Brettanomyces in wine - [email protected]

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Reprinted with Permission from: Proceedings of the 5th International Symposium on Cool Climate
Viticulture and Oenology, 16-20 January 2000, Melbourne, Australia
Brettanomyces in Wine
Thomas Henick-Kling1, Christoph Egli, Jonathan Licker, Craig Mitrakul, Terry E. Acree
1
Associate Professor for Enology, Cornell University, Department of Food Science and Technology,
Geneva, NY 14456-0462, USA.
The Brettanomyces / Dekkera yeasts can be found in fermenting must and in wine. Typically they
grow after alcoholic and malolactic fermentation during storage of wine in tank, barrel, or bottle.
They contribute characteristic ‘bretty’ flavors which are described as smoky, barnyard, plastic, burnt
plastic, vinyl, Bandaid ™, and creosote. They can also contribute a ‘metallic’ bitterness. Compounds
which are responsible for brett flavor in wine include 4-ethyl phenol, 4-ethyl guaiacol, isovaleric acid,
and unidentified "burnt plastic" compound.
Descriptive sensory evaluations show an inverse
relationship between fruity and bretty flavor perception. The brett aromas in some wines are considered
a positive attribute, especially when present at low concentration. Often these flavors are considered a
defect. The wine’s varietal and regional flavor characteristics might be completely masked by these
flavors and the wine can be unpleasantly bitter. 4-ethyl phenol is used by some wineries as an
indicator compound for the activity of Brettanomyces. Yet some wines having a strong brett flavors do
contain none or very little 4-ethyl phenol. Preliminary studies show that 4-ethyl phenol is formed all
through growth of Brettanomyces. Thus this compound can be used to confirm the presence of bretty
flavors. The search for indicator compounds formed during early stages of growth of B. continues.
The Brettanomyces yeasts can produce the characteristic brett flavors when growing at low cell density
of several hundred to several thousand cells per mL. The fact that these yeasts are often present at low
cell numbers and that they are slow growing makes detection difficult. Genetic analyses of wine
isolates have shown that only strains of B. bruxellensis grow in wine. Semi-selective plating is
performed prior to genetic analysis on media including cycloheximide. The development of DNA
based probes has made it possible to reliably identify the yeast once it is plated on a nutrient agar plate.
Further developments of specific probes and of probe application techniques will improve the
specificity and speed of detection.
What are of Brettanomyces / Dekkera yeasts?
The management of Brettanomyces is fast becoming one of the most important winemaking issues.
Brettanomyces/Dekkera yeasts are reported to be involved in spoilage by off-flavor production in beer
and wine (Anonymous 1998, Chatonnet et al. 1992, Chatonnet et al. 1995, Edlin et al. 1995, Kuniyuki
et al. 1984, Spaepen & Verachtert 1982). In wine these yeasts typically grow in low cell numbers after
completion of the alcoholic and malolactic fermentation during aging of wine in barrels and bottles.
The aroma characteristics of their spoilage-causing metabolites were described as burnt plastic, smoky,
barnyard, horse sweat, leather, and wet wool (Boulton et al. 1996, Chatonnet et al. 1992, Fugelsang
1997, Heimoff 1996, Heresztyn 1986, Ibeas et al. 1996, Licker et al. 1998). To some people the
presence of some of these aroma characteristics can be a positive aroma attribute. To others, even the
slightest hint of Brettanomyces character is cause for a wine’s rejection. Despite the traditional
negative connotations of 'brett' character, in some cases, the yeasts may add positive characters and
complexity to red wines (Heimoff 1996, Fugelsang 1997). These flavors though can become so
dominant that they completely mask the varietal and regional flavor characteristics of a wine. These
flavors which we call bretty here and which we will describe further in this paper should not to be
confused with mousiness (see discussion below). To be able to protect the wines against spoilage by
these yeasts and to possibly be able to positively integrate some of their flavor into some wines it is
important that winemakers are familiar with the Brettanomyces flavor characteristics and with its
physiology. Winemakers need new tools for reliable and early detection of Brettanomyces in their
wines before the wine quality is altered.
In 1960, van der Walt and van Kerken (1960) reported the ascospore formation in yeast strains
previously classified as the non-sporeforming yeasts Brettanomyces and proposed that a new genus be
created to accommodate the sporogenous forms. Van der Walt proposed the name Dekkera for the new
genus and reported two species, D. bruxellensis and D. intermedia (1964). Several taxonomic changes
have occurred since van der Walt's introduction of the new genus with additions and deletions to both
genera as the demonstration of spore formation and the absence of spore formation occurred.
Brettanomyces, the anamorph form of the genus Dekkera includes five species. B. nanus was added to
the four recognized species B. bruxellensis, B. anomalus, B. custersianus and B. naardenensis
following the renaming of Eeniella nana on the basis of rDNA sequence homology (Anonymous 1998,
Boekhout et al. 1994, Hoeben and Clark-Walker 1986, Yamada et al. 1995). Molecular studies of
species within this genus were initiated based on the mitochondrial genome (Hoeben and Clark-Walker
1986) and the rRNA genes (Molina et al. 1993). Molina et al. (1993) carried out restriction analysis of
the 18S rRNA from various Brettanomyces yeasts and found good correlation with isoenzyme
electrophoresis and DNA homology analysis. Further investigation of the ribosomal RNA genes on the
partial sequence level of the 18S and 26S rRNA was done by Yamada et al. (1994) and Cai et al.
(1996), who determined the sequence of various Brettanomyces and Dekkera yeast strains by PCR and
direct sequencing. Ibeas et al. (1996) used nested PCR to track one single strain in sherry wine.
Mitrakul et al. (1999) used RAPD-PCR to discriminate yeast strains within the species B. bruxellensis.
Although strain discrimination worked out well by RAPD-PCR in that study, results can be difficult to
reproduce using this method. Alternatively, ribosomal genes have been used extensively to give
information about the phylogenetic relationship of taxa. In many eukaryotes ribosomal genes in yeast
are organized as 18S-5.8S-26S operons which are tandemly repeated 50 to 200 times per haploid
genome (Planta and Raué 1989). The two internal transcribed spacers (ITS1 and ITS2) separate the 18S
and the 26S from the 5.8S rRNA gene. Conserved portions of rRNA genes are useful for inferring
phylogenetic relationships between strains. ITS regions are less conserved due to less evolutionary
constraints (Musters at el. 1990) and therefore can be used to discriminate species within some genera.
Ribosomal DNA restriction fragments length polymorphism and comparative sequence analysis of the
two internal transcribed spacer (ITS) regions located between the ribosomal RNA genes was carried
out using Brettanomyces/Dekkera yeast reference strains and wine isolates (Egli et al. 2000). Both
ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make
them suitable as specific identification target sites. Specific oligonucleotides were designed for each
Brettanomyces/Dekkera species and evaluated for specificity and reliability using reference strains and
wine isolates. Use of these specific primers allowed for species-specific discrimination.
Brettanomyces/Dekkera yeast isolates from wine were shown to uniquely belong to the species B.
bruxellensis.
The traditional methods of identification as described by van der Walt and Yarrow (1984) and Barnett
et al. (1990) are laborious and time consuming. Identification takes 3 to 4 weeks and often results are
ambiguous and can be unreliable (Mitrakul et al. 1999). The development of molecular techniques for
yeast identification has substantially reduced the time for identification with improved reliability
through the direct analysis of DNA, thus identification will not vary with the physiological state
(Dowhanick, T. M. 1995, Ness et al. 1993). Included among the molecular techniques for
identification are DNA fingerprinting methods. DNA fingerprinting describes any procedure which
provides a unique profile of DNA for a given organism (Meaden 1990).
Mitrakul et al. (1998) and Egli et al. (2000) isolated and described Brettanomyces from 3 Cabernet
Sauvignon wines which were characterized by tasting panels as having no, some, and strong bretty
flavor characteristics (Licker et al. 1999). Using the molecular methods of karotyping, restriction
fragment length polymorphism (RFLP), and RAPD-PCR analysis the isolates were indentified as
Brettanomyces / Dekkera bruxellensis (Fig. 1). To test the specificity of the OPA-03 primer in the
RAPD-PCR assay for Brettanomyces yeast, several other yeasts which are abundant in the must or
wine were taken as controls. Included were Candida guilliermondii (lane 13), Pichia anomala (lane
14), Hanseniaspora uvarum (lane 15), and Saccharomyces cerevisiae (lane 16). Of these yeasts
Hanseniaspora uvarum has the ability to grow in the presence of cycloheximide as do
Brettanomyces/Dekkera yeasts (Barnett et al. 1990; Fugelsang 1997). None of the fingerprints of these
yeasts was similar to those obtained for Brettanomyces/Dekkera yeasts (Fig. 1B) confirming the
usefulness of primer OPA-03 for detection and discrimination purposes. Similar patterns were found
for all isolates of the three vintages 1989, 1992, and 1994 matching with D. bruxellensis reference
strains (Fig. 1C). Further investigations showed that over the years, different strains of Brettanomyces
evolved in the wines of this winery (Egli, unpublished). Each wine typically contained one strain of
Brettanomyces (Mitrakul et al. 1999).
A
M 1 2 3 4 5 6 7 8 9 10 11 12 c
bp
26 45
16 05
11 98
67 6
51 7
46 0
39 6
35 0
B
bp
26 45
16 05
11 98
67 6
51 7
46 0
39 6
35 0
M 13 14 15 16 c
C
M 89 92 94 c
bp
26 45
16 05
11 98
67 6
51 7
46 0
39 6
35 0
Figure 1. Species determination of Brettanomyces/Dekkera strains and wine isolated yeasts using
RAPD-PCR fingerprints generated with primer OPA-03. (A) Lanes 1-7 represent strains of the species
D. bruxellensis (lane 1, D. bruxellensis; lane 2, B. custersii; lane 3, D. abstinens; lane 4, B.
bruxellensis; lane 5, B. intermedius; lane 6, B. lambicus; lane 7, D. intermedia), lanes 8-10 represent
strains of the species D. anomala (lane 8, D. anomala; lane 9, B. claussenii; lane 10, B. anomalus), and
lanes 11 and 12 represent single strains of the species D. custersiana (B. custersianus) and D.
naardenensis (D. naardenensis), respectively. (B) Lane 13 represents Candida guilliermondii; lane 14,
Pichia anomala; lane 15, Hanseniaspora uvarum; and lane 16, S. cerevisiae. (C) Lanes 89, 92, and 94
represent single isolated colonies from the 1989, 1992, and 1994 wine samples, respectively. The
patterns were identical for all ten isolated strains in each vintage. Only one strain is shown for that
reason. Lanes M show the D-15 DNA marker, and lanes c the negative control containing no DNA.
(Mitrakul et al. 1998)
Occurrence of Brettanomyces
In 1930 Krumbholz and Tauschanoff (1933) isolated the yeast Mycotorula intermedia from a French
grape must; Custer (1940) reclassified it as Brettanomyces bruxellensis. In 1911 from Wädenswil,
Osterwalder (1912) isolated and identified the yeast Monilia vini in Swiss apple wine; Schanderl
(1950) and Schanderl & Draczynski (1952) presumed it to be a Brettanomyces species; based on the
physiological evidence of Osterwalder, van der Walt & van Kerken (1958b) characterized it as
Brettanomyces intermedius. In the early 1950's Schanderl (1950) and Schanderl & Draczynski (1952)
of Geisenheim reported the first uncontested occurrence of Brettanomyces in bottled wine (van der
Walt and van Kerken, 1958a) ; the isolation was from a German sparkling wine. Since then
Brettanomyces yeast have been reported in almost every winemaking region of the world (Licker et al.
1998).
Although Brettanomyces/Dekkera yeasts are typically not included in the genera of yeasts found on the
surface of grapes (Fleet & Heard 1993), it is quite common for these yeasts to be found in the winery
(Chatonnet et al. 1995, Fugelsang 1997, Licker 1999). We have indications that Brettanomyces can
already occur on some grapes. Studies currently underway will be testing this hypothesis. Researchers
isolated Brettanomyces in fermenting grape must from around the world: France (Krumbholz and
Tauschanoff, 1933; Domercq, 1956); Germany (Schanderl, 1950);Italy (Florenzano,1951; Trioli,
1996), South Africa (van der Walt and van Kerken, 1961); Uzbekistan (Mavlani, 1968); New Zealand
(Wright and Parle, 1974); and Spain (Mateo et al., 1991; Querol et al., 1990). Brettanomyces
populations were found to be a minority in the microflora of fermenting musts. Domercq (1956)
detected two Brettanomyces cultures out of 80 red; no cultures were isolated out of 38 white grape
musts sampled from French wineries. With a refinement of isolation procedures Brettanomyces were
isolated consistently from fermentations in 6 of 15 New Zealand wineries, and occasionally from 4 of
the 15 (Wright and Parle, 1974). In total they were isolated in 33 of 124 fermenting white and red
musts in New Zealand.
The alteration of wines by Brettanomyces/ Dekkera yeasts typically occurs during barrel aging of wine
prior to bottling (Chatonnet 1995). These yeasts have caused wine spoilage in wine producing areas
around the world (Fugelsang 1997, Boulton et al. 1996, Chatonnet et al. 1992, Heimoff 1996,
Heresztyn 1986, Ibeas et al. 1996).
However, little is known about these yeasts and rapid and reliable detection of these yeasts is needed
before positive or negative aromas and flavors can be attributed to particular strains.
Kunkee and Amerine (1970) wrote of the problems associated with yeast detection: "The major
problem of studies on yeast ecology results from the methods used for isolating the microflora. So
many different techniques have been used that comparisons of frequency of occurrence must be made
with caution". Van der Walt & van Kerken (1961) were initially unable to isolate Brettanomyces from
musts and winery equipment using customary media and techniques. It can take a week or longer
before colonies are visible, a far longer incubation time compared to Saccharomyces and other yeast.
Custer (1940) was first to note the characteristically slow growth of the genera. Saccharomyces,
Kloeckera, Metschnikowia, Pichia, Candida, or "wild" yeast develop earlier in culture thereby
hindering detection of Brettanomyces. Early mold development inhibits detection by covering the
media surface. Brettanomyces may go undetected; plates may be discarded before colonies develop.
Addition of 100 ppm cycloheximide (actidione), 10 ppm thiamin, and 0.5% calcium carbonate to
media aids in the selective detection of Brettanomyces yeast (Phaff and Starmer, 1980).
Another problem of Brettanomyces detection in the winery is large variations in barrel to barrel
populations. In a 45 week barrel sampling study of stored Cabernet Sauvignon wine, Blazer and
Schleußner (1995) determined it was necessary to stir barrels before plating. Measured populations
increased after stirring - in some cases by 10-fold or more; in others, detection depended on stirring.
Fugelsang (1993) stated that wood cooperage is the most frequently cited source of Brettanomyces
within the winery. It must be remembered though that Brettanomyces can enter a winery with the
grapes. Wineries are encouraged by some enologists in the United States to destroy Brettanomycesinfected barrels to avoid further contamination within the winery (Boulton et al., 1996; Heimoff, 1996).
Another problem is barrel to barrel variations within the same lot. In a recent two-year Californian
winery investigation (Frey et al., 1996; Heimoff, 1996), new oak, stainless steel, and previously
Brettanomyces infected barrels were filled with Cabernet Sauvignon inoculated with 4 cells/mL of a
Brettanomyces culture. Similar barrels were filled with sterilized wine. All of the wooden barrels
(new & old containing inoculated & sterile wine) developed Brett. populations. Brettanomyces
populations were undetectable in all of the stainless steel barrels.
Flavors associated with Brettanomyces yeasts
Brettanomyces yeasts and their characteristic flavors were first described in English beers (Licker et al.
1998). To obtain the "English character" flavor production in beer Claussen (1904) stressed "a general
rule cannot be given for all cases, but the quality of Brettanomyces to be added must be regulated by
local circumstances, more especially by the time the beer has to be stored and by the temperature of the
storing room." A Brettanomyces inoculation with a wort of 1.055 specific gravity and a room
temperature of 24-27 °C would achieve the "English" character.
Shimwell confirmed these
conditions: a 1.060 specific gravity was essential to achieve a "vinous" wine-like flavor (Shimwell,
1947); in contrast, a beer under 1.050 would produce an unpalatable and turbid beer with an
objectionable, insipid flavor and aroma (Shimwell, 1938) . As Shimwell (1947) noted, Brettanomyces
can behave "as a desirable organism in one beer and an undesirable one at one and the same brewery".
Clearly, in winemaking practice we are not yet at the stage where we could utilize the flavor
characteristics of Brettanomyces as deliberately as was discribed by these two brewing researchers.
We still lack sufficient understanding to the different flavor characteristics, of the different strains of
Brettanomyces, of the conditions under which they grow in wine, and the conditions which affect the
type of flavors they produce.
We started our investigations of the flavor contribution of Brettanomyces yeasts by obtaining different
wines which were described by winemakers as being bretty. Licker et al. (1999) studied two groups of
commercial wines with suspected “Brett” character using two trained panels of judges. One group of
judges (A) were winemakers from New York State and experienced tasters from the New York
Agricultural Experiment Station. The second panel (B) was an international group of enologists
including members from Australia, France, Germany, Switzerland, and the USA. The first group of
wines included four Cabernet Sauvignon and two Pinot Noir wines; the second included four Cabernet
Sauvignon wines. All were evaluated by sensory descriptive analysis and GC/MS 4-ethyl phenol
analysis. Characteristic “Brett” aromas such as plastic, burnt plastic, Band-aid™, cow manure,
barnyard, and horse sweat were summarized by panel A as ‘plastic’. For panel B ‘plastic’ included
only plastic, burnt plastic, and Band-aid™ odors. Dry manure and sweaty/animal were separate
descriptors.
In both groups, the wines were differentiated by univariate analysis of variance
(ANOVA) and multivariate discriminant analysis (DA) by two descriptors: plastic and fruity. Figure 2
shows the clear separation of the 3 Cabernet Sauvignon wines, the youngest wine being the most fruity
and least plastic (bretty), the oldest being the most plastic (bretty) and least fruity. The 1992 wine was
characterized by both some fruit and some plastic, the brett aromas appeared to give it some spicy
aroma character. The a priori “Brett” observations from the winemakers proved to be a consistent
predictor of Brett character for all wines. In these wines the intensity of perceived brett character
agreed with the 4-ethyl phenol concentrations. The “strong Brett” wines were the older vintage wines
with higher 4-ethyl phenol concentrations, higher plastic and lower fruity mean scores. Wines with
“maybe some” and “no Brett” had the lower 4-ethyl phenol concentrations and more fruit with less
plastic scores. This investigation shows that aroma modifications by Brettanomyces yeasts can be
reliably detected and quantified with trained tasters. Further investigations into the chemical basis of
the ‘Brett’ aromas will allow us to use chemical indicators to detect activity of these yeasts early.
1
Odor activ ity
100 %
2
4
.
9 8
5 6
3
12
Odor activ ity
Odor activ ity
92CS
15
14
b
2
3
7
5
9
8
10
14
1
4
6
15 1
1
100 %
89 CS
7
11 10
13
1
100 %
0
a
12
13
3
2
4
5
7
11
15
700
10
14
8 6 912
1
3
Retentio n Ind ex (OV101)
1500
~ 94CS
plastic
~
smoky
~ ~
~
~~~
~
~
~~ ~
c
fruity
DF1 = 87%
~
spicy
Figure 2. Scatterplot of the canonical scores for the two discriminant functions based on data from
panel A (1989, 1992, and 1994 Cabernet Sauvignon produced in the same winery from the same
vineyard sources).
Figure 3. Odor spectrum gas chromatograms on an OV101 column - the top 15 odor active
compounds: (a) "no Brett", (b) "medium Brett", (c) "high Brett". Numbers on the chromatogram refer
to the chemical structures in figure 4. an OV101 column - the top 15 odor active compounds: (a) "no
Brett", (b) "medium Brett", (c) "high Brett". Numbers on the chromatogram refer to the chemical
names in Table 1.
Comparing the odor spectrum gas chromatograms of the three wines (Fig. 3 and Table 1), in a general
effect was observed. 'Floral', 'fruity' compounds were the dominant odorants in the "no Brett" wine
while 'rancid', 'plastic' odors accounted for 1/3 or less of the odor activity; in the "medium Brett" wine,
the 'floral', 'fruity' compounds decrease to 1/2 or less of the odor activity while the 'rancid', 'plastic'
compounds increase to 2/3; in the "high Brett" wine, the 'rancid', 'plastic' compounds were the
dominant odorants while the 'floral', 'fruity' compounds were far less dominant. The 'floral' odorant
identified as 2-phenyl ethanol was the dominant compound in "no Brett" and "medium Brett" wines; in
the "high Brett" wine, it was equally as dominant as isovaleric acid and the unknown compound. The
'fruity' odorant ß-damascenone was equally dominant among the three wines; for this reason, it should
not be considered as a contributor to "Brett" aroma.
Odor/Compound
0
Odor activity +/- S. E.
50
100
rancid, isovaleric acid
burning tires, cis-2-nonenal
burning tires, trans-2-nonenal
plastic, guaiacol
plastic, 4-ethyl phenol
plastic, ethyl decanoate
plastic, unknown
C94CS
C92CS
C89CS
Figure 4. Comparison of the odor activity of 7 dominant bretty odor components in Cabernet
Sauvignon wines from 3 different vintages from the same winery (GCO analysis, Licker 1999).
The odor active compounds were ranked from 1-15, 1 being the most odor active compound. The
numbers above each peak in the chromatogram correspond to the compound and associated descriptor
in the table.
Isovaleric acid (3-methyl butanoic acid) was found to be the dominant odorant in the "high Brett" wine
as detected by CharmAnalysis. The odor described by the GCO sniffer was 'rancid'; the chemical
identity of the odorant was confirmed by GC-MS. This acid is produced in wine by yeast as a
metabolic byproduct of protein (99). Volatile phenolic compounds, such as 4-ethyl guaiacol, guaiacol,
and 4-ethyl phenol, were also among the dominate odor active compounds in this wine; however, the
individual contribution by each of the three phenolics was half or less than the odor activity of
isovaleric acid. The chemical identity of the second most dominate odorant in the "high Brett" wine,
identified as 'plastic' by CharmAnalysis, remains 'unknown'.
Bretty and mousy flavors
Bretty flavors should not be confused with mousy flavors. When we refer to bretty flavors we refer to
aromas such as burnt plastic, bandaid™, barnyard, manure, creosote, wet wool, sweaty leather, and a
bitter, matallic-like taste. These are the terms with which California and New York winemakers
described bretty flavors (Licker et al. 1998, Licker et al. 1999). In tasting many wines with different
intensities of brett flavors only once have we tasted a wine that was bretty and mousy. On the other
hand we have tasted many wines which were mousy but not bretty. Mousiness is a distinct flavor
which is perceived retronasally, it is almost never smelled. It is described as the aroma of mouse or
rodent urine, in some mild cases it has been described as black walnut like. While the smoky,
barnyardy, sweaty aromas are found attractive by some people, the mousy flavor is always strongly
rejected by people who perceive it. Heresztyn (1986) described tetrahydropyridines formed by some
isolates of Brettanomyces and by some lactobacilli as the chemicals responsible for the mousy taint.
The taint was produced by each class of microorganisms only in the presence of lysine and ethanol.
Researchers at the Australian Wine Research Institute (Henschke 1996a) have since identified two
additional compounds associated with mousiness and produced by both Brettanomyces and
Lactobacillus species: 2-acetyl-1-pyrroline (ACPY) and ethyltetrahydropyridine (ETPY). In tainted
wines ETPY was found at concentrations below its high odor threshold, thereby contributing little to
the sensory character of mousiness. The two acetyl-tetrahydropyridines and ACPY were reported to be
the main mousy taint compounds. It is still unclear how the Brettanomyces implicated in the mousy
taint relate to the many Brettanomyces strains isolated from bretty wines which were not mousy.
Clearly more research is necessary to compare wine descriptions from different countries and to
understand which strains of Brettanomyces, under what conditions, can form the mousy flavors in
wine.
Physiology of Brettanomyces yeasts – conditions which favor their growth and ways in which
winemakers can limit their growth
Even if some infection by Brettanomyces and some amount of bretty flavors is attractive in some wines
in other wines this contribution of Brettanomyces to the wine flavor profile is not desired at all.
Therefore the winemaker must constantly look out for signs of infection (presence of Brettanomyces or
appearance of characteristic bretty aromas). Then infected and non-infected wines can be kept separate
and treated as necessary. We still lack very good ecological studies which show the path of infection
with Brettanomyces yeasts in the winery. Now that we have the molecular biological tools to identify
individual strains of Brettanomyces (Mitrakul et al. 1998, Egli et al. 1999) we can design studies which
tell us where exactly a particular strain in a particular wine came from. Brettanomyces can be brought
in with the grapes particularly damaged grapes contain a large number of microorgansims.
Also,apparently more commonly, Brettanomyces is spread to new batches of wine from other batches
of infected wine or from contaminated barrels and other winery equipment. It appears that
Brettanomyces yeast are not very competitive with the main fermentative yeast Saccharomyes. We
don’t know the nutrient requirements or the range of pH, temperature, and alcohol concentration over
which Brettanomyces can grow. Why is Brettanomyces found almost always in red wines and almost
never in white? We do know that these yeasts are sensitive to SO2, they can be removed by
appropriately tight filters (1 micrometer and smaller) and they are killed by ozone and dimethyl
dicarbamate (DMDC, Velcorin™).
Good cellar hygiene is the first step in controlling infection of Brettanomyces. Equipment, tanks and
barrels, the cellar environment should be kept clean at all times. New and used barrels can bring
Brettanomyces into a winery. Batches of wine infected with Brettanomyces should be kept separate
and monitored carefully (add SO2 or filter if yeast population rises to high cell density, more than 10 2
cells per mL). Before blending an infected batch with another wine remove the yeast by filtration or
kill the yeast with Velcorin™. Carefully sanitize the container in which the infected wine was stored.
Several studies indicate that Brettanomyces can grow in the grape must.
This is important to
remember when skin contact or cold soaking is used. To reduce the risk of Brettanomyces growth
before alcoholic fermentation, damaged grapes should be avoided, SO2 should be added (20-50 mg/L).
After cold soak the must should be warmed quickly to suitable fermentation temperature (for most
wine yeast 16-20°C) and a yeast starter culture should be added once the must has been warmed. A
good active fermentation with Saccharomyces cerevisiae apparently suppresses growth of
Brettanomyces. After completion of alcoholic fermentation the wine again is under increased risk of
infection by Brettanomyces. To limit this risk, malolactic fermentation should be induced quickly, best
with the addition of a starter culture. After completion of malolactic fermentation the wine should be
racked and sulfited or a least sulfited (to maintain 0.8 mg/L molecular SO2). Maintaining adequate
free SO2 during barrel or tank aging of wine will prevent in most cases Brett growth. According to
Chatonnet et al., (1993), the only way to limit the growth of Brettanomyces in red wines aged in oak
barrels is to maintain a sufficient concentration of free sulfur dioxide (SO 2) throughout the aging
process. At least 7 g/barrel of SO2 gas should be used to disinfect empty barrels. Filled wine barrels
should receive 20 to 25 mg/L of free SO2, 30 to 35 mg/L in the hot summer. These concentrations
should be maintained throughout the aging process to limit Brettanomyces development (Chatonnet et
al., 1993). Scott (1995) showed yeast and bacteria could penetrate the porous cellular structure of oak
barrels and establish active permanent sub-surface mixed cultures even after cleaning. Variations in
wine composition (pH, anthocyanin concentration, nutrient content, and temperature) can affect SO 2
treatment in wine (Rose, 1987; Smith, 1996). Brettanomyces can endure SO2 treatment in barrels
(Hock, 1990). The yeast survives treatment in areas of limited SO 2 contact: around bung holes, in the
oak, and in the yeast sediment (lees). Of course in the case of red wines it is often not possible or
desirable to maintain high free SO2 concentrations during wine aging. In this case the wines must be
monitored regularly for presence of Brettanomyces yeasts and for flavor change.
Today, monitoring the concentration of 4-ethylphenol is still the most convenient method to monitor
many batches of wine. In most cases an increase of 4-ethylphenol indicates presence and activity of
Brettanomyces. Not always though is the presence of Brettanomyces indicated by 4-ethylphenol and
not always when 4-ethylphenol is found can live Brettanomyces be found. The main disadvantage is
that once we find increased concentrations of 4-ethylphenol in a wine the wine already is noticeably
bretty. Thus the 4-ethylphenol content only confirms what a trained winemaker can taste. Preliminary
data from Zoecklein, Whiton, and Fugelsang (personal communication 1999) indicate that 4ethylphenols is formed continually while live cells of Brettanomyces are present in wine (Fig. 5). The
4-ethylphenol concentration increases with cell number and as long as live yeast are present (including
decline phase of growth, in this case after). We should have other indicator chemicals which are
formed by Brettanomyces during early stages of infection, before the typical brett aromas are
developed. Also we need microbiological detection tests which are easier, quicker, and more reliable
than the plating tests and identification tests. A direct assay for Brettanomyces cells or DNA in the
wine is needed. This test should be quick (minutes) so that it can be used during wine transfers.
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