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Handbook of EnologyVolume 1
The Microbiology of Wine and Vinifications2nd Edition
Pascal Ribéreau-GayonDenis DubourdieuBernard Donèche
Aline Lonvaud
Faculty of EnologyVictor Segalen University of Bordeaux II,
Talence, France
Original translation by
Jeffrey M. Branco, Jr.Winemaker
M.S., Faculty of Enology, University of Bordeaux II
Revision translated by
Christine RychlewskiAquitaine Traduction, Bordeaux, France
Innodata0470010355.jpg
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Handbook of EnologyVolume 1
The Microbiology of Wine and Vinifications2nd Edition
-
Handbook of EnologyVolume 1
The Microbiology of Wine and Vinifications2nd Edition
Pascal Ribéreau-GayonDenis DubourdieuBernard Donèche
Aline Lonvaud
Faculty of EnologyVictor Segalen University of Bordeaux II,
Talence, France
Original translation by
Jeffrey M. Branco, Jr.Winemaker
M.S., Faculty of Enology, University of Bordeaux II
Revision translated by
Christine RychlewskiAquitaine Traduction, Bordeaux, France
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Copyright 2006 John Wiley & Sons Ltd, The Atrium, Southern
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Library of Congress Cataloging-in-Publication Data:
Ribéreau-Gayon, Pascal.[Traité d’oenologie. English]Handbook
of enology / Pascal Ribéreau-Gayon, Denis Dubourdieu, Bernard
Donèche ; original translation by Jeffrey M. Branco, Jr.—2nd
ed. /translation of updates for 2nd ed. [by] Christine
Rychlewski.
v. cm.Rev. ed. of: Handbook of enology / Pascal Ribéreau Gayon
. . . [et al.].
c2000.Includes bibliographical references and index.Contents: v.
1. The microbiology of wine and vinificationsISBN-13:
978-0-470-01034-1 (v. 1 : acid-free paper)ISBN-10: 0-470-01034-7
(v. 1 : acid-free paper)
1. Wine and wine making—Handbooks, manuals, etc. 2. Wine and
winemaking—Microbiology—Handbooks, manuals, etc. 3. Wine and
winemaking—Chemistry—Handbooks, manuals, etc. I. Dubourdieu, Denis.
II.Donèche, Bernard. III. Traité d’oenologie. English. IV.
Title.
TP548.T7613 2005663′.2—dc22
2005013973
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British
Library
ISBN-13: 978-0-470-01034-1 (HB)ISBN-10: 0-470-01034-7 (HB)
Typeset in 10/12pt Times by Laserwords Private Limited, Chennai,
IndiaPrinted and bound in Great Britain by Antony Rowe Ltd,
Chippenham, WiltshireThis book is printed on acid-free paper
responsibly manufactured from sustainable forestryin which at least
two trees are planted for each one used for paper production.
http://www.wiley.com
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ContentsRemarks Concerning the Expression of Certain Parameters
of Must and Wine Composition viiPreface to the First Edition
ixPreface to the Second Edition xiii
1 Cytology, Taxonomy and Ecology of Grape and Wine Yeasts 1
2 Biochemistry of Alcoholic Fermentation and Metabolic Pathways
of Wine Yeasts 53
3 Conditions of Yeast Development 79
4 Lactic Acid Bacteria 115
5 Metabolism of Lactic Acid Bacteria 139
6 Lactic Acid Bacteria Development in Wine 161
7 Acetic Acid Bacteria 183
8 The Use of Sulfur Dioxide in Must and Wine Treatment 193
9 Products and Methods Complementing the Effect of Sulfur
Dioxide 223
10 The Grape and its Maturation 241
11 Harvest and Pre-Fermentation Treatments 299
12 Red Winemaking 327
13 White Winemaking 397
14 Other Winemaking Methods 445
Index 481
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Remarks Concerning the Expressionof Certain Parameters of
Mustand Wine CompositionUNITS
Metric system units of length (m), volume (l) andweight (g) are
exclusively used. The conversion ofmetric units into Imperial units
(inches, feet, gal-lons, pounds, etc.) can be found in the
followingenological work: Principles and practices of wine-making,
R.B. Boulton, V.L. Singleton, L.F. Bissonand R.E. Kunkee, 1995, The
Chapman & HallEnology Library, New York.
EXPRESSION OF TOTAL ACIDITYAND VOLATILE ACIDITY
Although EC regulations recommend the expres-sion of total
acidity in the equivalent weight of tar-taric acid, the French
custom is to give this expres-sion in the equivalent weight of
sulfuric acid. The
more correct expression in milliequivalents perliter has not
been embraced in France. The expres-sion of total and volatile
acidity in the equivalentweight of sulfuric acid has been used
predomi-nantly throughout these works. In certain cases,
thecorresponding weight in tartaric acid, often used inother
countries, has been given.
Using the weight of the milliequivalent of thevarious acids, the
below table permits the conver-sion from one expression to
another.
More particularly, to convert from total acidityexpressed in
H2SO4 to its expression in tartaricacid, add half of the value to
the original value(4 g/l H2SO4 → 6 g/l tartaric acid). In the
otherdirection a third of the value must be subtracted.
The French also continue to express volatileacidity in
equivalent weight of sulfuric acid. Moregenerally, in other
countries, volatile acidity is
Desired Expression
Known Expression meq/l g/l g/l g/lH2SO4 tartaric acid acetic
acid
meq/l 1.00 0.049 0.075 0.060
g/l H2SO4 20.40 1.00 1.53 1.22
g/l tartaric acid 13.33 0.65 1.00
g/l acetic acid 16.67 0.82 1.00
Multiplier to pass from one expression of total or volatile
acidity to another
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viii Remarks Concerning the Expression of Certain Parameters of
Must and Wine Composition
expressed in acetic acid. It is rarely expressedin
milliequivalents per liter. The below table alsoallows simple
conversion from one expression toanother.
The expression in acetic acid is approximately20% higher than in
sulfuric acid.
EVALUATING THE SUGARCONCENTRATION OF MUSTS
This measurement is important for tracking grapematuration,
fermentation kinetic and if necessarydetermining the eventual need
for chaptalization.
This measurement is always determined byphysical, densimetric or
refractometric analysis.The expression of the results can be given
accord-ing to several scales: some are rarely used, i.e.degree
Baumé and degree Oechsle. Presently, twosystems exist (Section
10.4.3):
1. The potential alcohol content (titre alcoomét-raque
potential or TAP, in French) of mustscan be read directly on
equipment, which isgraduated using a scale corresponding to 17.5or
17 g/l of sugar for 1% volume of alcohol.Today, the EC recommends
using 16.83 g/l asthe conversion factor. The ‘mustimeter’ is
ahydrometer containing two graduated scales:one expresses density
and the other gives adirect reading of the TAP. Different
methodsvarying in precision exist to calculate the TAPfrom a
density reading. These methods take var-ious elements of must
composition into account(Boulton et al., 1995).
2. Degree Brix expresses the percentage of sugarin weight. By
multiplying degree Brix by 10,the weight of sugar in 1 kg, or
slightly lessthan 1 liter, of must is obtained. A conversiontable
between degree Brix and TAP exists inSection 10.4.3 of this book.
17 degrees Brixcorrespond to an approximate TAP of 10% and20
degrees Brix correspond to a TAP of about12%. Within the alcohol
range most relevant toenology, degree Brix can be multiplied by
10
and then divided by 17 to obtain a fairly goodapproximation of
the TAP.
In any case, the determination of the Brix or TAPof a must is
approximate. First of all, it is notalways possible to obtain a
representative grapeor must sample for analysis. Secondly,
althoughphysical, densimetric or refractometric measure-ments are
extremely precise and rigorously expressthe sugar concentration of
a sugar and water mix-ture, these measurements are affected by
other sub-stances released into the sample from the grapeand other
sources. Furthermore, the concentrationsof these substances are
different for every grapeor grape must sample. Finally, the
conversion rateof sugar into alcohol (approximately 17 to 18
g/l)varies and depends on fermentation conditions andyeast
properties. The widespread use of selectedyeast strains has lowered
the sugar conversion rate.
Measurements Using Visibleand Ultraviolet SpectrometryThe
measurement of optic density, absorbance, iswidely used to
determine wine color (Volume 2,Section 6.4.5) and total phenolic
compounds con-centration (Volume 2, Section 6.4.1). In theseworks,
the optic density is noted as OD, OD 420(yellow), OD 520 (red), OD
620 (blue) or OD 280(absorption in ultraviolet spectrum) to
indicate theoptic density at the indicated wavelengths.
Wine color intensity is expressed as:
CI = OD 420 + OD 520 + OD 620,Or is sometimes expressed in a
more simplifiedform: CI = OD 420 + OD 520.
Tint is expressed as:
T = OD 420OD 520
The total phenolic compound concentration isexpressed by OD
280.
The analysis methods are described in Chapter 6of Handbook of
Enology Volume 2, The Chemistryof Wine.
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Preface to the First EditionWine has probably inspired more
research andpublications than any other beverage or food. Infact,
through their passion for wine, great scientistshave not only
contributed to the development ofpractical enology but have also
made discoveriesin the general field of science.
A forerunner of modern enology, Louis Pasteurdeveloped
simplified contagious infection mod-els for humans and animals
based on his obser-vations of wine spoilage. The following
quoteclearly expresses his theory in his own words:‘when profound
alterations of beer and wine areobserved because these liquids have
given refugeto microscopic organisms, introduced invisibly
andaccidentally into the medium where they thenproliferate, how can
one not be obsessed by thethought that a similar phenomenon can and
mustsometimes occur in humans and animals.’
Since the 19th century, our understanding ofwine, wine
composition and wine transformationshas greatly evolved in function
of advances in rel-evant scientific fields i.e. chemistry,
biochemistry,microbiology. Each applied development has leadto
better control of winemaking and aging con-ditions and of course
wine quality. In order tocontinue this approach, researchers and
winemak-ers must strive to remain up to date with the
latestscientific and technical developments in enology.
For a long time, the Bordeaux school of enologywas largely
responsible for the communication ofprogress in enology through the
publication ofnumerous works (Béranger Publications and laterDunod
Publications):
Wine Analysis U. Gayon and J. Laborde (1912);Treatise on Enology
J. Ribéreau-Gayon (1949);
Wine Analysis J. Ribéreau-Gayon and E. Peynaud(1947 and 1958);
Treatise on Enology (2 Volumes)J. Ribéreau-Gayon and E. Peynaud
(1960 and1961); Wine and Winemaking E. Peynaud (1971and 1981); Wine
Science and Technology (4 volu-mes) J. Ribéreau-Gayon, E. Peynaud,
P. Ribéreau-Gayon and P. Sudraud (1975–1982).
For an understanding of current advances inenology, the authors
propose this book Handbookof Enology Volume 1: The Microbiology of
Wineand Vinifications and the second volume of theHandbook of
Enology Volume 2: The Chemistry ofWine: Stabilization and
Treatments.
Although written by researchers, the two vol-umes are not
specifically addressed to this group.Young researchers may,
however, find these booksuseful to help situate their research
within a par-ticular field of enology. Today, the complexity
ofmodern enology does not permit a sole researcherto explore the
entire field.
These volumes are also of use to students andprofessionals.
Theoretical interpretations as wellas solutions are presented to
resolve the problemsencountered most often at wineries. The
authorshave adapted these solutions to many different sit-uations
and winemaking methods. In order to makethe best use of the
information contained in theseworks, enologists should have a broad
understand-ing of general scientific knowledge. For example,the
understanding and application of molecularbiology and genetic
engineering have becomeindispensable in the field of wine
microbiology.Similarly, structural and quantitative physiochem-ical
analysis methods such as chromatography,
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x Preface to the First Edition
NMR and mass spectrometry must now bemastered in order to
explore wine chemistry.
The goal of these two works was not to createan exhaustive
bibliography of each subject. Theauthors strove to choose only the
most relevant andsignificant publications to their particular field
ofresearch. A large number of references to Frenchenological
research has been included in theseworks in order to make this
information availableto a larger non-French-speaking audience.
In addition, the authors have tried to conveya French and more
particularly a Bordeaux per-spective of enology and the art of
winemaking.The objective of this perspective is to maximizethe
potential quality of grape crops based on thespecific natural
conditions that constitute their ‘ter-roir’. The role of enology is
to express the char-acteristics of the grape specific not only to
varietyand vineyard practices but also maturation condi-tions,
which are dictated by soil and climate.
It would, however, be an error to think that theworld’s greatest
wines are exclusively a result oftradition, established by
exceptional natural con-ditions, and that only the most ordinary
wines,produced in giant processing facilities, can ben-efit from
scientific and technological progress.Certainly, these facilities
do benefit the most fromhigh performance installations and
automation ofoperations. Yet, history has unequivocally shownthat
the most important enological developmentsin wine quality (for
example, malolactic fermenta-tion) have been discovered in ultra
premium wines.The corresponding techniques were then applied toless
prestigious products.
High performance technology is indispensablefor the production
of great wines, since a lackof control of winemaking parameters can
easilycompromise their quality, which would be less ofa problem
with lower quality wines.
The word ‘vinification’ has been used in thiswork and is part of
the technical language ofthe French tradition of winemaking.
Vinificationdescribes the first phase of winemaking. It com-prises
all technical aspects from grape maturityand harvest to the end of
alcoholic and some-times malolactic fermentation. The second
phaseof winemaking ‘winematuration, stabilization and
treatments’ is completed when the wine is bottled.Aging
specifically refers to the transformation ofbottled wine.
This distinction of two phases is certainly theresult of
commercial practices. Traditionally inFrance, a vine grower farmed
the vineyard andtransformed grapes into an unfinished wine. Thewine
merchant transferred the bulk wine to his cel-lars, finished the
wine and marketed the product,preferentially before bottling. Even
though mostwines are now bottled at the winery, these long-standing
practices have maintained a distinctionbetween ‘wine grower
enology’ and ‘wine mer-chant enology’. In countries with a more
recentviticultural history, generally English speaking, thevine
grower is responsible for winemaking andwine sales. For this
reason, the Anglo-Saxon tradi-tion speaks of winemaking, which
covers all oper-ations from harvest reception to bottling.
In these works, the distinction between ‘vinifi-cation’ and
‘stabilization and treatments’ has beenmaintained, since the first
phase primarily concernsmicrobiology and the second chemistry. In
thismanner, the individual operations could be linkedto their
particular sciences. There are of course lim-its to this approach.
Chemical phenomena occurduring vinification; the stabilization of
wines dur-ing storage includes the prevention of
microbialcontamination.
Consequently, the description of the differentsteps of enology
does not always obey logic asprecise as the titles of these works
may leadto believe. For example, microbial contaminationduring
aging and storage are covered in Vol-ume 1. The antiseptic
properties of SO2 incited thedescription of its use in the same
volume. This lineof reasoning lead to the description of the
antioxi-dant related chemical properties of this compoundin the
same chapter as well as an explanation ofadjuvants to sulfur
dioxide: sorbic acid (antisep-tic) and ascorbic acid (antioxidant).
In addition,the on lees aging of white wines and the result-ing
chemical transformations cannot be separatedfrom vinification and
are therefore also coveredin Volume 1. Finally, our understanding
of pheno-lic compounds in red wine is based on complexchemistry.
All aspects related to the nature of the
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Preface to the First Edition xi
corresponding substances, their properties and theirevolution
during grape maturation, vinification andaging are therefore
covered in Volume 2.
These works only discuss the principles ofequipment used for
various enological operationsand their effect on product quality.
For example,temperature control systems, destemmers, crushersand
presses as well as filters, inverse osmosismachines and ion
exchangers are not described indetail. Bottling is not addressed at
all. An in-depthdescription of enological equipment would merit
adetailed work dedicated to the subject.
Wine tasting, another essential role of thewinemaker, is not
addressed in these works.Many related publications are, however,
readilyavailable. Finally, wine analysis is an essential toolthat a
winemaker should master. It is, however, notcovered in these works
except in a few particular
cases i.e. phenolic compounds, whose differentfamilies are often
defined by analytical criteria.
The authors thank the following people whohave contributed to
the creation of this work:J.F. Casas Lucas, Chapter 14, Sherry; A.
Brugi-rard, Chapter 14, Sweet wines; J.N. de Almeida,Chapter 14,
Port wines; A. Maujean, Chapter 14,Champagne; C. Poupot for the
preparation ofmaterial in Chapters 1, 2 and 13; Miss F. Luye-Tanet
for her help with typing.
They also thank Madame B. Masclef in particu-lar for her
important part in the typing, preparationand revision of the final
manuscript.
Pascal Ribéreau-GayonBordeaux
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Preface to the Second EditionThe two-volume Enology Handbook was
pub-lished simultaneously in Spanish, French, and Ital-ian in 1999
and has been reprinted several times.The Handbook has apparently
been popular withstudents as an educational reference book, as
wellas with winemakers, as a source of practical solu-tions to
their specific technical problems and sci-entific explanations of
the phenomena involved.
It was felt appropriate at this stage to preparean updated,
reviewed, corrected version, includingthe latest enological
knowledge, to reflect the manynew research findings in this very
active field. Theoutline and design of both volumes remain thesame.
Some chapters have changed relatively littleas the authors decided
there had not been any sig-nificant new developments, while others
have beenmodified much more extensively, either to clarifyand
improve the text, or, more usually, to includenew research findings
and their practical applica-tions. Entirely new sections have been
inserted insome chapters.
We have made every effort to maintain the sameapproach as we did
in the first edition, reflectingthe ethos of enology research in
Bordeaux. We useindisputable scientific evidence in
microbiology,biochemistry, and chemistry to explain the detailsof
mechanisms involved in grape ripening, fermen-tations and other
winemaking operations, aging,and stabilization. The aim is to help
winemakersachieve greater control over the various stages
inwinemaking and choose the solution best suitedto each situation.
Quite remarkably, this scientificapproach, most intensively applied
in making thefinest wines, has resulted in an enhanced capac-ity to
bring out the full quality and character of
individual terroirs. Scientific winemaking has notresulted in
standardization or leveling of quality.On the contrary, by making
it possible to correctdefects and eliminate technical
imperfections, ithas revealed the specific qualities of the
grapesharvested in different vineyards, directly related tothe
variety and terroir, more than ever before.
Interest in wine in recent decades has gonebeyond considerations
of mere quality and takenon a truly cultural dimension. This has
led somepeople to promote the use of a variety of tech-niques that
do not necessarily represent significantprogress in winemaking.
Some of these are sim-ply modified forms of processes that have
beenknown for many years. Others do not have a suf-ficiently
reliable scientific interpretation, nor aretheir applications
clearly defined. In this Hand-book, we have only included
rigorously testedtechniques, clearly specifying the optimum
con-ditions for their utilization.
As in the previous edition, we deliberatelyomitted three
significant aspects of enology: wineanalysis, tasting, and winery
engineering. In viewof their importance, these topics will each
becovered in separate publications.
The authors would like to take the opportunityof the publication
of this new edition of Volume 1to thank all those who have
contributed to updatingthis work:
— Marina Bely for her work on fermentationkinetics (Section 3.4)
and the production ofvolatile acidity (Sections 2.3.4 and
14.2.5)
— Isabelle Masneuf for her investigation of theyeasts’ nitrogen
supply (Section 3.4.2)
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xiv Preface to the Second Edition
— Gilles de Revel for elucidating the chemistryof SO2,
particularly, details of combinationreactions (Section 8.4)
— Gilles Masson for the section on rosé wines(Section 14.1)
— Cornelis Van Leeuwen for data on the impactof vineyard water
supply on grape ripening(Section 10.4.6)
— André Brugirard for the section on Frenchfortified wines—vins
doux naturels (Section14.4.2)
— Paulo Barros and Joa Nicolau de Almeida fortheir work on Port
(Section 14.4.3)
— Justo. F. Casas Lucas for the paragraph onSherry (Section
14.5.2)
— Alain Maujean for his in-depth revision of thesection on
Champagne (Section 14.3).
March 17, 2005
Professor Pascal RIBEREAU-GAYONCorresponding Member of the
InstituteMember of the French Academy of Agriculture
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1
Cytology, Taxonomy and Ecologyof Grape and Wine Yeasts
1.1 Introduction 11.2 The cell wall 31.3 The plasmic membrane
71.4 The cytoplasm and its organelles 111.5 The nucleus 141.6
Reproduction and the yeast biological cycle 151.7 The killer
phenomenon 191.8 Classification of yeast species 221.9
Identification of wine yeast strains 35
1.10 Ecology of grape and wine yeasts 40
1.1 INTRODUCTION
Man has been making bread and fermented bev-erages since the
beginning of recorded history.Yet the role of yeasts in alcoholic
fermentation,particularly in the transformation of grapes intowine,
was only clearly established in the middleof the nineteenth
century. The ancients explainedthe boiling during fermentation
(from the Latinfervere, to boil) as a reaction between
substances
that come into contact with each other duringcrushing. In 1680,
a Dutch cloth merchant, Antonievan Leeuwenhoek, first observed
yeasts in beerwort using a microscope that he designed andproduced.
He did not, however, establish a rela-tionship between these
corpuscles and alcoholicfermentation. It was not until the end of
the eigh-teenth century that Lavoisier began the chemicalstudy of
alcoholic fermentation. Gay-Lussac con-tinued Lavoisier’s research
into the next century.
Handbook of Enology Volume 1 The Microbiology of Wine and
Vinifications P. Ribéreau-Gayon, D. Dubourdieu, B. Donèche and A.
Lonvaud 2006 John Wiley & Sons, Ltd
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2 Handbook of Enology: The Microbiology of Wine and
Vinifications
As early as 1785, Fabroni, an Italian scientist, wasthe first to
provide an interpretation of the chem-ical composition of the
ferment responsible foralcoholic fermentation, which he described
as aplant–animal substance. According to Fabroni, thismaterial,
comparable to the gluten in flour, waslocated in special utricles,
particularly on grapesand wheat, and alcoholic fermentation
occurredwhen it came into contact with sugar in the must. In1837, a
French physicist named Charles Cagnardde La Tour proved for the
first time that the yeastwas a living organism. According to his
findings,it was capable of multiplying and belonged to theplant
kingdom; its vital activities were at the baseof the fermentation
of sugar-containing liquids.The German naturalist Schwann confirmed
his the-ory and demonstrated that heat and certain chem-ical
products were capable of stopping alcoholicfermentation. He named
the beer yeast zucker-pilz, which means sugar
fungus—Saccharomycesin Latin. In 1838, Meyen used this
nomenclaturefor the first time.
This vitalist or biological viewpoint of the roleof yeasts in
alcoholic fermentation, obvious tous today, was not readily
supported. Liebig andcertain other organic chemists were convinced
thatchemical reactions, not living cellular activity,were
responsible for the fermentation of sugar.In his famous studies on
wine (1866) and beer(1876), Louis Pasteur gave definitive
credibilityto the vitalist viewpoint of alcoholic fermentation.He
demonstrated that the yeasts responsible forspontaneous
fermentation of grape must or crushedgrapes came from the surface
of the grape;he isolated several races and species. He
evenconceived the notion that the nature of the yeastcarrying out
the alcoholic fermentation couldinfluence the gustatory
characteristics of wine. Healso demonstrated the effect of oxygen
on theassimilation of sugar by yeasts. Louis Pasteurproved that the
yeast produced secondary productssuch as glycerol in addition to
alcohol and carbondioxide.
Since Pasteur, yeasts and alcoholic fermen-tation have incited a
considerable amount ofresearch, making use of progress in
microbiology,
biochemistry and now genetics and molecularbiology.
In taxonomy, scientists define yeasts as unicel-lular fungi that
reproduce by budding and binaryfission. Certain pluricellular fungi
have a unicellu-lar stage and are also grouped with yeasts.
Yeastsform a complex and heterogeneous group foundin three classes
of fungi, characterized by theirreproduction mode: the sac fungi
(Ascomycetes),the club fungi (Basidiomycetes), and the imper-fect
fungi (Deuteromycetes). The yeasts found onthe surface of the grape
and in wine belong toAscomycetes and Deuteromycetes. The
haploidspores or ascospores of the Ascomycetes class arecontained
in the ascus, a type of sac made fromvegetative cells. Asporiferous
yeasts, incapable ofsexual reproduction, are classified with the
imper-fect fungi.
In this first chapter, the morphology, repro-duction, taxonomy
and ecology of grape andwine yeasts will be discussed. Cytology is
themorphological and functional study of the struc-tural components
of the cell (Rose and Harrison,1991).
Fig. 1.1. A yeast cell (Gaillardin and Heslot, 1987)
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Cytology, Taxonomy and Ecology of Grape and Wine Yeasts 3
Yeasts are the most simple of the eucaryotes.The yeast cell
contains cellular envelopes, acytoplasm with various organelles,
and a nucleussurrounded by a membrane and enclosing thechromosomes.
(Figure 1.1). Like all plant cells,the yeast cell has two cellular
envelopes: thecell wall and the membrane. The periplasmicspace is
the space between the cell wall andthe membrane. The cytoplasm and
the membranemake up the protoplasm. The term protoplastor
sphaeroplast designates a cell whose cellwall has been artificially
removed. Yeast cellularenvelopes play an essential role: they
contributeto a successful alcoholic fermentation and releasecertain
constituents which add to the resultingwine’s composition. In order
to take advantage ofthese properties, the winemaker or enologist
musthave a profound knowledge of these organelles.
1.2 THE CELL WALL
1.2.1 The General Roleof the Cell Wall
During the last 20 years, researchers (Fleet, 1991;Klis, 1994;
Stratford, 1999; Klis et al., 2002) havegreatly expanded our
knowledge of the yeast cellwall, which represents 15–25% of the dry
weightof the cell. It essentially consists of polysaccha-rides. It
is a rigid envelope, yet endowed with acertain elasticity.
Its first function is to protect the cell. Withoutits wall, the
cell would burst under the internalosmotic pressure, determined by
the compositionof the cell’s environment. Protoplasts placed inpure
water are immediately lysed in this manner.Cell wall elasticity can
be demonstrated by placingyeasts, taken during their log phase, in
a hypertonic(NaCl) solution. Their cellular volume decreasesby
approximately 50%. The cell wall appearsthicker and is almost in
contact with the membrane.The cells regain their initial form after
being placedback into an isotonic medium.
Yet the cell wall cannot be considered an inert,semi-rigid
‘armor’. On the contrary, it is a dynamicand multifunctional
organelle. Its composition andfunctions evolve during the life of
the cell, in
response to environmental factors. In addition toits protective
role, the cell wall gives the cellits particular shape through its
macromolecularorganization. It is also the site of moleculeswhich
determine certain cellular interactions suchas sexual union,
flocculation, and the killerfactor, which will be examined in
detail later inthis chapter (Section 1.7). Finally, a number
ofenzymes, generally hydrolases, are connected tothe cell wall or
situated in the periplasmic space.Their substrates are nutritive
substances of theenvironment and the macromolecules of the cellwall
itself, which is constantly reshaped duringcellular
morphogenesis.
1.2.2 The Chemical Structureand Function of the
ParietalConstituents
The yeast cell wall is made up of two prin-cipal constituents:
β-glucans and mannoproteins.Chitin represents a minute part of its
composi-tion. The most detailed work on the yeast cellwall has been
carried out on Saccharomyces cere-visiae —the principal yeast
responsible for thealcoholic fermentation of grape must.
Glucan represents about 60% of the dry weightof the cell wall of
S. cerevisiae. It can bechemically fractionated into three
categories:
1. Fibrous β-1,3 glucan is insoluble in water,acetic acid and
alkali. It has very few branches.The branch points involved are
β-1,6 linkages.Its degree of polymerization is 1500. Underthe
electron microscope, this glucan appearsfibrous. It ensures the
shape and the rigidity ofthe cell wall. It is always connected to
chitin.
2. Amorphous β-1,3 glucan, with about 1500glucose units, is
insoluble in water but solublein alkalis. It has very few branches,
like thepreceding glucan. In addition to these fewbranches, it is
made up of a small number ofβ-1,6 glycosidic linkages. It has an
amorphousaspect under the electron microscope. It givesthe cell
wall its elasticity and acts as an anchorfor the mannoproteins. It
can also constitute anextraprotoplasmic reserve substance.
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4 Handbook of Enology: The Microbiology of Wine and
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3. The β-1,6 glucan is obtained from alkali-insoluble glucans by
extraction in acetic acid.The resulting product is amorphous, water
sol-uble, and extensively ramified by β-1,3 glyco-sidic linkages.
Its degree of polymerization is140. It links the different
constituents of thecell wall together. It is also a receptor site
forthe killer factor.
The fibrous β-1,3 glucan (alkali-insoluble) proba-bly results
from the incorporation of chitin on theamorphous β-1,3 glucan.
Mannoproteins constitute 25–50% of the cellwall of S.
cerevisiae. They can be extracted fromthe whole cell or from the
isolated cell wallby chemical and enzymatic methods.
Chemicalmethods make use of autoclaving in the pres-ence of alkali
or a citrate buffer solution atpH 7. The enzymatic method frees the
manno-proteins by digesting the glucan. This methoddoes not
denature the structure of the mannopro-teins as much as chemical
methods. Zymolyase,obtained from the bacterium Arthrobacter
luteus,is the enzymatic preparation most often used toextract the
parietal mannoproteins of S. cerevisiae.This enzymatic complex is
effective primarilybecause of its β-1,3 glucanase activity. The
actionof protease contaminants in the zymolyase com-bine, with the
aforementioned activity to liberatethe mannoproteins. Glucanex,
another industrialpreparation of the β-glucanase, produced by a
fun-gus (Trichoderma harzianum), has been recentlydemonstrated to
possess endo- and exo-β-1,3 andendo-β-1,6-glucanase activities
(Dubourdieu andMoine, 1995). These activities also facilitate
theextraction of the cell wall mannoproteins of theS. cerevisiae
cell.
The mannoproteins of S. cerevisiae have amolecular weight
between 20 and 450 kDa. Theirdegree of glycosylation varies.
Certain ones con-taining about 90% mannose and 10% peptides
arehypermannosylated.
Four forms of glycosylation are described(Figure 1.2) but do not
necessarily exist at thesame time in all of the mannoproteins.
The mannose of the mannoproteins can consti-tute short, linear
chains with one to five residues.
They are linked to the peptide chain by O-glycosyllinkages on
serine and threonine residues. Theseglycosidic side-chain linkages
are α-1,2 and α-1,3.
The glucidic part of the mannoprotein can alsobe a
polysaccharide. It is linked to an asparagineresidue of the peptide
chain by an N -glycosyllinkage. This linkage consists of a double
unit ofN -acetylglucosamine (chitin) linked in β-1,4. Themannan
linked in this manner to the asparagineincludes an attachment
region made up of a dozenmannose residues and a highly ramified
outerchain consisting of 150 to 250 mannose units.The attachment
region beyond the chitin residueconsists of a mannose skeleton
linked in α-1,6with side branches possessing one, two or
threemannose residues with α-1,2 and/or α-1,3 bonds.The outer chain
is also made up of a skeleton ofmannose units linked in α-1,6. This
chain bearsshort side-chains constituted of mannose residueslinked
in α-1,2 and a terminal mannose in α-1,3. Some of these side-chains
possess a branchattached by a phosphodiester bond.
A third type of glycosylation was describedmore recently. It can
occur in mannoproteins,which make up the cell wall of the yeast. It
consistsof a glucomannan chain containing essentiallymannose
residues linked in α-1,6 and glucoseresidues linked in α-1,6. The
nature of the glycan–peptide point of attachment is not yet clear,
but itmay be an asparaginyl–glucose bond. This type ofglycosylation
characterizes the proteins freed fromthe cell wall by the action of
a β-1,3 glucanase.Therefore, in vivo, the glucomannan chain mayalso
comprise glucose residues linked in β-1,3.
The fourth type of glycosylation of yeast manno-proteins is the
glycosyl–phosphatidyl–inositolanchor (GPI). This attachment between
the ter-minal carboxylic group of the peptide chain anda membrane
phospholipid permits certain manno-proteins, which cross the cell
wall, to anchorthemselves in the plasmic membrane. The regionof
attachment is characterized by the followingsequence (Figure 1.2):
ethanolamine-phosphate-6-mannose-α-1,2-mannose-α-1,6-mannose-α-1,4-glucosamine-α-1,6-inositol-phospholipid.
A C-phospholipase specific to phosphatidyl inositoland therefore
capable of realizing this cleavage
-
Cytology, Taxonomy and Ecology of Grape and Wine Yeasts 5
6M[M 6M 6M 6M ]n 6M 6M
2
M
2
M
2
M
2
M2
M
2
M
2
M
3
M
3
M
3
M
P
M
3
M
2
M3
M
3
M
2
M P2
M
3
2 3
M M
MP 6M6
Mβ 4 GNAcβ 4 GNAcβ NH Asn
3M 3M 2M 2M O Ser/Thr
(G,M) Xxx
lipid P Ins 6 GN 4 M 6 M 2 M 6 P (CH2)2 NH C O
Fig. 1.2. The four types of glucosylation of parietal yeast
mannoproteins (Klis, 1994). M = mannose; G = glucose;GN =
glucosamine; GNAc = N-acetylglucosamine; Ins = inositol; Ser =
Serine; Thr = threonine; Asn = asparagine;Xxx = the nature of the
bond is not known
was demonstrated in the S. cerevisiae (Flick andThorner, 1993).
Several GPI-type anchor manno-proteins have been identified in the
cell wall ofS. cerevisiae.
Chitin is a linear polymer of N -acetylglucos-amine linked in
β-1,4 and is not generally found inlarge quantities in yeast cell
walls. In S. cerevisiae,chitin constitutes 1–2% of the cell wall
and isfound for the most part (but not exclusively) inbud scar
zones. These zones are a type of raisedcrater easily seen on the
mother cell under theelectron microscope (Figure 1.3). This
chitinic scaris formed essentially to assure cell wall integrityand
cell survival. Yeasts treated with D polyoxine,an antibiotic
inhibiting the synthesis of chitin, arenot viable; they burst after
budding.
The presence of lipids in the cell wall has notbeen clearly
demonstrated. It is true that cell walls
Fig. 1.3. Scanning electron microscope photograph
ofproliferating S. cerevisiae cells. The budding scars onthe mother
cells can be observed
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6 Handbook of Enology: The Microbiology of Wine and
Vinifications
prepared in the laboratory contain some lipids(2–15% for S.
cerevisiae) but it is most likelycontamination by the lipids of the
cytoplasmicmembrane, adsorbed by the cell wall during
theirisolation. The cell wall can also adsorb lipids fromits
external environment, especially the differentfatty acids that
activate and inhibit the fermentation(Chapter 3).
Chitin are connected to the cell wall or sit-uated in the
periplasmic space. One of themost characteristic enzymes is the
invertase (β-fructofuranosidase). This enzyme catalyzes
thehydrolysis of saccharose into glucose and fruc-tose. It is a
thermostable mannoprotein anchoredto a β-1,6 glucan of the cell
wall. Its molecularweight is 270 000 Da. It contains
approximately50% mannose and 50% protein. The periplasmicacid
phosphatase is equally a mannoprotein.
Other periplasmic enzymes that have been notedare β-glucosidase,
α-galactosidase, melibiase, tre-halase, aminopeptidase and
esterase. Yeast cellwalls also contain endo- and exo-β-glucanases
(β-1,3 and β-1,6). These enzymes are involved in thereshaping of
the cell wall during the growth andbudding of cells. Their activity
is at a maximumduring the exponential log phase of the
populationand diminishes notably afterwards. Yet cells in
thestationary phase and even dead yeasts containedin the lees still
retain β-glucanases activity intheir cell walls several months
after the completionof fermentation. These endogenous enzymes
areinvolved in the autolysis of the cell wall during the
ageing of wines on lees. This ageing method willbe covered in
the chapter on white winemaking(Chapter 13).
1.2.3 General Organization of the CellWall and Factors Affecting
itsComposition
The cell wall of S. cerevisiae is made up of anouter layer of
mannoproteins. These mannopro-teins are connected to a matrix of
amorphous β-1,3glucan which covers an inner layer of fibrous β-1,3
glucan. The inner layer is connected to a smallquantity of chitin
(Figure 1.4). The β-1,6 glucanprobably acts as a cement between the
two lay-ers. The rigidity and the shape of the cell wallare due to
the internal framework of the β-1,3fibrous glucan. Its elasticity
is due to the outeramorphous layer. The intermolecular structure
ofthe mannoproteins of the outer layer (hydrophobiclinkages and
disulfur bonds) equally determinescell wall porosity and
impermeability to macro-molecules (molecular weights less than
4500). Thisimpermeability can be affected by treating thecell wall
with certain chemical agents, such asβ-mercaptoethanol. This
substance provokes therupture of the disulfur bonds, thus
destroying theintermolecular network between the
mannoproteinchains.
The composition of the cell wall is stronglyinfluenced by
nutritive conditions and cell age.The proportion of glucan in the
cell wall increases
Cytoplasm
Cytoplasmic membrane
Mannoproteins and β-1,3 amorphous glucan
β - 1,3 fibrous glucan
Cell wall
Periplasmic space
External medium
Fig. 1.4. Cellular organization of the cell wall of S.
cerevisiae
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Cytology, Taxonomy and Ecology of Grape and Wine Yeasts 7
with respect to the amount of sugar in the cul-ture medium.
Certain deficiencies (for example,in mesoinositol) also result in
an increase in theproportion of glucan compared with
mannopro-teins. The cell walls of older cells are richer inglucans
and in chitin and less furnished in manno-proteins. For this
reason, they are more resistantto physical and enzymatic agents
used to degradethem. Finally, the composition of the cell wall
isprofoundly modified by morphogenetic alterations(conjugation and
sporulation).
1.3 THE PLASMIC MEMBRANE
1.3.1 Chemical Compositionand Organization
The plasmic membrane is a highly selective barriercontrolling
exchanges between the living cell andits external environment. This
organelle is essentialto the life of the yeast.
Like all biological membranes, the yeast plasmicmembrane is
principally made up of lipids andproteins. The plasmic membrane of
S. cerevisiaecontains about 40% lipids and 50% proteins.Glucans and
mannans are only present in smallquantities (several per cent).
The lipids of the membrane are essentiallyphospholipids and
sterols. They are amphiphilicmolecules, i.e. possessing a
hydrophilic and ahydrophobic part.
The three principal phospholipids (Figure 1.5)of the plasmic
membrane of yeast are phos-phatidylethanolamine (PE),
phosphatidylcholine(PC) and phosphatidylinositol (PI) which
repre-sent 70–85% of the total. Phosphatidylserine (PS)and
diphosphatidylglycerol or cardiolipin (PG) areless prevalent. Free
fatty acids and phosphatidicacid are frequently reported in plasmic
membraneanalysis. They are probably extraction artifactscaused by
the activity of certain lipid degradationenzymes.
The fatty acids of the membrane phospholipidscontain an even
number (14 to 24) of carbon atoms.The most abundant are C16 and C18
acids. Theycan be saturated, such as palmitic acid (C16) andstearic
acid (C18), or unsaturated, as with oleic
acid (C18, double bond in position 9), linoleic acid(C18, two
double bonds in positions 9 and 12) andlinolenic acid (C18, three
double bonds in positions9, 12 and 15). All membrane phospholipids
sharea common characteristic: they possess a polar orhydrophilic
part made up of a phosphorylatedalcohol and a non-polar or
hydrophobic partcomprising two more or less parallel fatty
acidchains (Figure 1.6). In an aqueous medium, thephospholipids
spontaneously form bimolecularfilms or a lipid bilayer because of
their amphiphiliccharacteristic (Figure 1.6). The lipid bilayers
arecooperative but non-covalent structures. Theyare maintained in
place by mutually reinforcedinteractions: hydrophobic interactions,
van derWaals attractive forces between the hydrocarbontails,
hydrostatic interactions and hydrogen bondsbetween the polar heads
and water molecules.The examination of cross-sections of
yeastplasmic membrane under the electron microscopereveals a
classic lipid bilayer structure with athickness of about 7.5 nm.
The membrane surfaceappears sculped with creases, especially
duringthe stationary phase. However, the physiologicalmeaning of
this anatomic character remainsunknown. The plasmic membrane also
has anunderlying depression on the bud scar.
Ergosterol is the primary sterol of the yeast plas-mic membrane.
In lesser quantities, 24 (28) dehy-droergosterol and zymosterol
also exist (Figure1.7). Sterols are exclusively produced in the
mito-chondria during the yeast log phase. As with phos-pholipids,
membrane sterols are amphipathic. Thehydrophilic part is made up of
hydroxyl groupsin C-3. The rest of the molecule is
hydrophobic,especially the flexible hydrocarbon tail.
The plasmic membrane also contains numerousproteins or
glycoproteins presenting a wide rangeof molecular weights (from 10
000 to 120 000).The available information indicates that the
orga-nization of the plasmic membrane of a yeast cellresembles the
fluid mosaic model. This model,proposed for biological membranes by
Singer andNicolson (1972), consists of two-dimensional solu-tions
of proteins and oriented lipids. Certain pro-teins are embedded in
the membrane; they arecalled integral proteins (Figure 1.6). They
interact
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8 Handbook of Enology: The Microbiology of Wine and
Vinifications
R' C O
O
CH
H2C O P
O
O−
O CH2 CH2 NH3+
Phosphatidyl ethanolamine
R C
O
O
R' C
O
O
CH2
CH
H2C O P
O
O−
O CH2 C
H
COO−
NH3+
Phosphatidyl serine
OHOH
H H
O
H
OHH
H
HO
OH H
P
O
O
O−
CH2
HC
H2C
O
O C
C
O
O
R'
R
Phosphatidyl inositol
R C O
O
CH2
CHOCR'
O H2C O P
O
O−
O CH2 CH2 N+(CH3)3
Phosphatidyl choline
R C
O
O CH2
CHOCR'
O H2C O P
O
O−
O CH2 C CH2 O P
O
O−
O CH2
HC O
H2C O
C
C R
O
R'
O
Diphosphatidyl glycerol (cardiolipin)
R C
O
O CH2
Fig. 1.5. Yeast membrane phospholipids
strongly with the non-polar part of the lipid bilayer.The
peripheral proteins are linked to the precedentby hydrogen bonds.
Their location is asymmetrical,at either the inner or the outer
side of the plasmicmembrane. The molecules of proteins and
mem-brane lipids, constantly in lateral movement, arecapable of
rapidly diffusing in the membrane.
Some of the yeast membrane proteins have beenstudied in greater
depth. These include adenosinetriphosphatase (ATPase), solute
(sugars and amino
acids) transport proteins, and enzymes involved inthe production
of glucans and chitin of the cellwall.
The yeast possesses three ATPases: in the mito-chondria, the
vacuole, and the plasmic membrane.The plasmic membrane ATPase is an
integral pro-tein with a molecular weight of around 100 Da.
Itcatalyzes the hydrolysis of ATP which furnishesthe necessary
energy for the active transport ofsolutes across the membrane.
(Note: an active
-
Cytology, Taxonomy and Ecology of Grape and Wine Yeasts 9
Polar head: phosphorylated alcohol
Hydrocarbon tails: fatty acid chains
a
b
Fig. 1.6. A membrane lipid bilayer. The integralproteins (a) are
strongly associated to the non-polarregion of the bilayer. The
peripheral proteins (b) arelinked to the integral proteins
transport moves a compound against the concen-tration gradient.)
Simultaneously, the hydrolysis ofATP creates an efflux of protons
towards the exte-rior of the cell.
The penetration of amino acids and sugarsinto the yeast
activates membrane transport sys-tems called permeases. The general
amino acid
permease (GAP) contains three membrane proteinsand ensures the
transport of a number of neutralamino acids. The cultivation of
yeasts in the pres-ence of an easily assimilated nitrogen-based
nutri-ent such as ammonium represses this permease.
The membrane composition in fatty acids andits proportion in
sterols control its fluidity. Thehydrocarbon chains of fatty acids
of the membranephospholipid bilayer can be in a rigid and
orderlystate or in a relatively disorderly and fluid state. Inthe
rigid state, some or all of the carbon bondsof the fatty acids are
trans. In the fluid state,some of the bonds become cis. The
transitionfrom the rigid state to the fluid state takes placewhen
the temperature rises beyond the fusiontemperature. This transition
temperature dependson the length of the fatty acid chains and
theirdegree of unsaturation. The rectilinear hydrocarbonchains of
the saturated fatty acids interact strongly.These interactions
intensify with their length. Thetransition temperature therefore
increases as thefatty acid chains become longer. The doublebonds of
the unsaturated fatty acids are generallycis, giving a curvature to
the hydrocarbon chain(Figure 1.8). This curvature breaks the
orderly
H3C
CH3
CH3
CH3
H3C
HO
H3C
H3C
CH3
CH3
CH2
H3C
HO
H3C
H3C
CH3
CH3
H3C
HO
H3C
H
Ergosterol (24) (28) Dehydroergosterol
Zymosterol
Fig. 1.7. Principal yeast membrane sterols
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10 Handbook of Enology: The Microbiology of Wine and
Vinifications
Stearic acid (C18, saturated)
Oleic acid (C18, unsaturated)
Fig. 1.8. Molecular models representing the three-di-mensional
structure of stearic and oleic acid. The cisconfiguration of the
double bond of oleic acid producesa curvature of the carbon
chain
stacking of the fatty acid chains and lowers thetransition
temperature. Like cholesterol in the cellsof mammals, ergosterol is
also a fundamentalregulator of the membrane fluidity in
yeasts.Ergosterol is inserted in the bilayer perpendicularlyto the
membrane. Its hydroxyl group joins, byhydrogen bonds, with the
polar head of thephospholipid and its hydrocarbon tail is
insertedin the hydrophobic region of the bilayer. Themembrane
sterols intercalate themselves betweenthe phospholipids. In this
manner, they inhibitthe crystallization of the fatty acid chains at
lowtemperatures. Inversely, in reducing the movementof these same
chains by steric encumberment, theyregulate an excess of membrane
fluidity when thetemperature rises.
1.3.2 Functions of the PlasmicMembrane
The plasmic membrane constitutes a stable,hydrophobic barrier
between the cytoplasm andthe environment outside the cell, owing to
its
phospholipids and sterols. This barrier presents acertain
impermeability to solutes in function ofosmotic properties.
Furthermore, through its system of permeases,the plasmic
membrane also controls the exchangesbetween the cell and the
medium. The function-ing of these transport proteins is greatly
influencedby its lipid composition, which affects membranefluidity.
In a defined environmental model, thesupplementing of membrane
phospholipids withunsaturated fatty acids (oleic and linoleic)
pro-moted the penetration and accumulation of certainamino acids as
well as the expression of the gen-eral amino acid permease (GAP),
(Henschke andRose, 1991). On the other hand, membrane sterolsseem
to have less influence on the transport ofamino acids than the
degree of unsaturation ofthe phospholipids. The production of
unsaturatedfatty acids is an oxidative process and requires
theaeration of the culture medium at the beginningof alcoholic
fermentation. In semi-anaerobic wine-making conditions, the amount
of unsaturated fattyacids in the grape, or in the grape must,
probablyfavor the membrane transport mechanisms of fattyacids.
The transport systems of sugars across the mem-brane are far
from being completely elucidated.There exists, however, at least
two kinds of trans-port systems: a high affinity and a low
affinitysystem (ten times less important) (Bisson, 1991).The low
affinity system is essential during the logphase and its activity
decreases during the station-ary phase. The high affinity system
is, on the con-trary, repressed by high concentrations of
glucose,as in the case of grape must (Salmon et al., 1993)(Figure
1.9). The amount of sterols in the mem-brane, especially
ergosterol, as well as the degreeof unsaturation of the membrane
phospholipidsfavor the penetration of glucose in the cell. Thisis
especially true during the stationary and declinephases. This
phenomenon explains the determininginfluence of aeration on the
successful completionof alcoholic fermentation during the yeast
multi-plication phase.
The presence of ethanol, in a culture medium,slows the
penetration speed of arginine and glucoseinto the cell and limits
the efflux of protons
-
Cytology, Taxonomy and Ecology of Grape and Wine Yeasts 11
00
0
0
0
0.0 0.1 0.2 0.3 0.4 0.5 0.60
1
2
3
4
5
6
high affinity transportsystem activity
Length of the fermentation as a decimal of total time
Glu
cose
pen
etra
tion
spee
d (m
mol
/h/g
dry
wei
ght) low affinity
transportsystem activity
Fig. 1.9. Evolution of glucose transport system activityof S.
cerevisiae fermenting a medium model (Salmonet al., 1993). LF =
Length of the fermentation as adecimal of total time GP = Glucose
penetration speed(mmol/h/g of dry weight) 0 = Low affinity
transportsystem activity ∗ = High affinity transport
systemactivity
resulting from membrane ATPase activity (Alexan-dre et al.,
1994; Charpentier, 1995). Simulta-neously, the presence of ethanol
increases thesynthesis of membrane phospholipids and
theirpercentage in unsaturated fatty acids (especiallyoleic).
Temperature and ethanol act in synergy toaffect membrane ATPase
activity. The amount ofethanol required to slow the proton efflux
decreasesas the temperature rises. However, this modifica-tion of
membrane ATPase activity by ethanol maynot be the origin of the
decrease in plasmic mem-brane permeability in an alcoholic medium.
Therole of membrane ATPase in yeast resistance toethanol has not
been clearly demonstrated.
The plasmic membrane also produces cellwall glucan and chitin.
Two membrane enzymesare involved: β-1,3 glucanase and chitin
syn-thetase. These two enzymes catalyze the poly-merization of
glucose and N -acetyl-glucosamine,derived from their activated
forms (uridinediphosphates—UDP). The mannoproteins areessentially
produced in the endoplasmic reticulum
(Section 1.4.2). They are then transported by vesi-cles which
fuse with the plasmic membraneand deposit their contents at the
exterior of themembrane.
Finally, certain membrane proteins act as cel-lular specific
receptors. They permit the yeast toreact to various external
stimuli such as sexual hor-mones or changes in the concentration of
externalnutrients. The activation of these membrane pro-teins
triggers the liberation of compounds such ascyclic adenosine
monophosphate (cAMP) in thecytoplasm. These compounds serve as
secondarymessengers which set off other intercellular reac-tions.
The consequences of these cellular mecha-nisms in the alcoholic
fermentation process meritfurther study.
1.4 THE CYTOPLASM AND ITSORGANELLES
Between the plasmic membrane and the nuclearmembrane, the
cytoplasm contains a basiccytoplasmic substance, or cytosol. The
organelles(endoplasmic reticulum, Golgi apparatus, vacuoleand
mitochondria) are isolated from the cytosol bymembranes.
1.4.1 Cytosol
The cytosol is a buffered solution, with a pHbetween 5 and 6,
containing soluble enzymes,glycogen and ribosomes.
Glycolysis and alcoholic fermentation enzymes(Chapter 2) as well
as trehalase (an enzyme cat-alyzing the hydrolysis of trehalose)
are present.Trehalose, a reserve disaccharide, also cytoplas-mic,
ensures yeast viability during the dehydrationand rehydration
phases by maintaining membraneintegrity.
The lag phase precedes the log phase in asugar-containing
medium. It is marked by a rapiddegradation of trehalose linked to
an increase intrehalase activity. This activity is itself
closelyrelated to an increase in the amount of cAMP inthe
cytoplasm. This compound is produced by amembrane enzyme, adenylate
cyclase, in response
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12 Handbook of Enology: The Microbiology of Wine and
Vinifications
to the stimulation of a membrane receptor by anenvironmental
factor.
Glycogen is the principal yeast glucidic reservesubstance.
Animal glycogen is similar in structure.It accumulates during the
stationary phase in theform of spherical granules of about 40 µm
indiameter.
When observed under the electron microscope,the yeast cytoplasm
appears rich in ribosomes.These tiny granulations, made up of
ribonucleicacids and proteins, are the center of proteinsynthesis.
Joined to polysomes, several ribosomesmigrate the length of the
messenger RNA. Theytranslate it simultaneously so that each
oneproduces a complete polypeptide chain.
1.4.2 The Endoplasmic Reticulum,the Golgi Apparatusand the
Vacuoles
The endoplasmic reticulum (ER) is a doublemembrane system
partitioning the cytoplasm. It islinked to the cytoplasmic membrane
and nuclearmembrane. It is, in a way, an extension of thelatter.
Although less developed in yeasts than inexocrine cells of higher
eucaryotes, the ER hasthe same function. It ensures the addressing
ofthe proteins synthesized by the attached ribosomes.As a matter of
fact, ribosomes can be either freein the cytosol or bound to the
ER. The pro-teins synthesized by free ribosomes remain in
thecytosol, as do the enzymes involved in glycolysis.Those produced
in the ribosomes bound to the ERhave three possible destinations:
the vacuole, theplasmic membrane, and the external
environment(secretion). The presence of a signal sequence
(aparticular chain of amino acids) at the N -terminalextremity of
the newly formed protein determinesthe association of the initially
free ribosomes inthe cytosol with the ER. The synthesized
proteincrosses the ER membrane by an active transportprocess called
translocation. This process requiresthe hydrolysis of an ATP
molecule. Having reachedthe inner space of the ER, the proteins
undergo cer-tain modifications including the necessary excisingof
the signal peptide by the signal peptidase. Inmany cases, they also
undergo a glycosylation.
The yeast glycoproteins, in particular the struc-tural, parietal
or enzymatic mannoproteins, con-tain glucidic side chains (Section
1.2.2). Some ofthese are linked to asparagine by N
-glycosidicbonds. This oligosaccharidic link is constructed inthe
interior of the ER by the sequential additionof activated sugars
(in the form of UDP deriva-tives) to a hydrophobic, lipidic
transporter calleddolicholphosphate. The entire unit is transferred
inone piece to an asparagine residue of the polypep-tide chain. The
dolicholphosphate is regenerated.
The Golgi apparatus consists of a stack ofmembrane sacs and
associated vesicles. It is anextension of the ER. Transfer vesicles
transportthe proteins issued from the ER to the sacs of theGolgi
apparatus. The Golgi apparatus has a dualfunction. It is
responsible for the glycosylationof protein, then sorts so as to
direct them viaspecialized vesicles either into the vacuole or
intothe plasmic membrane. An N-terminal peptidicsequence determines
the directing of proteinstowards the vacuole. This sequence is
present inthe precursors of two vacuolar-orientated enzymesin the
yeast: Y carboxypeptidase and A proteinase.The vesicles that
transport the proteins of theplasmic membrane or the secretion
granules, suchas those that transport the periplasmic invertase,are
still the default destinations.
The vacuole is a spherical organelle, 0.3 to3 µm in diameter,
surrounded by a single mem-brane. Depending on the stage of the
cellularcycle, yeasts have one or several vacuoles. Beforebudding,
a large vacuole splits into small vesi-cles. Some penetrate into
the bud. Others gatherat the opposite extremity of the cell and
fuseto form one or two large vacuoles. The vacuo-lar membrane or
tonoplast has the same generalstructure (fluid mosaic) as the
plasmic membranebut it is more elastic and its chemical
com-position is somewhat different. It is less richin sterols and
contains less protein and glyco-protein but more phospholipids with
a higherdegree of unsaturation. The vacuole stocks someof the cell
hydrolases, in particular Y carboxypep-tidase, A and B proteases, I
aminopeptidase,X-propyl-dipeptidylaminopeptidase and
alkalinephosphatase. In this respect, the yeast vacuole can
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Cytology, Taxonomy and Ecology of Grape and Wine Yeasts 13
be compared to an animal cell lysosome. Vacuolarproteases play
an essential role in the turn-overof cellular proteins. In
addition, the A proteaseis indispensable in the maturation of other
vacuo-lar hydrolases. It excises a small peptide sequenceand thus
converts precursor forms (proenzymes)into active enzymes. The
vacuolar proteases alsoautolyze the cell after its death.
Autolysis, whileageing white wine on its lees, can affect wine
qual-ity and should concern the winemaker.
Vacuoles also have a second principal function:they stock
metabolites before their use. In fact,they contain a quarter of the
pool of the aminoacids of the cell, including a lot of arginine as
wellas S-adenosyl methionine. In this organelle, thereis also
potassium, adenine, isoguanine, uric acidand polyphosphate
crystals. These are involvedin the fixation of basic amino acids.
Specificpermeases ensure the transport of these metabolitesacross
the vacuolar membrane. An ATPase linkedto the tonoplast furnishes
the necessary energyfor the movement of stocked compounds
againstthe concentration gradient. It is different from theplasmic
membrane ATPase, but also produces aproton efflux.
The ER, Golgi apparatus and vacuoles canbe considered as
different components of aninternal system of membranes, called the
vacuome,participating in the flux of glycoproteins to beexcreted or
stocked.
1.4.3 The MitochondriaDistributed in the periphery of the
cytoplasm, themitochondria (mt) are spherically or
rod-shapedorganelles surrounded by two membranes. Theinner membrane
is highly folded to form cristae.The general organization of
mitochondria is thesame as in higher plants and animal cells.
Themembranes delimit two compartments: the innermembrane space and
the matrix. The mitochon-dria are true respiratory organelles for
yeasts. Inaerobiosis, the S. cerevisiae cell contains about50
mitochondria. In anaerobiosis, these organellesdegenerate, their
inner surface decreases, and thecristae disappear. Ergosterol and
unsaturated fattyacids supplemented in culture media limit
thedegeneration of mitochondria in anaerobiosis. In
any case, when cells formed in anaerobiosis areplaced in
aerobiosis, the mitochondria regain theirnormal appearance. Even in
aerated grape must,the high sugar concentration represses the
synthe-sis of respiratory enzymes. As a result, the mito-chondria
no longer function. This phenomenon,catabolic glucose repression,
will be described inChapter 2.
The mitochondrial membranes are rich in
phos-pholipids—principally phosphatidylcholine,
phos-phatidylinositol and phosphatidylethanolamine(Figure 1.5).
Cardiolipin (diphosphatidylglycerol),in minority in the plasmic
membrane (Figure 1.4),is predominant in the inner mitochondrial
mem-brane. The fatty acids of the mitochondrial phos-pholipids are
in C16:0, C16:1, C18:0, C18:1.In aerobiosis, the unsaturated
residues predomi-nate. When the cells are grown in
anaerobiosis,without lipid supplements, the short-chain satu-rated
residues become predominant; cardiolipinand
phosphatidylethanolamine diminish whereasthe proportion of
phosphatidylinositol increases. Inaerobiosis, the temperature
during the log phase ofthe cell influences the degree of
unsaturation of thephospholipids- more saturated as the
temperaturedecreases.
The mitochondrial membranes also containsterols, as well as
numerous proteins and enzymes(Guerin, 1991). The two membranes,
inner andouter, contain enzymes involved in the synthesis
ofphospholipids and sterols. The ability to synthesizesignificant
amounts of lipids, characteristic of yeastmitochondria, is not
limited by respiratory deficientmutations or catabolic glucose
repression.
The outer membrane is permeable to mostsmall metabolites coming
from the cytosol since itcontains porine, a 29 kDa transmembrane
proteinpossessing a large pore. Porine is present inthe
mitochondria of all the eucaryotes as wellas in the outer membrane
of bacteria. Theintermembrane space contains adenylate kinase,which
ensures interconversion of ATP, ADP andAMP. Oxidative
phosphorylation takes place in theinner mitochondrial membrane. The
matrix, on theother hand, is the center of the reactions of
thetricarboxylic acids cycle and of the oxidation offatty
acids.
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14 Handbook of Enology: The Microbiology of Wine and
Vinifications
The majority of mitochondria proteins are codedby the genes of
the nucleus and are synthesized bythe free polysomes of the
cytoplasm. The mito-chondria, however, also have their own
machineryfor protein synthesis. In fact, each mitochon-drion
possesses a circular 75 kb (kilobase pairs)molecule of
double-stranded AND and ribosomes.The mtDNA is extremely rich in A
(adenine) andT (thymine) bases. It contains a few dozen genes,which
code in particular for the synthesis of cer-tain pigments and
respiratory enzymes, such ascytochrome b, and several sub-units of
cytochromeoxidase and of the ATP synthetase complex. Somemutations
affecting these genes can result in theyeast becoming resistant to
certain mitochondrialspecific inhibitors such as oligomycin. This
prop-erty has been applied in the genetic marking ofwine yeast
strains. Some mitochondrial mutantsare respiratory deficient and
form small colonieson solid agar media. These ‘petit’ mutants are
notused in winemaking because it is impossible toproduce them
industrially by respiration.
1.5 THE NUCLEUS
The yeast nucleus is spherical. It has a diameterof 1–2 mm and
is barely visible using a phasecontrast optical microscope. It is
located near theprincipal vacuole in non-proliferating cells.
Thenuclear envelope is made up of a double membraneattached to the
ER. It contains many ephemeralpores, their locations continually
changing. Thesepores permit the exchange of small proteinsbetween
the nucleus and the cytoplasm. Contraryto what happens in higher
eucaryotes, the yeastnuclear envelope is not dispersed during
mitosis.In the basophilic part of the nucleus, the crescent-shaped
nucleolus can be seen by using a nuclear-specific staining method.
As in other eucaryotes, itis responsible for the synthesis of
ribosomal RNA.During cellular division, the yeast nucleus
alsocontains rudimentary spindle threads composed ofmicrotubules of
tubulin, some discontinuous andothers continuous (Figure 1.10). The
continuousmicrotubules are stretched between the twospindle pole
bodies (SPB). These corpuscles arepermanently included in the
nuclear membrane and
Discontinous tubules
Continuoustubules
Nucleolus
Cytoplasmicmicrotubules
Chromatin
Pore
Spindle pole body
Fig. 1.10. The yeast nucleus (Williamson, 1991). SPB =Spindle
pole body; NUC = Nucleolus; P = Pore; CHR =Chromatin; CT =
Continuous tubules; DCT = Discon-tinuous tubules; CTM = Cytoplasmic
microtubules
correspond with the centrioles of higher organisms.The
cytoplasmic microtubules depart from thespindle pole bodies towards
the cytoplasm.
There is little nuclear DNA in yeasts comparedwith higher
eucaryotes—about 14 000 kb in ahaploid strain. It has a genome
almost three timeslarger than in Escherichia coli, but its
geneticmaterial is organized into true chromosomes. Eachone
contains a single molecule of linear double-stranded DNA associated
with basic proteinsknown as histones. The histones form
chromatinwhich contains repetitive units called nucleosomes.Yeast
chromosomes are too small to be observedunder the microscope.
Pulse-field electrophoresis (Carle and Olson,1984; Schwartz and
Cantor, 1984) permits the sep-aration of the 16 chromosomes in S.
cerevisiae,whose size range from 200 to 2000 kb. Thisspecies has a
very large chromosomic polymor-phism. This characteristic has made
karyotypeanalysis one of the principal criteria for the
iden-tification of S. cerevisiae strains (Section 1.9.3).The
scientific community has nearly establishedthe complete sequence of
the chromosomic DNAof S. cerevisiae. In the future, this detailed
knowl-edge of the yeast genome will constitute a powerfultool, as
much for understanding its molecular phys-iology as for selecting
and improving winemakingstrains.
The yeast chromosomes contain relatively fewrepeated sequences.
Most genes are only present