Sparkling Wine Production: January 2011

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31
CHAPTER
Sparkling Wine Production
Philippe Jeandet, Yann Vasserot, Gérard Liger-Belair and Richard Marchal
Laboratory of Enology and Applied Chemistry
Unité de Recherche sur la Vigne et le Vin de Champagne, Research Unit N°2069
University of Reims, Faculty of Science, PO Box 1039, 51687 Reims cedex 02, France
1. INTRODUCTION
The chapter describes Sparkling winemaking with emphasis on champagne. A wine is called sparkling
if it is surcharged with carbon dioxide (not less than 5 g/L at 20°C). There are four types of sparkling
wines out of which type I includes excess carbon dioxide produced by fermentation of residual sugar
from the primary fermentation. The type II includes those with excess CO2 produced from malo-lactic
fermentation, the third type is characterized by excess CO2 produced from fermentation of sugar added
after the fermentation and most of the sparkling wines of the world are produced by this method. In the
IVth type of wine, excess CO2 is added and includes the carbonated and crackling wines. Amongst the
sparkling wines, champagne is the most famous. The focus has been made specifically on three points
of particular relevance for Champagne in this chapter. Firstly, the difficulty in initiating the malolactic
fermentation in Champagne wines makes it necessary to have more information dealing with the
nutritional requirements and the metabolism of lactic acid bacteria under wine conditions. This topic is
given detailed consideration in a separate section of this chapter. Secondly, Champagne would not be
Champagne without the magical presence of bubbles. Effervescence, which is related to gas discharging
from a liquid by bubbling, is the main characteristic of Champagne wines, namely the quality of the
product is often related to the size of bubbles formed in the flute. There is thus, the need for specific
research in what we call “the Physics of effervescence in Champagne”. This aspect is covered as an
integral part of this contribution. Finally, a major aspect in appreciating a Champagne, concerns its
foam: firstly, the foam appearing when and after pouring the glass and, secondly, the foam ring formed
on the liquid surface called the collar which is the result of bubbles formed in the glass. Some
physicochemical parameters of foam will also be described as how each step of the winemaking process
can modify qualitatively and quantitatively, the foaming properties of Champagne wines.
2 Handbook of Enology: Principles, Practices and Recent Innovations
2. SPARKLING WINES
Several methods are available to transform the base wine into a sparkling wine. These are as follows:
the methode champenoise, the Cremants from France and Luxembourg, the methode traditionnelle
(formerly the methode champenoise) e.g. used for Cavas, the transfer method, the methode ancestrale
(Limoux, Gaillac) also including the Dioise method and the bulk method (Cuve Close).
2.1 Champenoise method

Elaboration of Sparkling Wines from France and Luxembourg uses the champenoise method for both
the base wine and the prise de mousse. Differences occur with the Champagne winemaking especially
when considering the separation of juices after pressing the whole grapes, which often remains virtual.42
The second phase, that is, second fermentation and aging in the bottle, is very close to that of Champagne
wines except for the fact that Cremants only have nine months aging in contact with lees. Cava elaboration
theoretically, uses the methode traditionnelle (formerly the methode champenoise), but the base wine is
rarely obtained after pressing whole grapes, which differs from the Champagne method. The most
commonly used technique for making the base wine is rather that of a traditional white wine.42 The
second phase is perfectly controlled as for Champagne or Cremants. Aging on lees usually takes 9
months for Cava wines.
2.2 The transfer method

In the transfer method, the wine (obtained by traditional white winemaking) is fermented and aged on
lees in the bottle (as for the methode traditionnelle) but there are no constraints of riddling (a crucial,
expensive and lengthy operation) and disgorging. After bottle fermentation and proper aging, the
bottles are automatically emptied into a steel tank without degassing since the wine is maintained at an
isobarometric pressure. At this stage, the dosage can be directly added in the tank (scheme 1) but
winemakers generally prefer adding the dosage in another steel tank after having filtered the wine
(scheme 2).6,42 After standing several days, the wine is filtered and bottled (scheme 1) or just bottled
(scheme 2). All operations are carried out under a carbon dioxide atmosphere using isobarometric
bottling. Advantages of the transfer method are the suppression of the lengthy operation of remuage
and the fact that the dosage is more uniform. However, this method is expensive, energy-consuming
and a risk of oxidation of the wine does really exist.6
2.3 Methode ancestrale

The methode ancestrale, which is an elaborating method but very difficult to control, has been developed
in the vineyards of Limoux and Gaillac. The base wine is made with whole grapes (mainly of the
Mauzac variety) or through a traditional white winemaking using a semi-fermented wine. In fact,
sugars are used for both the primary alcoholic fermentation and the second fermentation. At different
steps of the elaboration process, it is essential to stop fermentation each time it starts to accelerate. In
this way, refrigeration (until 0°C), sulfiting, depletion of yeast nutrients (using settling, fining, filtration
or centrifugation) are repeated as many times as necessary to regulate or stop the activity of yeast. The
wine is then, filtered and kept at 0°C until springtime. At this step, the second fermentation takes place
Sparkling Wine Production 3
(2-3 months) in the bottle (at rigorously controlled temperatures) with yeast and remaining sugars of
the semi-fermented wine. Riddling and disgorging (without dosage) then occur, but the wine can also
be sold with a slight yeast deposit at the bottom of the bottle. These wines receive the Blanquette
Methode Ancestrale Appellation of Controlled Origin.25
For the Dioise method, the same principles of winemaking as for the ancestrale method are used, that
is, elaboration of a semi-fermented wine from the Muscat à petits grains variety, filtration and
refrigeration at 0°C, second fermentation in the bottle (using sugars remaining after the first fermentation).
To increase the extraction of aroma compounds, pectinolytic enzymes are added to Muscat grape
berries in the crusher. This imposes fining treatments since the must obtained presents high turbitidies
ranging from 1000 to 1500 NTU. The flotation technique is used for clarification of the must.69 After
the second fermentation is stopped by refrigerating the cellar, bottles are emptied into a steel tank
maintained at an isobarometric pressure by CO2 to avoid degassing (as for the transfer method). After
filtration, the wine is bottled using isobarometric bottling. The final alcoholic content of the Clairette
de Die is of approximately 7.5° with 40 to 50 g/L residual sugars. The elaboration of Asti spumante
follows the same process and is made from the same grape variety.
2.4 Bulk method

The bulk method (cuve close) is a simpler and more cost-effective technique that has been developed to
obtain ordinary wines of low prices. It is also called as tank method or charmat process. The second
fermentation does not take place in the bottle but the base wine is sent to a reinforced steel fermentation
tank able to contain several hundreds hectoliters of wine. Yeast and sugars are added and the wine is
maintained at a temperature of 20 to 25°C. The prise de mousse duration does not exceed 10 days. The
second fermentation is stopped by a light sulfiting and by refrigerating the wine at-2°C. After having
been cold-stabilized at-5°C for several days, the wine is filtered at a low temperature and then, bottled
using isobarometric bottling. One disadvantage of this method is that there is no aging of the wine as
the wine contact with lees is insufficient.
3. CHAMPAGNE: PREAMBLE AND PRODUCTION TECHNOLOGY
3.1 Champagne
Champagne is undoubtedly the most prestigious effervescent wine throughout the world and up to 300
millions bottles of this wine are produced per year. The traditions have made these sparkling wines
symbols of lifestyle, unique and authentic symbols for any form of celebration. Champagne would not
be Champagne without the magical presence of the bubbles linked to the CO2 formed during the
secondary fermentation, which is a characteristic of the methode champenoise. Champagne is defined
as bottle fermented sparkling wine as in its production secondary fermentation takes place in bottle.
The various steps involved in champagne producting include base wine production, sugaring, yeasting,
bottling, blending stabilization, rampage, disgrifting. A proper blending of base wines takes place
before secondary fermentation.
3.2 The Grapes Variety for Champagne

Champagne is made from three main varieties, Pinot Noir, a black grape variety that gives Champagne
wines their aromas of red fruits, as well as their strength and body, Pinot Meunier, another black
cultivar characterized by its suppleness and spiciness, giving Champagne wines their roundness and
fragrance and finally, Chardonnay a white grape variety, providing Champagne wines with their finesse,
as well as their floral and, in some instances, mineral notes very typical of the Champagnes Blancs de
Blancs, that is, Champagnes made only with Chardonnay grapes.
The Champagne vineyard belongs to the so-called septentrional (Northern) vineyards and this particular
location leads to cold-growing conditions, making difficult the maturation process (ripening of grape
berries). The stage of maturity of grape fruits is a fundamental parameter which determines the quality
of wines. Thus, knowledge of the physiological and biochemical events of the maturation process is of
great interest. It was shown, namely, that studies in the changes in sugar and organic acid contents of
grape berries during ripening in the Champagne vineyards would be useful for estimating the harvest
date.76,77
3.3 Base Wine Preparation

Champagne wines are produced in two steps: the base wine is made first, after which a second
fermentation is initiated in the bottle (Prise de mousse). Champagne is typically a white wine whose
grapes are hand-harvested and pressed very lightly in the way of limiting the extraction of phenolic
compounds from the skins and the stems, namely, to avoid the diffusion of color (anthocyanins) into
the must. Less than three quarters of this wine are indeed obtained from the two black varieties, Pinot
Noir and Pinot Meunier, the other quarter being obtained from the white variety, Chardonnay. In order
to obtain high quality musts, proper picking and pressing conditions are required. For example, sorting
to remove rot-attacked grape clusters is needed during picking, since it has been shown that gray mold
caused by the phytopathogenic fungus, Botrytis cinerea Pers., can have deleterious effects on the
foaming properties of Champagne wines.74 For more details on the production of base wine for champagne
or sparkling wines, the readers may refer to a separate chapter on Red and White Production of this
book. Grapes must be transported as intact as possible to avoid skin maceration in the juice and oxidation
phenomena. The entire grape clusters are pressed lightly (with pressures ranging from 1.5 to 2 bars)
without crushing. Strong regulations enacted in 1993 have determined the extraction yield of juice:
4000 kg of grape bunches produce 25.50 hl of must, the first 20.50 hl constituting the cuvee, the last 5
hl constituting the taille. Only the cuvee will be used for producing quality Champagnes; the taille is
destined for distillation or vinted apart from the cuvée to be possibly used for blending. The taille is
characterized, as compared to the cuvee, by a decrease in the total acidity (tartaric and malic acids), an
increase in the mineral and phenolics concentrations together with an increase in the pH. Finally, the
taille is a heavier and less fine product than the cuvee, explaining why it is rarely used for winemaking.
As soon as the must is extracted, SO2 is added at doses varying from 3 to 8 g/hl, mainly depending on
the pressing fraction (cuvée or taille) and the level of gray mold in the vineyard. Musts with turbitidies
of 200 to 400 NTU are clarified by centrifugation or after static settling, debourbage (18 to 24 h at
temperatures ranging from 6 to 15°C) using tannins, bentonite or casein (+ bentonites) as fining
agents. During static settling, some must proteins, pectin together with mineral cations and phenolics
are removed. The mechanism by which proteins are removed from musts or the wine by the use of
bentonites, that is, interactions between bentonites and proteins begin to be precisely understood.37,38,39
3.4 Primary Fermentation

Once musts are clarified, the alcoholic fermentation occurs after inoculation (10-15 g/hl) with selected
strains of Saccharomyces cerevisiae cv bayanus (mainly DV 10, IOC and Levuline CHP strains).
Champagne wines are generally, though not always, chaptalized. Alcoholic fermentation usually takes
place in stainless steel tanks at temperatures ranging from 15 to 18°C (sometimes fermentation is
conducted at lower temperatures, e.g. 13°C). Since musts or wines may be stained by anthocyanins in
the case of “botrytized” or overmature grapes, charcoal is commonly used as an agent for reducing
color in colored musts and wines. Charcoal which has deleterious effects on the organoleptic and
foaming properties of Champagne wines may advantageously be replaced by yeast lees recovered after
alcoholic fermentation of Chardonnay musts.106 Yeast lees are able to adsorb some of the volatile sulfur
compounds found in wines, as well.84,110
3.5 Malolectic fermentation

Malolactic fermentation of base wines is now widely used in Champagne to avoid the occurrence of
this fermentation during bottle fermentation (prise de mousse) or during the aging of wines on laths,
which may lead to an increase in the volatile acidity and the appearance of lactic notes in wine tasting.
Malolactic fermentation is achieved by inoculating lactic acid bacteria (Oenococcus oeni) with a
fermenting starter containing 107 cells/ml. In some Champagne Houses, malolactic fermentation is not
practiced to keep freshness and fruity characteristics in the wine and could be avoided by using high
SO2 concentrations (8 to 10 g/hl). Some work has also been done to study the malolactic fermentation
inhibiting capabilities of lysozyme (added at the must or the wine level) and its possible effects on
Champagne wine foamabilty.67,68 Because of the difficulty to easily initiate malolactic fermentation
especially in highly acidic wines such as Champagne wines, studies have focussed on the nutritional
requirements and the metabolism of lactic acid bacteria under wine conditions,21,22,32,107,108 an aspect
discussed later in this chapter.
3.6 Clarification
After completion of the malolactic fermentation, wines undergo clarification treatments (static settling
or centrifugation + fining including the use of bentonite, gelatin + [tannins or silica gel], casein +
bentonite, charcoal + bentonite, fish gelatins or wheat gluten.70,71) Because of the situation of crisis
linked to the bovine spongiform encephalopathy, leading the public and winemakers to lose their
confidence in the use of animal proteins in Enology (gelatins, egg proteins, casein,…), several research
works were started to study the possibility to substitute animal proteins by those originating from
plants in fining treatments.70,71
3.7 Blending
The practice of blending wines from different grape varieties, different origins (crus) and different
years (reserve wines) is essential to maintain the quality of Champagne and the House style unique to
every Champagne producer. Reserve wines are kept two or three years, (sometimes on lees) in tanks at
12-13°C and protected from oxygen; reserve wines may also be kept for longer time (i.e. ten years).
The Brut Non-Vintage and the Demi-Sec Champagnes are usually a blend of wines from several years,
from different grape varieties and a number of crus. The Brut, especially, is a product very typical of
the wine producer. Vintage Champagnes are produced exclusively from the wines of a single harvest.
3.8 Stabilization
Stabilization of wine with respect to potassium hydrogen tartrate (KHT) is a critical point in winemaking
in Champagne. Before being stabilized, wines may be filtered on a simple continuous earth filter;
wines obtained at this stage are called vins clairs. The stability usually required in Champagne wines
corresponds to the temperature of-4°C. Briefly, potassium hydrogen tartrate stabilization is obtained
by treating the wine with artificial cold using different technologies, namely, slow cold stabilization
without KHT crystal seedling, rapid cold stabilization including KHT crystal seedling by the static
contact process or by the dynamic continuous process.75 Work has been done to search whether the very
expensive treatment of wines with artificial cold could be advantageously replaced by the addition of
inhibitors of the crystallization process of potassium hydrogen tartrate such as metatartaric acid or
carboxymethylcellulose (C.M.C.). Such inhibitors increase the width of the supersaturation field of
KHT in the wine, thus delaying tartrate salts precipitation in the bottle.19
Once the wine is stabilized with respect to both KHT and colloids, it is filtered (on earth filters or
cellulose cartridges). Wines obtained at this stage are called vins de base champenois.
3.9 Secondary fermentation and bottle aging

The secondary fermentation in the bottle (the so-called prise de mousse) may thus occur by adding the
liqueur de tirage, that is saccharose (18-24 g/l according to the CO2 concentration desired), immobilized
or freely suspended yeast (inoculation with 106 cells/ml), diamonium phosphates and riddling adjuvants
(bentonite, alginate). The yeast used is acclimatized for lower temperature fermentation and with
alcohol tolerance in the base wine. After capping with a crown stopper, the bottles are placed horizontally
on laths (sur lattes) or directly in crates. This secondary fermentation is a slow (duration: six to eight
weeks) and steady low-temperature fermentation (11-12°C) characteristic of the so-called prise de
mousse. The time interval between addition of the liqueur de tirage and expedition must be, for
Champagne, of at least 15 months. After the secondary fermentation has been completed, Champagne
wines undergo a long maceration time (1.5 to 8 years or even more !) on yeast lees. There is a wine
enrichment in compounds synthesized and released by yeast. Autolysis, that is, enzymatic self-degradation
process of yeast cell constituents that takes place immediately after cell death at the end of fermentation,
also occurs during aging. It consists in the release of some yeast constituents such as amino-acids,
peptides, nucleotides, mannoproteins and aroma compounds that would influence wine sensory
properties. Autolysis has an unique occurrence and significance during production of Champagne
wines that must have at least 12 months in contact with yeast in the bottle.16,29 Gas exchanges may also
occur during aging, specifically, there is a loss of CO2 and O2 is able to penetrate in the bottle through
the crown stopper. Thus, a slight oxidation takes place during aging of Champagne wines on lees.
During bottle aging, some sensory defects could appear. This is especially the case when wines are
submitted to light exposure (even in the bottle). Light may be responsible for the vitamin B2-related
photodegradation of methionine, leading to the formation of volatile sulfur compounds such as
methanethiol and dimethyldisulfide (DMDS), which give the wine cooked cauliflower or wet wool
smells (the so-called gouts de lumiere).79
3.10 Remuage
Subsequent sedimentation and removal of yeast requires the time-consuming and expensive procedure
of riddling (remuage), that is, frequent but controlled turning of the slanted, inverted bottles to bring
the yeast sediment together with adjuvants (bentonites or alginates) down to the cork. Nowadays,
economic pressures have shortened times of remuage down to 3 to 4 days with freely suspended yeast
and even two days with agglomerating cells. Otherwise, the use of immobilized yeast cells instead of
inoculation with freely suspended yeast for this secondary fermentation would considerably simplify
the riddling process.23 The disadvantage of shortening times of remuage is that the wines obtained are
sometimes not clear, containing particles in suspension and termed voltigeurs, which were proved to be
of mineral origin (i.e. bentonites added in the liqueur de tirage).46
3.11 Disgorging
Once the deposit (yeast + adjuvant) is concentrated against the crown cap, disgorging can occur. This
operation together with dosage, corking and wire-capping is done automatically only for bottle or
demie-bottles. Flasks of higher capacity, that is, Magnum, Jeroboam, Mathusalem…are still disgorged
manually. During disgorging, the inverted bottle is partially immersed in a low-temperature brine,
freezing the top of the bottle and entrapping the yeast + adjuvant deposit, which is removed in the form
of ice. The liqueur d’expedition or dosage consisting of sugar (for a final concentration in the bottle of
6-50 g/l saccharose) and antioxidants (SO2, citric acid or ascorbic acid) is then added. During this
operation, there is a loss in the CO2 pressure ranging from 0.5 to 0.8 bar. Finally, the bottle is corked.
As non effervescent wines, Champagnes are affected by corkiness. It has been estimated that at least
0.5 to 2% of bottled Champagnes present corkiness, a percentage of real concern to the wine industry.105
The protective effect of a composite cork stopper on Champagne wine pollution with 2,4,6-
trichloroanisole, the main component responsible for corkiness, is now being studied.109
As can be seen from this short description, Champagne winemaking is a long and complex process.
Quality regulations have built this very famous wine throughout three centuries.
4. MALOLACTIC FERMENTATION IN SPARKLING WINE PRODUCTION

Malolactic fermentation is nowadays a very important stage in sparkling wine production. 4 The conversion
of the dicarboxylic acid, malate, into the monocarboxylic acid, lactate and carbonic gaz by lactic acid
bacteria decreases the acidity of the wine and increases its pH. This fermentation is important in wines
which are produced from grapes grown in cool climates, such as Champagne wines, which often have
a high organic acid (tartrate plus malate) content and low pH. In addition, malolactic fermentation
(MLF) makes these wines microbiologically more stable43,50 and some metabolism of these micro-
organisms, such as diacetyl or ethyl lactate production, can change favourably the flavour of the
wine.92 Malolactic fermentation should occur before bottling in order to prevent subsequent bacterial
development which would form undesirable deposits.104 For these reasons, and because improved sanitary
conditions in winemaking have resulted in the production of wines in which malolactic fermentation
may not occur spontaneously or can be very unpredictable, many winemakers tried to induce it with
starter cultures developed from freeze-dried or frozen Oenococcus œni biomass.27,31,48,50,51 Nevertheless,
despite the use of these starter cultures, malolactic fermentation remains very often difficult to induce
and more specially in Champagne wines. Difficulties of inducing MLF are usually attributed to the
cumulative inhibitory effects of low pH, high alcohol and SO2 contents of wines.13,113 Nevertheless,
these difficulties could also have as their origin the deficiency or the imbalance of Champagne wines in
some nutrients. Among these nutrients, the free amino acids could be of great significance. In fact, they
are, along with short peptides, the most important source of nitrogen for the growth of wine lactic acid
bacteria and most of them are either required or are stimulatory to the growth of Oenococcus œni.34,101
4.1 Effect of Amino Acids
4.1.1 Effect on bacterial growth

Amino acids have been classified into three groups according to the extent of bacterial growth in each
deficient medium. From 0% to 10% of growth, the amino acid is considered as essential, from 10% to
50%, favourable, and over 50%, indifferent. The number of essential amino acids varies from 5 (MC1)
to 11 (NCBF 1707) as in Table 1. It is noticeable that the indigenous MC strains have lower requirements
than the reference strains and such a result may indicate that they are better able to grow in stringent
conditions as in wine. L-arginine, L-glutamic acid, L-isoleucine and L-tryptophan are essential for the
growth of all the strains tested as well as L-methionine, except for the strain MC1. Strains MC1, MC2,
MC4 and NCFB 1823 need L-cystein for optimal development and L-leucine is essential for reference
strains. It has been shown that whatever Oenococcus œni strains used, L-arginine, L-isoleucine, L-
glutamic acid and L-tryptophan were essential for bacterial growth.34,86 These authors discussed the
effect of L-cysteine, L-methionine or L-leucine deficiencies on bacterial growth. L-leucine was found
to be essential for 45% of Oenococcus œni strains tested by Garvie34 and for 75% of those tested by
Peynaud and Domercq.86 L-cysteine was essential for 50% of the strains of Peynaud and Domercq86
and for 100% of those of Garvie.34 The same graduated response is observed for the strains tested in
our studies also. Unlike Tracey and Britz101 who reported that Oenococcus œni did not have an absolute
requirement for tyrosine, other authors considered that L-phenylalanine and tyrosine were also essential
for bacterial growth. But our studies did not confirm this assessment, except in the case of NCFB 1707.
In fact, this reference strain shows a specific behavior since it also requires L-histidine, L-lysine, L-
phenylalanine, L-tyrosine and L-valine for growth. Though L-valine is not an essential amino acid,
except for Oenococcus œni NCFB 1707, its absence markedly limited bacterial growth. The same
phenomenon is observed to a lesser extend with L-aspartic acid or L-leucine. Such a favourable effect
of L-aspartic acid on Oenococcus œni growth is in good agreement with the results obtained on a
bacterial strain isolated from Argentina wines.64,95 It could result from the ability of Oenococcus œni to
synthesize an essential amino acid, L-isoleucine, from L-aspartic acid.
Surprisingly, Glycine, L-proline or L-serine deficiencies enhance the growth of Oenococcus oeni. Such
a result, which has yet been reported for L-proline33 may be interpreted as an inhibitory effect of these
amino acids. They could act as competitors in the transport of an essential amino acid. Such a phenomenon
has already been noted for L-valine transport in a Leuconostoc mesenteroides sp. dextranicum strain.35
4.1.2 Effect on D-glucose fermentation

Between 80 and 100% of the D-glucose was consumed in the complete medium (i.e. with all amino
acids) by every strain. Absence of an essential amino acid such as L-arginine, L-glutamic acid,
L-isoleucine, L-tryptophan or L-methionine, inhibits D-glucose utilization (Figure 1). When the amino
acid deficiency limited bacterial growth, as it was the case for L-histidine, L-lysine and L-valine
(Table 1), D-glucose was only partially metabolized. In most of the media used, the consumption of
Fig. 1. D-glucose consumed. Results are the ratio of amounts of D-glucose consumed in one amino acid-
deficient medium (indicated at the bottom) and D-glucose degraded in the complete medium. They are
given in percentages. (a) Strains MC1, MC2, MC4; (b) Strains NCFB 1707, NCFB 1674, NCFB 1823.
Source: Ref. No. 33.
D-glucose was correlated with the amount of growth, confirming D-glucose as the main carbon source
for growth of Oenococcus œni as established earlier.17,87 Nevertheless, such a result is not observed in a
medium without L-aspartic acid for all the strains studied or in a medium without L-leucine in the case
of MC strains. In fact, in these media the final growth is noticeably reduced (Table 1) though
D-glucose consumption is the same as in the complete medium (Figure 1).
Oenococcus œni is heterofermentative and it is15 shown that 1 g of D-glucose produced 0.5 g of
D-lactic acid, 0.33 g of ethanol and acetate, 0.09g of carbonic gas and 0.08 g of biomass. D-glucose
fermentation and end products formation was studied using the MC2 strain. In the complete medium,
D-glucose was entirely fermented and, since we obtained almost the theoretical yield of D-lactic acid
production, this fermentation appears to be mainly realized by the heterofermentative pathway.
Table 1. Growth of six Oenococcus œni strains in different synthetic media after incubation for 9 days.
Percentages of the ratios of absorbance at 650nm observed for the deficient medium and the
complete one.
Strains
Amino acid omitted in MC1 MC2 MC4 NCFB NCFB NCFB
the culture medium 1707 1823 1674
None 100 100 100 100 100 100
L-alanine 101 92 91 90 94 109
L-arginine 3 3 3 0 2 0
L-aspartic acid 61 71 64 52 77 74
L-cysteine 7 7 15 97 2 88
L-glutamic acid 1 8 2 0 1 0
Glycine 102 104 108 103 115 102
L-histidine 93 77 84 1 92 91
L-isoleucine 4 5 5 0 0 0
L-leucine 71 69 65 0 2 0
L-lysine 57 98 89 2 59 43
L-methionine 16 8 9 2 6 4
L-phenylalanine 100 91 99 1 109 91
L-proline 113 108 101 92 116 106
L-serine 95 111 111 85 102 89
L-threonine 87 111 77 95 94 93
L-tryptophan 7 6 2 0 5 0
L-tyrosine 92 86 89 0 96 86
L-valine 30 35 32 1 20 15
Strains MC1, MC2 and MC4 were isolated from champagne wines by Möet & Chandon laboratory.
Following amino acid deficiency, D-glucose metabolism is modified and the modifications obtained
can be classified in four types (Figure 2):
Type I: medium without L-threonine or L-tyrosine: the D-lactic acid production was nearly the same
as in the complete medium.
Type II: medium without glycine, L-alanine or L-phenylalanine: the D-lactic acid production was less
extended and an increase in the synthesis of other products was noted.
Type III: medium without L-proline or L-lysine : the D-lactic acid production was markedly reduced
and the amount of other products was higher than in the complete medium.
Fig. 2. End-products distribution of heterolactic metabolism of D-glucose for strain MC2. The amino acid
deficiency is indicated at the bottom of the disk: (n), percentage of biomass production; (p), percentage
of D-lactic acid production; ( ), percentage of other products. Source: Ref. No. 33.
Type IV: medium without L-aspartic acid, L-leucine or L-serine: an overproduction of D-lactic was
observed. It was accompanied by an underproduction of biomass in the case of L-aspartic acid or
L-leucine deficiencies.
The underproduction of D-lactic acid caused by deficiencies in amino acids from types II and III could
be interpreted as a negative effect of the deficiency on the heterolactic pathway. The cell could use
D-glucose for synthesizing other products. These could be ethanol, acetate or products needed to make
up the amino acid deficiency. Nevertheless, HPLC profiles obtained with the culture samples of these
groups of media showed no great differences with those of the complete medium and further experiments
are needed to investigate whether D-glucose is used for anything other than heterolactic products.
More surprising was the overproduction of D-lactic acid from D-glucose in the absence of L-aspartic
acid, L-leucine or L-serine in the culture medium. The phenomenon could be related to low biomass
synthesis, D-glucose being used for the synthesis of D-lactic acid rather than biomass. Another hypothesis
is that other unknown substrates, not catabolized in the complete medium, lead to the production of
D-lactic acid in these particular cases of deficiency. The experiments of Pilone and Kunkee,87 carried
out in a similar context, supported this hypothesis.
4.1.3 Effect on L-Malic acid consumption

In the complete medium, the consumption of L-malic acid varied with the strain used. MC2 and NCFB
1707 strains were the best strains able to metabolize L-malic acid while MC1 and MC4 strains had
weak L-malic acid degradation activities (Table 2). Such differences between Oenococcus œni strains
have been extensively described earlier also.15
In most cases of amino acid deficiency, the amount of L-malic acid consumed was reduced (Figure 3).
When no growth occurred, as in a medium without L-arginine, L-glutamic acid, L-isoleucine or
L-tryptophan, only a weak degradation activity was noted, may be due to the residual malolactic
activity of the inoculum. When the growth was largely reduced, as was the case of absence of L
histidine, L-methionine, L-valine or L-cysteine, only a small amount of L-malic acid was degraded.
Such results do not agree with those of observed earlier.101 It was effectively noted that L-malic acid
could be entirely consumed though bacterial growth was reduced following amino acid deficiency.
Table 2. D-glucose and L-malic consumption (g/l) in the medium with all amino acids after culture for 9
days. Initial concentrations of D-glucose and L-malic acid were respectively 8 g/l and 5 g/l.
Oenococcus œni strains

NCFB NCFB NCFB
MC1 MC2 MC4 1707 1823 1674
D-glucoseconsumed (g/l) 6.99 7.94 7 7.82 6.61 8
L-malic acidconsumed (g/l) 0.38 5 0.65 4.92 2.53 1.91

Fig. 3. L-malic acid consumed. Results are the ratio of L-malic acid consumed in each culture medium
deficient in one amino acid (indicated at the bottom) and L-malic acid degraded in the complete medium.
They are expressed as percentages. Source: Ref. No. 33.
More surprising is the fact that L-proline, glycine, L-phenylalanine or L-tyrosine deficiencies reduced
L-malic acid consumption although growth occurred, as in the complete medium. Studies conducted
on resting cells, indicate that some amino acids were able to stimulate specifically L- malic acid
consumption in Oenococcus œni strains.85 These four amino acids could act at the different stages of
malolactic conversion: inlet of L-malic acid,18 malolactic enzyme reaction or malolactic enzyme
biosynthesis, outlet of L-lactic acid. Nevertheless, since low L-malic acid utilization (low L-lactic acid
production) is related with low D-lactic acid production from D-glucose in the absence of
L-phenylalanine, L-proline or glycine, it may be assumed that, even if the export system of D-lactic
acid and L-lactic acid are different, the lack of these amino acids could limit the transport of lactic acid
across the cell membrane.
4.2 Effect of an Excessive Concentration of One Amino Acid
4.2.1 Effect on bacterial growth

When cultures were performed with L-malic acid, most of the amino acids tested had no effect on
bacterial growth (Figure 4). Nevertheless, two of them, L-aspartic acid and L-isoleucine, showed an inhibitory
effect against all the bacterial strains used. Even when the results obtained for the indigenous strains Oenococcus
œni 8406 and Oenococcus œni 8403 are quite identical, it is noticeable that the reference strain Oenococcus
œni NCFB 1707 was found be more sensitive to amino acid inhibition. Its growth is, in fact, more strongly
inhibited by L-aspartic acid and L-isoleucine. Moreover, it is also inhibited by L-serine and L-tryptophan.
It is interesting to note that despite their inhibitory effect at high concentrations, L-isoleucine and L-
tryptophan are essential amino acids for Oenococcus œni NCFB 1707.32
However, some of the amino acid like L-arginine and L-threonine, are able to slightly stimulate the
growth of the two indigenous strains. When L-malic acid is omitted in the culture medium (Figure 5),
the inhibitory character of L-aspartic acid and L-isoleucine is intensified and all the other amino acids,
with the exception of L-glutamic acid, show a stimulatory effect on bacterial growth.
Fig. 4. Effects of an excessive amount of one amino acid on the growth of Oenococcus œni strains.
Cultures were performed with malic acid at 37.28 mmol/l. Source: Ref. No. 21.
Fig 5. Influence of malic acid on the effect of an excessive amount of one amino acid on the growth of
Oenococcus œni 8403. Malic acid was used at 37,28 mmol/l. Source: Ref. No. 31.
4.2.2 Bacterial growth stimulation by amino acids

Bacterial growth stimulation by high concentrations of L-arginine can result from the susceptibility of
this amino acid to ATP synthesis through its deamination via the arginine deiminase pathway.61
Nevertheless, it is clear that the high concentrations of L-threonine modify D-glucose metabolism with
an underproduction of D-lactic acid (Table 3). So it was hypothesized that more D-glucose was used
for biomass production.
Table 3. Effect of an excessive amount of one amino acid on D-glucose consumption and D-lactic acid
formation by Oenococcus œni 8403 (D-glucose initial concentration: 44.4 mmol/l).
Reference L-arginine L-threonine L-tyrosine L-glycine

Absorbance max.(650 nm) 0.155 0.374 0.316 0.354 0.337
D-glucose consumed (mmol/l) 11.1 27.9 21.7 19.5 21.1
D-lactic acid produced (mmol/l) 9.7 nd 12 nd nd
D-glucose/D-lactiqueratio 1.15 nd 1.81 nd nd
4.2.3 Bacterial growth inhibition by amino acids.

The D-form of aspartic acid, formed from L-aspartic acid by an aspartate racemase, is an important
component in the peptidoglycan layer of bacterial cell walls.82 Then, L-aspartic acid is an important
amino acid for lactic acid bacteria and is, with L-glutamic acid and L-arginine, one of the most
consumed amino-acids. Nevertheless, as illustrated in Figure 6 for the reference strain Oenococcus œni
NCFB 1707, high concentrations of L-aspartic acid inhibit bacterial growth, resulting in a decrease in
both the maximum biomass production and the maximum growth rate (mmax). Thus, when L-aspartic
acid concentration increased from 0.3 mmol/l to 10 mmol/l, the maximum biomass production decreased
from 0.15 to 0.07 mg/ml and mmax from 0.19 to 0.11 h.-1 Since fumaric acid is known for its bactericidal
activity against lactic acid bacteria,87 the inhibitory effect of high concentrations of L-aspartic acid
could be due to the deamination of this amino acid, through an aspartase activity, into fumaric acid and
ammonia. Occurrence of an aspartase activity has been reported in Escherichia coli,28 though, at the
present time, no evidence of existence of an activity really in lactic acid bacteria is documented. The
inhibitory of L-aspartic acid effects could also be its involvement in antagonistic interactions with the
Fig. 6. Effects of varying L-aspartic acid concentration on the growth of Oenococcus œni NCFB 1707.
Basal medium with L-aspartic acid mmol/l: (p), 0; (•), 0.15; (p), 0.3; ),6; (x),10. Source: Ref. No. 107.
consumption of essential amino acids. Such antagonistic interactions between amino acids have been
reported for Leuconostoc mesenteroides.35,81 Further inhibitory effect on bacterial growth was all the
more reduced since the global amino acid concentration in the culture medium is increased (Figure 7).
Even though, 10 mmol/l of L-aspartic acid reduced the maximum biomass production of Oenococcus
œni NCFB 1707 by 67.1% in a medium in which all other amino acids are used at 0.15 mmol/l (Figure
7A). Such a biomass production is only reduced by 5.4% in a medium in which all the other amino
acids are used at 1.2 mmol/l (Figure 7D). The same phenomenon has also been observed for the
indigenous strains Oenococcus œni 8403 and Oenococcus œni 8406.21 It can be concluded that the
inhibitory effect of this amino acid is not due to its high concentration but because it is in excess with
respect to other amino acids and supports the antagonistic interactions hypothesis.
Fig. 7. Effects of global amino acid content on the inhibitory effect of L-aspartic acid on the growth of
Oenococcus œni NCFB 1707. (°), L-aspartic acid used at 10 mmol/l; (•), L-aspartic acid used at the same
concentration as the other amino acids of the culture medium (i.e. 0.15 mmol/l for A, 0.3 mmol/l for B, 0.6
mmol/l for C and 1.2 mmol/l for D). Source: Ref. No. 107.
Only L-glutamic acid is able to reduce significantly the inhibitory effect of L-aspartic acid on bacterial
growth with almost the same efficaciousness as that obtained in a culture medium in which all the
amino acids are used at 1.2 mmol/l (Table 4). Since L-glutamic acid is an essential amino acid for
Oenococcus œni NCFB 170732 and the transport of this amino acid was found to be inhibited competitively
by L-aspartic acid in other strains of lactic acid bacteria80,98 it is hypothesized that an excess of L-
aspartic acid inhibits Oenococcus œni growth by preventing the bacterial use of L-glutamic acid. This
hypothesis has been confirmed by transport assays with labelled (14C)-L-glutamic acid. So when the L-
aspartic acid concentration increased from 0 to 0.033 mmol/l, the initial rate of L-glutamic acid transport
was reduced from 0.47 pmol/min/mg dry mass to 0.06 pmol/min/mg dry mass (Figure 8) as was
Table 4. Effect of different amino acids on Oenococcus œni NCFB 1707 growth inhibition by
L-aspartic acid.
Amino acid added Growth inhibition (%) Amino acid added Growth inhibition (%)
None 100 L-lysine 83
L-alanine 72.5 L-methionine 82
L-arginine 77 L-phenylalanine 83
L-cysteine 67 L-proline 78
L-glutamic acid 9.2 L-serine 75
Glycine 77 L-threonine 85
L-histidine 75 L-tryptophan 70
L-isoleucine 80 L-tyrosine 63
L-leucine 90 L-valine 90
Fig. 8. Rate of L-glutamic acid transport by glucose-energised cells of Oenococcus œni NCFB 1707
in the absence of L-aspartic acid, (x); and in the presence of 33 µmol/l of L-aspartic acid, (•).
observed with the indigenous strain Oenococcus œni 840321. As L-glutamic acid transport was inhibited
competitively by L-aspartic acid (Ki = 2.13 µM at pH 7), it indicated that L-aspartic acid and
L-glutamic acid share the same transport system.
4.3 Effect on L-malic acid and D-glucose consumption

L-malic acid consumption is hardly affected by an excessive amount of one amino acid. In fact,
whatever the amino acid considered, L-malic acid remains entirely metabolized at the end of the
bacterial growth even if its consumption rate decreased, as it can be obtained with high concentrations
of L-aspartic acid and L-isoleucine.21
The effect of an excessive amount of one amino acid on D-glucose fermentation is more evident and
varies with the presence or the absence of L-malic acid.21 When cultures are performed with L-malic
acid, D-glucose metabolism is not modified by an excessive amount of one amino acid and this is still
in good agreement with Cavin’s empirical equation.15 Nevertheless, in the case of an excessive amount
of L-aspartic acid, L-isoleucine and, to a lesser extent, L-glutamic acid (amino acids which are all
susceptible to inhibit bacterial growth) D-glucose fermentation is not complete.
When L-malic acid is omitted in the culture medium, an excessive amount of L-arginine and L-
threonine modifies D-glucose metabolism with an underproduction of D-lactic acid. Though it reduces
D-glucose fermentation, a high concentration of L-aspartic acid does not modify D-glucose metabolism,
except in the case of Oenococcus œni 1707 where it leads to an overproduction of acetic acid.107
4. FOCUS ON BUBBLE DYNAMICS IN CHAMPAGNE WINES

Upon pouring Champagne in a glass and looking to as what is happening in such a little space it would
be interesting to bubbles being born on several spots of the glass wall, detaching and then rising in-line
toward the free surface into the form of elegant bubble trains, as so many tiny hot-air balloons. While
collapsing at the free surface, bubbles would also emit a typical crackling sound and produce a cloud of
tiny droplets that pleasantly tickle the taster’s nostrils. Effervescence livens up Champagne, sparkling
wines, beers and many other carbonated beverages. Without bubbles, Champagne and sparkling wines
are unrecognizable which the beer would flat.
Small bubbles rising through the liquid, as well as a bubble ring (the so-called collerette) at the
periphery of a flute poured with champagne, are the hallmark of this traditionally festive wine, and
even if there is no scientific evidence yet to correlate the quality of a Champagne with the fineness of
its bubbles, people nevertheless often make a connection between them. It has now become an important
stake in the Champagne research area to achieve the perfect petite bubble. Therefore, a few years ago,
we decided together with colleagues from the University of Reims and from the research department of
Champagne Moet and Chandon, to examine more closely the behavior of bubbles in carbonated beverages.
Our goal was to detect, illustrate and finally understand better the role played by each of the numerous
parameters involved in the bubbling way in a process. It was found, for example, that striking differences
distinguish ascending Champagne bubbles from their beer counterparts. The simple but close observation
of a glass poured with carbonated beverages also recently revealed an unexplored and visually appealing
phenomenon. Here the three main steps of a bubble’s life are summarized, i.e., the bubble birth, the
bubble ascent and the bursting of a bubble at the free surface of the liquid.3,53,54,55,56,57,58,59,60 Our results
were all obtained in real consuming conditions, in glasses poured with standard commercial Champagne
wines.
5.1 The bubble Genesis

In the case of Champagne, sparkling wines and beer97, the main gas responsible for bubble production
is carbon dioxide, which is produced by yeast during the fermentation in the closed bottle. Yeast
convert sugars to alcohol and carbon dioxide molecules. According to Henry’s law, an equilibrium
progressively establishes between CO2 molecules dissolved into the liquid and CO2 molecules into the
vapor phase in the headspace under the cork. When the bottle or can is opened, the CO2 pressure in the
vapor phase suddenly falls. The thermodynamic equilibrium of the closed container is broken, and the
liquid becomes supersaturated with CO2 molecules. To recover a new stable thermodynamic state
corresponding to the atmospheric pressure, CO2 molecules must escape from the supersaturated liquid.
When poured into a glass, two mechanisms enable dissolved CO2 molecules to escape from the
supersaturated liquid medium: diffusion through the free surface of the liquid, and bubble formation.
However, to cluster into the form of bubble embryos, dissolved CO2 molecules need to push their way
through the liquid molecules strongly linked by the so-called Van der Waals attractive forces. Henceforth,
bubble formation is characterized by an energy barrier to overcome. It requires very high supersaturating
ratios, which are totally unrealistic in the case of carbonated beverages. In weakly supersaturated
liquids, such as Champagne, sparkling wines and carbonated beverages in general, bubbles need pre-
existing gas cavities60 with radii of curvature greater than a critical radius in order to overcome the
nucleation energy barrier and grow freely. In bubbly, the critical radius below which bubble production
becomes impossible is sub-micrometric, around 0.2 µm. As a result, bubble formation spots on the
wall of a glass poured with carbonated beverage necessarily reveals tiny pre-existing gas cavities
greater than this critical radius.
To have access to bubble production sites, a high-speed video camera fitted with a microscope objective
was pointed at the base of each investigated bubble train. Hundreds of different “bubble nurseries”
were carefully observed. Contrary to general assumption, nucleation sites are not located on irregularities
of the glass itself. The length-scale of glass irregularities is far below the critical radius of curvature
required for bubble nucleation. Nucleation sites are located on impurities stuck on the glass wall. It was
shown that most of nucleation sites are hollow and roughly cylindrical exogenous cellulose fibers
coming from the surrounding air or remaining from the wiping process.60 Because of geometrical
properties, such particles can not be completely wetted by the liquid and are able to entrap gas pockets
during the filling of a glass. Four typical nucleation sites found in a glass poured with champagne are
displayed in Plate 69. Gas pockets trapped inside the particles clearly appear. Dissolved CO2 molecules
migrate into the gas pocket. A bubble appears and grows rooted to its nucleation site because of
capillary forces. Finally, the increasing buoyancy induces its detachment, thus providing an opportunity
for a new bubble to nucleate, grow-rooted to its nucleation site and detach at the same size, and so on,
until bubble production stops through lack of dissolved gas. Pre-existing gas cavities trapped inside
particles stuck on the glass wall provide bubble production in carbonated beverages. This cycle of
bubble production at a given nucleation site is characterized by its “bubbling” frequency, that is, the
number of bubbles produced per second.60 This clockwork and repetitive bubble production from
nucleation sites can be easily underscored by the naked eye by lighting bubble trains with a stroboscope.
By equaling the flash frequency of strobe lighting with the frequency of the cycle of bubble production,
the corresponding bubble train appears “frozen”. The time needed to reach the moment of bubble
detachment depends on the geometrical properties of the given nucleation site. Now, since a collection
of particle shapes and sizes exists on the glass wall, the bubbling frequency may also vary from one site
to another. Since the kinetics of the bubble growth also depends on the dissolved CO2 molecule content,
differences in the bubble formation frequencies may be observed from one beverage to another. For
example, in Champagne wines where the dissolved gas content is approximately three times higher
than in beer, the most active nucleation sites emit up to about thirty bubbles per second, whereas in
beer, nucleation sites emit up to only about ten bubbles per second.
5.2 The Bubble Rise

A short examination of the typical regular bubble train presented in Plate 70 is already very instructive.
It clearly appears that bubbles grow during ascent. This bubble growth during ascent is caused by a
continuous diffusion of dissolved CO2 molecules through the bubble interface.54,55,56,59 While expanding,
bubbles increase their buoyancy, causing them to continuously accelerate and separate from each others
on their way up.
Beers and sparkling wines are obviously not pure liquids. They contain, in addition to alcohol and
dissolved CO2 molecules, many other organic compounds which may show surface activity (mostly
composed of proteins and glycoproteins in Champagne wines and beers). Like soap molecules, such
molecules have a water-soluble and a water-insoluble part. They are called “surfactants”. Surfactants
prefer to gather around the surface of a bubble, the water-insoluble part stuck out of the liquid into the
gas-filled bubble, rather than staying into the liquid bulk. The role of this surfactant coating around gas
bubbles becomes crucial when buoyancy induces their detachment from the wall impurities and forces
them to plough their way through the liquid molecules. Adsorbed surface-active molecules stiffen a
bubble by forming a sort of shield on its surface.56,59 Actually, surfactants encountered along the bubble
path progressively adsorb at the surface of a rising bubble, thus increasing the immobile area of the
bubble surface. According to fluid dynamics, a bubble rigidified by surfactants and rising through a
liquid runs into more resistance than a bubble presenting a more flexible skin free from surface-active
materials. Therefore, the drag coefficient experienced by a bubble of fixed radius rising in a surfactant
solution progressively increases, thus decreasing its velocity of rise to a minimal value when the bubble
interface gets completely contaminated. In ultra-pure water free from surfactants, a millimetric bubble
rises at a velocity close to 30 cm/s. In water with little amounts of proteins (only of order of several
mg/l), the bubble velocity progressively decreases as soon as the bubble interface traps proteins, to
finally reach a final velocity of order of 15 cm/s (twice less than in pure water !) when the bubble
interface is completely rigidified by a protein coating.
The case of rising and expanding bubbles is a little bit more subtle than that of bubbles of fixed radii.
In the case of a rising and expanding bubble, since the bubble expands during its rise through the
supersaturated liquid, the bubble interface continuously increases, and therefore continuously offers
newly created surface to the adsorbed surface-active materials. Expanding bubbles therefore experience
two opposing effects. If the rate of dilatation of the growing bubble overcomes the rate at which
surface-active molecules stiffen the bubble surface, a bubble progressively cleans its interface. It means
that the ratio of the bubble surface covered by surfactants to the bubble surface free from surfactants
decreases. On the contrary, if this ratio increases, the bubble surface inexorably gets contaminated by
a surfactant monolayer and progressively becomes rigid.
By measuring the drag coefficients experienced by expanding Champagne and beer bubbles during
their way up and by comparing them with data found in the huge scientific literature dealing with
bubbles of various sizes ascending through various liquids, we provided evidence that beer bubbles
showed a behavior very close to that of rigid spheres. On the contrary, Champagne, sparkling wines
and soda’s bubbles were found to present a more flexible interface during ascent. This is not a surprising
result, since beer contains much higher amounts of surface-active macromolecules (of order of several
hundreds mg/l) likely to be adsorbed at a bubble interface than Champagne (only several mg/l). 59
Furthermore, since the gas content is lower in beer, growth rates of beer bubbles are lower than those
of Champagne. As a result, the “cleaning” effect due to the bubble’s expansion may be too weak to
avoid the rigidification of the beer bubble interface. In Champagne, sparkling wines and sodas, there is
too little amount of surface-active materials and bubbles grow too quickly to succumb to the same
tragic fate. In fact, because the drag experienced by a rising bubble is all the more high since the
coating of surfactants is well developed, bubbles of the same size are rising slower in beer than in the
other carbonated beverages.
5.3 The Bubble Collapse at the Free Surface

A few seconds after being born on a glass wall impurity a few centimeters below the liquid surface,
bubbles reach the free surface with a close millimetric diameter. As an iceberg, a millimetric gas
bubble at the free surface of a liquid only slightly emerges. Most of the bubble volume lies below the
liquid surface. The emerged part of the bubble, the bubble-cap, is a spherical portion of liquid film
which progressively gets thinner due to drainage. Actually, a bubble-cap which has reached a critical
thickness becomes sensitive to vibrations, thermal gradients and finally ruptures. In 1959, two physicists,
Taylor and Culick (see,57,58) independently examined the disintegration process of thin liquid sheets.
They found that a hole appears in the bubble-cap which very quickly propagates propelled by surface
tension forces. After the disintegration of the bubble-cap, which occurs on a time scale of 10 to 100 µs
for millimetric bubbles, a complex hydrodynamic process ensues which induces the collapse of the
submerged part of the bubble. We tried to freeze this process, for bubbles collapsing at the surface of
a glass poured with Champagne, by using high-speed macrophotography.
A reconstructed time sequence illustrating six stages of the collapse of a Champagne bubble is
presented in Plate 71. A brief description of each frame follows57,58: between the frame 1 and 2, the
thin liquid film, which constitutes the emerged part of the bubble, has just ruptured. During this
extremely brief initial phase, the bulk shape of the bubble has been frozen by the flashlight and a
nearly millimetric open cavity remains in the liquid surface. While collapsing, the bubble cavity
gives rise to an high-speed liquid jet above the free surface (frames 3 and 4). Near the base of the
liquid jet in frame 4, one can distinguish an extremely small bubble (around 100 µm), probably
entrapped during the collapsing process. Due to its own velocity, this upward liquid jet becomes
unstable. A capillary wave develops along the jet (the well-known Rayleigh-Plateau instability which
clearly appears in frame 5). In frame 6, the liquid jet finally breaks-up into droplets called jet drops.
The combined effects of inertia and surface tension give detaching jet drops various and often
amazing shapes. Finally, in frame 7, droplets ejected by the parent bubble recover a quasi-spherical
shape. Due to surface excitations following bubble collapse, capillary wave trains centered on the
bursting bubble are propagating at the free surface. On the right side of the central bubble in frame
7, the tiny bubble entrapped during collapse can be observed. Since hundreds of bubbles are bursting
every second during the first minutes that follows the pouring of some carbonated beverage, one can
conclude that the liquid surface is literally spiked with such cone-shaped structures, unfortunately
too short-lived to be observed to the naked eye.
At a millimetric scale, such a violent hydrodynamic phenomenon which leads to the projection of an
high-speed liquid jet is driven by the capillary pressure gradients arising around the open cavity frozen
at the free surface after the disintegration of the bubble-cap.58 Immediately after the rupture of the
bubble-cap, sides of the cavity become a region of positive curvature. It ensues a ring of high pressure
on the sides of the open cavity. At the same time, due to a negative curvature, a low pressure zone exists
around the underside of the cavity. As a result, fluid is rapidly drawn from the sides to the axis of
symmetry. The underside of the cavity becomes a region of high pressure which pushes fluid upward to
produce the liquid jet.
The liquid jet that follows a bubble collapse strikingly resembles, in miniature, that one can observe as
a drop impacts the surface of a pool of liquid. Harold Edgerton (1903-1990),99 the twentieth century
master of stop-action photography, invented and developed the electronic flash. He popularized “high-
speed events” by photographing everything, from flying bullets and instants of sport actions, to drops
of liquid impacting surfaces. His most famous snapshot, the coronet made by a drop of milk, was seen
by millions of people throughout the world. Walt Disney studios will even inspire from Edgerton’s
snapshots, in “Bambi”, to make rain drops more realistic. Hervé Lemaresquier, a Colleague from the
University of Reims, reproduced the pioneering work of Edgerton and froze the different stages
experienced by a milk drop impacting on a free surface of milk.58 Shape details of the two various
liquid jets, produced during a bubble collapse and during a drop impact are displayed in Plate 72.
Despite noticeable differences in time and length scales, hydrodynamic structures arising after a drop
impact are clearly very close to those that follow a bubble collapse.
In addition to aesthetic considerations, bubbles bursting at the free surface impart feel to Champagne
wines, beers and many other beverages. Jet drops are ejected up to several centimeters above the free
surface with a velocity of several meters per second. Nociceptors (pain receptors) of the nose are thus
stimulated during tasting, as are receptors in the mouth when bubbles burst over the tongue. Furthermore,
in addition to these mechanical stimulation, bubbles bursting at the free surface are also expected to
play a major role in flavor release. Actually, due to their molecular structure, many aromatic compounds
of carbonated beverages show surface activity. Bubbles rising and expanding in the liquid bulk act as
aromatic molecules traps and drag such surface-active molecules along their way up. Such molecules
progressively concentrate at the free surface at much higher amounts than those found in the liquid
bulk. Bubbles collapsing at the free surface are thus expected to spray in the air a cloud of tiny droplets
over-concentrated in potentially aromatic molecules, thus highlighting flavors of Champagne, sparkling
wines and many other beverages. In a near future, we will try to quantify this flavor release effect for
each of the numerous aromatic molecules found in a Champagne wine.
A nice parallel can also be drawn between the fizz in a bubbly and the “fizz of the ocean”. Bursting
bubbles entrained by the omnipresent breaking of waves also act as miniature microtomes to skim-off
the surface layer and eject it into the atmosphere in the form of jet drops. In the early seventies,
oceanographers found that droplets ejected into the air contained much higher amounts of particles and
surface-active materials than those found in the bulk of the water directly below, thus participating to
the global air/sea exchanges.
Moreover, despite the large body of research concerned with collapsing bubble dynamics, the close-up
observation of bubbles collapsing at the free surface of Champagne recently revealed an unexplored
and visually appealing phenomenon. As far as we know, and surprising as it may seem, most of the
previous studies were conducted with single bubbles collapsing at free surfaces. Now, for a few seconds
after pouring, the free surface is completely covered with a monolayer composed of quite monodisperse
millimetric bubbles collapsing close to each others. Effervescence in carbonated beverages thus, ideally
lends to a preliminary work with bubbles collapsing close to each others.
Since the collapsing process of a millimetric cavity at the free surface of a liquid interface is very short
(2-3 ms), very few photographs froze snapshots of the collapsing process, in a bubble monolayer.57
Photographs displayed in Plate 73a-73b, for example, were taken immediately after the rupture of a
bubble-cap in the bubble monolayer. Clusters of bubbles can be observed, where bubbles are strongly
deformed toward a bubble-free central area, leading to unexpected and visually appealing flower-
shaped structures. A few time sequences of the whole process have also been captured with the high-
speed video camera. One is presented in Plate 74. Between frame 1 and frame 2, the bubble-cap of a
bubble ruptured and left an open cavity at the free surface (the submerged part of the bubble).
Paradoxically, adjacent bubble-caps are sucked and not blown-up by bursting bubbles, contrary to
what could have been thought at first glance. Capillary pressure gradients around the open cavity left
by the ruptured bubble-cap are, once more, the driving forces of this violent sucking process.57 But,
contrary to the single bubble collapse case, liquid flows appear in the thin film of adjacent bubble-caps.
With a lot of patience and a little bit of chance, we also froze oblique views of this violent and short-
lived sucking process. It can be seen, in Plates 75 & 76, that bubble-caps adjacent to the bubble-free
central zone are literally stretched toward the lowest part of the cavity left by a central bursting bubble.
During this sudden stretching process, adjacent bubble-caps areas significantly increase. A systematic
image analysis of numerous time sequences conducted with an high-speed video camera demonstrated
an average increase of approximately 15 % of bubble-cap areas adjacent to a central collapsing bubble.57
Stresses in the bubble-caps of adjoining bubbles were evaluated during the sucking process and found
to be of order of 105 to 106 dyn/cm.2 By comparison, in previous studies, numerical models conducted
to stresses of order of (only) 104 dyn/cm2 in the boundary layer around single millimetric collapsing
bubbles. Therefore, stresses in the bubble-caps of bubbles adjacent to millimetric collapsing cavities
should be, at least, one order of magnitude higher than those observed around single collapsing cavities.
While absorbing the energy released during collapse, as tiny “air-bags” would do, adjoining bubble-
caps store this energy into the thin liquid film of emerging bu8bble-caps, leading finally to stresses
higher than those observed in the boundary layer around single millimetric collapsing bubbles. As far
as we know, no results concerning the collateral effects on adjoining bubbles of bubbles collapsing in
a bubble monolayer had been reported up to now. Contrary to what could have been thought at first
glance, effervescence in carbonated beverages turned out to be a fantastic tool to investigate a first
approach of the physical chemistry of rising, expanding and collapsing bubble dynamics.
6. CHAMPAGNE WINE FOAMING PROPERTIES

The popularity of Champagne is linked to the elegance of its sparkling and foaming properties. Consumers
appreciate both a regular and durable effervescence, leading to a foam ring composed of small bubbles
on the liquid surface. Before smelling and tasting a Champagne wine, consumers will pay attention to
the appearance of the foam. In the light of this, it is important to study both the compounds involved
in this phenomenon and enological treatments able to modify the quality of foam.
6.1 Methods Used to Measure Tri-dimensional Foam

Until 1988, scientific studies on the foaming properties of Champagne wines were still scarce. It was
thus necessary to develop apparatus enabling researchers to directly quantify such a phenomenon. The
experimental methods used in our laboratories have led to the development of an apparatus called
‘Mosalux’ (a sparging procedure) and an artificial viewing equipment (A.V.E.) capable of measuring
the foam when pouring the wine as well as the collar in the glass.
6.1.1 Foaming Properties as Determined with the Mosalux System.

The Mosalux system (Figure 9) is adapted from that described by Rudin94 and allows measurements of
the increase with time of the height of a wine foam column submitted to a definite effervescence.78 A
glass cylinder (4 cm in diameter and 40 cm long) placed on a glass frit (pore size 16 - 40 µm) is filled
with the base wine to be analyzed (100ml). All wines are filtered 0.45 or 1.2 µm before foam
measurements. Carbon dioxide is injected into the glass cylinder through the glass-frit with a constant
rate of gas flow (7 l/h) and under a constant pressure (100kPa). Foam height (measured in millimeters)
is controlled by photoelectric cells (infra-red beams). Graphics are traced with one point for every five
seconds (Figure 10). Each point is the average of three values. For each series, all measurements are
made the same day to reduce dispersion of the values. The foamability (mm) corresponds to the
maximal foam height reached by the column of foam. Foam stability is the foam height (mm) after 8
min sparging.
The Mosalux apparatus has also been used91 to determine other parameters such as the expansion of
foam E, the Bikerman coefficient7 S (lifetime of a bubble in dynamic conditions when formation and
destruction of bubbles are balanced) and the lifetime of the foam LF (Figure 11). Foam expansion E is
defined as the ratio of the foam volume V80 (measured at t=80s) on the initial liquid volume Vliq. With
this procedure, E is not the maximal expansion but rather a standardized maximal expansion. The
lifetime LF expressed in seconds is calculated as follows:
¥
Lf = 1/H0 x ò80 H dt = area under curve/initial foam height = A / H0
Fig. 9. Principle of the Mosalux functioning (sparging procedure).
Fig. 10. Typical foaming properties of a Champagne wine using the Mosalux.
Fig. 11. Typical foam evolution during 0 to 80 s and after gas stop (after 80 s).
The Bikerman coefficient S is obtained with a gas flow Q=10 l/h. Carbon dioxide is stopped only after
a constant height of foam HS has been obtained for two minutes. S is defined as the ratio of the foam
volume (when a constant height is reached) on the gas flow Q.
Most experiments were made with Champagne base wines (still wines) rather than Champagne because
the natural effervescence makes the experiments more difficult to realize and to interpret, as well. This
is not a drawback since it has been shown that the foaming properties of the Champagne follow the
same trends as still wines.78
6.1.2 Artificial Viewing Equipment (AVE)
6.1.2.1 Video system

The system has been described in details in previous studies.62,111 Briefly, two video cameras captured
large-scale and small-scale side views allowing quantification of foam breakdown (collapse) just after
expansion in the glass (Figure 12). Calibration parameters were determined using an accurately sized
Fig. 12. Artificial viewing equipment used to determine foam parameters.

spot giving the resolution along the camera axes; horizontal resolution was 0.11 mm/pixel and vertical
resolution 0.15 mm/pixel. Ambient temperature was controlled (20 ± 1°C). The system was optimized
as follows: the thickness of the foam vs time is always linear. The slope of the straight section expresses
the speed at which foam thickness (Fs) increases in pixels/50 ms. The height of liquid vs time is also
linear. The slope of the straight section expresses the speed at which the height of liquid (Ls) increases,
in pixels/50ms. Fs and Ls include all parameters analyzed in previous works. Fs makes the estimation of
the expansion of foam possible. Ls characterizes stability of the foam, directly depending on the drainage.
The time of Champagne pouring PT (time in seconds, i.e. the time necessary to fill the glass) and the
liquid appearance time in the glass LT (time in seconds) (just after the pouring, the glass only contains
foam; the liquid appears when bubbles collapse) are used to analyze the different stages of pouring.
Larger the PT, the more stable the foam and higher the LT, longer the drainage. The AVE measures the
height of the collar at different times after the pouring began (H80 for example is the height after 80 s).
6.1.2.2 Pouring Robot, Bottles and Glasses Used with the AVE System
This apparatus was developed to standardize the method of pouring Sparkling wines. The Champagne
is poured into the glass with a robot (Figure 13). Before experiments, bottles are placed at +8°C for 16
hours and left at this temperature after each pouring. Five mechanical pouring are made for each bottle
which required no more than one hour. Pouring is facilitated by a small glass cylinder being placed on
the bottle-neck. The glass cylinder is washed in the same way as the wine glass. A single black glass is
used. Before use, the glass is washed in cold and hot water, and is extensively rinsed with deionized
water before drying in a “defatted,” filtered (0.2µm) air stream. This washing procedure is done after
each analysis. An observation window is left in the glass for the two lateral cameras (Figure 12), for the
video camera giving the information to the pouring robot (camera 3, Figure 13) and for laser lighting.
The laser light (l = 670 nm) is placed at 45° with regard to the lateral camera. The pencil of ray lights
the window of the glass (5 mm). Parameters are dictated to the robot as follows : the robot begins to
detect foam and liquid heights when the foam reaches 10.3 mm (31 pixels). The robot stops the
pouring when the liquid height reaches 38.6 mm (115 pixels). The minimum foam height (Minfoam)
Fig. 13. Robot controlling the pouring of the Champagne in the glass.
at the end of the pouring must be 66.6 mm (200 pixels); the maximum foam height at the end of the
pouring (Maxfoam) depends on Minfoam. The difference between these two parameters is 6.6 mm (20
pixels). The maximum foam height before overflowing is 103.3 mm (310 pixels). When the glass is
filled, a sound is emitted. The glass is then moved manually to analyze the foam with the AVE.
6.2 Tensio-Active Compounds and Foaming Properties
6.2.1 Proteins and Macromolecular Colloids

Despite their low concentration in Sparkling wines (ranging from 4 to 16 mg/l), previous works have
shown that proteins are largely involved in the foaming properties of Champagne wines. Indeed, a
good correlation (r = 0.845 with n = 31) between foamability (as determined using the Mosalux
apparatus) and the wine protein content was obtained,78 as determined using the direct Bradford method.10
Another example of the relationship existing between the protein content and the foamability of
Champagne wines has also been described11 which showed that a foam wine in which the protein
concentration is increased by 20 % compared to that of the corresponding base wine, has a higher
foamability and an increased plateau height. Simultaneously, it has also been shown that the protein
concentration of a Champagne wine constitutes a limiting factor for the foaming properties of a wine.63
By mixing base wines with either ultrafiltrates or ultraconcentrates (MWCO at 10 kDa), it is demonstrated
that the control of foam stability after stopping the gas sparging is strictly correlated with the protein
concentration of a considered wine. Nevertheless, proteins have various effects on the foamability of
wines. It was observed that hydrophobic proteins are more concentrated in a foam wine though
hydrophilic proteins are less concentrated.12
Champagne wines (generally speaking all white wines) contain a large proportion of proteins and
glycoproteins originating from the grape berry20,44,66,83,89,112 while others come from yeast.20,30,52,112 Most
of them have pI ranging from 2.5 to 4.5 and molecular masses ranging from 12 to 65 kDa. 12 The
presence of a grape invertase of 62/64 kDa66 may also be mentioned. This invertase is a N-glycoprotein
originating from the plant, as demonstrated by using immunological methods. This enzyme keeps its
activity in wine and presents a high hydrophobicity (1050 kcal/100 amino-acid residues) and a pI
of 3.9.66 More recently, plant lectins showing an hemagglutining activity which is inhibited by
p-aminophenyl-b-D-glucoside5 have also been characterized.
6.2.2 Fatty Acids

The main physical processes which lead to the breakdown of foam are film gravity drainage, bubble
coalescence and disproportionation (Ostwald ripening). In beer, small amounts of lipids may cause
rapid foam collapse. Several researchers indicated that not only the presence of lipids but also their
physical state and the lipids/beer contact time were important.49,90 The effect of lipids on the foaming
properties of Champagne wines has also been studied using a similar scientific approach26 to answer the
question whether the foam behavior is modified when lipids and free fatty acids are added to base
wines at various alcohol concentrations. Investigations were made at two different levels. At a global
level, the average foam life-time, expansion and the average bubble lifetime were quantified. At a local
level, static surface tension measurements were made to understand which physical processes govern
foam behavior.
At first, the typical lipidic composition of three Champagne wines (standard blends of Chardonnay,
Pinot Noir, and Pinot Meunier) were determined following a modified Folch procedure.102 The lipid
fractions were characterized by thin layer chromatography and quantified by gas chromatography
analysis.102 The lipid content (C16-C20) average value was 308 mg/l total free fatty acids. Then, the
influence of an addition of commercial lipid mixtures (to mimic the previous average lipid composition
of Champagne) on the foam’s global properties of base wines was investigated. Aliquots of this lipid
solution were added to wines until their lipid concentration reached twice or six times their initial
values. This was made for various alcohol contents using wines obtained as follows: a still wine was
evaporated (volume decrease of 70%) and ultra pure water was added to restore the initial volume.
This new low-alcohol matrix was again evaporated (50%) to provide a wine with 1.4% alcohol. Then,
appropriate aqueous alcoholic solutions were added in order to obtain wines of 2%, 5% and 11.3% v/
v alcohol each. The 11.3% value is typical for still wines. The reference liquids were wines without
lipid addition but with the same alcohol content. Aqueous alcoholic solutions (provide 2%, 5%, and
11.3%, v/v) were obtained by mixing ultra pure water with ethanol.
Foam characteristics were determined with the Mosalux apparatus. Measurements were carried out
within 30 min after preparation of the solutions and/or after three days of gently shaking at 11°C. All
glassware was systematically treated with fresh sulfochromic acid, and rinsed first with a large amount
of ultra pure water and then with experimental solutions (e.g. wines, hydroalcoholic solutions or lipid
mixtures).
Obviously, the foam numbers (Lf, S and E) decreased in the presence of lipids when foaming occurred
30 min after the wine preparation (Figure 14). No concentration dependence was observed within
experimental errors. Surprisingly, all the foam values were restored after three days interaction.
A major result was the higher tolerance capacity of the base wine towards lipids even at high concentrations
(six times the initial lipid concentration in wine). The changes in foam characteristics may be attributed
to changes in the lipid’s physical state with time. These results were qualitatively consistent with
Robert’s model on beers.90 But investigations of the latter were carried out on free fatty acids and
glycerides only, and the chemical composition (for example the ratio lipids/proteins) of beer is very
different from that of wine. Another major difference between beer and wine is the alcohol content. It
may be reasonable to assume that Champagne foam numbers obtained after three days of lipids and
wine interaction were essentially linked to the alcohol content (Figure 14). In fact, with a very high
alcohol content (compared to beer) lipids are well dissociated (molecularly dissolved). Ethanol governs
the gas/liquid interface and thus, the foam behavior. So, the effect of commercial lipids in wine could
not be measured because they do not seem to be active at the interface.
In order to corroborate this hypothesis, three base wines with different alcohol contents were prepared.
After three days of interaction, the curves of Lf vs lipid concentration (Figure 15a), showed that lipids
did not have much effect on the average time of foamlife for a given alcohol content. From Figure 15c,
it seems that expansion was slightly enhanced without concentration-dependence effect. In contrast,
Fig. 14. Effects of addition of lipids on foam lifetime (a), Bikerman coefficient(b) and foam expansion (c).
Alcohol content: 11.3% (V/V) ; age of the solution: - - x - - : 30 min.; ¾ x¾: 3 days.
the Bikerman coefficient values (Figure 15b) remained unchanged at any lipid concentration in the
presence of 11.3% ethanol while for 2% and 5% alcohol wines, the S values were dependent on the
presence of lipids (the highest at 5%). Moreover, Lf and E were obviously maximal and S minimal
when the alcohol concentration was 5%. From these observations two conclusions can be pointed out.
The lipid effect occured only if the alcohol concentration was low enough to allow it and, for a high
alcohol content, the foam behavior was mainly governed by ethanol concentration.
With the Mosalux apparatus, LF values depend on the fractionation of surface-active agents and, more
precisely, on molecules which are adsorbed at the interface during sparging. LF depends on the alcohol
content and, clearly for the experimentation duration, lipid molecules which are less surface active and
diffuse more slowly than ethanol molecules have no time to be adsorbed. The Bikerman coefficient S
was only measured when the rates of foam formation and destruction were balanced. This was achieved
after a few minutes of gas sparging. The differences observed between samples with and without lipids
(at 2% and 5% ethanol) showed that the physical and chemical equilibria had time to establish when S
was measured. Therefore, lipids were present and active at the interface. As long as lipids were not
molecularly dissolved, they were present as some tiny droplets in the solution (Figure 16). When some
Fig. 15. Effects of addition of lipids (3 days) on foam lifetime (a), Bikerman. coefficient (b), and foam
expansion (c). Alcoholic contents (v/v) are 2% , 5% and 11.3%.
fat reaches a film interface, it spreads there and it simultaneously drags some adjacent liquid. This
causes a drainage of the film to a critical thickness below which it becomes so thin that it can rupture.
This “spreading mechanism” (Figure 16) was first described by Ross.93
Some physical interpretation of the data can be provided by the determination of static surface tensions
of the solutions. Results are given in Table 5 for three samples with and without lipid addition (five
times). Measurements were made after equilibration of the surface (10 min after pouring). Obviously,
alcohol has a strong effect on the interface when we compare the surface tension values of hydroalcoholic
solutions with those of pure water (ds1). The comparison between wine surface tensions at 2, 5 and
11.3% ethanol and references (2, 5, 11.3% aqueous alcoholic solutions) indicated the effect of surface-
active materials. Higher the alcohol content, the less surface-active the lipids (ds2) would be. There is
a competition between alcohol and lipid molecules to be adsorbed at the interface. Clearly, alcohol is
the main chemical constituent which governs the gas/liquid interface.
Finally, the lipid effect on the interface can be appreciated after comparison of the surface tension
values of various matrices with and without six times the lipid content of wines (ds3). From these
Fig .16. The spreading mechanism.
Table 5. Influence of alcohol concentration and lipids on the surface tension of wines.
2% ethanol wine 5% ethanol wine 11.3% ethanol wine

mN/m mN/m mN/m mN/m mN/m mN/m
Hydroalcoholic 65.1 ds1 = 7.4 58.6 ds1 = 14 49.4 ds1 = 23
solution
Wine surfactants effect 51.3 ds2 = 13.8 51.3 ds2 = 7.3 47.9 ds2 = 1.5
Added lipids effect 54.2 ds3 = -2.9 49.6 ds3 = 1.7 47.7 ds3 = 0.2
s(H2O) = 72.5 mN/m

ds1 = spure water -shydroalcoholc solution
ds2 = shydroalcoholc solution -swine without added lipids
ds3 = swine without added lipids -swine with added lipids
measurements, it was deduced that lipids could adsorb at the interface and have an effect on the surface
tension only when the ethanol content is relatively low (2 and 5%). For 11.3% wines with
ds3 = 0.2mN.m-1, no effect of lipids was displayed as was with the case of S with the Mosalux apparatus.
6.2.3 Ethanol
The surface pressure (P) of ethanol in water at a volume of 11.3 % is 23 mN/m though the surface
pressure of ethanol in wine is 24.5 mN/m.26 The difference between the last value and the first one
corresponds to the contribution of the other surfactants present in wine to the surface pressure, i.e.
P = 1.5 mN/m. This value of 1.5 mN/m becomes 13.8 mN/m for the same wine but with a concentration
of ethanol of 2 % (v/v) after partial dealcoholisation. This clearly shows the impact of the ethanol
content on the adsorption of surface-active compounds at the gas-liquid interface. After adding 1.3 %
ethanol to a base wine-simulating a bottle fermentation-it was observed78 that the foamability decreased
by 50 %. From these data that it can be seen that the gas-liquid interface is essentially linked to the
presence of ethanol.
6.3 Enological Practices, different Factors and Sparkling Wine Foam Quality
The ability of Champagne wine to form a persistent collar is an important feature in terms of product
attractiveness for the consumer. Knowledge of enological practices capable of modifying Sparkling
wine foaming properties is thus important for winemakers.
6.3.1 Lysozyme Treatments on Champagne Base Wines

It is now known that proteins are largely involved in the stabilization of foam in Champagne wines,
despite their low concentration. In the field of Enology, hen egg lysozyme is used to prevent malolactic
fermentation because Oenococcus oeni species are sensitive to the lytic action of this enzyme.1,36 It has
been searched whether this enzyme can reduce the lactic acid bacteria flora in wines after completion
of the malolactic fermentation. Quantities added to musts and wines ranged from 250 to 1000
mg/l.1,36,88 Lysozyme was shown to exhibit foaming properties depending on the structure of the protein
and the concentration of the bulk protein solution.100 For this reason, it was important to estimate the
effect of lysozyme on Champagne base wine foaming characteristics. Both the Pinot Noir and the
Chardonnay varieties were tested. Conclusions were nearly the same for both varieties, and only the
results concerning a Chardonnay wine are presented hereafter.
Lysozyme was added to the must after static settling67 while the natrium bentonite (soaked in water to
swell 24 hours before its utilization) was added to the wine after the alcoholic fermentation (Figure
17). Treatments with bentonite alone diminished foamability and foam stability (Table 6). After an
addition of 30 g/hl bentonite, the Chardonnay wine becomes unstable and the foam stability could not
be measured as was the case with the wine treated with bentonite after an addition of lysozyme. For an
addition of lysozyme in the must, changes in wine foamability are hardly perceptible (+ 3%). An
increase in lysozyme concentration could be the cause of small changes in foamability. Differences in
foam stability were also very small. Thus, wine foaming properties do not seem to be correlated to the
Fig. 17. Description of the Chardonnay wine production. First experiment: lysozyme is added to the must
and bentonite (or charcoal) treatments are made on the wine.
Table 6. Foamability, foam stability, lysozyme and protein concentrations in wines produced
according to the first experiment (see Figure 17). Lysozyme was added to the musts and bentonite
treatment was made on the wines.
Wines Foamability Foam stability Lysozyme Protein
content
(see treatments Difference Difference Difference Difference (mg/l) (mg/l)
fig. 25) mm compared compared mm compared compared
to the to the to the to the
control bentonite control bentonite
wine treated wine wine treated wine
Control (A) 144 – 32 – 0 11.8
50 g/hl Lysozyme 148 + 3% – 32 0% – 263 –
(addition to the
must) (B)
30 g/hl 104 – 28% unstable 0 3.2
Bentonite (C)
50 g/hl 125 – 13% + 20% unstable – –
Lysozyme + 30
g/hl Bentonite (G)
80 g/hl 55 – 62% 26 – 19% 0 1.5
Bentonite (D)
50 g/hl 117 – 19% + 113% unstable 24 –
Lysozyme + 80 (-91%)
g/hl Bentonite (H)
wine protein content when an exogenous protein is added to the wine. In contrast, previous experiments
performed with wines from the same region63,78 clearly demonstrated a correlation between the wine
protein content and wine foaming properties. These discrepancies are linked to protein characteristics.
Studies on the kinetics of adsorption of proteins at the air-water interface from bulk mixtures are still
scarce.2,14,45,114 Molecular factors that affect preferential adsorption of proteins at the air-water interface
are unknown, although it is intuitively understood that differences in the surface hydrophobicity or
hydrophilicity, as well as the rate of adsorption might be involved. In wine, yeast and grape proteins
cover a large range of MW and pHi. Some of them are only composed of amino acids whilst others are
glycosylated.66,112 Differences between the wine proteins may thus result in different foaming behaviors.
It is not the case for lysozyme because features of this protein are very unique on a chemical point of
view: e.g. a low molecular mass (14.3 kDa), a high pHi (10.4) and a compact tridimensional structure
(four disulphide bonds). Lysozyme is a highly ordered, rigid, hydrophilic and positively charged
protein.40 The kinetics of adsorption of lysozyme and bovine serum albumin (BSA) as a single component
and in 1:1 mixture experiments has been studied.2 In the single-component system, a long induction
period for lysozyme adsorption was observed, which indicates that there is an energy barrier for its
adsorption at the air-water interface. Unlike lysozyme, BSA in the single-component system did not
exhibit a lag time for adsorption. If we now consider the wine, lysozyme is unable to significantly
improve foam stability and foamability. This is probably due to a competition phenomenon between
endogenous wine proteins and lysozyme. This may also be linked to the energy barrier and the lag time
for lysozyme adsorption, thus largely limiting the presence of lysozyme at the air-wine interface.
The Chardonnay wine originating from the lysozyme-treated must plus wine bentonite treatment presents
a higher foamability than the wine treated only with bentonite (Table 6). When wine proteins are
partially removed (30 g/hl bentonite), increase in foamability due to the presence of lysozyme does not
exceed 20% maximum. For a bentonite treatment of 80 g/hl, the Chardonnay wine protein content
(measured according to a modified Bradford method72 to correct interferences due to ethanol and
phenolic compounds, essentially) was only 1.5 mg/l and the increase in foamability when lysozyme
was added became considerable (+113%). Thus, lysozyme treatment seems to be of particular interest
when a drastic bentonite treatment is necessary. The lysozyme content (as determined by RP-HPLC
one month after the enrichment in wine and four months after its addition in the must) shows a very
significant decrease in this enzyme when bentonite is used (-91%). Thus, lysozyme was not responsible
for the observed increase in foamability. This compound rather seems to play a protective effect
towards endogenous wine proteins originating from the grape berry and yeast. Lysozyme is probably
removed from wine because of its high isoelectric point (equal to 10.4). At pH around 3, it bears a
positive net charge and is thus, more easily adsorbed by bentonite (who bears an overall negative net
charge) than endogenous proteins whose isoelectric points essentially range between 3 and 4.5.12,66
In the second experiment, 67 control wines were enriched in lysozyme after bentonite treatment
(Figure 18). Bentonite at a concentration of 150 g/hl completely destroyed the Chardonnay wine
foamability (Table 7) with a decrease of 80%. After an addition of 20 or 80 g/hl lysozyme, the wine
foamability still decreased by 78% compared to the control. Lysozyme was absolutely unable to restore
foaming properties of the treated wine. The same phenomenon was observed for foam stability (-40%
for the bentonite-treated wine and-37% for the same wine + lysozyme) (Table 7).
In sum, one can say that lysozyme has a protective effect on wine foaming properties when added
before bentonite treatment. This compound can restore correct foamability even when the
“deproteinization” treatment is severe. Lysozyme treatment seems to be of particular interest when a
bentonite treatment is needed. However, increase in foamabilily obtained when the bentonite treatment
is done at the wine level before the addition of lysozyme, is poor or nil. Finally, lysozyme cannot
suppress modifications of foaming properties induced by bentonite.
6.3.2 Effects of Botrytis cinerea Infection

Some of the proteins secreted by B. cinerea, the causal agent for gray mold in the vineyard, have
enzymatic activities (proteases in particular103) and their presence in grape berries may result in alterations
of the resulting musts and wines.65 Vinifications with healthy grapes and grapes infected by B. cinerea
have been experimented on since 1993 at the Comite Interprofessionnel du Vin de Champagne.8,9
Sensorial evaluations were systematically organized to appreciate differences within wines vinted with
healthy and infected grapes, respectively. During pouring the Champagne in the glass, it was always
noted a complete absence of foam (collar) in Champagne wines that originate from infected grapes, in
Fig. 18. Description of the Chardonnay and Pinot Noir wine production. Second experiment : bentonite or
charcoal is added to the wine and the lysozyme treatment is made on the centrifuged wine.
Table 7. Foamability, foam stability and lysozyme content in wines produced according to the second
experiment (see Figure 18). The wines were treated with bentonite and lysozyme was added after
deproteinization.
Wines Foamability Foam stability Lysozyme

content
(see treatments mm Difference mm Difference (mg/l)
fig. 26) %compared %compared
to the to the
control bentonite control bentonite
wine treated wine wine treated wine
Control (K) 125 — 37 — 0
150 g/hl 25 – 80 — 22 – 40 — 0
Bentonite (L)
150 g/hl 28 – 78 + 12 23 – 38 +4 169
Bentonite+
20g/hl
Lysozyme (M)
150 g/hl 28 – 78 + 12 24 – 35 +9 798
Bentonite+
80g/hl
Lysozyme (N)
spite of normal effervescence (nucleation of bubbles). To precisely estimate the effects of B. cinerea
grape berry infection, Champagne wines originating from grapes (cv. Chardonnay, Pinot Noir and
Pinot Meunier) differing in their level of B. cinerea infection (20 to 40% ) were obtained. Wines were
analyzed using the sparging procedure (Mosalux) and the AVE system, 36 months after the beginning
of the second alcoholic fermentation in the bottle (prise de mousse in the Methode champenoise).74
6.3.2.1 Current Analyses

Champagnes vinted with healthy grapes had higher concentrations of tartaric acid than those vinted
with infected grapes. This explains in part higher pH values (between 0.2 and 0.4 unit) and lower total
acidity (between 0.6 and 1.4 g H2SO4/l) for “botrytized” Champagnes. Lower concentrations in tartaric
acid led to poor tartaric stabilizations and a very high potassium content. The presence of gluconic acid
in infected wines was another characteristic of B. cinerea metabolism (Table 8). The three control
wines showed differences in their current analyses. Nevertheless, the consequences of B. cinerea infection
were the same for the three varieties studied.
6.3.2.2 Foaming Characteristics with AVE

For each variety, Champagne wines gave Fs nearly equal values, might be whatever the level of infection.
Moreover, values were approximately the same for the three varieties. The speed at which the foam
increases seems to be relatively constant. The speed at which the height of liquid Ls increases in the
glass largely depends on the level of infection for the Chardonnay and the Pinot Noir wines (Figure
19). For the Chardonnay wines, the difference between the control and the 20% infected wine was
+43%. The increase became very important (+269%) for an infection level of 40%. For the Pinot Noir
wines, an infection level of 20% caused an enormous increase of Ls (+627%), reflecting a poor foam
stability (Figure 17). The difference between 20% and 40% infection was only 33%. For these two
grape varieties, B. cinerea infection was shown to be very detrimental to Champagne appearance
Table 8. Champagne wine current analyses (36 months after the second
alcoholic fermentation in the bottle).
Varieties Chardonnay Pinot noir Pinot meunier

% infection 0% 20% 40% 0% 20% 40% 0% 20% 40%
pH 3.15 3.31 3.52 3.14 3.19 3.34 3.07 3.11 3.28
Total acidity 4.2 3.6 3.2 4.6 4.5 4.0 5.2 4.6 3.8
(g/l H2SO4)
Volatile acidity 0.26 0.31 0.28 0.26 0.34 0.31 0.27 0.25 0.30
(g/l H2SO4)
Total SO2 (mg/l) 37 38 38 41 40 38 34 38 40
Tartaric acid (g/l) 3.4 2.4 2.1 4.0 3.4 3.0 4.2 3.0 2.3
+
K (mg/l) 344 496 710 389 450 562 373 389 537
Gluconic acid (g/l) 0.03 0.12 0.22 0.03 0.10 0.24 0.02 0.08 0.22
Fig. 19. Influence of B. cinerea infection on the speed at which the height of liquid (Ls) increases in the
glass during Champagne pouring. Ls characterizes the stability of the foam.
during pouring. Surprisingly, for Pinot Meunier wines, the effect of moldiness was not discernible. Ls
values were low and indicated a slow drainage (Figure 19). B. cinerea infection slightly diminishes the
time of liquid appearance LT in the glass for the Chardonnay and the Pinot Meunier Champagne wines
(Figure 20). At the opposite, the effect of B. cinerea infection was very marked for the Pinot Noir
wines as the infection level reached 20%, showing a very rapid drainage. The time of pouring, PT, also
considerably diminished when B. cinerea infection reached 20% for Pinot Noir (-74%) and for Pinot
Meunier (-58%) wines (Figure 21). When B. cinerea infection reached 40%, PT values were close to
the values obtained for an infection level of 20%. Moldiness influence was less perceptible for the
Chardonnay wine (-13% for an infection level of 20%). The PT value for the control wine was also low
in comparison with those of Pinot Noir and Pinot Meunier control wines (Figure 21). Moldiness
Fig. 20. Influence of B. cinerea infection on the time necessary for liquid appearance (LT) in the glass.
Fig. 21. Influence of B. cinerea infection on the time of champagne pouring (PT) in the glass.
considerably altered the foam height observed 80 seconds (H80) after the beginning of pouring the
glass: -83% for the Pinot Noir wine and-89% for the Pinot Meunier wine for an infection level of 20%
(Figure 22). For an infection level of 40%, the collar height was still worse (-90% for these two
varieties). For Chardonnay Champagne wines, values are more difficult to compare because of the
very small collar observed for the control wine (Figure 22). The same conclusion was drawn 140 s
after the beginning of pouring.
6.3.2.3 Champagne Foamability Using the Sparging Technique

Champagne wines obtained from “botrytized” grapes were compared with Champagne wines vinted
with healthy grapes. Foamability suffered considerable damages for 20% B. cinerea infection. Decreases
in foamability were approximately the same for the three studied wines (-60 to –65%) as compared
Fig. 22. Influence of B. cinerea infection on the collar height 80 s (H80) after the beginning of pouring.
with the control Champagne wines. Injuries were still more marked if the infection level reached 40%
(-68 to –76% in foamability). With the sparging technique, the effervescence is standardized. Three
similar decreases in foamability were observed for three identical levels of infection. It was not the
case for AVE where wines with similar infection levels can present various degassing kinetics.
The speed at which the height of liquid Ls increases and the time of liquid appearance LT in the glass
largely depends on the level of infection. Generally speaking, B. cinerea infection was very detrimental
to Champagne appearance during pouring the glass. Moldiness considerably altered the foam height
observed a few minutes after the beginning of pouring. These are important features in terms of
product attractiveness for the consumer. For all wines, the foamability observed under standardized
effervescence, also suffers considerable damages for 20% B. cinerea infection. Fortunately, climatic
conditions in the Champagne area during maturation and harvest rarely allow such a high level of B.
cinerea infection.
6.3.3 Fining and Charcoal Treatment

After alcoholic fermentation, the wine is a colloidal solution and suspension. The particle density,
which is close to that of the wine, the electrical repulsion forces and the diffusion phenomena lead to
a very slow and insufficient spontaneous clarification. For these reasons, organic and mineral fining
agents are still commonly used in the Champagne area to clarify still white wines. Champagne base
wines elaborated with Pinot grapes are sometimes slightly pink colored as described earlier. Then, a
charcoal treatment has to be combined with bentonite fining. Each step of the Champagne winemaking
process modifies both the qualitative and quantitative levels of wine tensio-active compounds. Different
studies have precisely estimated the modifications in the foaming properties induced by various
enological processes such as fining and discoloration treatments.
6.3.3.1 Bentonite Fining

Bentonites are swelling clays used as fining agents (bentonites possess a two-dimensional crystalline
structure based on staked layers of tetrahedra, octahedras, and tetrahedra connected to each other by
oxygen bridge).41 This treatment has been experimented in several studies and the conclusions drawn
are always the same. Only two examples are given in this section. The foamability of wines obtained
from musts treated with bentonite at a concentration of 50 g/hl decreased by 40 to 60 %.78 Correlatively,
the protein content of the same wines diminished by 20 to 60 %. In another example, the foamability
of a Pinot Noir still wine regularly decreased when the dose of bentonite was increased (Figure 23).
Other results have previously been presented in the section 4.3.1 dedicated to the effects of lysozyme
and bentonite treatment on wine foam characteristics.67
6.3.3.2 Tannins/Gelatin Fining

In contrast, techniques such as fining with gelatine-tannins (or gelatine-silica gel) significantly increased
the foaming properties of the corresponding wines when compared to the non-treated wines.73 This
fining is more and more forsaken in Champagne wine elaboration.
Fig. 23. Influence of bentonite and charcoal treatments on Champagne base wine foamability.
6.3.3.3 Charcoal Treatment

In the Champagne area, Pinot Noir and Pinot Meunier grape berries constitute approximately 3/4 of all
the bunches. For this reason, Champagne is sometimes slightly pink coloured. Plant charcoal (resulting
from plant calcinations) is then used in Enology for wine discoloration. It can also be employed to
diminish off-flavors in white wines. Treatments with charcoal always diminish foamability (Table 9)
for Chardonnay and Pinot Noir wines.67 The decrease in foamability for charcoal concentrations varying
from 30 to 80 g/hl is smaller (23 and 31% for Pinot Noir wines, for example) than the difference
observed between the two same doses of bentonite, respectively (20 and 59% for the same wine),
probably because of a lower protein adsorption. Decreases in foamability were fairly different for the
two wines (23 and 39% for the Pinot Noir wine and for the Chardonnay wine, respectively using 30g/
hl charcoal). Foam stability was unchanged even when 80 g/hl charcoal were used (Table 9); nevertheless,
treatments with charcoal never generate the foam instability currently observed with bentonite. When
the charcoal treatment was 80 g/hl, protein contents were 10.1 mg/l for the Pinot Noir wine and 10.4
mg/L for the Chardonnay wine, respectively. This indicates the poor affinity of charcoal for endogenous
Table 9. Foamability, foam stability, lysozyme and protein concentrations (mg/l) in wines produced
according to the first experiment (see Figure 17). Lysozyme was added to the musts and charcoal
treatment was made on the wines.
Foamability Foam stability

Wines mm Difference mm Difference Protein
(see treatments fig. 25) compared compared (mg/l)
tothe to the
control wine controlwine
Control (A) 124 — 37 — 14.9
Pinot Noir 30 g/hl Charcoal (E) 96 – 23% 36 – 3% 13.0
80 g/hl Charcoal (F) 85 – 31% 34 – 8% 10.1
Control (A) 144 — 32 — 11.8
Chardonnay 30 g/hl Charcoal (E) 88 – 39% 34 + 6% 11.4
80 g/hl Charcoal (F) 69 – 52% 33 + 3% 10.4
wine proteins. In another example, as for bentonite treatments, the foamability of a Pinot Noir still
wine regularly decreases when the dose of charcoal increases (Figure 23). We also observed that
bubbles in a wine treated with charcoal were larger than the control wine bubbles. This characteristic
is not appreciatedby Champagne or Sparkling wine consumers.
6.3.4 Filtration
After spontaneous clarification and fining, the turbidity of the wine is generally too high for bottling
or for the second in-bottle fermentation and it is necessary to filter it (except for some rare wines
vinified and matured in barrels). Filtration is important for the brilliance of white wines, but results
presented hereafter show that this step does not modify the turbidity of wines.
Foam expansion (E) is defined as the ratio of the foam volume to the liquid volume under standard
conditions. In Figure 24, it is clear that foam formation depends on filtration. Actually, during foam
formation, the films are still young and thick; they are drained mainly by gravity. Assuming that
bubble nucleation is constant, the difference of slopes (during foam formation) should be due to a
different rate of film rupture. These different coalescence rates can be explained as follows. During
foam formation, bubbles trap substances to stabilize their interfaces.24,40 If a lack of these components
exists, e.g. proteins and particles, that is, bentonite,46 yeast,96 the young films are not stable; the surface
tension is high and coalescence takes place easily.47
For the foam stability (LF), it is obvious that the fewer the surface-active agents, the shorter the average
lifetime of the foam. It is important to remember that this value (LF) represents the foam lifetime once
the gas bubbling has been stopped and the gravity drainage is acting. Surprisingly, as for the Bikerman
coefficient (S), LF does not vary so much and a maximum range of 1.6 seconds is obtained for wine B
(no significant differences). Foam stability is highly dependent on the physical environment for wines:
(1) under gravity drainage, the foam stability is characterized by LF and is particle (colloids or surfactants)
Fig. 24. Typical curves of foam evolution as a function of filtration porosity.

dependent; and (2) under steady dynamic conditions, the foam stability is characterized by S and is not
particle (or colloid) dependent.
An explanation for these results is proposed. At the beginning of foam formation, the physico-chemical
equilibrium has no time to establish. This means that during the first 80 seconds, the foam films are not
yet stabilized because it takes some time for the surface-active components to diffuse toward the film
interface. As for the foam behavior under steady dynamic conditions (after 5 minutes of sparging), it
is roughly the same for all cases. The quantity of alcohol is likely to be so high that particle (or colloid)
adsorption at the gas/liquid interface is hindered. Actually, the value is typical of foam in which the
bulk composition is mainly represented by a protein and a particle-free hydroalcoholic solution.
In order to corroborate this hypothesis, foaming experiments were made where wines were replaced by
a model solution and a pure hydroalcoholic solution without particles and macromolecules
(Table 10). Model systems give very different values for E and LF ; this shows that particles and
macromolecules greatly influence the foam short-term behavior. On the other hand, wines, model
solutions and not hydroalcoholic solutions are characterized by a similar value of S. This clearly
demonstrates that “major compounds” (tartaric acid, glycerol, …) influence foam behavior (comparisons
between model solutions and hydroalcoholic solutions). Except for their size, the distinction between
particles and colloids is not obvious. Experiments have clearly shown that all foam properties decreased
when the colloid or particle content decreased. In wine, it is established that particles stabilize the gas/
liquid interface.
Table 10. Analytical composition of wines, model solutions, hydroalcoholic solutions

and their foam values.
Wines Modelsolution Hydroethanolicsolution

Alcohol (% v/v) 11.2 – 11.6 11.5 11.5
Tartaric acid (g/l) 3–6 3.5 —
Lactic acid (g/l) 0–7 4.8 —
Glycerol (g/l) 4–7 4.7 —
+
K (mg/l) 300 – 700 450 —
+
Mg (mg/l) 50 – 90 78 —
Ca++ (mg/l) 60 – 120 83 —
Na+ (mg/l 5 – 15 483** —
pH 3 – 3.3 3.0 6.5
Cationic strength (mol/l) 0.01 – 0.05 0.027 —
E 1–2 0.51 0.2
LF (sec) 30 – 100 8.0 7.2
S (sec) 13 – 20 13.5 6.5
*excess of sodium due to pH adjustment.

7. CONCLUSION AND FUTURE TREND

Based on discussion on the chapter it can be assumed that the winemaking process can considerably
affect the foaming properties of Champagne wines and has to be carefully considered. It is clear that
particles and/or colloids are limiting factors to foam behavior. These should greatly influence the
winemaking process since winemakers daily use filtration techniques. A better knowledge of colloid
and particle patterns is thus of great interest. Lysozyme has a protective effect on wine foaming properties
when added before that bentonite treatment. This compound can restore correct foamability even when
the “deproteinization” treatment is severe. Information concerning the factors and components controlling
foamability and foam stability of Champagne or Sparkling wines is thus, of considerable interest to the
winemaker.
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2.1 Champenoise method

2.2 The transfer method

2.3 Methode ancestrale

2.4 Bulk method

3. CHAMPAGNE: PREAMBLE AND PRODUCTION TECHNOLOGY

3.2 The Grapes Variety for Champagne

3.3 Base Wine Preparation

3.4 Primary Fermentation

3.5 Malolectic fermentation

3.9 Secondary fermentation and bottle aging

4. MALOLACTIC FERMENTATION IN SPARKLING WINE PRODUCTION

4.1 Effect of Amino Acids

4.1.1 Effect on bacterial growth

4.1.2 Effect on D-glucose fermentation

4.1.3 Effect on L-Malic acid consumption

Oenococcus œni strains

Source: Ref. No. 33.

4.2 Effect of an Excessive Concentration of One Amino Acid

4.2.1 Effect on bacterial growth

4.2.2 Bacterial growth stimulation by amino acids

Reference L-arginine L-threonine L-tyrosine L-glycine

Source: Ref. No. 21.

4.2.3 Bacterial growth inhibition by amino acids.

Source: Ref. No. 22.

4.3 Effect on L-malic acid and D-glucose consumption

4. FOCUS ON BUBBLE DYNAMICS IN CHAMPAGNE WINES

5.1 The bubble Genesis

5.2 The Bubble Rise

5.3 The Bubble Collapse at the Free Surface

6. CHAMPAGNE WINE FOAMING PROPERTIES

6.1 Methods Used to Measure Tri-dimensional Foam

6.1.1 Foaming Properties as Determined with the Mosalux System.

Fig. 9. Principle of the Mosalux functioning (sparging procedure).

6.1.2 Artificial Viewing Equipment (AVE)

6.1.2.1 Video system

Fig. 12. Artificial viewing equipment used to determine foam parameters.

6.2 Tensio-Active Compounds and Foaming Properties

6.2.1 Proteins and Macromolecular Colloids

6.2.2 Fatty Acids

Fig .16. The spreading mechanism.

2% ethanol wine 5% ethanol wine 11.3% ethanol wine

s(H2O) = 72.5 mN/m

6.3.1 Lysozyme Treatments on Champagne Base Wines

6.3.2 Effects of Botrytis cinerea Infection

Wines Foamability Foam stability Lysozyme

6.3.2.1 Current Analyses

6.3.2.2 Foaming Characteristics with AVE

Varieties Chardonnay Pinot noir Pinot meunier

6.3.2.3 Champagne Foamability Using the Sparging Technique

6.3.3 Fining and Charcoal Treatment

6.3.3.1 Bentonite Fining

6.3.3.2 Tannins/Gelatin Fining

6.3.3.3 Charcoal Treatment

Foamability Foam stability

Fig. 24. Typical curves of foam evolution as a function of filtration porosity.

Table 10. Analytical composition of wines, model solutions, hydroalcoholic solutions

Wines Modelsolution Hydroethanolicsolution

*excess of sodium due to pH adjustment.

7. CONCLUSION AND FUTURE TREND