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Creation and Use of a Brettanomyces Aroma Wheel

A Publication of the American Society for Enology and Viticulture
discovery into practice 1:1 (2017) page 12
1Depar tment of Viticulture and
Enology, Universit y of Californ ia,
Davis, One Shields Avenue, Davis,
CA 95616; and 2Codexis,, 200
Penobscot Drive, Redwood Cit y,
CA 94063.
*Corresponding author
 
Manusc ript submitted Apr 2016,
revised Aug 2016, accepted Aug
Copyr ight © 2017 by the
American Soc iety for Enology
and Vit icult ure. All rights
Creation and Use of a Brettanomyces Aroma Wheel
C.M. Lucy Joseph,1
Elizabeth Albino,2
and Linda F. Bisson3*
Cite this article:
Joseph CML, Albino E and
Bisson LF. 2017. Creation
and use of a Brettanomyces
Aroma Wheel. Catalyst 1:12-
Goals: The ability of the yeast Brettanomyces to produce negative aroma attri
butes from grape phenolic precursors is well known. However, this yeast syn
valued or deemed positive in certain wine st yles or matrices. To better under
stand the spectrum of positive and negative aroma traits associated with the
presence of Brettanomyces in wine, we created an aroma wheel to categorize
and describe the variety of aroma impacts of this yeast on the basis of analyses
model controlled environment.
Key Findings:
• The Brettanomyces aroma wheel categorizes many of the aroma descriptors
associated with wine spoilage by Brettanomyces.
ated with microbial infection of both Brettanomyces and lactic acid bacteria
• There is no evidence for a Brettanomyces strain that produces only positive
characters in wine.
Impact and Significance: A comprehensive Brettanomyces aroma wheel was
generated with different strains under controlled product–precursor conditions
and tested for broader utility in commercial winemaking. Thirty commercial
wines described by wine critics using t wo or more of the identical or similar
terms to those found on the Brettanomyces aroma wheel were obtained from a
retail outlet. All wines purchased had evidence of presence of Brettanomyces,
LAB, or both. The inner circle, or general categories of the wheel, were used more
less generally used as commercial wine descriptors. The terms presented on
the aroma wheel can therefore be used to determine if a wine is likely to have
sensory characteristics contributed by Brettanomyces or LAB. This appeared to
“earthy,” “chemical,” and “animal.” Thus, the wheel can be used to identify wines
with a strong, but not necessarily negative, microbial signature.
Key words: Brettanomyces, ethylphenol, lactic acid bacteria, odor-active compound,
wine aroma
The yeast Brettanomyces was initially found in beer in the early 1900s by
Claussen1, but much more research has since been conducted on its role as a
spoilage agent in wine. Brettanomyces
acters in South African white wines by van der Walt and van Kerken2. Seminal
studies by Heresztyn, Chatonnet et al., and Licker et al.3
primary compounds associated with the Brettanomyces taint and investigated
the conditions required to produce them. These volatile phenolic taints are
derived from grape precursors. The two most commonly formed compounds,
Brettanomyces Aroma Wheel – 13
A Publication of the American Society for Enology and Viticulture
discovery into practice 1:1 (2017)
described as “animal,” “barnyard,” or “sweaty horse”
or as “medicinal,” “BandAidTM,” or “smok y.” However,
more recent work showed that a wider variety of aro
ma compounds are produced by Brettanomyces from
different substrates and different chemical conditions
in the wine4. Some of these characters would also be
generally described as negative, such as “rancid” and
“burning tires,” whereas others are positive, that is,
Anecdotal reports of Brettanomyces found in cer
tain wineries giving only positive aroma character
istics prompted winemakers to express interest in
the characterization of these strains and the identi
Brettanomyces strains that could be safely
used as inocula. To address this interest, we screened
the 99 independent isolates of Brettanomyces that ex
isted in the Universit y of California Davis Department
of Viticulture and Enology Wine Yeast and Bacteria
Culture Collection to assess the spectrum of end
products produced5. We conducted these studies in
a standard synthetic medium supporting the growth
of Brettanomyces with and without the presence of
ethanol6. The use of a synthetic medium enabled full
control of the type and level of precursors used as
supplements and facilitated product analysis by gas
by the yeast using panelists. These studies were aug
mented by analyses of Brettanomyces in actual red
  
characters present in these simple growth solutions
as well as in the wine studies5. 
categorization of the aroma descriptors the panel
ists used, and to distinguish the different strain and
substrate effects5, we created a Brettanomyces aroma
During these studies, winemakers sent wines for
analysis that contained high levels of volatile phenols
as determined by chemical analysis, sometimes an
order of magnitude above the published level of de
tection7, but that did not display the common aroma
traits associated with these compounds by nose. The
wines were sent in hopes that the strains isolated
would prove useful as inocula for wine production.
The isolated strains were also included in our pre
vious studies to determine if these strains produced
masking compounds, either alternative aroma charac
ters made by the yeast or enhanced from the grape.
An alternative explanation, however, was that the ma
trix of the wine, and not the yeast, was responsible
conditions and in different wine matrices, all of these
strains could produce sensorially detectable levels of
volatile phenols.
A wide variety of Brettanomyces strains were exam
ined under different substrate and oxygen conditions
5 B. bruxellensis
strains studied could make the compounds 4EP and
the two cinnamic acids, coumaric and ferulic acid, are
present. However, in the presence of other substrates
under other conditions, such as amino acids under
higher oxygen, these strains can also make a wide vari
ous studies, the major metabolic compounds produced
are often not aroma active, and those that contribute
to the aroma are often produced in very low amounts5.
However, the major sensorially active compounds are
often closely related to the major products produced
by Brettanomyces5.
pounds either as being major volatile compounds or
as compounds that were detectable, using humans as
aroma detectors in collaboration with the gas chro
dent on the substrate that was in the medium and the
dependent only on the substrate availability, and an
did not depend on either the strain or the substrate
used in the test and were produced by Brettanomyces
under all conditions5.
The Brettanomyces aroma wheel created with the
panelists during these studies potentially represented
aroma impacts of Brettanomyces in commercial wines.
We assessed the validit y of this aroma wheel for the
chasing wines that had been described by wine critics
in terms consistent with those found on the Brettano-
myces aroma wheel and evaluating those wines for the
presence of Brettanomyces
in an extension program with more than 100 wine
makers in attendance, with tastings of wines spiked
with various substrates and strains of Brettanomyces
14 – Joseph et al.
A Publication of the American Society for Enology and Viticulture
discovery into practice 1:1 (2017)
and obtaining assessments of aroma characters from
the audience. This paper presents the aroma wheel and
the validation thereof, using marketplace wines select
ed on the basis of commercial descriptions matching
multiple terms on the wheel.
Major Observations and
We determined these descriptive terms in a synthetic
Table 1 Chemical compounds produced by different strains of Brettanomyces bruxellensis and the aromas they produce.
Chemical compound
and CAS number
Type of
product Aroma*
2-Ethyl-1-hexanol 104-76-7 Ester Yes No Yes Citrus, floral
2-Methyl-1-butanol 137-32-6 Alcohol Yes No Yes Canned fruit, plastic
3-Methyl-1-butanol (isoamyl) 123-51-3 Alcohol No No Yes Banana, whiskey, chemical
4-Ethyl guaiacol 2785-89-9 Phenolic Yes No Yes Smoky, clove, spice, phenolic
4-Ethyl phenol 123-07-9 Phenolic Yes No Yes Phenolic, creosote, band-aid
Ethyl 2-methyl butyrate 7452-79-1 Ester Yes Yes Yes Mint, citrus, green apple
Phenethyl alcohol 60-12-8 Alcohol, ester No No Yes Floral, rose
1-Decanol 112-30-1 Alcohol Yes Yes Yes Waxy, floral, orange
1-Octanol 111-87-5 Alcohol Yes Yes Yes Citrus, waxy, aldehydic, floral
2-Methyl butyric acid 116-53-0 Fatty acid, ester Yes Yes Yes Blue cheese, rancid
2-Nonanone 821-55-6 Ketone No No Yes Fruity, soapy, herbaceous
3-Methyl butyric acid (isovaleric)
Fatty acid, ester Yes Yes Yes Sweaty feet, cheese
Acetic acid 64-19-7 Organic acid No Yes Yes Vinegar, sour
β-Farnesene 18794-84-8 Terpene Yes Yes Yes Woody
Butanol 71-36-3 Alcohol Yes No Yes Alcohol
Decanoic acid 334-48-5 Fatty acid Yes No Yes Rancid, sour, fatty
Ethyl acetate 141-78-6 Ester No Yes Yes Pear, apple, nail polish remover
Ethyl decanoate 110-38-3 Ester Yes Yes Yes Fruity, apple, waxy
Ethyl dodecanoate 106-33-2 Ester Yes Yes Yes Soapy, rum, clean
Ethyl isobutyrate 97-62-1 Ester Yes No Yes Fruity, rum
Ethyl octanoate 106-32-1 Ester No No Yes Fruity, pineapple, apricot
Ethyl tetradecanoate 124-06-1 Ester Yes No Yes Waxy, violet
Isobutyric acid 79-31-2 Fatty acid Yes Yes Yes Rancid, cheese
Octanoic acid 124-07-2 Fatty acid Yes No Yes Rancid, cheesy
Pentanoic acid 109-52-4 Fatty acid No No Yes Putrid, rancid, sweat, cheese,
Phenethyl acetate 103-45-7 Ester Yes Yes Yes Floral, rose, honey
Phenethyl propionate 103-52-6 Ester No Yes Yes Musty, floral, yeasty
Phenylacetaldehyde 122-78-1 Aldehyde No Yes Yes Floral, honey
2-Methoxy-4-vinylphenol 7786-61-0 Phenolic Yes Yes No Woody, cedar, roasted nuts
4-Methoxyphenethyl methanol
Alcohol, ester Yes Yes No Floral, balsamic, fruit, anise
Amyl-octanoate 638-25-5 Fatty acid Yes Yes No Wine, elderflower, orris
Bisabolene 495-62-5 Terpene Yes Yes No Woody, citrus, tropical fruit, green
Butyric acid 107-92-6 Fatty acid Yes Yes No Fruity, cheesy, acetic
Ethyl butyrate 105-54-4 Ester Yes Yes No Tutti-frutti, pineapple, cognac
Ethyl isovalerate 108-64-5 Ester Yes No No Fruity, esters, sharp, pineapple
Ethyl valerate 539-82-2 Ester Yes Yes No Tropical fruit, strawberry, pineapple
Heptanoic acid 11-14-08 Fatty acid Yes Yes No Fatty, animal
Isoamyl alcohol 125-51-3 Alcohol No No No Fruity, banana, whiskey
Nonanal 124-19-6 Aldehyde Yes Yes No Citrus, waxy, melon, aldehydic
Ocimene 502-99-8 Terpene Yes Yes No Fruity, floral, wet cloth
Octyl butyrate 110-39-4 Fatty acid, ester Yes Yes No Fruity, oily, fresh or green, earthy
Pentyl formate 638-49-3 Ester Yes Yes No Fruity, unripe banana, earthy
Phenethyl formate 104-62-1 Ester Yes Yes No Floral, green, watercress, hyacinth
Undecanoic acid 112-37-8 Fatty acid No Yes No Creamy, fatty coconut
*Descriptors derived from The Good Scents Company website:
Brettanomyces Aroma Wheel – 15
A Publication of the American Society for Enology and Viticulture
discovery into practice 1:1 (2017)
compounds by pure cultures of Brettanomyces. Dur
ing discussions with panelists evaluating the synthetic
samples, we organized the outer wheel descriptors
the smoky character often associated with Brettano-
myces presence in wine was split into two terms—a
smoked meat character under “savory” and a burned
beans character under “rotten and putrid,” but the
matrix effects will determine how these characters
are perceived in wines, often modulating the more
extreme characteristics. The “dairy” characteristics
for multiple strains of Brettanomyces, but are rarely
noted for Brettanomyces
production conditions. The thresholds of detection in
synthetic media will obviously differ from those in
wines, and detection of the same level of a given com
pound will vary across different wines. These descrip
tors may be used as a guide for the types of descriptive
terms that indicate microbial impact on wine aroma.
To test the validity of the Brettanomyces arom a
wheel, we took advantage of the wine descriptions
posted by an online wine retailer, K&L Wine Merchants
and reviews from a number of sources in a searchable
format for wines available for purchase. We searched
wines that were described with multiple terms on
the wheel as candidates for assessing the presence of
Brettanomyces. We also noted whether these wines
were described with terms related to those on the aro
ma wheel. None of these wines were described using
descriptors associated with Brettanomyces spoilage,
and none of these aroma traits were detected in our
own sensory analyses of these wines. We were more
interested in determining whether other Brettanomy-
presence of this yeast in commercial wines. We also
or had any other pertinent processing information (i.e.,
cessing of the selected wines was variable, and none
of the reported winemaking procedures was strongly
known about the widespread infections of wine with
Brettanomyces, this observation was not surprising.
by wine critics, such as “Asian spice,” as this descrip
tor seemed a hybrid of our tamarind and savory terms
when the panel evaluated these wines. We then pur
chased the 30 wines from a retailer to test for the pres
ence of Brettanomyces
Figure 1 The completed aroma wheel
using descriptors for Brettanomyces in
synthetic wine and determined by a trained
panel of judges. Twelve general categories
are broken down to between three and
seven more specic terms. The reverse of
the wheel (not shown) shows photomicro-
graphs of B. bruxellensis and summarizes
some of the information given here.
16 – Joseph et al.
A Publication of the American Society for Enology and Viticulture
discovery into practice 1:1 (2017)
We used four different methods to determine the
presence of Brettanomyces
anomyces. The initial microscopic and plating exami
nation on Wallerstein Laboratory Differential nutrient
and de Man, Rogosa, and Sharpe medium (
nystatin for bacteria revealed that 21 of the wines
had viable LAB, and another six had viable Brettano-
to have both viable Brettanomyces and LAB. A num
ber of these wines, that is, those that had not initially
yielded microbial isolates, were also tested with qPCR
for Brettanomyces
The qPCR and HPLC analyses were performed by the
analy tical wine lab at Treasury Wine Estates in Napa,
Table 2 Wines selected for the descriptors used on the Brettanomyces Aroma Wheel.
No. Vintage Variety
Brett qPCR
present Descriptors
11996 Red Bordeaux
blend, France
Pediococcus <10 11.8 6.5 LAB Smoke and earth Earthy, savory
22004 Red Bordeaux
blend, France
6900 13.7 6.9 LAB, Brett Cedar, tobacco,
underbrush, smoke/
burning embers, toast,
cigar smoke, asian
Savory, spicy,
32005 Red, Rioja,
NGb1300 12.5 7.0 Brett Umami, soy sauce,
Savory, earthy
42006 Sangiovese
Grosso, Italy
Lactobacillus 2200 13.6 7.8 LAB, Brett Cooked meat, tar,
smoke, new leather,
forest floor, and root
chemical, spicy,
52007 Sangiovese,
1600 12.5 7.5 LAB, Brett Musky, black truffle,
roses, spice, tobacco,
ginger, cola, leather,
and game
Earthy, floral,
spicy, woody,
savory, animal
62007 Red Bordeaux
blend, Australia
Brettanomyces 13,000 12.9 7.2 Brett Spice box, violets,
pencil lead, leather,
warm earth, and sweet
Spicy, floral,
woody, savory,
72008 Tempranillo,
Lactobacillus 1600 12.7 7.3 LAB, Brett Beef jerky, meaty,
spicy, savory
Savory, spicy
82008 Red Bordeaux
blend, France
Lactobacillus <10 10.8 6.8 LAB Smoke, pencil
Savory, woody
92009 Rhone blend,
Lactobacillus <10 10.5 7.0 LAB Mineral, spicy, black
tea, roasted mesquite,
graphite, charcoal,
truffle, leather, and
Earthy, spicy,
10 2009 Cabernet
LAB Scorched earth/burning
pencil shavings, rose,
gardenia and violet,
oriental spice, flinty
mineral, graphite,
tobacco, and crushed
Savory, earthy,
woody, floral,
11 2009 Pinot noir,
Pediococcus LAB Asian spices, meat
stock, soy, mineral,
umami, incense, and
Spicy, savory,
12 2009 Charbono,
Pediococcus LAB Root beer, minerality Spicy, earthy,
13 2009 Grenache-Mataro-
Shiraz blend,
Lactobacillus LAB Spicecake, funk, and
earth plus a little tar
Spicy, earthy,
14 2010 Grenache-Syrah-
Mourvedre blend,
<10 1529 450 LAB, Brett Spices, smoked
meat, acacia flowers,
graphite, scorched
Spicy, savory,
floral, earthy
(continued on page 17)
Brettanomyces Aroma Wheel – 17
A Publication of the American Society for Enology and Viticulture
discovery into practice 1:1 (2017)
CA. The results of these analyses indicated that three
additional wines also had Brettanomyces. One of the
indicating probable Brettanomyces cont amination at
Brettanomyces contami
both Brettanomyces
the wines chosen using the descriptors from the Brett-
anomyces aroma wheel tested positive for microbial
spoilage organisms.
Although these terms were developed using pure
cultures of Brettanomyces, many of the wines showed
evidence of LAB but not of Brettanomyces. This unex
pected result may mean that both of these classes of
organisms can produce the same aroma compounds
from amino acid precursors as has previously been re
ported for the mousy trait derived from lysine9. This is
also consistent with our observation of the dairy traits
found with Brettanomyces. However, these were com
mercial wines, and we cannot rule out the possibility
that Brettanomyces was present at some point during
the lifespan of the wines that tested negative for this
yeast in our study.
Table 2 (continued) Wines selected for the descriptors used on the Brettanomyces Aroma Wheel.
No. Vintage Variety
Brett qPCR
present Descriptors
15 2011 Syrah, France Lactobacillus,
LAB Smoky, meatiness,
singed vanilla,
tobacco, violet, and
Savory, woody,
spicy, floral
16 2003 Syrah, California Pediococcus LAB Smoked meat, loamy
soil, violets, lavender
Savory, floral,
17 2010 Cabernet franc,
Brettanomyces 7342 96 Brett Spicy, savory herbs Spicy, savory
18 2009 Red Bordeaux
blend, France
NG <10 335 77 Brett Mineral, cedar, violets,
and lavender
Earthy, floral
19 2007 Pinot noir,
Pediococcus <10 9 6 LAB Funky/wild, game,
earth, forest floor,
loam, truffles, spice,
Animal, earthy,
20 1999 Pinot noir,
Lactobacillus LAB Umami, violets, forest
floor, chinese five
spice, and mushroom
Savory, earthy,
floral, spicy
21 2001 Tempranillo-
Granache blend,
NG 1200 12.4 7.2 Brett Singed plum, balsamic,
sandalwood, potpourri
Savory, woody,
spicy, floral
22 2004 Tempranillo,
Lactobacillus LAB Leather, cinnamon,
ox-blood, smoked meat
Savory, spicy,
23 2007 Cabernet franc,
New Zealand
Brettanomyces Brett Cooked meats, wild
game, violets, herbs,
spice, and mineral
Savory, animal,
spicy, earthy,
24 2009 Red Bordeaux
blend, France
Brettanomyces <10 768 124 Brett Black tea, earth,
mocha, spice, tobacco,
and mineral
Earthy, woody,
25 2009 Mourvedre-Syrah-
Grenache blend,
Lactobacillus LAB Exotic asian spices,
sandalwood, crushed
rock, mineral, and
Spicy, woody,
26 2009 Syrah, France Lactobacillus LAB Mineral, smoky, spice Earthy, savory,
27 2009 Cabernet franc,
Brettanomyces 11,000 869 313 Brett Gravel and sand Earthy
28 2010 Garnacha, Spain Lactobacillus LAB Roasted herbs, spice,
and woodsmoke
Savory, spicy,
29 2010 Mourvedre-
Grenache Rhone
blend, France
Lactobacillus LAB Rose, lavender, asian
spices, forest floor,
and truffle, tarry
Floral, spicy,
earthy, chemical
30 2010 Merlot-Cabernet
Sauvignon blend
Pediococcus LAB Earth, tobacco, and
roasted cedar
Earthy, woody,
a4EP, 4-ethyl phenol; 4EG, 4-ethyl guaiacol; LAB, lactic acid bacteria; Brett, Brettanomyces.
bNG = No growth.
Blank spaces indicate not determined.
18 – Joseph et al.
A Publication of the American Society for Enology and Viticulture
discovery into practice 1:1 (2017)
Broader Impact
We analyzed these data to look for correlations be
tween the presence of LAB or Brettanomyces and the
  
grounds for these wines differed in each case. The
variabilit y in microbial population was not the only
difference among the wines. Moreover, the absence of
a particular class of organisms does not necessarily
mean that members of that class were never present
during the life of the wine and merely indicates that
uct. Another issue with any analysis is the number of
eight had BrettanomycesBrett-
anomyces and LAB. Despite these issues, we could see
some general trends in the data.
The categories spicy, savory, and earthy accounted
for most of the classes of descriptors used for the wines.
 
monly, and only rarely were the animal and “chemical”
unacceptable nature of wines with these aroma char
 
masked in the wine. The absence of fruity may be more
a result of the selection procedure we used, as those
descriptors were too commonly used to be valuable as
selection criteria. The division of the aroma categories
by type of microbial contamination typically followed
Brettanomyces 
Half of the wines with Brettanomyces
the wines with both LAB and Brettanomyces
 
was seen for the woody descriptor. High numbers of
wines with the savory and spicy descriptors had ei
ther Brettanomyces
myces had these two types of descriptors. The earthy
descriptor was also highly prevalent in all the wines;
occur exclusively with LAB, but none that occurred
exclusively with Brettanomyces. One descriptor that
stood out for LAB was Asian spice, which also in
ing that character. A few other descriptors occurred
less often, but still were exclusively associated with
and tar. The low number of samples and the variabilit y
metabolic origins among the community of microbes
crobial contamination.
The Brettanomyces aroma wheel was generated
as a consequence of multiple analyses of the growth
of this yeast in a synthetic matrix with and without
Brettanomyces can generate an array of aroma com
nol derivatives. The wheel was developed in consulta
tion with two panels and was validated by selecting
a set of commercial wines that were described using
Figure 2 The percentage of the wines with a
given microbial contaminant (Brettanomyces,
lactic acid bacteria [LAB], or both) that were
described as having a specic aroma charac-
teristic. For example, 100% of wines with both
Brettanomyces and LAB and 77% of wines
with LAB alone were described as spicy. The
information was compiled from Table 2.
Brettanomyces Aroma Wheel – 19
A Publication of the American Society for Enology and Viticulture
discovery into practice 1:1 (2017)
multiple terms on the wheel. Although the presence
of Brettanomyces
wines, the remainder contained LAB, Lactobacillus, and
Pediococcus, suggesting that LAB can produce similar
compounds in wine or, alternatively, that Brettanomy-
ces had been present in these wines at some point but
was no longer detectable at the time of the evaluation.
The Brettanomyces aroma wheel represents a useful
Experimental Design
The work presented here builds on a series of previous
ly published studies that generated an array of terms
for the description of synthetic media inoculated with
different strains of Brettanomyces with and without
Those publications contain more detailed descriptions
of the sensory analyses conducted, and we summa
rize them here. A set of 99 Brettanomyces strains were
evaluated for aroma characteristics in minimal media,
either unsupplemented or supplemented with phenyl
alanine, tyrosine, or tryptophan. Aroma terms for the
99 strains were aggregated, for a total of 2646 obser
vations and 90 unique terms. Judges met as a group
afterward to clarify terminology in order to aid in de
putrid” rather than “fruity” or “spicy.” Panel discus
sions were used to group terms into 1 of 13 classes
of related descriptors. Panelists agreed on the overall
groupings of their terms, and judges were encouraged
to use their own terms. There was no communication
among the panelists during the initial sample analy
ses and no effort was made to force agreement among
the individual panelists in the terms used. All samples
received randomized three digit codes as assigned by
Strains were presented in groups of three or four (i.e.,
whom had previous experience with Brettanomyces
aroma evaluation. Judges were asked to provide a list
of descriptors for each sample; to rate the aroma pro
tive and negative;” and to apply an overall intensity
rating on a scale of one through nine.
Descriptors that were used by more than a single
panelist during the analyses of both synthetic and ac
tual wine samples were compiled for consideration in
groupings were changed from 13 to 12 by the inclusion
tation.” More general descriptors for certain aroma
example, fuel and gasoline were combined into a single
term, and “boiled cabbage” was used for “pot stickers,”
extension program focused on identifying the impacts
of Brettanomyces in wine. Commercial wines deliber
ately made with Brettanomyces were evaluated during
this program as a series of Merlot wines that we had
produced through inoculation with different Brettano-
myces strains. Attendees were asked to evaluate the
wines, write descriptors, and then consult the Brett-
anomyces aroma wheel to determine if the descriptors
no new terms were suggested, and none were sug
gested for removal.
Estates, Tom Collins, and Josh Miles for qPCR and HPLC
wine analysis.
References and Footnotes
1. Claussen NH. 1904. On a method for the applicat ion of
2. 
    
causing turbidity on South African table wines. Ant Leeu
3.   Metabolism of volat ile phenolic
compounds from hydroxycinnamic ac ids by Brett-
Chatonnet P, Dubourdieu D and Boidron JN. 1995. The
 Brettanomyces/Dekkera sp. yeasts and lactic
acid bacteria on the et hylphenol content of red wine.
Am J Enol Vitic 
    
“Bret t” (Brettanomyces 
tion. InACS Symposium Series,
vol. 714.   
American Chemical Society, Washington DC .
4. 
 
study of the aroma of six premium quality Spanish aged
20 – Joseph et al.
A Publication of the American Society for Enology and Viticulture
discovery into practice 1:1 (2017)
  Brettanomyces
bruxellensis and other yeast species during the initia l stages
   
  The development
    
     
 
5. Albino EA . 2011. A survey of Brettanomyces/Dekkera
strains for differences in aroma production. T hesis,
University of California, Davis.
 
Production of volatile compounds by wine strains of Brett-
anomyces bruxellensis grown in the presence of dif ferent
 
Brettanomyces bruxellensis
 
6.  
   
of Brettanomyces bruxellensis strains isolated from wines.
7. Chatonnet P, Dubourdieu D, Boidon JN and Pons M. 1992. The
 WLD medium is Wallerstein Laboratory Differential me
dium and is commercially available. This medium allows
select ion against S. cerevisiae because it contains the
antibiot ic cycloheximide. Brettanomyces is resist ant to
cycloheximide and will g row on this medium display ing
a distinctive colony morphology. MRS (de Man, Rogosa
the lactic acid bacteria and is used to detect these organ
isms in wine.
9.   
pyridines by species of Brettanomyces and Lactobacillus
... Assessors were instructed to avoid hedonic terms. To facilitate aroma term generation, the Brettanomyces and red wine aroma wheel were provided during all evaluation sessions, as well as a list of terms generated after the first session [40,41]. FP was completed weekly for 42 days, starting on Day 0 immediately after inoculation, for a total of seven evaluation sessions. ...
... To determine the extent of spoilage in the inoculated wines, the terms generated by the FP were categorized into two categories, traditional spoilage or non-spoilage terms. Traditional spoilage terms were identified using the categories with negative connotations (dairy, fermentation, earthy, chemical/solvent, rotten and putrid, animal, savory, and veggie) from the Brettanomyces aroma wheel [41]. Additional terms, such as geranium and mousy, were included based on their association with Lactobacillus and Pediococcus infections [3,12]. ...
... Both replicates of Brettanomyces I1A and F3 were located in the same quadrant and were described as smoky, spicy, floral, chemical, and cherry. These sensory characteristics are often associated with a Brettanomyces infection, especially at the earlier stages when the metabolites are at concentrations that may contribute to wine complexity [41]. The FP data at Day 28 were analyzed using GPA to visualize the assessor term usage in a consensus map ( Figure 6). ...
Wine faults, often caused by spoilage microorganisms, are considered negative sensory attributes, and may result in substantial economic losses. The objective of this study was to use the electronic tongue (e-tongue) and flash sensory profiling (FP) to evaluate changes in red wine over time due to the presence of different spoilage microorganisms. Merlot wine was inoculated with one of the following microorganisms: Brettanomyces bruxellensis, Lactobacillus brevis, Pediococcus parvulus, or Acetobacter pasteurianus. These wines were analyzed weekly until Day 42 using the e-tongue and FP, with microbial plate counts. Over time, both FP and e-tongue differentiated the wines. The e-tongue showed a low discrimination among microorganisms up to Day 14 of storage. However, at Day 21 and continuing to Day 42, the e-tongue discriminated among the samples with a discrimination index of 91. From the sensory FP data, assessors discriminated among the wines starting at Day 28. Non-spoilage terms were used to describe the wines at significantly higher frequency for all time points until Day 42, at which point the use of spoilage terms was significantly higher (p < 0.05). These results suggest that application of these novel techniques may be the key to detecting and limiting financial losses associated with wine faults.
... ity regarding the production of other aroma-active molecules such as esters, fatty acids, terpenes, phenolic compounds, N-heterocycle, (see Brettanomyces aroma wheel, Joseph et al., 2017;Serra Colomer, Funch, et al., 2020;Tyrawa et al., 2019). Although some environmental factors may impact ester production (e.g., ethanol and pcoumaric acid concentrations Conterno et al., 2013;Joseph et al., 2007), esterase activity is also strain-dependent (Holt et al., 2018;Spaepen & Verachtert, 1982;Steensels et al., 2015;Verstrepen et al., 2003). ...
Full-text available
Human-associated microorganisms are ideal models to study the impact of environmental changes on species evolution and adaptation because of their small genome, short generation time, and their colonization of contrasting and ever-changing ecological niches. The yeast Brettanomyces bruxellensis is a good example of organism facing anthropogenic-driven selective pressures. It is associated with fermentation processes in which it can be considered either as a spoiler (e.g. winemaking, bioethanol production) or as a beneficial microorganism (e.g. production of specific beers, kombucha). Besides its industrial interests, noteworthy parallels and dichotomies with Saccharomyces cerevisiae propelled B. bruxellensis as a valuable complementary yeast model. In this review, we emphasize that the broad genetic and phenotypic diversity of this species is only beginning to be uncovered. Population genomic studies have revealed the co-existence of auto- and allotriploidization events with different evolutionary outcomes. The different diploid, autotriploid and allotriploid subpopulations are associated with specific fermented processes, suggesting independent adaptation events to anthropized environments. Phenotypically, B. bruxellensis is renowned for its ability to metabolize a wide variety of carbon and nitrogen sources, which may explain its ability to colonize already fermented environments showing low-nutrient contents. Several traits of interest could be related to adaptation to human activities (e.g. nitrate metabolization in bioethanol production, resistance to sulphite treatments in winemaking). However, phenotypic traits are insufficiently studied in view of the great genomic diversity of the species. Future work will have to take into account strains of varied substrates, geographical origins as well as displaying different ploidy levels to improve our understanding of an anthropized yeast's phenotypic landscape.
... Brettanomyces yeast can produce alcohol, but are well known for their ability to produce a cornucopia of pleasant and unpleasant flavor compounds. Producing flavors that range from "fruity" and "spicy" to "horse blanket" and "urine", these yeasts are equal parts pest and gift [33]. In some beer styles, like sours and lambics, these yeasts are responsible for the many unique flavors associated with those beverages, but in many others, they are considered a flaw or a contaminant [34]. ...
Full-text available
As microbreweries have flourished and craft beer brewing has expanded into a multibillion-dollar industry, the ingredients and techniques used to brew beer have changed and diversified. New brewing ingredients and techniques have led to increased concern over biogenic amines in the final product. Biogenic amine composition and concentration in beer, as well as the changes to the protein and amino acid content when adjuncts are used, have received little attention. A complex biochemical mixture, the proteins, amino acids, and biogenic amines undergo a variety of enzymatic and non-enzymatic catabolic, proteolytic, and oxidative reactions during brewing. As biogenic amines in fermented food receive increased scrutiny, evaluating knowledge gaps in the evolution of these compounds in the beer brewing process is critical.
... Brettanomyces yeast can produce alcohol but they are well known for their ability to produce a cornucopia of pleasant and unpleasant flavor compounds. Producing flavors that range from "fruity" and "spicy" to "horse blanket" and "urine", these yeasts are equal parts pest and gift (Joseph, Albino, and Bisson 2017). In some beer styles, like sours and lambics, these yeasts are responsible for the many unique flavors associated with those beverages, but in many others, it is considered a flaw or a contamination (Hersh 1996). ...
For many years the only beer that was commercially available in the United States were simple lagers and ales made primarily of barley (and other cereals), water, hops, and yeast. These beer varieties were simple and quick to produce with high levels of consistency. In the last 30 years the rise in craft and microbreweries have dramatically changed the landscape of brewing and beer. As smaller breweries rose in popularity so did the use of unique ingredients such as vegetables, fruits, herbs, meat, etc. along with higher gravity mashes and long barrel aging times. All these ingredients have a profound effect on the chemical makeup of a beer, adding a variety of organic acids, antimicrobials, amino acids, etc. These compounds become important when examining the influences of what may be the most important ingredient in beer (and all alcoholic beverages), yeast. Yeast, specifically Saccharomyces yeast (though many other genera are capable of this), consume simple carbohydrates within the unfermented beer and produce carbon dioxide and alcohol. While these fermentation biproducts are the most well-known compounds produced by yeast, there are many others. During development (log phase) and times of stress/carbohydrate starvation, yeast can often consume proteins and amino acids and produce biogenic amines. Biogenic amines (BA’s) are small nitrogenous bases produced through the enzymatic decarboxylation of amino acids by living organisms. Examples of biogenic amines include histamine, serotonin, tyramine, and spermine (though many others exist). These compounds are important and powerful signaling molecules used by every living creature on Earth. While endogenous biogenic amines produced within the body are critical to survival, exogenous biogenic amines in a consumed food product can be highly dangerous; biogenic amines can cause a variety of symptoms ranging from headaches to hyper/hypotension to pseudo-anaphylaxis. While all living things create these compounds, the levels in most food products are too low to cause any symptoms: The only products with potentially dangerous levels of biogenic amines are fermented foods as bacteria and fungi used to make these products can also produce biogenic amines. While the biogenic amine content in certain fermented foods like cheese, sausages, and lacto-fermented vegetables has been well studied, formation of biogenic amines in beer remains largely under-examined. The three main objectives of this research were: 1., to perform an extensive review of the literature to ascertain analytical methods, types, levels, and sources of biogenic amines in beer, 2., to develop a quick and effective high performance liquid chromatography (HPLC) method to examine the biogenic amine content in beer, and 3., use this HPLC method to test a variety of commercial beer sample to examine what effect (if any) these new microbrewery recipes had on the biogenic amine profile of the final beverage. Method development focused primarily on the tagging of BA’s using the Waters AccQ Rapid Amine Tag (AccQ Tag Ultra, 2014) and analysis using an Agilent Organic Acids column. In total, more than 17 HPLC methodologies were tested and only one effectively resolved a mixed amine standard solution. Despite this successful test using a mixed standard, no mobile phase adjustment was able to effectively separate biogenic amines from interfering compounds (free amino acids or other nitrogenous compounds) in a beer sample. It was ultimately decided that moving forward, an effective extraction method, involving sample cleanup and capable of separating biogenic amines from highly soluble peptides and amino acids would be necessary for successful analysis.
... Studies on Brettanomyces yeasts showed that this group of yeasts is not only a source for deterioration of the sensory qualities of drinks, but in some cases can also provide beneficial aromas for craft and specialty beverages [6]. The Brettanomyces aroma wheel was recently developed [14]. Ethyl acetate, lactate, hexanoate, and octanoate produced by Brettanomyces yeasts contributes to tropical fruit and pineapple-like flavors of lambic and gueuze beers. ...
Full-text available
Brettanomyces naardenensis is a spoilage yeast with potential for biotechnological applications for production of innovative beverages with low alcohol content and high attenuation degree. Here, we present the first annotated genome of B. naardenensis CBS 7540. The genome of B. naardenensis CBS 7540 was assembled into 76 contigs, totaling 11,283,072 nucleotides. In total, 5168 protein-coding sequences were annotated. The study provides functional genome annotation, phylogenetic analysis, and discusses genetic determinants behind notable stress tolerance and biotechnological potential of B. naardenensis.
... Although the importance of Brettanomyces species in wine, beer and bioethanol fermentation is acknowledged (9,(24)(25)(26), the molecular and biochemical features of these species required for brewing are poorly understood. Thus, the aim of this work is to review the current knowledge of the molecular and biochemical pathways, as well as the biotechnological potential of these yeasts in the brewing industry, with a particular focus on aromatic compound biosynthesis. ...
Brettanomyces is a semi‐domesticated yeast that is a crucial component of lambic beers and is increasingly attracting the attention of the brewing industry. Brettanomyces display Saccharomyces‐like features, such as a positive Crabtree effect, ethanol synthesis and tolerance to harsh environments. Additionally, Brettanomyces exhibit β‐glucosidase and esterase activities, the production of phenolic compounds and tetrahydropyridines, together with the ability to ferment dextrins and breakdown cellobiose from wooden casks. Although the importance of Brettanomyces species is documented in the production of different beer styles, the molecular and biochemical features of these species required for brewing are poorly understood. Therefore, this work reviews the current knowledge of the molecular biology and biochemistry underlying the performance of Brettanomyces in the brewing industry. © 2019 The Institute of Brewing & Distilling
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Saccharomyces cerevisiae remains the baker’s yeast of choice in the baking industry. However, its ability to ferment cereal flour sugars and accumulate CO2 as a principal role of yeast in baking is not as unique as previously thought decades ago. The widely conserved fermentative lifestyle among the Saccharomycotina has increased our interest in the search for non-conventional yeast strains to either augment conventional baker’s yeast or develop robust strains to cater for the now diverse consumer-driven markets. A decade of research on alternative baker’s yeasts has shown that non-conventional yeasts are increasingly becoming important due to their wide carbon fermentation ranges, their novel aromatic flavour generation, and their robust stress tolerance. This review presents the credentials of non-conventional yeasts as attractive yeasts for modern baking. The evolution of the fermentative trait and tolerance to baking-associated stresses as two important attributes of baker’s yeast are discussed besides their contribution to aroma enhancement. The review further discusses the approaches to obtain new strains suitable for baking applications
Occupations in wine industry in Japan can be categorized into several clusters. The standard and method of the tasting differ depending on each purpose of tasting. Even in the same cluster the methods and flavor terminology has not been standardized. However it is essential for the development of Japanese wine industries to share the evaluation of the odor among different wine clusters. Therefore, it is important to select flavor terminology by the person of each cluster and develop Japanese wine aroma wheel.
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Saccharomyces cerevisiae remains the baker’s yeast of choice in the baking industry. However, its ability to ferment cereal flour sugars and accumulate CO2 as a principal role of yeast in baking is not as unique as previously thought decades ago. The widely conserved fermentative lifestyle among the Saccharomycotina has increased our interest in the search for non-conventional yeast strains to either augment conventional baker’s yeast or develop robust strains to cater for the now diverse consumer-driven markets. A decade of research on alternative baker’s yeasts has shown that non-conventional yeasts are increasingly becoming important due to their wide carbon fermentation ranges, their novel aromatic flavour generation, and their robust stress tolerance. This review presents the credentials of non-conventional yeasts as attractive yeasts for modern baking. The evolution of the fermentative trait and tolerance to baking-associated stresses as two important attributes of baker’s yeast are discussed besides their contribution to aroma enhancement. The review further discusses the approaches to obtain new strains suitable for baking applications.
Brettanomyces is a genus of ‘rogue’ yeasts that may grow in red wines and, very occasionally, white and sparkling wines. By far the most prevalent species is Brettanomyces bruxellensis. The compounds metabolised by the yeast give off‐odours including those of antiseptic, sweat, stables, and vomit. These compounds include 4‐ethyphenol, 4‐ethylguaiacol, and isovaleric acid. The yeast may grow in wine during production, maturation, and/or storage, and off‐odours may emerge years after the wine has been bottled. Growth often occurs during the barrel maturation of wines. Prevention may be effected by the formulation of a control strategy and implementation of appropriate winemaking procedures. Treatment of affected wine prior to bottling is a two‐stage process, involving removal of viable yeast cells, and reduction in the level of volatile phenols.
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Barnyard, horse sweat, Band-aid, burnt plastic, wet animal, wet leather: all have been used to describe an aroma or flavor characteristics in some wines deemed "Bretty". The organisms cited for the production of this character are the yeasts of the genus Brettanomyces and Dekkera. In the literature, 4-ethyl phenol and 4-ethyl guaicol are the identified volatile phenolic compounds associated with this off-odor in wine. Included in this report is a review of "Brett" flavor and results from our recent study on wines identified by their respective wine makers as having "Brett" character. In wines with "Brett" character, sensory profiles showed an increase in plastic odors and a decrease in fruit odors. Analysis by gas chromatography-olfactometry (GCO) revealed two predominate odor-active compounds: isovaleric acid and a second unknown compound; other identified odor-active compounds included guaiacol. 4-ethyl guaiacol, 4-ethyl phenol, 2-phenyl ethanol, ß-damascenone, isoamyl alcohol, ethyl decanoate, cis-2-nonenal and trans-2-nonenal. Using the technique CharmAnalysis for GCO analysis, along with gas chromatography-mass spectrometry (GC-MS), odor-active compounds were identified by their respective Kovàts retention indices.
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A survey of 95 Brettanomyces strains was undertaken to identify strains that consistently give positive aroma characteristics (e.g., spicy, fruity, and floral). These strains were grown in a defined medium, and five human evaluators rated each strain according to aroma quality, and their ratings were coupled with a solid phase micro-extraction with gas chromatography (SPME GC-MS) analysis. None of the strains yielded universally positive aromas for the evaluators. A further characterization of nine of these strains grown with both aromatic amino acids (phenylalanine, tryptophan, and tyrosine) and hydroxycinnamic acids (caffeic, p-coumaric, and ferulic acids) indicated that low levels of compounds that were most important in differentiating the strains may contribute to a positive sensorial perception of Brettanomyces strain aromas under these conditions. To define the components associated with positive Brettanomyces character, the volatile aroma compounds produced by five Brettanomyces bruxellensis strains were analyzed. Compound detecting in SPME GC-MS was coupled with olfactory analysis (SPME GC-MS-O), using nine individual evaluators to identify compounds most associated with Brettanomyces character and to assess the breadth of descriptive terms used by different individuals for the same compound. Twenty-two compounds were identified as having an impact on aroma, including the well-known ethylphenols and vinylphenols, as well as several fatty acids, alcohols, esters, terpenes, and an aldehyde. © 2015 by the American Society for Enology and Viticulture. All rights reserved.
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The production of volatile metabolic products was determined for five Brettanomyces bruxellensis isolates from wine when grown in a defined medium with and without the hydroxycinnamic acids caffeic, coumaric, and ferulic or the aromatic amino acids phenylalanine, tryptophan, and tyrosine. The aim of the study was to determine the relationship between substrates and end products and to define strain differences in the production of volatile compounds. In the presence of coumaric and ferulic acids, all strains produced very similar metabolic products, primarily 4-ethylphenol (4EP) and 4-ethylguaiacol (4EG), respectively. There was a more pronounced effect of strain with the other substrates, and a variety of compounds with the potential to impact wine aroma were detected. Growth of Brettanomyces in the defined medium, with and without coumaric acid, under varying oxygen concentrations was also studied to determine the effect of this compound on growth parameters. The highest concentrations of 4EP were found under anaerobic conditions. Coumaric acid also had a significant positive affect on growth of Brettanomyces at 25% air saturation levels. At full aeration, coumaric acid addition showed little to no impact on growth or 4EP formation. Significantly higher concentrations of acetic acid were formed in the presence of coumaric acid, suggesting that 4EP formation may aid in the recycling of oxidized cofactor NAD+.
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Brettanomyces bruxellensis isolates, primarily from wine, were selected for characterization by geo- graphic diversity, vintage year of isolation, and variety of wine. A total of 47 isolates were characterized geneti- cally by sequencing a portion of the 26S rDNA gene. Physiological differences were tested in a subset of 35 iso- lates. The majority of the strains characterized by the 26S rDNA sequence were grouped into one of six clusters. Some similarities of physiology were noted, but many traits were highly variable and did not cluster into the same groupings identified by the DNA analysis. Statistical analysis found 4-ethylphenol and 4-ethylguaiacol produc- tion to be the most discriminant characters.
A fraction of glycosidic precursors extracted from different non-floral grapes has been reconstituted with a synthetic must and the must has been fermented in duplicate by yeasts belonging to different genera previously selected by their high glycosidase activity (Saccharomyces cerivisiae, Saccharomyces bayanus; S. cerevisiae x S. bayanus, Brettanomyces bruxellensis, Hanseniaspora uvarum, Kloeckera apiculata, Torulaspora delbrueckii and Debaryomyces carsonii). Fermentation was allowed to take place for 3 weeks, but only was complete for Saccharomyces yeasts. The wines obtained were analyzed by sensory analysis and by gas chromatography and gas chromatography–mass spectrometry to determine the sensory descriptors and the aroma composition. The results have shown that the yeast genus exerts a critical influence on the levels of most varietal aroma compounds, affecting to all families coming from precursors, including nor-isoprenoids, terpenols, benzenoids, volatile phenols, vanillins and lactones. Leaving aside ethylphenols and vinylphenols, most aroma compounds are produced at relatively low concentrations, but in numbers enough to likely cause a sensory effect.
Worldwide wine production has been significantly affected by Brettanomyces bruxellensis spoilage. This alteration, sometimes referred to as “Brett character”, results in the production of several volatile compounds and a large spectrum of flavours and aromas. Ethylphenols (namely 4-ethylphenol and 4-ethylguaiacol) are the best-known markers of this defect with a commonly used aggregate detection threshold of about 400 μg/l. Fifty-one Bordeaux red wines were tasted with the aim of wine profiling for commercial purposes. Ethylphenol concentrations of wines were very poorly correlated to the corresponding tasting notes. Sensory analysis was employed to demonstrate the complexity of “Brett character”. A masking effect of isobutyric acid and isovaleric acid on the detection of ethylphenols in wine was proven. This partly explained the poor correspondence between ethylphenol concentrations and presence of “Bretty” descriptors.
The aroma of six premium quality Spanish red wines has been studied by quantitative gas chromatography-olfactometry (GC-O) and techniques of quantitative chemical analysis. The GC-O study revealed the presence of 85 aromatic notes in which 78 odorants were identified, two of which-1-nonen-3-one (temptatively) and 2-acetylpyrazine-are reported in wine for the first time. Forty out of the 82 quantified odorants may be present at concentrations above their odor threshold. The components with the greatest capacity to introduce differences between these wines are ethyl phenols produced by Brettanomyces yeasts (4-ethylphenol, 4-ethyl-2-methoxyphenol, and 4-propyl-2-methoxyphenol), 2,5-dimethyl-4-hydroxy-3(2H)-furanone (furaneol), (Z)-3-hexenol, thiols derived from cysteinic precursors (4-methyl-4-mercaptopentan-2-one, 3-mercaptohexyl acetate, and 3-mercaptohexanol), some components yielded by the wood [(E)-isoeugenol, 4-allyl-2-methoxyphenol, vanillin, 2-methoxyphenol (guaiacol), and (Z)-whiskylactone], and compounds related to the metabolism (2-phenylethanol, ethyl esters of isoacids, 3-methylbutyl acetate) or oxidative degradation of amino acids [phenylacetaldehyde and 4,5-dimethyl-3-hydroxy-2(5H)-furanone (sotolon)]. The correlation between the olfactometric intensities and the quantitative data is, in general, satisfactory if olfactometric differences between the samples are high. However, GC-O fails in detecting quantitative differences in those cases in which the olfactive intensity is very high or if odors elute in areas in which the odor chromatogram is too complex.
Aims: Wine is the product of complex interactions between yeasts and bacteria in grape must. Amongst yeast populations, two groups can be distinguished. The first, named non-Saccharomyces (NS), colonizes, with many other micro-organisms, the surface of grape berries. In the past, NS yeasts were primarily considered as spoilage micro-organisms. However, recent studies have established a positive contribution of certain NS yeasts to wine quality. Amongst the group of NS yeasts, Brettanomyces bruxellensis, which is not prevalent on wine grapes, plays an important part in the evolution of wine aroma. Some of their secondary metabolites, namely volatile phenols, are responsible for wine spoilage. The other group contributing to wine aroma, which is also the main agent of alcoholic fermentation (AF), is composed of Saccharomyces species. The fermenting must is a complex microbial ecosystem where numerous yeast strains grow and die according to their adaptation to the medium. Yeast-yeast interactions occur during winemaking right from the onset of AF. The aim of this study was to describe the interactions between B. bruxellensis, other NS and Saccharomyces cerevisiae during laboratory and practical scale winemaking. Methods and results: Molecular methods such as internal transcribed spacer-restriction fragment length polymorphism and polymerase chain reaction and denaturing gradient gel electrophoresis were used in laboratory scale experiments and cellar observations. The influence of different oenological practices, like the level of sulphiting at harvest time, cold maceration preceding AF, addition of commercial active dry yeasts on B. bruxellensis and other yeast interactions and their evolution during the initial stages of winemaking have been studied. Brettanomyces bruxellensis was the most adapted NS yeast at the beginning of AF, and towards the end of AF it appeared to be more resistant than S. cerevisiae to the conditions of increased alcohol and sugar limitation. Conclusions: Among all NS yeast species, B. bruxellensis is better adapted than other wild yeasts to resist in must and during AF. Moreover, B. bruxellensis appeared to be more tolerant to ethanol stress than S. cerevisiae and after AF B. bruxellensis was the main yeast species in wine. Significance and impact of the study: Brettanomyces bruxellensis interacts with other yeast species and adapts to the wine medium as the dominant yeast species at the end of AF. Contamination of B. bruxellensis might take place at the beginning of malolactic fermentation, which is a critical stage in winemaking.