Handbook of enology.

Remarks Concerning the Expression
of Certain Parameters of Must
and Wine Composition
UNITS
Metric system units of length (m), volume (l) and
weight (g) are exclusively used. The conversion of
metric units into Imperial units (inches, feet, gal-
lons, pounds, etc.) can be found in the following
enological work: Principles and practices of wine-
making, R.B. Boulton, V.L. Singleton, L.F. Bisson
and R.E. Kunkee, 1995, The Chapman & Hall
Enology Library, New York.
EXPRESSION OF TOTAL ACIDITY
AND 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 per
liter has not been embraced in France. The expres-
sion of total and volatile acidity in the equivalent
weight of sulfuric acid has been used predomi-
nantly throughout these works. In certain cases, the
corresponding weight in tartaric acid, often used in
other countries, has been given.
Using the weight of the milliequivalent of the
various acids, the below table permits the conver-
sion from one expression to another.
More particularly, to convert from total acidity
expressed in H
2
SO
4
to its expression in tartaric
acid, add half of the value to the original value
(4 g/l H
2
SO
4
→ 6 g/l tartaric acid). In the other
direction a third of the value must be subtracted.
The French also continue to express volatile
acidity in equivalent weight of sulfuric acid. More
generally, in other countries, volatile acidity is
Desired Expression
Known Expression meq/l g/l g/l g/l
H
2
SO
4
tartaric acid acetic acid
meq/l 1.00 0.049 0.075 0.060
g/l H
2
SO
4
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
viii Remarks Concerning the Expression of Certain Parameters of Must and Wine Composition
expressed in acetic acid. It is rarely expressed
in milliequivalents per liter. The below table also
allows simple conversion from one expression to
another.
The expression in acetic acid is approximately
20% higher than in sulfuric acid.
EVALUATING THE SUGAR
CONCENTRATION OF MUSTS
This measurement is important for tracking grape
maturation, fermentation kinetic and if necessary
determining the eventual need for chaptalization.
This measurement is always determined by
physical, densimetric or refractometric analysis.
The expression of the results can be given accord-
ing to several scales: some are rarely used, i.e.
degree Baum
´
e and degree Oechsle. Presently, two
systems exist (Section 10.4.3):
1. The potential alcohol content (titre alcoom´et-
raque potential or TAP, in French) of musts
can be read directly on equipment, which is
graduated using a scale corresponding to 17.5
or 17 g/l of sugar for 1% volume of alcohol.
Today, the EC recommends using 16.83 g/l as
the conversion factor. The ‘mustimeter’ is a
hydrometer containing two graduated scales:
one expresses density and the other gives a
direct reading of the TAP. Different methods
varying in precision exist to calculate the TAP
from 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 sugar
in weight. By multiplying degree Brix by 10,
the weight of sugar in 1 kg, or slightly less
than 1 liter, of must is obtained. A conversion
table between degree Brix and TAP exists in
Section 10.4.3 of this book. 17 degrees Brix
correspond to an approximate TAP of 10% and
20 degrees Brix correspond to a TAP of about
12%. Within the alcohol range most relevant to
enology, degree Brix can be multiplied by 10
and then divided by 17 to obtain a fairly good
approximation of the TAP.
In any case, the determination of the Brix or TAP
of a must is approximate. First of all, it is not
always possible to obtain a representative grape
or must sample for analysis. Secondly, although
physical, densimetric or refractometric measure-
ments are extremely precise and rigorously express
the sugar concentration of a sugar and water mix-
ture, these measurements are affected by other sub-
stances released into the sample from the grape
and other sources. Furthermore, the concentrations
of these substances are different for every grape
or grape must sample. Finally, the conversion rate
of sugar into alcohol (approximately 17 to 18 g/l)
varies and depends on fermentation conditions and
yeast properties. The widespread use of selected
yeast strains has lowered the sugar conversion rate.
Measurements Using Visible
and Ultraviolet Spectrometry
The measurement of optic density, absorbance, is
widely used to determine wine color (Volume 2,
Section 6.4.5) and total phenolic compounds con-
centration (Volume 2, Section 6.4.1). In these
works, 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 the
optic 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 simplified
form: CI = OD 420 + OD 520.
Tint is expressed as:
T =
OD 420
OD 520
The total phenolic compound concentration is
expressed by OD 280.
The analysis methods are described in Chapter 6
of Handbook of Enology Volume 2, The Chemistry
of Wine.
Preface to the First Edition
Wine has probably inspired more research and
publications than any other beverage or food. In
fact, through their passion for wine, great scientists
have not only contributed to the development of
practical enology but have also made discoveries
in the general field of science.
A forerunner of modern enology, Louis Pasteur
developed simplified contagious infection mod-
els for humans and animals based on his obser-
vations of wine spoilage. The following quote
clearly expresses his theory in his own words:
‘when profound alterations of beer and wine are
observed because these liquids have given refuge
to microscopic organisms, introduced invisibly and
accidentally into the medium where they then
proliferate, how can one not be obsessed by the
thought that a similar phenomenon can and must
sometimes occur in humans and animals.’
Since the 19th century, our understanding of
wine, wine composition and wine transformations
has greatly evolved in function of advances in rel-
evant scientific fields i.e. chemistry, biochemistry,
microbiology. Each applied development has lead
to better control of winemaking and aging con-
ditions and of course wine quality. In order to
continue this approach, researchers and winemak-
ers must strive to remain up to date with the latest
scientific and technical developments in enology.
For a long time, the Bordeaux school of enology
was largely responsible for the communication of
progress in enology through the publication of
numerous works (B
´
eranger Publications and later
Dunod Publications):
Wine Analysis U. Gayon and J. Laborde (1912);
Treatise on Enology J. Rib
´
ereau-Gayon (1949);
Wine Analysis J. Rib
´
ereau-Gayon and E. Peynaud
(1947 and 1958); Treatise on Enology (2 Volumes)
J. Rib
´
ereau-Gayon and E. Peynaud (1960 and
1961); Wine and Winemaking E. Peynaud (1971
and 1981); Wine Science and Technology (4 volu-
mes) J. Rib
´
ereau-Gayon, E. Peynaud, P. Rib
´
ereau-
Gayon and P. Sudraud (1975–1982).
For an understanding of current advances in
enology, the authors propose this book Handbook
of Enology Volume 1: The Microbiology of Wine
and Vinifications and the second volume of the
Handbook of Enology Volume 2: The Chemistry of
Wine: Stabilization and Treatments.
Although written by researchers, the two vol-
umes are not specifically addressed to this group.
Young researchers may, however, find these books
useful to help situate their research within a par-
ticular field of enology. Today, the complexity of
modern enology does not permit a sole researcher
to explore the entire field.
These volumes are also of use to students and
professionals. Theoretical interpretations as well
as solutions are presented to resolve the problems
encountered most often at wineries. The authors
have adapted these solutions to many different sit-
uations and winemaking methods. In order to make
the best use of the information contained in these
works, enologists should have a broad understand-
ing of general scientific knowledge. For example,
the understanding and application of molecular
biology and genetic engineering have become
indispensable in the field of wine microbiology.
Similarly, structural and quantitative physiochem-
ical analysis methods such as chromatography,
x Preface to the First Edition
NMR and mass spectrometry must now be
mastered in order to explore wine chemistry.
The goal of these two works was not to create
an exhaustive bibliography of each subject. The
authors strove to choose only the most relevant and
significant publications to their particular field of
research. A large number of references to French
enological research has been included in these
works in order to make this information available
to a larger non-French-speaking audience.
In addition, the authors have tried to convey
a French and more particularly a Bordeaux per-
spective of enology and the art of winemaking.
The objective of this perspective is to maximize
the potential quality of grape crops based on the
specific natural conditions that constitute their ‘ter-
roir’. The role of enology is to express the char-
acteristics of the grape specific not only to variety
and vineyard practices but also maturation condi-
tions, which are dictated by soil and climate.
It would, however, be an error to think that the
world’s greatest wines are exclusively a result of
tradition, 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 from
high performance installations and automation of
operations. Yet, history has unequivocally shown
that the most important enological developments
in wine quality (for example, malolactic fermenta-
tion) have been discovered in ultra premium wines.
The corresponding techniques were then applied to
less prestigious products.
High performance technology is indispensable
for the production of great wines, since a lack
of control of winemaking parameters can easily
compromise their quality, which would be less of
a problem with lower quality wines.
The word ‘vinification’ has been used in this
work and is part of the technical language of
the French tradition of winemaking. Vinification
describes the first phase of winemaking. It com-
prises all technical aspects from grape maturity
and harvest to the end of alcoholic and some-
times malolactic fermentation. The second phase
of winemaking ‘winematuration, stabilization and
treatments’ is completed when the wine is bottled.
Aging specifically refers to the transformation of
bottled wine.
This distinction of two phases is certainly the
result of commercial practices. Traditionally in
France, a vine grower farmed the vineyard and
transformed grapes into an unfinished wine. The
wine merchant transferred the bulk wine to his cel-
lars, finished the wine and marketed the product,
preferentially before bottling. Even though most
wines are now bottled at the winery, these long-
standing practices have maintained a distinction
between ‘wine grower enology’ and ‘wine mer-
chant enology’. In countries with a more recent
viticultural history, generally English speaking, the
vine grower is responsible for winemaking and
wine 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 been
maintained, since the first phase primarily concerns
microbiology and the second chemistry. In this
manner, the individual operations could be linked
to their particular sciences. There are of course lim-
its to this approach. Chemical phenomena occur
during vinification; the stabilization of wines dur-
ing storage includes the prevention of microbial
contamination.
Consequently, the description of the different
steps of enology does not always obey logic as
precise as the titles of these works may lead
to believe. For example, microbial contamination
during aging and storage are covered in Vol-
ume 1. The antiseptic properties of SO
2
incited the
description of its use in the same volume. This line
of reasoning lead to the description of the antioxi-
dant related chemical properties of this compound
in the same chapter as well as an explanation of
adjuvants 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 separated
from vinification and are therefore also covered
in Volume 1. Finally, our understanding of pheno-
lic compounds in red wine is based on complex
chemistry. All aspects related to the nature of the
Preface to the First Edition xi
corresponding substances, their properties and their
evolution during grape maturation, vinification and
aging are therefore covered in Volume 2.
These works only discuss the principles of
equipment used for various enological operations
and their effect on product quality. For example,
temperature control systems, destemmers, crushers
andpressesaswellasfilters,inverseosmosis
machines and ion exchangers are not described in
detail. Bottling is not addressed at all. An in-depth
description of enological equipment would merit a
detailed work dedicated to the subject.
Wine tasting, another essential role of the
winemaker, is not addressed in these works.
Many related publications are, however, readily
available. Finally, wine analysis is an essential tool
that a winemaker should master. It is, however, not
covered in these works except in a few particular
cases i.e. phenolic compounds, whose different
families are often defined by analytical criteria.
The authors thank the following people who
have 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 of
material 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, preparation
and revision of the final manuscript.
Pascal Rib
´
ereau-Gayon
Bordeaux
Preface to the Second Edition
The 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 with
students as an educational reference book, as well
as 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 prepare
an updated, reviewed, corrected version, including
the latest enological knowledge, to reflect the many
new research findings in this very active field. The
outline and design of both volumes remain the
same. Some chapters have changed relatively little
as the authors decided there had not been any sig-
nificant new developments, while others have been
modified much more extensively, either to clarify
and improve the text, or, more usually, to include
new research findings and their practical applica-
tions. Entirely new sections have been inserted in
some chapters.
We have made every effort to maintain the same
approach as we did in the first edition, reflecting
the ethos of enology research in Bordeaux. We use
indisputable scientific evidence in microbiology,
biochemistry, and chemistry to explain the details
of mechanisms involved in grape ripening, fermen-
tations and other winemaking operations, aging,
and stabilization. The aim is to help winemakers
achieve greater control over the various stages in
winemaking and choose the solution best suited
to each situation. Quite remarkably, this scientific
approach, most intensively applied in making the
finest wines, has resulted in an enhanced capac-
ity to bring out the full quality and character of
individual terroirs. Scientific winemaking has not
resulted in standardization or leveling of quality.
On the contrary, by making it possible to correct
defects and eliminate technical imperfections, it
has revealed the specific qualities of the grapes
harvested in different vineyards, directly related to
the variety and terroir, more than ever before.
Interest in wine in recent decades has gone
beyond considerations of mere quality and taken
on a truly cultural dimension. This has led some
people to promote the use of a variety of tech-
niques that do not necessarily represent significant
progress in winemaking. Some of these are sim-
ply modified forms of processes that have been
known for many years. Others do not have a suf-
ficiently reliable scientific interpretation, nor are
their applications clearly defined. In this Hand-
book, we have only included rigorously tested
techniques, clearly specifying the optimum con-
ditions for their utilization.
As in the previous edition, we deliberately
omitted three significant aspects of enology: wine
analysis, tasting, and winery engineering. In view
of their importance, these topics will each be
covered in separate publications.
The authors would like to take the opportunity
of the publication of this new edition of Volume 1
to thank all those who have contributed to updating
this work:
— Marina Bely for her work on fermentation
kinetics (Section 3.4) and the production of
volatile acidity (Sections 2.3.4 and 14.2.5)
— Isabelle Masneuf for her investigation of the
yeasts’ nitrogen supply (Section 3.4.2)
xiv Preface to the Second Edition
— Gilles de Revel for elucidating the chemistry
of SO
2
, particularly, details of combination
reactions (Section 8.4)
— Gilles Masson for the section on ros
´
ewines
(Section 14.1)
— Cornelis Van Leeuwen for data on the impact
of vineyard water supply on grape ripening
(Section 10.4.6)
— Andr
´
e Brugirard for the section on French
fortified wines— vins doux naturels (Section
14.4.2)
— Paulo Barros and Joa Nicolau de Almeida for
their work on Port (Section 14.4.3)
— Justo. F. Casas Lucas for the paragraph on
Sherry (Section 14.5.2)
— Alain Maujean for his in-depth revision of the
section on Champagne (Section 14.3).
March 17, 2005
Professor Pascal RIBEREAU-GAYON
Corresponding Member of the Institute
Member of the French Academy of Agriculture
1
Cytology, Taxonomy and Ecology
of Grape and Wine Yeasts
1.1 Introduction 1
1.2 The cell wall 3
1.3 The plasmic membrane 7
1.4 The cytoplasm and its organelles 11
1.5 The nucleus 14
1.6 Reproduction and the yeast biological cycle 15
1.7 The killer phenomenon 19
1.8 Classification of yeast species 22
1.9 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 into
wine, was only clearly established in the middle
of the nineteenth century. The ancients explained
the boiling during fermentation (from the Latin
fervere, to boil) as a reaction between substances
that come into contact with each other during
crushing. In 1680, a Dutch cloth merchant, Antonie
van Leeuwenhoek, first observed yeasts in beer
wort using a microscope that he designed and
produced. He did not, however, establish a rela-
tionship between these corpuscles and alcoholic
fermentation. It was not until the end of the eigh-
teenth century that Lavoisier began the chemical
study 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 2nd Edition P. Rib
´
ereau-Gayon, D. Dubourdieu, B. Don
`
eche and
A. Lonvaud 
2006 John Wiley & Sons, Ltd ISBN: 0-470-01034-7
2 Handbook of Enology: The Microbiology of Wine and Vinifications
As early as 1785, Fabroni, an Italian scientist, was
the first to provide an interpretation of the chem-
ical composition of the ferment responsible for
alcoholic fermentation, which he described as a
plant–animal substance. According to Fabroni, this
material, comparable to the gluten in flour, was
located in special utricles, particularly on grapes
and wheat, and alcoholic fermentation occurred
when it came into contact with sugar in the must. In
1837, a French physicist named Charles Cagnard
de La Tour proved for the first time that the yeast
was a living organism. According to his findings,
it was capable of multiplying and belonged to the
plant kingdom; its vital activities were at the base
of 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 alcoholic
fermentation. He named the beer yeast zucker-
pilz, which means sugar fungus—Saccharomyces
in Latin. In 1838, Meyen used this nomenclature
for the first time.
This vitalist or biological viewpoint of the role
of yeasts in alcoholic fermentation, obvious to
us today, was not readily supported. Liebig and
certain other organic chemists were convinced that
chemical 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 credibility
to the vitalist viewpoint of alcoholic fermentation.
He demonstrated that the yeasts responsible for
spontaneous fermentation of grape must or crushed
grapes came from the surface of the grape;
he isolated several races and species. He even
conceived the notion that the nature of the yeast
carrying out the alcoholic fermentation could
influence the gustatory characteristics of wine. He
also demonstrated the effect of oxygen on the
assimilation of sugar by yeasts. Louis Pasteur
proved that the yeast produced secondary products
such as glycerol in addition to alcohol and carbon
dioxide.
Since Pasteur, yeasts and alcoholic fermen-
tation have incited a considerable amount of
research, making use of progress in microbiology,
biochemistry and now genetics and molecular
biology.
In taxonomy, scientists define yeasts as unicel-
lular fungi that reproduce by budding and binary
fission. Certain pluricellular fungi have a unicellu-
lar stage and are also grouped with yeasts. Yeasts
form a complex and heterogeneous group found
in three classes of fungi, characterized by their
reproduction mode: the sac fungi (Ascomycetes),
the club fungi (Basidiomycetes), and the imper-
fect fungi (Deuteromycetes). The yeasts found on
the surface of the grape and in wine belong to
Ascomycetes and Deuteromycetes. The haploid
spores or ascospores of the Ascomycetes class are
contained in the ascus, a type of sac made from
vegetative cells. Asporiferous yeasts, incapable of
sexual reproduction, are classified with the imper-
fect fungi.
In this first chapter, the morphology, repro-
duction, taxonomy and ecology of grape and
wine yeasts will be discussed. Cytology is the
morphological 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)
Cytology, Taxonomy and Ecology of Grape and Wine Yeasts 3
Yeasts are the most simple of the eucaryotes.
The yeast cell contains cellular envelopes, a
cytoplasm with various organelles, and a nucleus
surrounded by a membrane and enclosing the
chromosomes. (Figure 1.1). Like all plant cells,
the yeast cell has two cellular envelopes: the
cell wall and the membrane. The periplasmic
space is the space between the cell wall and
the membrane. The cytoplasm and the membrane
make up the protoplasm. The term protoplast
or sphaeroplast designates a cell whose cell
wall has been artificially removed. Yeast cellular
envelopes play an essential role: they contribute
to a successful alcoholic fermentation and release
certain constituents which add to the resulting
wine’s composition. In order to take advantage of
these properties, the winemaker or enologist must
have a profound knowledge of these organelles.
1.2 THE CELL WALL
1.2.1 The General Role
of the Cell Wall
During the last 20 years, researchers (Fleet, 1991;
Klis, 1994; Stratford, 1999; Klis et al., 2002) have
greatly expanded our knowledge of the yeast cell
wall, which represents 15–25% of the dry weight
of the cell. It essentially consists of polysaccha-
rides. It is a rigid envelope, yet endowed with a
certain elasticity.
Its first function is to protect the cell. Without
its wall, the cell would burst under the internal
osmotic pressure, determined by the composition
of the cell’s environment. Protoplasts placed in
pure water are immediately lysed in this manner.
Cell wall elasticity can be demonstrated by placing
yeasts, taken during their log phase, in a hypertonic
(NaCl) solution. Their cellular volume decreases
by approximately 50%. The cell wall appears
thicker and is almost in contact with the membrane.
The cells regain their initial form after being placed
back into an isotonic medium.
Yet the cell wall cannot be considered an inert,
semi-rigid ‘armor’. On the contrary, it is a dynamic
and multifunctional organelle. Its composition and
functions evolve during the life of the cell, in
response to environmental factors. In addition to
its protective role, the cell wall gives the cell
its particular shape through its macromolecular
organization. It is also the site of molecules
which determine certain cellular interactions such
as sexual union, flocculation, and the killer
factor, which will be examined in detail later in
this chapter (Section 1.7). Finally, a number of
enzymes, generally hydrolases, are connected to
the cell wall or situated in the periplasmic space.
Their substrates are nutritive substances of the
environment and the macromolecules of the cell
wall itself, which is constantly reshaped during
cellular morphogenesis.
1.2.2 The Chemical Structure
and Function of the Parietal
Constituents
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 cell
wall has been carried out on Saccharomyces cere-
visiae —the principal yeast responsible for the
alcoholic fermentation of grape must.
Glucan represents about 60% of the dry weight
ofthecellwallofS. cerevisiae.Itcanbe
chemically 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. Under
the electron microscope, this glucan appears
fibrous. It ensures the shape and the rigidity of
the cell wall. It is always connected to chitin.
2. Amorphous β-1,3 glucan, with about 1500
glucose units, is insoluble in water but soluble
in alkalis. It has very few branches, like the
preceding glucan. In addition to these few
branches, it is made up of a small number of
β-1,6 glycosidic linkages. It has an amorphous
aspect under the electron microscope. It gives
the cell wall its elasticity and acts as an anchor
for the mannoproteins. It can also constitute an
extraprotoplasmic reserve substance.

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