and Escot,
2002; Saucier et al., 2002) by preventing the precipitation of anthocyanins and tannins (Escot

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et al., 2001; Francois et al., 2007) and reducing the oxidation process on polyphenols
(Salmon, 2005), but other authors reported no improvements in wine colour properties when
ageing over-lees was developed (d l i - l et al., 2015; Loira et al., 2013; Rodrigues
et al., 2012). Our results are partially in agreement with both data, since Td showed both the
lowest CI and TPI value decreases and also the highest mannoprotein release values and Mp
showed lower TPI decreases and also higher mannoprotein values when compared with the
other ageing over-lees assays. However, other factors, such us cell wall adsorption, could be
also contributing to the final colour properties of wines, since Lt showed the lowest
enhancement in mannoprotein but final CI values similar to Td assay.
Degradation of pigments present in wine or their development into other compounds, which,
in both cases, can lead to lower colour indices, have been described before (Palomero et al.,
2009). Furthermore, other authors have reported before adsorption of anthocyanins
phenomena related to yeast structure (Morata et al., 2003). In this case, a higher decrease in
AC using M. pulcherrima lees has been observed when compared with the other assays that
performed ageing over-lees, but no significant differences could be established among the
other assays.
Amino acids
Higher levels in amino acids were reported for the treatments that performed ageing over-lees.
Although this increase was observed in all the trials, due to the cell lysis process, the
differences obtained in their amino acid profile could be related with the different amino
acidic content of the different yeast strain used (Vaughan-Martini et al., 1979). Table 3 shows
statistical differences among the different amino acids concentration at levels of some units in
mg/l, except for the case of alanine and asparagine, where differences up to tens in mg/l were
observed. Mp assay reached higher significant levels than all the other treatments in histidine,

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aspartic acid, alanine, arginine, phenylalanine, isoleucine, leucine, serine and tyrosine. Td
repetitions showed higher levels in histidine and tryptophan while Lt showed higher levels in
lysine and threonine. Lt reported higher final levels in alanine, lysine and serine (Table 3).
CT007 and 3D produced the highest concentrations in leucine and threonine and 3D also
scored the highest value in ornithine.
The statistical differences reported in histidine, phenylalanine, ornithine and tyrosine show
that ageing over-lees processes could increase the content of biogenic amine precursors that
could be metabolized to biogenic amines by the action of microorganisms with decarboxylase
enzymes activities (Alcaide-Hidalgo et al., 2007; Lehtonen, 1996; Ribéreau-Gayon et al.,
2006; Smit et al., 2008).
Biogenic amines
Histamine is the most studied biogenic amine, although it is not the most abundant in wine,
because of their likely occurring health risks such as headaches, low blood pressure, heart
palpitations, oedema, vomiting (Moreno-Arribas and Polo, 2008a). Other biogenic amines
such as Tyramine or putrescine may also influence human health (Jansen et al., 2003; Kanny
et al., 2001; Maynard et al., 1996; Moreno-Arribas and Polo, 2008a; Romano et al., 2007;).
The final levels of histamine were always lower than 1 mg/l (Table 4). A histamine value of 2
mg/L is considered the most restricted level in some countries due to food safety legislation
(Lehtonen, 1996; Martuscelli et al., 2013). It has been described that most biogenic amines
are produced during malolactic fermentation and wine ageing (Alcaide-Hidalgo et al., 2007;
Benito et al., 2015; Lonvaud-Funel, 1999). Nevertheless, our results prove that a controlled
ageing over-lees process, without any deviation performed by lactic acid bacteria, does not
produce higher levels of biogenic amines than a regular control, even in the cases where the
amino acids precursors were increased. Thus, the previous increases reported in the literature
could be related to preservation conditions and bacteria presence, as several factors can

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influence the presence of biogenic amines (Del Petre et al., 2009; Marqués et al., 2008;
Moreno-Arribas and Polo, 2008b). Reductions in biogenic amines were reported (Table 4)
depending on the biogenic amine and yeast strain. Treatments Mp and 3D were more
effective in removing biogenic amines. Other authors have reported other non-
Saccharomyces species such as Hanseniaspora vineae as highly effective reducing histamine
contents (Medina et al., 2013).
Analytical determination of volatile compounds
Slight differences were found among the studied volatile compounds (Table 5), especially for
3D treatment, that show statistical differences in 1-propanol and 2-methyl-butanol with the
other assays. Nevertheless, since the final higher alcohol content was below 300 mg/l in all
c s s, his f c s ’ i fl c iv ly wi q ality (Rapp and Mandery, 1986). Other
authors have also reported differences in higher alcohols (Loira et al., 2013) after an ageing
over-lees process. It has been also reported increases in ethyl lactate (Loira et al., 2013) and
in 2-Phenyl-ethanol (Liu et al., 2015) after an ageing over-lees process. This phenomenon
was not observed in this work, so it could depend on specific strains.
Sensory analysis
The most significant differences were found in mouth volume, persistence and structure (Fig.
2). These factors could depend on mannoprotein content (Belda et al., 2015) in the case of T.
delbrueckii treatment that could have influenced other parameters such as preference or
overall impression. Also, M. pulcherrima together with S. cerevisiae 3D as high producers of
mannoproteins scored relatively higher values in mouth volume, persistence and structure.
However, Mp assay showed the lowest values in preference and overall impression. Thus, it
indicates that mannoprotein release during wine ageing over-lees is an important factor but
there are many others that also pose an important influence in wine perception.

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Conclusions
Overall, we can conclude that the aged over-lees processes and the different strains used
influenced, in a significant way, some of the studied wine quality parameters. Since not too
many differences have been detected in the analytical aroma profile among assays, several
differences were observed in their sensorial analysis. T. delbrueckii released higher amounts
of mannoproteins that not only improved the wine mouthfeel properties but also seems to
contribute to its colour stabilization. All the trials increased the amino acids content of wines.
Furthermore, biogenic amines are generated from their amino acids precursors, mainly by
microbial decarboxylation. The origin, detection, and quantification of biogenic amines in
wine are extremely important for oenology, because of their health risks. In spite of the fact
that some strains increased biogenic amines precursors, according to our results, we cannot
conclude that an ageing process is directly related to biogenic amines increases. However, an
exhaustive microbiological control should be performed during these processes to avoid the
presence of biogenic amines related bacteria. Finally, different sensory profiles of wines were
observed depending on the strain used for ageing over-lees, and they were mainly related
with mannoproteins content. In conclusion, the use of certain non-Saccharomyces and non-
conventional S. cerevisiae strains lees during ageing of wines could be a successful
postfermentative alternative to improve the sensorial characteristics of young wines and to
produce more distinctive wines.
Acknowledgements
Funding for the research in this paper was provided by Agrovin S.A, under the framework of
the project IDI20130192-ENZIOXIVIN (Centre for Industrial Technological Development-
CDTI, Spain). The authors are very grateful to the accredited laboratory Estación Enológica

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de Haro directors, Montserrat Iñiguez and Elena Meléndez for performing the amino acids
and biogenic amines analyses.
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Figure legends
Figure 1. Mannoprotein content (milligrams per liter of mannose) of the initial control wine
(Control) and of the wines aged over-lees with S. cerevisiae CT007 (CT007), S. cerevisiae
3D (3D), L. thermotolerans NS-G-32 (Lt), M. pulcherrima NS-EM-34 (Mp) and T.
delbrueckii NS-TD (Td). Results represent the mean±SD for three replicates. Bars marked
with the same letter showed no significant differences (p<0.05).

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Figure 2. Taste and olfactory attribute scores for the initial control wine (Control) and for the
final wines aged over-lees with S. cerevisiae CT007 (CT007), S. cerevisiae 3D (3D), L.
thermotolerans NS-G-32 (Lt), M. pulcherrima NS-EM-34 (Mp) and T. delbrueckii NS-TD
(Td). Means marked with the same letter showed no significant differences (p<0.05).

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Table 1. Final analysis after ageing over-lees with S. cerevisiae CT007 (CT007), L. thermotolerans (Lt), S. Cerevisiae 3D (3D), M.
pulcherrima (Mp) and T. delbrueckii (Td).
Compounds Control CT007 Lt 3D Mp Td

L-Lactic Acid (g/L) 1.62 ± 0.13 1.68 ± 0.15 1.65 ± 0.16 1.69 ± 0.15 1.64 ± 0.14 1.62 ± 0.17
L-Malic Acid (g/L) 0.24 ± 0.07 0.29 ± 0.09 0.25 ± 0.11 0.24 ± 0.07 0.27 ± 0.08 0.24 ± 0.07
Acetic Acid (g/L) 0.38 ± 0.09 0.4 ± 0.12 0.38 ± 0.07 0.41 ± 0.08 0.39 ± 0.09 0.36 ± 0.13
Glucose+Fructose
0.32 ± 0.11 0.41 ± 0.09 0.36 ± 0.08 0.50 ± 0.16 0.32 ± 0.06 0.38 ± 0.09
(g/L)
Glycerol (g/L) 8.34 ± 0.18 8.12 ± 0.13 8.22 ± 0.20 8.36 ± 0.14 8.31 ± 0.13 8.29 ± 0.16
pH 3.51 ± 0.03 3.53 ± 0.06 3.51 ± 0.05 3.54 ± 0.06 3.52 ± 0.04 3.53 ± 0.05
Urea (mg/L) 14.51 ± 1.18 15.32 ± 2.14 14.31 ± 1.99 14.76 ± 1.87 15.13 ± 1.66 14.28 ± 2.06
Citric Acid (g/L) 0.41 ± 0.07 0.43 ± 0.09 0.39 ± 0.10 0.38 ± 0.09 0.42 ± 0.11 0.45 ± 0.08
Ethanol (% v/v) 14.51 ± 0.14 14.46 ± 0.13 14.58 ± 0.17 14.55 ± 0.14 14.53 ± 0.11 14.52 ± 0.16
Results represent the mean±SD for three replicates. No significant differences were observed among assays in the evaluated parameters
(p<0.05).
Table 2. Final colour analysis after ageing over-lees with S. cerevisiae CT007 (CT007), L. thermotolerans (Lt), S. Cerevisiae 3D (3D), M.
pulcherrima (Mp) and T. delbrueckii (Td).
Colour Measurements
Control CT007 Lt 3D Mp Td
(Absorbance Units)
420nm 5.07±0.09b 4.73±0.16a 4.75±0.18a 4.86±0.12ab 4.75±0.15a 4.81±0.14a
520nm 6.37±0.08c 5.75±0.14a 5.96±0.16ab 6.10±0.16b 5.89±0.11ab 6.17±0.10b
620nm 0.84±0.01a 0.83±0.02a 0.84±0.02a 0.86±0.02a 0.83±0.02a 0.84±0.02a
CI 12.28±0.07c 11.31±0.13a 11.55±0.15ab 11.77±0.13b 11.47±0.12a 11.88±0.11b
TPI 43,3±0,00d 32,33±0,12a 33,87±0,95c 32,87±0,49ab 33,6±0,36bc 33,93±0,71c
AC 158±0,00c 135,33±5,03b 127,67±4,04a 133,67±3,51b 130±0,00ab 132±2,65ab
Results represent the mean±SD for three replicates. Means in the same row with the same letter are not significantly different (p<0.05).

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Table 3. Final amino acids analysis after ageing over-lees with S. cerevisiae CT007 (CT007), L. thermotolerans (Lt), S. Cerevisiae 3D
(3D), M. pulcherrima (Mp) and T. delbrueckii (Td).

Histidine (mg/l) 2.54±0.00a 3.63±0.35b 3.92±0.47b 3.78±0.42b 5.92±0.55c 6.94±0.72c
Aspartic acid (mg/l) 4.82±0.00a 6.29±0.56c 6.53±0.61c 6.48±0.54c 8.79±0.93d 5.14±0.18b
Alanine (mg/l) 24.09±0.00a 28.44±1.28c 30.18±1.52c 29.11±1.33c 38.28±1.99d 25.42±0.24b
Arginine (mg/l) 26.53±0.00a 30.68±2.43bc 34.53±2.12c 31.18±2.31bc 38.26±2.45d 28.45±0.92b
Asparagine (mg/l) 16.86±0.00a 20.18±1.56b 18.24±1.12b 20.26±1.27b 28.52±2.24c 20.54±1.62b
Phenylalanine (mg/l) 2.48±0.00a 4.77±0.23c 4.58±0.19c 4.89±0.56c 6.77±0.51d 3.43±0.14b
Glycine (mg/l) 2.89±0.00a 5.16±0.59b 4.67±0.51b 5.26±0.61b 5.78±0.72b 4.95±0.63b
Tryptophan (mg/l) 1.78±0.00a 3.72±0.23b 3.56±0.21b 3.92±0.38b 3.82±0.36b 5.16±0.56c
Isoleucine (mg/l) 1.56±0.00a 3.15±0.24c 3.64±0.32c 3.21±0.23c 4.87±0.46d 2.43±0.18b
Lysine (mg/l) 14.12±0.00a 18.12±1.78b 25.36±2.56c 19.26±1.96b 23.21±2.16c 18.14±1.12b
Leucine (mg/l) 2.76±0.00a 5.62±1.08c 3.98±0.42b 5.98±0.92c 6.12±1.26c 3.72±0.35b
Ornithine (mg/l) 2.46±0.00a 5.14±0.42c 4.82±0.38c 5.55±0.48c 5.26±0.32c 3.22±0.21b
Serine (mg/l) 2.15±0.00a 3.98±0.31b 4.89±0.64bc 4.11±0.36b 5.86±0.58c 4.02±0.35b
Tyrosine (mg/l) 2.74±0.00a 5.13±0.42b 5.55±0.51b 5.38±0.43b 6.82±0.62c 5.16±0.53b
Threonine (mg/l) 14.24±0.00a 19.44±1.18c 18.92±1.11c 19.86±1.22c 16.11±1.02b 16.03±0.98b
Results represent the mean±SD for three replicates. Means in the same row with the same letter are not significantly different (p<0.05).
Table 4. Final biogenic amines analysis after ageing over-lees with S. cerevisiae CT007 (CT007), L. thermotolerans (Lt), S. Cerevisiae 3D
(3D), M. pulcherrima (Mp) and T. delbrueckii (Td).

Histamine (mg/l) 0.98 ± 0.00b 0.86 ± 0.03 a 0.89± 0.05 a 0.83 ± 0.03a 0.81± 0.03a 0.88 ± 0.04a
Tyramine (mg/l) 1.34 ± 0.00 c 1.20 ± 0.04 ab 1.27 ± 0.03 b 1.16 ± 0.05 a 1.14 ± 0.04 a 1.26 ± 0.03 b
Phenylethylamine (mg/l) n.d. n.d. n.d. n.d. n.d. n.d.
Putrescine (mg/l) 3.12± 0.00 c 2.75± 0.06 ab 2.81 ± 0.05 ab 2.71 ± 0.04 a 2.66 ± 0.05 a 2.82 ± 0.06b
Cadaverine (mg/l) n.d. n.d. n.d. n.d. n.d. n.d.
Results represent the mean±SD for three replicates. Means in the same row with the same letter are not significantly different (p<0.05), n.d:
not detected.

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Table 5. Final analysis of volatile compounds (mg/L) after ageing over-lees with S. cerevisiae CT007 (CT007), L. thermotolerans (Lt), S.
Cerevisiae 3D (3D), M. pulcherrima (Mp) and T. delbrueckii (Td).

Acetaldehyde 14.22±1.67a 15.42±2.02a 13.86±1.48a 12.98±1.78a 15.38±2.13a 14.37±1.55a
Ethyl lactate 42.26±3.48a 45.52±3.52a 41.34±2.77a 40.11±3.02a 47.22±3.75a 42.98±3.16a
Ethyl acetate 29.45±1.85a 30.06±1.54a 28.76±1.91a 28.14±1.74a 31.26±2.12a 29.89±1.98a
Diacetyl 3.78±0.44a 3.69±0.48a 3.82±0.56a 3.22±0.38a 3.64±0.68a 3.66±0.52a
Isoamyl acetate 2.37±0.32a 2.34±0.38a 2.46±0.35a 2.46±0.48a 2.23±0.39a 2.41±0.51a
1-Propanol 37.82±3.52a 35.46±3.61ab 37.06±3.33a 30.82±2.25b 38.13±3.68a 34.35±3.68ab
Isobutanol 41.32±3.82a 37.62±3.33a 41.24 ±3.16a 35.62±3.76a 42.87±3.58a 38.14±2.88a
3-Methyl-butanol 34.33±2.13a 33.78±1.98a 34.46±2.44a 33.12±1.18a 34.69±2.06a 33.03±1.24a
2-Methyl-butanol 86.38±4.36a 82.24±4.55ab 85.79±3.98a 75.16±3.88b 87.54±4.58a 83.34±4.26ab
Isobutyl acetate n.d. n.d. n.d. n.d. n.d. n.d.
Ethyl butyrate n.d. n.d. n.d. n.d. n.d. n.d.
2-Phenyl-ethanol 24.58±2.96a 22.93±3.08a 25.33±3.12a 25.78±3.46a 21.72±3.21a 26.22±3.88a
2- Phenyl ethyl acetate 3.26±0.39a 3.22±0.44a 3.02±0.41a 3.48±0.52a 3.06±0.47a 3.56±0.48a
Hexanol 3.48±0.26a 3.28±0.32a 3.52±0.34a 3.16±0.29a 3.58±0.42a 3.21±0.37a
Results represent the mean±SD for three replicates. Means in the same row with the same letter are not significantly different (p<0.05), n.d:
not detected.

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7. DISCUSIÓN

197

198
Discusión
7. DISCUSIÓN GENERAL
El proceso de elaboración de vino, a pesar su origen antrópico, constituye uno de los

principales hábitats y reservorios naturales de las levaduras, tanto en el viñedo como en el
proceso de fermentación (Pretorius, 2000). La diversidad de especies de levaduras asociadas
al proceso de vinificación lo convierte en un interesante foco de estudios de ecología
microbiana, así como de la influencia que sobre la misma tienen tanto factores climáticos y
oro-geográficos, como las distintas prácticas vitivinícolas (Lachance y Stramer, 1998; Liu et
al., 2015). Esta diversidad microbiana, unida a la complejidad nutricional de un sustrato como
el mosto de uva, cuya metabolización determinará en parte la composición final del vino,
hacen de la enología una interesante ciencia para el estudio y aplicación de la diversidad
metabólica asociada a especies y cepas de levaduras (Belda et al., 2016a).
7.1. Diversidad microbiana y metabólica asociada al proceso de fermentación: estudio

filo-funcional de levaduras de interés enológico
El microbioma asociado a un vino o viñedo está en la base del concepto de terroir

microbiano, que pretende dar explicación a la incidencia de los microorganismos en el perfil
sensorial de los vinos de una determinada región vitivinícola (Bokulich et al., 2014; Gilbert et
al., 2014). Los estudios poblacionales llevados a cabo para el establecimiento de estos
conceptos han arrojado resultados notables respecto a la complejidad microbiana del proceso
de fermentación vínica (Liu et al., 2015). Un total de 93 especies de levadura pertenecientes a
30 géneros distintos han sido identificadas en ambientes enológicos (Barata et al., 2008;
2012; Bisson y Joseph, 2009), de las cuales aproximadamente 25 han sido directamente
relacionadas con el proceso de vinificación (Jolly et al., 2014) aunque su papel en el proceso
y, por tanto, su potencial aplicación han sido escasamente estudiados.
En este contexto, el objetivo inicial de este trabajo, recogido en su primer capítulo,

consistió en el estudio de la diversidad de especies de levaduras asociadas a distintas zonas
vitivinícolas de España (D.O. Ribera del Duero, D.O. Rueda y D.O. Tierra de León) y la

199
Discusión
posterior caracterización de sus propiedades metabólicas en lo referente a la producción de

enzimas de interés enológico (β-glucosidasa, β-D-xilosidasa, α-L-arabinofuranosidasa, β-
liasa, proteasa, pectinasa, celulasa y sulfito reductasa). El establecimiento de una colección
amplia de 770 levaduras pertenecientes a 15 especies distintas permitió el estudio de la
variabilidad metabólica inter- e intraespecífica para las ocho actividades enzimáticas citadas
previamente (Belda et al., 2016a).
El diseño del muestreo y la metodología de aislamiento de levaduras fueron adaptados a

los objetivos generales del trabajo consistentes en la obtención de una diversidad razonable de
especies de levaduras no-Saccharomyces de interés enológico para su caracterización
enzimática y su posterior aplicación en fermentaciones combinadas con Saccharomyces
cerevisiae. Por ello, se procuró evitar el aislamiento tanto de esta última como de otras
especies de levaduras basidiomicetes y del hongo levaduriforme Aureobasidium pullulans, de
metabolismo eminentemente oxidativo y con una abundancia relativa elevada en la
comunidad microbiana presente en las uvas. Considerados estos elementos que pudieran
condicionar los datos de diversidad microbiana de la colección establecida, se pudo
comprobar la existencia de unos datos poblacionales globales (considerando los 4 orígenes
muestreados) similares a lo descrito en otros trabajos, destacando la dominancia de especies
de los géneros Hanseniaspora, Metschnikowia y Lachancea (Cocolin et al., 2000; Pinto et al.,
2015). Éstas suponen más del 85% de la población de levaduras aisladas en este trabajo,
pertenecientes a 12 géneros distintos, siendo la especie H. uvarum responsable de más del
50% de dicha población, de acuerdo a lo obtenido en trabajos de enfoque similar (Beltrán et
al., 2002; Wang et al., 2015).
A excepción del comportamiento de las especies dominantes, H. uvarum,

Metschnikowia sp. (agrupando M. pulcherrima y M. fructicola) y Lachancea thermotolerans,
la población de levaduras aisladas en las bodegas muestreadas en la D.O. Ribera del Duero
(EM y PDC) presentó una composición y evolución distinta en los años 2013 y 2014,
manteniéndose relativamente estable en la primera de ellas (EM) y variando
significativamente la otra (PDC), en la que pudo aislarse una diversidad mayor en la vendimia
2014. La gran cercanía geográfica de ambas bodegas (zonas de viñedo muestreadas) y, por
tanto, la similitud de condiciones climáticas y orográficas hace pensar que las prácticas
agrícolas o la casuística microclimática pudieron determinar este comportamiento diferencial.

200
Discusión
En los muestreos realizados en la D.O. Rueda, se observó el fenómeno contario y la

diversidad de levaduras aislada en la vendimia de 2013 fue considerablemente mayor que la
observada en 2014.
En el caso de la bodega muestreada en la D.O. Tierra de León, se registra gran

diversidad microbiana obtenida en un solo año de muestreo y con un número moderado de
aislamientos (73 aislamientos), pudiendo identificarse un total de 10 especies distintas. En
este caso, y aunque nuevamente no existen evidencias concluyentes de su relación con la
diversidad poblacional, destacan las características microclimáticas del año 2012 (un año
extraordinariamente seco) en el que fue muestreada la bodega de la D.O. Tierra de León con
respecto a los años 2013 y 2014 en los que se muestrearon las otras bodegas. Una menor
humedad durante el desarrollo de la uva y particularmente durante su vendimia conlleva un
menor desarrollo de poblaciones invasivas de hongos filamentosos lo que facilita el desarrollo
y asilamiento de las de una mayor diversidad de especies de levaduras.
La caracterización de las propiedades enzimáticas del conjunto de la colección

establecida permitió observar, ahora sí, patrones fenotípicos intraespecíficos característicos de
las distintas regiones y bodegas muestreadas. Considerando las 8 actividades enzimáticas
evaluadas, el uso adecuado de herramientas estadísticas de clustering nos permitió la
diferenciación a nivel funcional, de subgrupos de levaduras de la misma especie aislados en
distintos orígenes. Esta diferenciación fenotípica de las cepas de aisladas en distintos orígenes
sí está en consonancia con el concepto de terroir microbiano comentado anteriormente. La
relación potencial de estos patrones fenotípicos con las propiedades sensoriales de los vinos
llevó al desarrollo del concepto wine flavorome, como una evidencia más de la contribución
de la microbiota característica de cada zona vitivinícola a las propiedades sensoriales
diferenciadoras de los vinos de una región.
En cuanto a la distribución de las actividades enzimáticas a nivel interespecífico, se

pudieron establecer 3 grupos de actividades en función de su abundancia global, dependiendo
esta tanto del número de especies que muestran dicha actividad como de su abundancia
relativa en la población total. Así, destacan la actividad β-glucosidasa y proteasa como
actividades altamente distribuidas y contrariamente se sitúan las actividades α-L-

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Discusión
arabinofuranosidasa, pectinasa y celulasa restringidas a un bajo número de especies y, en

ocasiones, con baja representación en la comunidad de levaduras asociadas a mostos de uva.
La importancia de las enzimas glicosidasas como responsables de la liberación de

terpenos en vinos ha sido ampliamente estudiada, siendo estos los descriptores aromáticos
principales de ciertas variedades de uva como la moscatel, albariño o riesling (Marais, 1983).
Se ha descrito la producción de ciertas enzimas glicosidasas por parte de cepas de S.
cerevisiae, sin embargo la mayoría de ellas no presentan o presentan una actividad β-D-
glucosidasa muy limitada (Úbeda-Iranzo et al., 1998; Van Rensburg et al., 2005), necesaria
para la etapa final en la liberación de aromas terpénicos a partir de sus conjugados
glicosilados. Nuestros resultados mostraron que la mayoría de especies de levadura no-
Saccharomyces poseen en mayor medida esta actividad, de acuerdo con lo descrito en otros
trabajos (Fia et al., 2005), además de, en muchos casos, actividad β-D-xilosidasa e incluso α-
L-arabinofuranosidasa, estando esta última restringida a unas pocas especies. En este
contexto, la comunidad de levaduras no-Saccharomyces parecen ser un foco interesante de
estudio para su utilización como herramientas para incrementar la revelación del perfil
varietal de variedades de uva terpénicas o el revelado de aromas en variedades neutras.
En el presente estudio, destacó la producción de actividad β-D-glucosidasa de las

especies evaluadas del género Hanseniaspora, así como de Meyerozyma guilliermondii y
Wickerhamomyces anomalus. El uso de cepas de H. uvarum y W. anomalus para la liberación
de compuestos terpénicos en vinos ha sido previamente demostrada (Mendes-Ferreira et al.,
2001; Fernández-González et al., 2003; Mateo et al., 2011) destacándose su actividad en un
amplio rango de pH y con bajos índices de represión por glucosa (Mateo et al., 2011; López
et al., 2015). En este mismo sentido, la actividad β-D-xilosidasa mostrada por el grupo de
especies relacionadas S. cerevisiae, Torulaspora delbrueckii y Zygosaccharomyces bailii,
presenta, en la mayoría de cepas, un alto grado de represión por glucosa, limitando, de nuevo,
su uso para la liberación de terpenos en fermentaciones vínicas (Delcroix et al., 1994; Rosi et
al., 1994; Gueguen et al., 1995; Mateo y Di Stefano, 1997; Hernández et al., 2002; Mateo et
al., 2011).
Tanto la literatura científica como la actividad industrial indican también la capacidad

de ciertas cepas de M. pulcherrima para la liberación de terpenos glicosilados mediante la
producción de actividad α-L-arabinofuranosidasa (Fernández-González et al., 2003). Nuestros

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Discusión
resultados muestran que, si bien es cierto que la actividad β-D-glucosidasa se encuentra muy
ampliamente distribuida en el conjunto de cepas analizadas de Metschnikowia sp., la
producción de α-L-arabinofuranosidasa está restringida a unas pocas cepas.
En resumen, en lo referente a las actividades glicosidasas estudiadas y, de acuerdo con

lo previamente descrito por Manzanares et al. (1999), Pichia, Wickerhamomyces, y
Hanseniaspora constituyen géneros con altos grados de actividad. Adicionalmente, nuestro
trabajo destaca la producción de enzimas glicosidasas por parte de ciertas especies de
basidiomicetes ampliamente distribuidas en ambientes enológicos como son Rhodosporidium
toruloides y Cryptococcus amylolentus.
La actividad β-liasa, relacionada con la liberación de compuestos tiólicos mostró niveles

de abundancia moderados en la colección de levaduras estudiada. Aunque no puede
considerarse una actividad exclusiva de pocas especies, la mayoría de ellas muestran niveles
de actividad muy moderados, destacando la actividad de T. delbrueckii, K. marxianus y M.
guilliermondii. Aunque esta actividad ha sido ampliamente estudiada en S. cerevisiae, siendo
muy variable entre cepas (Howell et al., 2005; Thibon et al., 2008; Roncoroni et al., 2011;
Santiago y Gardner, 2015; Belda et al., 2016c;), su presencia en levaduras no-Saccharomyces
ha sido escasamente evaluada (Zott et al., 2011; Belda et al., 2016c). De forma análoga a lo
estudiado en el caso de las actividades glicosidasas y su represión por glucosa, y puesto que la
actividad β-liasa de S. cerevisiae presenta niveles elevados de represión catabólica por
nitrógeno, la caracterización de dicha actividad en levaduras no-Saccharomyces en diferentes
condiciones enológicas debe ser realizada para su aplicación como herramientas útiles en el
incremento del perfil tiólico de vinos blancos.
Las actividades proteasa, pectinasa y celulasa fueron evaluadas por su implicación en la

mejora de ciertas características tecnológicas de los vinos, fundamentalmente relacionadas
con el proceso de clarificación, extracción de compuestos fenólicos y prevención de la
quiebra proteica (Marangon et al., 2012; Belda et al., 2016b). La actividad proteasa mostró
gran abundancia en la colección de levaduras evaluada, sin embargo, un gran número especies
de notable interés enológico mostraron valores bajos o nulos de dicha actividad, como S.
cerevisiae, T. delbrueckii, Z. bailii o L. thermotolerans, entre otros. Por el contrario, el
conjunto de cepas evaluadas del género Metschnikowia, a excepción de las pertenecientes a la
especie M. viticola, mostraron niveles elevados de esta actividad, por lo que al margen

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Discusión
reconocida contribución a la mejora del perfil sensorial de los vinos, su uso como herramienta
de prevención de la quiebra proteica de vinos debe ser estudiada en futuros ensayos. En la
misma línea, y mostrando valores muy elevados de esta actividad, cabe destacar el
comportamiento de ciertas cepas de la especie W. anomalus, cuya contribución a la mejora del
perfil sensorial de los vinos también ha sido descrita con anterioridad (Domizio et al., 2011;
Izquierdo-Cañas et al., 2011).
En el extremo opuesto en cuanto a su abundancia se encuentran las actividades

pectinasa y celulasa en la colección de levaduras evaluada. La primera de ellas, si bien pudo
detectarse en aproximadamente el 50% de las cepas evaluadas de S. cerevisiae, su
funcionalidad en condiciones enológicas se encuentra de nuevo condicionada por la presencia
de glucosa (Radoi et al., 2005), contrariamente a lo observado en ciertas especies no-
Saccharomyces (Merín et al., 2011; Merín y Morata de Ambrosini, 2015). Nuestros resultados
muestran que, al margen de unas pocas cepas de T. delbrueckii, la presencia de actividad
pectinolítica parece estar reservada a las especies del género Metschnikowia y a A. pullulans
(única especie productora de actividad celulasa), habiéndose demostrado en este trabajo su
incidencia en el proceso de clarificación y extracción fenólica en vinos tintos (Belda et al.,
2016b).
Finalmente destaca la ausencia de actividad sulfito reductasa en la práctica totalidad de

especies no-Saccharomyces analizadas, a excepción de los elevados niveles de producción
mostrados por las especies del género Hanseniaspora.
7.2. Aplicación de levaduras pectinolíticas en maceración de vinos tintos
El uso de enzimas pectinolíticas en las fases de maceración, previas al comienzo de la

fermentación tumultuosa propiamente dicha, es una práctica habitual en enología para la
elaboración de vinos tintos. Su aplicación persigue el incremento de color, a través de la
extracción de polifenoles y antocianos, lográndose además, una mejor clarificación del vino
resultante (Van Rensburg y Pretorius, 2000; Merín y Morata de Ambrosini, 2015). La adición
de pectinasas se realiza en forma de preparados enzimáticos de origen fúngico Estos

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Discusión
preparados comerciales suelen consistir en una mezcla de enzimas con distintas actividades
(poligalacturonasa, pectín-liasa y pectín-metilesterasa), entre las que las poligalacturonasas
son las principales responsables de la actividad pectinolítica en vinos (Lang y Dornenburg,
2000). En este contexto, existe un interés científico e industrial en la búsqueda de levaduras
como fuente de enzimas pectinolíticas y su uso como herramientas biológicas en la mejora del
proceso de maceración. Por ello, en el presente trabajo se abordó la búsqueda y selección de
levaduras con actividad poligalacturonasa para su posterior aplicación como inóculos durante
la maceración prefermentativa para la mejora de los parámetros antes comentados. Además,
dado el interés en el desarrollo de los procesos de maceración prefermentativa en condiciones
de baja temperatura controlada para la mejora de la extracción y estabilización del color
(Merín y Morata de Ambrosini, 2015), se valoró la actividad de dichas actividades
enzimáticas aplicando procesos de maceración prefermentativa fría (12ºC) y convencional
(25ºC).
Los resultados mostrados en el primer capítulo de este trabajo confirman que la
presencia de actividad poligalacturonasa en levaduras de interés enológico está reservado a
unas pocas especies, fundamentalmente M. pulcherrima y A. pullulans (Belda et al., 2016a).
Los resultados de los trabajos mostrados en el segundo capítulo de este trabajo (Belda et al.,
2016b) demuestran la funcionalidad de ambas especies para la mejora de los parámetros de
calidad derivados de su actividad poligalacturonasa, fundamentalmente en condiciones de
maceración prefermentativa en frio (MPF). Esto parece indicar que el control de temperaturas
bajas en las etapas previas a la fermentación no sólo contribuye a la extracción de polifenoles
y antocianos por motivos químicos de solubilidad de la matriz acuosa como se venía creyendo
hasta el momento (Delteil, 2004, Hernández-Jiménez et al., 2012), sino que quizá un mayor
desarrollo de especies no-Saccharomyces, favorecido por las bajas temperaturas y el
consecuente retraso en el inicio de la fermentación alcohólica propiamente dicha (Mendoza et
al., 2009; Andorrá et al., 2010), contribuya también a este hecho mediante la actuación de las
actividades pectinolíticas provenientes de éstas, tanto en fermentaciones espontáneas como
mediante la inoculación de cepas no-Saccharomyces seleccionadas.
Los resultados mostrados en el mencionado trabajo (Belda et al., 2016b), prueban que la
incidencia de las levaduras pectinolíticas sobre los parámetros estudiados dependientes de las
mismas es más acentuada cuando se aplican en MPF., destacando los resultados mostrados
por M. pulcherrima. Ésta, en ensayos de fermentación secuencial junto con S. cerevisiae a

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Discusión
escala de laboratorio, logró incrementar el contenido en polifenoles y la extracción de

antocianos, así como los valores finales de intensidad de color en un 10%, 21% y 15%,
respectivamente, en comparación con el control exclusivamente inoculado con S. cerevisiae.
Asimismo, mostró una influencia positiva sobre la turbidez y el tiempo de filtración de los
vinos con reducciones en dichos valores del 57% y el 34%, respectivamente, aunque en este
caso, los valores similares mostrados por L. thermotolerans (levadura control sin actividad
poligalacturonasa), parecen indicar que existen otros factores, al margen de la actividad
pectinolítica de las levaduras, que contribuyen a este hecho.
Finalmente, se demostró la potencial aplicación de la cepa M. pulcherrima NS-EM-34

en la mejora de los parámetros analizados a escala semi-industrial obteniéndose incrementos
de la intensidad de color de los vinos superiores al 40% y del contenido en polifenoles
cercano al 20%, así como una reducción de la turbidez de los vinos del 42%. Estos resultados
mejoran significativamente lo mostrado en otros trabajos aplicando técnicas de MPF
(Panprivech et al., 2015) e incluso en el uso de S. cerevisiae cepas modificadas genéticamente
para la sobreexpresión del gen PGU1 codificante para una enzima poligalacturonasa en dicha
especie (Fernández-González et al., 2005; Radoi et al., 2005).
Si bien la incidencia de M. pulcherrima sobre la composición aromática y el contenido

en etanol de los vinos ha sido descrita con anterioridad (Parapouli et al., 2010; Rodríguez et
al., 2010; Sadoudi et al., 2012; Quirós et al., 2014; Contreras et al., 2015), este trabajo
describe por primera vez la incidencia de M. pulcherrima NS-EM-34 durante el proceso de
maceración prefermentativa en la mejora de los procesos de clarificación y extracción de
color. Así, fue posible confirmar mediante análisis estadístico de componentes principales
(PCA) que, en el estudio de un elevado número de parámetros de composición química del
vino, aquellos relacionados con dichos procesos son responsables de la diferenciación
analítica de los vinos fermentados exclusivamente con S. cerevisiae o en fermentación
secuencial con M. pulcherrima (Belda et al., 2016b).

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Discusión
7.3. Selección y aplicación de levaduras en fermentación para la mejora de las

propiedades sensoriales de los vinos
Como se muestra en los capítulos segundo y cuarto de este trabajo, las levaduras no sólo
tienen incidencia en la composición del vino durante de fermentación alcohólica. Durante
años, se han sucedido los trabajos de selección de levaduras S. cerevisiae para dotar a la
industria de inóculos con los que desarrollar las fermentaciones en bodega con garantías de
calidad y seguridad fermentativa (Pretorius, 2000). Estos inóculos eran seleccionados con dos
objetivos generales; finalizar eficientemente el proceso de fermentación del mosto y producir
vinos de alta calidad. A este respecto, la literatura clásica estableció dos grandes grupos de
propiedades a evaluar en los procesos de selección de cepas de S. cerevisiae como inóculo
(Zambonelli, 1998): propiedades tecnológicas (tolerancia al etanol, poder fermentativo,
resistencia al SO2, capacidad de crecimiento en medio líquido, crecimiento en amplio rango
de temperaturas, presencia de factor killer, etc.) y propiedades sensoriales (generación de
subproductos de la fermentación: ácido acético, glicerol, acetaldehído y alcoholes superiores;
producción de compuestos azufrados: H2S y SO2; y producción de enzimas hidrolíticas: β-
glucosidasa, esterasa, enzimas proteolíticas). Dado el elevado número de requisitos, la
presencia de cepas salvajes con una combinación óptima de propiedades tecnológicas y
sensoriales es muy baja (Rainieri y pretorius, 2000), por ello, la optimización en los procesos
de selección, mediante el desarrollo de métodos de screening metabólico de alto rendimiento,
parece ser la forma ideal de afrontar este reto (Figura 7).
Figura 7. Adaptación de métodos de detección de liberación de H2S a formato de alto rendimiento, con elevada
reproducibilidad y posibilidad de cuantificación por densitometrado. Adaptado de Belda et al. (2013). A) método
tradicional de detección por acetato de plomo; B) Detección en medio Biggy; C) Adaptación del método de
detección por acetato de plomo a formato de alto rendimiento que permite su cuantificación por densitometrado.

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Discusión
En este contexto, la primera parte del tercer capítulo de este trabajo, muestra el
desarrollo de un método de selección de levaduras con elevada actividad β-liasa, responsable
de la liberación de aromas tiólicos en vinos (Patente presentada a la Oficina Española de
Patentes y Marcas bajo el número de registro P-201500195). Esta enzima es responsable de la
liberación de los compuestos volátiles responsables del aroma tiólico de los vinos blancos, 3-
mercaptohexanol (3-MH) y 4-mercapto-4-metilpentan-2-ona (4-MMP), mediante la ruptura
de sus precursores no volátiles (cisteinilados) (Swiegers et al., 2009; Holt et al., 2012). Si
bien la liberación del primer compuesto no es responsabilidad exclusiva de un único gen,
aunque el gen STR3 de S. cerevisiae ha sido descrito como responsable mayoritario de tal
hecho (Holt et al., 2011, 2012), la liberación de 4-MMP desde su precursor cisteinilado ha
podido ser atribuida en su práctica totalidad a la acción del gen IRC7 en S. cerevisiae, cuya
inactivación ocasiona también una reducción considerable en la liberación de 3-MH
(Roncoroni et al., 2011). De las dos isoformas de IRC7 que han sido descritas, una de ellas
conteniendo una deleción que determina una disminución de actividad en la enzima, la
isoforma íntegra y, por tanto, más eficaz de la enzima, se encuentra muy poco presente entre
las cepas salvajes de S. cerevisiae, en niveles inferiores al 3% (Belda et al., 2016c). Por
primera vez, en este trabajo se describe la existencia de cepas con un genotipo heterocigoto
para el gen IRC7, cuyo fenotipo, aunque de menor actividad β-liasa, tiende a asemejarse al del
genotipo homocigoto para el gen íntegro, y su abundancia relativa en la población de cepas
salvajes evaluada fue del 9,4%. El análisis del genotipo IRC7 en una colección de 22 cepas de
levadura industriales (Agrovin S.A.) dio como resultado una distribución de 23% de cepas
homocigotas para el gen íntegro, 23% para el genotipo heterocigoto y 54% de cepas
homocigotas para el gen truncado. Este incremento en la proporción de cepas de genotipo
IRC7 íntegro, con respecto a las cepas salvajes evaluadas, es fácilmente explicable por el
filtro de calidad sensorial al que se someten las cepas de levadura para su selección previa a
su comercialización en las que los parámetros de calidad aromática juegan un papel decisivo
(Masneuf-Pomarède et al., 2002, 2006; Lee et al., 2008).
El umbral de percepción, extremadamente bajo, de estos compuestos tiólicos (3 ng/L

para 4-MMP y 60 ng/L para 3-MH) hace que, pequeños incrementos en la liberación de estos
compuestos puedan modular significativamente el perfil de aromas tiólicos de un vino, y por
tanto la selección de levaduras con alta actividad β-liasa constituye un reto de interés (Murat
et al., 2001). El medio de cultivo descrito en el primer apartado del capítulo tercero de este

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Discusión
trabajo presenta en su composición un sustrato químico comercial, la S-metil-cisteína (M-

6626, Sigma-Aldrich), de estructura y enlace C-S análogos a los de los precursores
cisteinilados naturales presentes en la uva para su uso como agente selectivo de levaduras en
función de su actividad β-liasa. Su presencia en el medio como única fuente de nitrógeno
permite seleccionar aquellas cepas capaces de usar el amonio derivado de su hidrólisis como
única fuente de nitrógeno. De esta forma, se pueden realizar procesos de aislamiento y
selección de levaduras con elevada actividad β-liasa en función de su capacidad de
crecimiento en el medio sólido descrito, incrementando las probabilidades de éxito en un
contexto de baja abundancia en la naturaleza. Además de la utilidad para la diferenciación
intraespecífica de S. cerevisiae en función de la integridad y funcionalidad de su gen IRC7,
este medio de cultivo permite la selección de levaduras no-Saccharomyces con elevada
actividad β-liasa, siempre con la precaución necesaria a la hora de establecer comparaciones a
nivel interespecífico dadas las diferencias basales de fitness entre especies. A este respecto,
los resultados mostrados en el trabajo de Belda et al. (2016c), destacan la elevada actividad β-
liasa mostrada por ciertas cepas de T. delbrueckii y K. marxianus, muy destacadas entre un
amplio rango de especies no-Saccharomyces de origen enológico evaluadas.
La incidencia de T. delbrueckii para el incremento del perfil tiólico de vinos blancos ha

sido recientemente descrita por Renault et al. (2016) quienes muestran un efecto sinérgico en
la liberación de 3-MH mediante la coinoculación de T. delbrueckii y S. cerevisiae en mostos
de la variedad Sauvignon blanc. Si bien sus resultados descartan la liberación de 4-MMP por
parte de T. delbrueckii, y han de ser interpretados con cautela por no detallarse el genotipo del
gen IRC7 de la cepa de S. cerevisiae utilizada (Zymaflore X5), este comportamiento parece
ser dependiente de cepa, ya que resultados recientes derivados de esta Tesis Doctoral
confirman la utilidad de la cepa T. delbrueckii Viniferm NS-TD para el incremento del perfil
tiólico de vinos blancos (variedad verdejo) mediante la liberación de 3-MH y, especialmente,
de 4-MMP (Figura 8).

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Discusión
Figura 8. Liberación de tioles varietales (ng/L) en fermentación de mosto de la variedad verdejo con cepas S.
cerevisiae con genotipo IRC7 funcional (S. cerevisiae Viniferm Revelacion (ScR)) y no funcional (S.
cerevisiae Viniferm Diana (ScD)) y su uso en inoculación secuencial con T. delbrueckii Viniferm NS-TD
(Td+R y Td+D, respectivamente). 3-MH (azul): 3-mercaptohexanol; 3-MHA (rojo): acetato de 3-
mercaptohexilo; 4-MMP (verde): 4-mercapto-4-metilpentan-2-ona. Datos procedentes de análisis ejecutados
por la compañía Nyseos (Montpellier, Francia). Letras diferentes sobre los valores del mismo parámetro
indican diferencias significativas entre ensayos en el correpondiente análisis ANOVA (p<0,05).
Probada la incidencia de T. delbrueckii Viniferm NS-TD en el perfil sensorial de la

variedad Verdejo, como una de las variedades blancas de mayor incidencia en la viticultura
española, se procedió a la caracterización de dicho impacto en la composición y propiedades
sensoriales en fermentaciones de vinos tintos de la variedad Tempranillo (Belda et al.,
2015b).
En este caso, se realizaron fermentaciones a escala de laboratorio (5 L) y a escala semi-

industrial (100 L) para el estudio de la evolución de parámetros analíticos básicos del vino así
como para la caracterización del perfil final de compuestos aromáticos volátiles. Así mismo
se llevó a cabo un seguimiento de la cinética de fermentación y la dinámica poblacional en los
ensayos, tanto exclusivamente inoculados con S. cerevisiae o T. delbrueckii por separado, o
en su inoculación conjunta de forma simultánea o secuencial. La inoculación simultánea no
mostró diferencias notables ni en cinética fermentativa ni en composición analítica de los
vinos comparada con la fermentación control inoculada exclusivamente con S. cerevisiae, lo
que puede explicarse por la rápida imposición de la población de ésta última especie de
acuerdo con lo observado en estudios similares con esta y otras especies no-Saccharomyces
(Azzolini et al., 2012; Oro et al., 2014). T. delbrueckii, a pesar de ser una de las levaduras no-

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Discusión
Saccharomyces con mayor poder fermentativo (Jolly et al., 2014), éste sigue siendo inferior al
mostrado por S. cerevisiae (Bisson y Kunkee 1991; Jolly et al., 2006), lo que junto a una
mayor demanda nutricional atribuida a las especies no-Saccharomyces (en términos de
consumo de recursos nitrogenados y vitaminas) y que puede dificultar la posterior actividad
por parte de S. cerevisiae en su inoculación secuencial (Romano et al., 2003) explicaría la
cinética fermentativa mas lenta mostrada en dicho ensayo. En cuanto a los parámetros
analíticos evaluados, destacó una ligera reducción en la acidez volátil de los vinos
fermentados con T. delbrueckii en inoculación secuencial, de acuerdo a lo previamente
descrito en la bibliografía (Moreno et al., 1991; Bely et al., 2008; Renault et al., 2009;
Azzolini et al., 2012). En cuanto al metabolismo de ácidos orgánicos, pudo observarse de
nuevo una ligera reducción en el contenido en ácido málico en los vinos fermentados en
presencia de T. delbrueckii Viniferm NS-TD, aunque esta parece ser una característica
dependiente de cepa, discrepando con los resultados de ligero incremento mostrados por la
cepa de T. delbrueckii evaluada por Sun et al. (2014). Más significativo es el incremento en el
contenido en ácido pirúvico observado en los vinos fermentados en presencia de T.
delbrueckii Viniferm NS-TD, observándose picos máximos de liberación muy superiores a los
observados en las fermentaciones inoculadas exclusivamente con S. cerevisiae. En el
metabolismo del ácido pirúvico no debe considerarse su valor final en el vino, si no el valor
máximo de producción obtenido, normalmente durante la fermentación tumultuosa, ya que
mas tarde este subproducto metabólico es consumido como fuente de carbono. Además, la
cantidad de ácido pirúvico liberado en fermentación ha sido relacionada con la formación de
pigmentos como la vitisina A que aportan estabilidad al color de los vinos (Morata et al.,
2003, 2012). Ensayos previos de selección de cepas de S. cerevisiae en base a su liberación de
ácido pirúvico, lograban el aislamiento de cepas productoras de valores máximos entre 60 y
132 mg/L que, en cualquier caso son inferiores a los valores máximos obtenidos en el uso
secuencial de la cepa T. delbrueckii Viniferm NS-TD en nuestro ensayo que alcanza valores
medios cercanos a 160 mg/L en las fermentaciones a escala de laboratorio. Así, en estos
ensayos puede observarse una relación directa entre la cantidad de ácido pirúvico liberada y la
intensidad de color final de los vinos siendo ésta significativamente superior en los vinos
inoculados con T. delbrueckii exclusivamente y en fermentación secuencial con S. cerevisiae.
En paralelo a este incremento en la liberación de ácido pirúvico, estas dos fermentaciones
mostraron niveles superiores de glicerol lo que contribuye a confirmar la mayor actividad de
la ruta gliceropirúvica en T. delbrueckii con respecto a la mayoría de cepas de S. cerevisiae

211
Discusión
(Ciani y Maccarelli, 1998; Renault et al., 2009). Así mismo, esta mayor liberación de glicerol
lleva asociada una ligera reducción en el contenido en etanol de los vinos así como de otros
alcoholes superiores volátiles. Este hecho puede relacionarse parcialmente con la mejor
valoración general atribuida por el panel de cata en la calidad general y aromática de los vinos
fermentados mediante inoculación secuencial con T. delbrueckii, ya que ha sido probado el
incremento en la calidad y complejidad aromática de los vinos con ligeras reducciones en su
contenido en etanol y otros alcoholes por tender estos a la monopolización del aroma general
de los vinos (Frost et al., 2015). No obstante, las pequeñas diferencias que se obtuvieron en
estos parámetros analíticos no justificaban del todo la diferencia en las puntaciones que el
panel de cata otorgó a los vinos en los que destacaban tanto a nivel general como, en especial,
en el volumen en boca, los vinos fermentados con T. delbrueckii en inoculación secuencial
con S. cerevisiae. Esto llevó a la valoración del contenido en manoproteínas de los vinos
fermentados a escala semiindustrial, siendo éstos los que mostraron las diferencias mas
patentes a nivel organoléptico. Como puede observarse en el trabajo de Belda et al. (2015b)
contenido en la segunda parte del tercer capítulo de esta Tesis Doctoral, el contenido en
manoproteínas fue el parámetro que mostró unas mayores diferencias entre los distintos
ensayos, incrementándose su concentración en las fermentaciones que contaron con más
tiempo de desarrollo y actuación de la cepa T. delbrueckii Viniferm NS-TD. Así, en paralelo a
lo mostrado en el apartado anterior en referencia al impacto de esta cepa sobre el perfil
aromático de vinos blancos mediante su potente actividad β-liasa, se pudo concluir que su
mayor aportación a la calidad de los vinos tintos estaba determinada por su liberación de
manoproteínas al vino, corroborando lo sugerido previamente por Domizio et al. (2014).
En base a estos resultados y dada la falta de información en referencia al uso de

levaduras no-Saccharomyces en crianza sobre lías, el presente trabajo abordó el estudio de la
influencia de algunas de las especies no-Saccharomyces de mayor implantación en la
industria enológica actual (T. delbrueckii, L. thermotolerans y M. pulcherrima) sobre el perfil
sensorial de vinos tintos durante la crianza sobre lías.

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Discusión
7.4. Aplicación de levaduras no convencionales en fases postfermentativas de crianza

sobre lías
El último objetivo del presente trabajo consistió en la evaluación de la influencia del

uso de lías de levaduras no-Saccharomyces y cepas industriales seleccionadas de S. cerevisiae
(S. cerevisiae Viniferm CT007 como cepa control de uso habitual en bodega y S. cerevisiae
Viniferm 3D, como cepa superproductora de manoproteínas, ambas de la colección de
Agrovin S.A.) sobre la composición analítica y el perfil sensorial de vinos tintos. En el trabajo
de Belda et al. (2016d) se evaluó la evolución de parámetros analíticos básicos, parámetros de
color, perfil aromático, composición de aminoácidos y contenido en manoproteínas de vinos
tintos tras un periodo corto de 4 meses en crianza sobre lías, siendo éste el mayor foco de
interés en el estudio. En primer lugar, cabe destacar que no se observaron diferencias ni
desviaciones en los parámetros enológicos básicos evaluados, lo que prueba el correcto
desarrollo del proceso de crianza sin intervención alguna de bacterias o levaduras
contaminantes ajenas al estudio. En referencia al contenido en manoproteínas de los vinos, y
como era de esperar, se observaron diferencias muy significativas entre los ensayos en crianza
sobre lías de las dos cepas de S. cerevisiae en estudio, siendo los valores mostrados por la
cepa S. cerevisiae Viniferm 3D, 2,7 veces superiores que los mostrados por la cepa S.
cerevisiae Viniferm CT007. En la misma línea y, en consonancia con lo obtenido en la
comparación de las mismas cepas en su uso en fermentación alcohólica, los valores mostrados
en el uso de T. delbrueckii Viniferm NS-TD fueron de aproximadamente el triple con respecto
a la cepa S. cerevisiae Viniferm CT007. M . pulcherrima NS-EM-34 logró un incremento del
doble en el contenido en manoproteínas con respecto a la cepa S. cerevisiae Viniferm CT007,
lejos de los valores mostrados por S. cerevisiae Viniferm 3D y T. delbrueckii Viniferm NS-
TD. Estos resultados están en consonancia con el estudio de liberación de manoproteínas
llevado a cabo por Domizio et al. (2014) que mostraba que ambas especies presentaban ratios
de liberación de manoproteínas por peso seco de pared celular muy superiores a la cepa de
referencia S. cerevisiae EC1118. A pesar de los resultados mostrados para la cepa M .
pulcherrima NS-EM-34 en crianza sobre lías, cabe destacar que, en estudios industriales
llevados a cabo en nuestro grupo de investigación, en el contexto del trabajo de Belda et al.
(2016b), esta cepa no mostró un incremento significativo del contenido en manoproteínas en
su uso como inóculo mixto en fermentación. En este sentido, del Barrio-Galán et al. (2015),
describen un comportamiento cepa-dependiente en los resultados de liberación de

213
Discusión
manoproteínas durante la fase de fermentación alcohólica y durante la crianza sobre lías. Sus
resultados sugieren que cepas altamente liberadoras de manoproteínas durante la
fermentación, no necesariamente lo son durante la crianza sobre lías y vice-versa. Esto es
comprensible atendiendo a los mecanismos que determinan ambos procesos y que no tienen
porque estar igualmente regulados en las distintas cepas o especies. En el caso de la liberación
de manoproteínas en fermentación, ésta está relacionada con la propia división celular o con
procesos de respuesta a estrés (Charpentier et al., 1986; Fleet, 1991), mientras que durante el
proceso de crianza sobre lías su liberación al medio está determinada por la degradación de
biopolímeros por acción de endo-hidrolasas inducidas durante el proceso de lisis celular
(Feuillat et al., 1989; Fornairon-Bonnefond et al., 2002). Así, mientras que en el caso de la
cepa T. delbrueckii Viniferm NS-TD ambos procesos parecen determinar la liberación de
manoproteínas al vino, en el caso de M. pulcherrima NS-EM-34, su contribución al contenido
en manoproteínas de los vinos parece estar limitado a la fase postfermentativa de crianza
sobre lías. En cuanto a la cepa L. thermotolerans NS-G-32, ésta mostro niveles ligeramente
inferiores a los de la cepa S. cerevisiae Viniferm CT007, en relación con lo mostrado también
por Domizio et al. (2014) durante la fermentación alcohólica. Si bien estos resultados,
meramente observacionales, dan idea sobre la potencial aplicación de estas cepas en procesos
industriales de crianza sobre lías, los mecanismos moleculares que determinan la liberación
de estos compuestos en las distintas especies de interés deben ser evaluados en profundidad.
El contenido en manoproteínas de los vinos parece ser el factor que determinó la preferencia
en calidad de los vinos determinada por el panel de cata en su análisis sensorial, si bien, pudo
comprobarse una clara preferencia en parámetros como estructura, volumen en boca o
impresión general en aquellos ensayos con mayores concentraciones de manoproteínas y dado
que no se observaron diferencias notables en el perfil analítico de composición aromática de
los diferentes ensayos.
El incremento del contenido en manoproteínas de los vinos es uno de los principales

objetivos en el desarrollo de procesos de crianza sobre lías, no obstante, este proceso presenta
una influencia notable sobre la estabilidad e intensidad de color y la composición en
polifenoles de los vinos (Palomero et al., 2009; Loira et al., 2013). Aunque se ha descrito una
relación entre la presencia de manoproteínas y la estabilización del color en los vinos (Feuillat
et al., 2001; Fuster y Escot, 2002; Saucier et al., 2002) por prevención de la precipitación de
antocianos y taninos (Escot et al., 2001; Francois et al., 2007) y del proceso de oxidación de

214
Discusión
los polifenoles (Salmon, 2005) nuestros resultados no pueden ser del todo explicados por este
hecho. Todos los ensayos de crianza sobre lías mostraron un descenso en los parámetros de
color evaluados tras los 4 meses de crianza, aunque este descenso fue menos acusado en los
ensayos que generaron un incremento en el contenido en manoproteínas (M. pulcherrima NS-
EM-34 y T. delbrueckii Viniferm NS-TD). No obstante, los datos de intensidad de color
obtenidos en el ensayo usando L. thermotolerans NS-G-32 (cepa poco productora de
manoproteínas durante la crianza sobre lías) fueron similares a los obtenidos en el ensayo con
T. delbrueckii Viniferm NS-TD, por lo que otros factores adicionales al contenido en
manoproteínas, como por ejemplo la adsorción de pigmentos a la pared celular de dichas
cepas, determinada por la porosidad, juegan un papel clave en el proceso (Gómez-Cordovés y
Gonzalez-San José, 1995; Morata et al., 2003).
Finalmente, se evaluó el contenido en aminas biógenas y aminoácidos, como

potenciales precursores de estas, en los vinos tras la crianza sobre lías. Las aminas biógenas y,
en particular la histamina por su mayor prevalencia, son estudiadas con interés en el vino dada
su implicación negativa en la salud humana (Moreno-Arribas y Polo, 2008).
El contenido en aminoácidos de una célula es propio de la cepa (Vaughan-Martini et al.,

1979) y, por tanto, contribuirá de distinta forma al perfil de aminoácidos del vino tras la lisis
de las lías durante la crianza. Así, el contenido en aminoácidos en los distintos ensayos
evaluados en este trabajo mostró diferencias notables, destacando los elevados valores
mostrados en el uso de las lías de M. pulcherrima NS-EM-34. Un mayor contenido en
histidina, fenilalanina y tirosina podría implicar un mayor riesgo de aparición de las aminas
biógenas de las que éstos son precursores por acción de la actividad descarboxilasa de ciertas
bacterias (Lehtonen, 1996; Ribéreau-Gayon et al., 2006; Alcaide-Hidalgo et al., 2007; Smit et
al., 2008). Sin embargo, el desarrollo de estos ensayos en condiciones higiénicas y de asepsia
adecuados permitió obtener valores, en los distintos ensayos de crianza sobre lías, inferiores a
los mostrados por el vino control de partida en consonancia con lo mostrado en otras especies
no-Saccharomyces como Hanseniaspora vineae (Medina et al., 2013) y sin superar en ningún
caso los límites de máximos establecidos en seguridad alimentaria (Martuscelli et al., 2013).
En resumen, los resultados obtenidos permiten demostrar la utilidad de la cepa T.

delbrueckii Viniferm NS-TD para la mejora de la calidad de vinos tintos en procesos cortos

215
Discusión
de crianza sobre lías, obteniendo niveles de manoproteínas ligeramente superiores a los

mostrados por la cepa comercial superproductora de manoproteínas S. cerevisiae Viniferm
3D, sin detrimento de la estabilidad del color de dichos vinos y sin poner en riesgo la
seguridad alimentaria del vino al no contribuir a la producción de aminas biógenas o sus
precursores aminoacídicos.
7.5. Perspectivas futuras
La información disponible sobre el metabolismo y la fisiología en fermentación de las

levaduras no-Saccharomyces aumenta a un ritmo considerable, existiendo una intensa
investigación al respecto. Sin embargo, este conocimiento dista todavía del disponible para S.
cerevisiae y, quizá, del necesario para poder convertirse en una realidad industrial. En este
contexto, los resultados de la presente Tesis Doctoral contribuyen al conocimiento general
sobre la fisiología de estas levaduras, así como a la información básica acerca de su
diversidad fenotípica en cuanto a la producción de enzimas de interés enológico. Estos
resultados abren las puertas a una línea de investigación futura acerca de las bases genéticas y
transcripcionales de la fisiología de estas levaduras, que permita una mejor comprensión y
aprovechamiento industrial de sus especiales características metabólicas.
Comprendida la base genética, en S. cerevisiae, de muchos de los procesos metabólicos

de interés en el proceso de fermentación vínica, el incremento exponencial de la información
genómica disponible sobre las distintas especies de levadura permite la búsqueda de genes,
ortólogos a los identificados en S. cerevisiae, para el estudio de su funcionalidad y regulación.
De esta forma se podrán estudiar en detalle los mecanismos de adaptación y respuesta de las
distintas especies de levadura de interés a las condiciones de fermentación vínica
(temperatura, nutrición nitrogenada, coinoculación S. cerevisiae/no-Saccharomyces)
permitiendo un uso racional y optimizado de las mismas y dotándolas, por tanto, de una
mayor presencia en la industria.

216

8. CONCLUSIONES

217

218
Conclusiones
8. CONCLUSIONES
1. El estudio poblacional realizado, considerando un total de 770 aislamientos de

levaduras no-Saccharomyces procedentes de tres Denominaciones de Origen
durante tres vendimias consecutivas, no demuestra la existencia de una
distribución de especies característica de dichos orígenes y sostenida en el tiempo,
mostrándose como determinantes las prácticas agrícolas y la casuística
microclimática de la zona. La agrupación en clusters intraespecíficos de las
levaduras aisladas considerando su origen, y en base a características fenotípicas
con incidencia en la calidad del vino, está en consonancia con el concepto de
terroir microbiano como comunidad de microorganismos asociada a un territorio y
determinante de las propiedades sensoriales de sus vinos.
2. La diversidad de especies de levaduras no-Saccharomyces asociadas al vino

constituye un espacio fenotípico a explorar para la aplicación de caracteres de su
fisiología que contribuyan a la calidad y complejidad de los vinos fermentados
exclusivamente con Saccharomyces cerevisiae. La ausencia en la mayoría de cepas
de S. cerevisiae de ciertas actividades enzimáticas (β-glucosidasa, β-liasa,
pectinasa) o su baja actividad en condiciones de fermentación vínica, hace
interesante el estudio de dichas actividades en especies no-Saccharomyces que
puedan presentar distintas regulaciones metabólicas y mecanismos de adaptación
al entorno fermentativo.
3. El uso de la cepa Metschnikowia pulcherrima NS-EM-34, seleccionada en base a

su actividad pectinolítica, permitió la mejora de ciertos parámetros de calidad de
los vinos tintos, incrementando sus índices de color y mejorando el rendimiento en
el proceso de clarificación. La aplicación de dicha cepa en maceración
prefermentativa en frío multiplicó sus efectos positivos sobre la calidad de los
vinos tintos elaborados.
4. El medio de cultivo desarrollado basado en el uso de S-metil-L-Cisteína (SMC)

como única fuente de nitrógeno y de estructura análoga a los precursores
cisteinilados naturales de los aromas tiólicos presentes en la uva, resultó útil para

219
Conclusiones
la selección de levaduras con alta actividad β-liasa y, por tanto, para discernir el
potencial de liberación de aromas tiólicos en cepas de levaduras S. cerevisiae y no-
Saccharomyces.
En S. cerevisiae, conocida la existencia del gen IRC7 como principal responsable

de la liberación de tioles a partir de sus precursores cisteinilados, el medio de
cultivo descrito permitió diferenciar, en base a su crecimiento, las cepas que en
homocigosis presentan el gen IRC7 delecionado y, por tanto, con poca capacidad
de liberación de aromas tiólicos, de las cepas que en homocigosis o heterocigosis
presentan el gen IRC7 intacto y, en consecuencia, con mayor actividad β-liasa.
Estas últimas están poco representadas en en poblaciones autóctonas de S.
cerevisiae, por lo que el desarrollo de este medio de cultivo permite dirigir los
procesos de selección de levaduras en base a su actividad β-liasa.
5. El uso de SMC como análogo del sustrato natural en el mosto de uva (Cisteína-4-
MMP) de las enzimas con actividad β-liasa, también permitió desarrollar un
método simplificado para cuantificar la actividad β-liasa de las levaduras
seleccionadas. Los productos de la actividad β-liasa sobre dicho sustrato
(metanotiol, y su dímero dimetildisulfuro) fueron detectados por cromatografía de
gases acoplada a espectrometría de masas con resultados análogos a los obtenidos
en la detección del compuesto volátil natural (4-MMP), pero simplificando la
metodología requerida para su valoración.
6. La cepa T. delbrueckii Viniferm NS-TD mostró los mayores niveles de actividad

β-liasa entre una amplia colección de levaduras no-Saccharomyces evaluada,
seguida por la cepa Kluyveromyces marxianus NS-PDC-99. La aplicación de T.
delbrueckii Viniferm NS-TD en fermentación secuencial con S. cerevisiae en
mosto fresco de la variedad Verdejo mostró un incremento muy significativo en la
liberación tanto de 3-MH como de 4-MMP con independencia del genotipo IRC7
presentado por la cepa de S. cerevisiae con la que se coinocule.
7. El uso de la cepa T. delbrueckii Viniferm NS-TD, en fermentación secuencial con

S. cerevisiae en vinos tintos, mostró una mejora significativa en su calidad

220
Conclusiones
sensorial. Esta mejora se atribuyó al notable incremento en el contenido en

manoproteínas. Así mismo, cabe destacar el mantenimiento de la acidez volátil de
estos vinos en comparación con el control exclusivamente inoculado con S.
cerevisiae, así como una ligera reducción de su contenido en ácido málico y de la
liberación de alcoholes superiores.
8. Se confirmó el interés de determinadas cepas de levaduras no-Saccharomyces en

la liberación de manoproteínas en fases postfermentativas de crianza sobre lías. En
ellas, destacó la cepa T. delbrueckii Viniferm NS-TD, superando ligeramente los
valores mostrados por la cepa superproductora de manoproteínas S. cerevisiae
Viniferm 3D, así como la cepa M. pulcherrima NS-EM-34 que, aunque en valores
notablemente menores, logró duplicar el contenido en manoproteinas de los vinos
tratados con la cepa control S. cerevisiae Viniferm CT007.

221

222

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UNIVERSIDAD COMPLUTENSE DE MADRID

FACULTAD DE CIENCIAS BIOLÓGICAS

Estudio filo-funcional de levaduras de interés

MEMORIA PARA OPTAR AL GRADO DE DOCTOR

Ignacio Belda Aguilar

Antonio Santos de la Sen

© Ignacio Belda Aguilar, 2016

FACULTAD DE CIENCIAS BIOLÓGICAS

DEPARTAMENTO DE MICROBIOLOGÍA III

Estudio filo-funcional de levaduras de

interés enológico para su aplicación

MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR:

IGNACIO BELDA AGUILAR

Dr. Antonio Santos de la Sen

Dra. Eva Navascués López-Cordón

FACULTAD DE CIENCIAS BIOLÓGICAS

DEPARTAMENTO DE MICROBIOLOGÍA III

Estudio filo-funcional de levaduras de

interés enológico para su aplicación

IGNACIO BELDA AGUILAR

FACULTAD DE CIENCIAS BIOLÓGICAS

DEPARTAMENTO DE MICROBIOLOGÍA III

Estudio filo-funcional de levaduras de

interés enológico para su aplicación

Tesis Doctoral presentada por D. Ignacio Belda Aguilar para

Dr. Antonio Santos de la Sen Dra. Eva Navascués López-Cordón

“In hard times, imagination is more important than knowledge”

Obligatorio comenzar por agradecer a Antonio Santos la dedicación en cuerpo y alma

En el capítulo personal, gracias a mi familia, siempre. Una fe ciega y un cariño

SUMMARY .......................................................................................................... XVII

1.1. Microbiología, enología e industria enológica ................................................ 3

1.2. Las levaduras en enología................................................................................. 4

1.2.1. Saccharomyces cerevisiae............................................................................. 8

1.2.2. Levaduras no-Saccharomyces ..................................................................... 10

1.2.2.1. Aplicaciones actuales............................................................................. 10

1.2.2.2. Perspectivas de investigación y aplicaciones futuras ............................ 14

1.3. Microbiología y elaboración de vino.............................................................. 15

1.3.1. Etapa prefermentativa.................................................................................. 15

1.3.2. Fermentación alcohólica.............................................................................. 16

1.3.3. Etapa post fermentativa .............................................................................. 18

1.4. Incidencia de las levaduras en la composición del vino ............................... 19

1.4.1. Consumo de azúcares y liberación de etanol............................................... 19

1.4.3.1. Ácidos volátiles..................................................................................... 21

1.4.3.2. Ácidos no volátiles................................................................................ 22

1.4.3.2.1. Ácido málico .................................................................................. 22

1.4.3.2.2. Ácido láctico................................................................................... 23

1.4.4. Compuestos nitrogenados............................................................................ 23

1.4.5. Compuestos aromáticos............................................................................... 25

1.4.5.2. Alcoholes superiores y ácidos grasos volátiles..................................... 26

1.4.5.4. Compuestos azufrados .......................................................................... 29

3.1. Estudio de la diversidad de especies de levaduras asociadas a distintas

regiones vitivinícolas y estudio inter- e intraespecífico de la producción de enzimas de

interés enológico. ................................................................................................................ 37

4.1. Aplicación de levaduras pectinolíticas en maceración prefermentativa para

la mejora tecnológica de vinos tintos ................................................................................ 83

5. CAPÍTULO 3 ........................................................................................................... 113

5.1. Desarrollo de un métodos rápido para la selección de levaduras con elevada

actividad β-liasa................................................................................................................ 115

5.2. Caracterización de la fisiología en fermentación de Torulaspora delbrueckii y