[ 2007 年 日本ブドウ・ワイン学会　招待講演要旨 ]

The Role of Brettanomyces in Wine Production

Linda F. Bisson1*, C. M. Lucy Joseph2, Donald O. Wirz3 and Bradford S. Kitson3

Brettanomyces (Dekkera) bruxellensis is ubiquitous yeast found in wines, beers, ciders, fruit drinks,

sodas and biofuel facilities. In wine this yeast is associated with a spectrum of characteristic aroma

compounds, most notably horsy or horse blanket and plastic or Band-Aid. These characters are derived from

the metabolism of phenolic compounds to vinyl phenols. Brettanomyces strains make a variety of other odor-

impacting compounds, some of which are considered positive, at least in small concentrations. Metabolites of

Brettanomyces are important components of the aroma profiles of some celebrated wines of France and other

older wine producing regions. A survey of 35 independent wine isolates of Brettanomyces from different

geographical regions was conducted to define the characters common to all strains. A descriptive analysis

revealed that the seven most common odor impacts were: Band-Aid, soy, horsy, earthy, leather, tobacco and

putrid. Individual strains displayed unique patterns of production of these compounds. Strains isolated from

California tended to be stronger in the earthy and putrid characters while strains from Europe were stronger in

horsy and Band-Aid. In a second study, the impact of supplementation with specific amino acids on the aroma

profile of Brettanomyces strains in minimal media was evaluated. In this study, additional characters, such as

sweaty, smoky, medicinal, cheesy and floral were detected. Samples were analyzed by GC/SPME/Olfactory to

determine if there were detectable differences in aroma profiles due to the Brettanomyces strain used or the

substrate added. Differences were associated with both the strains and the substrates added.

Key words: Brettanomyces, wine spoilage, vinyl phenol

IntroductionBrettanomyces is perhaps the most

controversial organism of wine production.

Brettanomyces bruxellensis, the anamorphic form of

Dekkera bruxellensis, is a spheroid, frequently ogival

ascomycete yeast that can form distinctive cell

shapes (Kurtzman and Fell 1998). It reproduces

vegetatively via multilateral budding and production

of pseudohyphae and non-septate mycelia are

common. The yeast is often readily distinguishable

microscopically from the wine yeast Saccharomyces

cerevisiae (Figure 1). Sporulation has not been

observed in the majority of wine isolates and these

isolates are therefore classified as Brettanomyces

bruxellensis. When sporulation does occur typically

one to four hat-shaped ascospores are found per

ascus (van der Walt and van Kerken 1960) and the

strain is classified instead as Dekkera bruxellensis.

The distinction between perfect or teleomorphic

strains (Dekkera) and imperfect or anamorphic

strains (Brettanomyces) is the direct observation of

spore formation. DNA sequence analysis suggests

that the two forms are identical to each other. The

1Professor, 2Collection curator and 3Graduate student Department of Viticulture and Enology, University of California, Davis, CA 94574

*Corresponding author [Phone: 1-530-752-3835; FAX: 1-530-752-0382; Email: lfbisson@ucdavis.edu

Acknowledgements: This research was supported by grants from the American Vineyard Foundation and from the California Competitive Grant Program for Research in Viticulture and Enology.

name Brettanomyces will be used to refer to these

yeasts throughout this manuscript, but the comments

and results obtained are directly applicable to

Dekkera as well.

Brettanomyces is also characterized by its unusual

metabolism. In the presence of molecular oxygen

Brettanomyces will ferment glucose and produce

acetic acid and carbon dioxide (CO2). The oxidation

of acetaldehyde to acetic acid reduces NAD+ to

NADH. Fermentation under aerobic conditions is

more rapid than under anaerobic conditions and this

metabolic phenomenon was termed the “negative

Pasteur effect”. This mode of metabolism was first

discovered by M.T.J. Custers, and it is also called the

“Custer’s Effect” (Scheffers 1966). Under the same

conditions, presence of high sugar concentration and

molecular oxygen, the yeast Saccharomyces forms

ethanol, a reduced end product, and CO2 which

serves to regenerate NAD+ from the NADH

generated during glucose catabolism. The production

of an oxidized end product such as acetic acid from

fermentation means that the cells will need other

mechanisms for the regeneration of NAD+, which

may involve molecular oxygen in the case of

Brettanomyces. Under anaerobic conditions

Brettanomyces can produce ethanol, so the yeast

does have the capability of balanced fermentation in

the absence of molecular oxygen.

Brettanomyces was first discovered in beer by

N.H. Claussen (Claussen 1904) and found to be

responsible for the “English character” of beers. The

genus name ‘brettano’ was coined from “British

brewing industry”. In beers fermented under specific

conditions, such as the Belgian or lambic beers, these

Brettanomyces characters are an important necessary

component of the overall odor of the product. In

shorter aged and less full bodied beers, Claussen

described the impact of Brettanomyces aroma

compounds as a “peculiar impure and sweet

mawkish taste”. Thus, from its discovery this yeast

has been associated with having both a positive and

negative odor impact on a product, depending upon

the odor matrix of that product and the specific

compounds and their ratios produced by the yeast.

Brettanomyces was subsequently isolated from

wine where it was also later shown to produce a wide

array of characters (van der Walt and van Kerken

1958). Volatile phenols are the chief class of classic

Brettanomyces odor impact compounds.

Brettanomyces decarboxylates hydroxycinnamic

acids (coumaric, ferulic and caffeic acid) producing

4-vinyl and 4-ethyl derivatives: 4-ethylphenol (4-

EP), 4-ethylguiacol (4-EG) and 4-ethylcatechol (4-

A

B

Figure 1 Photographs of Brettanomyces bruxellensis showing ogival cell shapes and pseudomycelia (Panel A) as compared to the ovoid budding cells of Saccharomyces cerevisiae (Panel B).

EC) respectively (Chatonnet et al. 1993; Hesford et

al. 2004; Heresztyn 1986; Steinke 1964). The

production of vinyl phenols is a two step enzymatic

process involving an initial decarboxylation of the

phenolic compound followed by reduction of the

vinyl phenol formed to the ethyl phenol (Figure 2).

Plastic, Band-Aid and chemical aromas have been

associated with 4-EP while clove and smoky aromas

have been attributed to 4-EG. Horsy characters have

been ascribed to 4-EC (Hesford et al. 2004). The

sensory thresholds for these compounds have been

determined (Chatonnet et al. 1993) and are presented

in Table 1. It has been observed that

Brettanomyces-infected wines contain higher levels

of isovaleric acid (IVA) than non-infected wines,

contributing a rancid aroma to red wines (Licker et

al. 1998). The presence of IVA and its recognition

threshold (Table 1) in wines has been determined

(Ferreira et al. 2000). There are also several fatty acid

esters, including ethyl-2-butyrate and ethyl

decanoate, that were found to be present in

Brettanomyces-infected red wines (Licker et al.

1998). Brettanomyces also has been shown to

produce compounds associated with the mousy taint

of wine. Heresztyn (1986) was the first to identify the

compounds, 2-acetyl-1,4,5,6-tetrahydropyridine and

2-acetyl-3,4,5,6-tetrahydropyridine, and organisms,

Brettanomyces and Lactobacillus, responsible for

mousiness in wines. These taints are derived from

metabolism of lysine. Subsequent research has

identified other compounds also associated with this

taint, 2-acetyl-1-pyrroline and

ethyltetrahydropyridine (Licker et al. 1998). A

detailed analysis of the odor-active compounds

produced by Brettanomyces has identified a wide

array of compounds, acids, alcohols, aldehydes,

esters, ketones and phenolic compounds in addition

to those described above (Licker et al. 1998). As

with beer, the impact of these compounds on the

overall aroma profile and perceived character of

wine depends upon the chemistry of the wine itself,

the matrix of other aroma compounds, and the actual

levels and nature of the end products produced by

Brettanomyces.

Unfortunately it is not easy to predict the odor

impact of Brettanomyces metabolism in a given wine

and difficult, if not impossible, to manipulate the

biological activities of this organism once it infects a

winery. Brettanomyces is commonly found as

resident flora of wineries (Peynod and Domercq

1956; van der Walt and van Kerken 1961),

particularly of wood surfaces found in barrels.

Table 1 Sensory Thresholds for Brettanomyces-Related Aroma Compounds

Compound Associated Aroma Threshold in Water

Threshold in Model Wine

Threshold in Red Wine

4-Ethylphenol Plastic, Band-aid 130 µg/L1 440 µg/L1 620 µg/L1

4-Ethylguaiacol Smoky, clove 25µg/L1 33 µg/L2

47 µg/L1 110 µg/L1

4-Ethylcatechol Horsy Nr3 Nr Nr

Isovlaeric acid Rancid, barnyard Nr 33.4 µg/L2 Nr

Tetrahydropyridines Mousy Nr Nr Nr

2-Acetyl-1-pyrroline Mousy Nr 1.49 µg/L2 Nr

Ethyl-2-methyl butarate Fruity Nr 18 µg/L2 Nr

1Chatonnet et al. 19932Ferreira et al, 20003Nr means not reported.

Figure 2 Pathway for the production of vinyl phenols by Brettanomyces.

Sanitation of porous substances like wood can be

challenging, and practices such as topping off of

barrels to reduce air exposure and head space may

lead to the spread of Brettanomyces throughout the

aging cellar of a winery. Brettanomyces has also been

found during primary fermentation in many wineries

but is present in low numbers (Licker et al 1998).

Since Brettanomyces grows more slowly than other

yeasts, identification of this organism based on

culturing may be difficult.

Genetic Diversity of Brettanomyces.

Brettanomyces or “Brett” as it is known in the wine

industry, has been isolated from all wine producing

regions on six continents (Conterno et al. 2006).

There are anecdotal reports of “good Brettanomyces”

strains that produce positive traits, grow rapidly

preventing the growth of other yeasts in barrel, and

that produce reduced levels of the objectionable

characters that this yeast is known to generate. While

several investigators have studied the relationship

between Brettanomyces characters and medium or

wine composition, the results are sometimes

inconsistent (Rose and Harrison 1971; Uscanga et al.

2000). This inconsistency implies genetic variation

exists across the species Brettanomyces bruxellensis.

A more comprehensive study was undertaken to

evaluate the genetic and physiological diversity of

Brettanomyces (Conterno et al. 2006). Yeasts were

selected for this study based on geographic diversity,

year of isolation and type of wine from which the

isolate was obtained. The 47 strains evaluated were

grouped into one of six clusters based upon sequence

analysis of the 26S rDNA region. Comparison of the

clustering of strains by DNA relatedness to

physiological traits revealed that some traits are

highly variable and have arisen across the DNA

clusters (Conterno et al. 2006). However, there were

some traits that did seem to correlate with genetic

grouping: level of production of 4-EP and 4-EG

under the same conditions, metabolism of citrate,

ethanol, glycerol, maltose, succinic acid and soluble

starch. There was a diversity of responses to growth

on other substrates. Striking differences in tolerance

to sulfite (SO2) and the ability to grow at low (cellar)

temperatures were also observed (Conterno et al.

2006). Thus, genetic variability is common in

Brettanomyces and likely driven by the specific

adaptive demands of the individual sources from

which it was isolated. This yeast is obviously highly

adaptive and resourceful in surviving adverse

environments.

Brettanomyces is also able to form biofilms,

which are layers of microbes that can coat surfaces

such as walls, tanks, hoses, and barrels. Biofilms are

difficult to get rid of and organisms in biofilms can

resist sanitation agents and survive. There is strain

variability in both the ability to form biofilms and the

resistance to sanitizing agents (Joseph et al. 2008).

The conclusion from these studies is that there is

significant genetic diversity among the strains

classified as Brettanomyces bruxellensis.

Management strategies for this organism will need to

be tailored to the specific metabolic activities of the

strain in question, which may prove quite difficult to

do under production conditions. Perhaps one day as

with the lactic acid bacteria, “good” strains of

Brettanomyces will indeed have been identified and

able to be used as specific inocula to control the

appearance of wild Brettanomyces isolates.

Bandaid

Horsey

Earthy

Soy

PutridControl

615

738

752 2030

2041

2046

2047

2048

2049

2050

2051

2052

2053

2054

2058

2059

2060

2062

2063

2065

2066

2067

2075

2076

2077

2078

2079

2080

2081

2082

2083

2085

2091

2092

2093

-3

-2

-1

0

1

2

3

-3 -2 -1 0 1 2 3

PC1 (41.4%)

PC2

(27.

9%)

Black: CAPink: CanadaLavender: NYBlue: MORed: FranceGreen: GermanyOrange: ChileDark Blue: NZBrown: BelgiumLight Green: Thailand

Descriptive Analysis of Brettanomyces Infected

Cabernet Sauvignon Wines. We also undertook an

analysis of the major descriptors used to characterize

a Cabernet Sauvignon wine following deliberate

inoculation with several different strains of

Brettanomyces bruxellensis (Wirz 2005). Thirty-five

strains of Brettanomyces were utilized and compared

to an uninoculated control of the same wine. After 46

days of incubation the wines were sterilely filtered

and bottled and a descriptive panel of 14 judges was

assembled and trained. Using an adapted consensus

method, seven terms were identified as describing the

wines: Band-Aid, earthy, horsy, leather, putrid, soy,

and tobacco. Principal component analysis of the

mean data indicated that the first two principle

components explained roughly 70% of the variation.

The first component separated wines that had no

obvious Brettanomyces characters from those that

did (Figure 3). In these cases the Brettanomyces

inoculum either was not successful in establishing in

the wines in spite of showing growth in the wine, or

the strains were slowly metabolizing. The second

principle component distinguished wines that had

more Band Aid from those that had more

earthy/putrid characters. Thus, in this wine some

strains appeared to generate more of the Band-Aid or

plastic notes while others were more earthy or foul

smelling. Thus, different strains will produce

different characters in the same base wine.

Interestingly, some of the wines that did not show

the signature or negative characters associated with

Brettanomyces actually appeared to be preferred

Figure 3 Principal component analysis of flavor attributes of wines inoculated with strains of Brettanomyces from divergent geographical regions.

even over the uninoculated control (Figure 3). This

suggests that the Brettanomyces inoculum did have

an effect on the wine, one that panelists could detect

even if the signature vinyl phenol characters were

absent.

Effect of nitrogen supplementation on

Brettanomyces in wine. The role of phenolic

compounds as precursors to the production of aroma

compounds in Brettanomyces has been well

documented experimentally. Of equal interest is the

spectrum of other compounds that are produced by

this yeast. The driving force for these metabolites

may well be the need to maintain the NAD+/NADH

balance of the cell while producing acetate in the

presence of oxygen. Therefore, the impact of

supplementation with various amino acids as sole

carbon or sole nitrogen source was evaluated in five

different strains from distinct genetic clusters using a

synthetic wine medium devoid of phenolics. L-

cysteine and glycine tended to be inhibitory towards

growth of all of the yeast strains (Kitson 2007).

Aromas common to all strains in the absence of

phenolic compounds were leather, yeasty, sweaty,

cheesy and floral. Floral aromas were strongest in

cultures supplemented with phenylalanine. The

impact of amino acid supplementation on the

Brettanomyces aroma profile in a Cabernet

Sauvignon wine was also evaluated. Of the five

strains evaluated, one retained media culturability

throughout growth in the wine. One of the other four

strains showed increases in cell biomass as

determined using molecular techniques (QPCR), but

was not culturable (Kitson 2007). The other three

strains were not culturable and showed little or no

growth by QPCR analysis. Volatile compound

profiles were evaluated using Solid-Phase

Microextraction (SPME). Statistical analysis

indicated that differences in volatile compound

production were dependent mainly on the strain

present, but there was an effect of nutrient

supplement as well.

The growth of all of the strains in the synthetic

wine media with low aeration was greatest with

asparagine, aspartic acid or tyrosine as the

supplemented amino acid. Interestingly, other amino

acids such as arginine showed good growth for some

strains (UCD615, UCD2058 and UCD2082) but

reduced growth for UCD2077 and UCD2091, as

compared to aspartic acid. Strain UCD2082 showed

good growth generally regardless of the

supplemented amino acid with the exceptions of

cysteine and glycine. Strain UCD2058 tended to

show the poorest growth with most amino acids as

sole nitrogen source. The other three strains showed

poorer growth with one or more of the following

amino acids as sole nitrogen source: alanine,

arginine, leucine, lysine, phenylalanine, serine,

threonine, or tryptophan (Kitson 2007). In minimal

media with full aeration, all of the strains showed

increased growth rates with valine and proline and

decreased growth rates with alanine, cysteine and

glycine. Some of the strains showed an increased

growth rate with glutamine and a decreased with

threonine. Thus the level of residual amino acids, in

combination with the specific strain of

Brettanomyces present and aeration practices, can

stimulate or restrict growth. All strains grew well

with mixtures of amino acids.

The SPME analysis identified a total of 63

peaks in these wines (Kitson 2007). Interestingly,

many of these peaks decreased in intensity in

samples inoculated with Brettanomyces as compared

to the control wine. Brettanomyces may generally

diminish the varietal characters of wine. The

reduction of some negative varietal or primary

fermentation characters may in part explain why

inoculated wines that did not show evidence of vinyl

phenol production were distinguishable from the

control wine in the Wirz (2005) study. Only two

compounds were characterized as being found only

in Brettanomyces infected wines: 4-ethylphenol and

4-ethylguaiacol. The amino acid treatments did not

reveal any aroma compounds specifically associated

with a particular amino acid. Higher rates of

browning were also noted in the wines inoculated

with Brettanomyces, suggesting that this yeast

impacts wine chemistry and the stabilization of wine

pigments.

Conclusions

Brettanomyces remains a controversial yeast in

wine production. Some winemakers highly prize the

ethyl phenol characters of Brettanomyces when

present in low concentration. Our study suggests that

Brettanomyces can produce some positive aroma

responses, through the direct synthesis of odor-

impact compounds and via the reduction of varietal

characteristics which may bring the aroma profile

into better balance. However, there is significant

strain variability across the species Brettanomyces

bruxellensis, and strain variability combined with the

inability to accurately quantitate wine precursor

compounds makes predicting the outcome of

Brettanomyces metabolism difficult in wine

production. The variability in response of this yeast

to inhibitory compounds such as sulfite and

sanitation agents makes it challenging to control

populations of this yeast in the winery. Further

research into the basic biology of Brettanomyces is

needed to better understand the factors driving the

production of both desired and undesired compounds

by this yeast.

Literature Cited

Chatonnet, P., D. Dubourdieu, J.N. Boidron, and M.

Pons.1992. The origin of ethylphenols in

wines. J. Sci. Food Agric. 60:165-178.

Claussen, N.H. 1904. On a method for the

application of Hansen’s pure yeast system in

the manufacturing of well-conditioned English

stock beers. J. Inst. Brewing. 10:308-331.

Conterno, L., C.M.L. Joseph, T.J. Arvik, T. Henick-

Kling, and L.F. Bisson. 2006. Genetic and

physiological characterization of

Brettanomyces bruxellensis strains isolated

from wines. Am. J. Enol. Vitic. 57:139-147.

Ferreira, V., R. Lopez, J.F. Cacho. 2000.

Quantitative determination of the odorants of

young red wines from different grape varieties.

J. Sci. Food Agric. 80:1659-1667,

Heresztyn, T. 1986a. Formation of substituted

tetrahydropyridines by species of

Brettanomyces and Lactobacillus isolated from

mousy wines. Am. J. Enol. Vitic. 37:127-132.

Heresztyn, T. 1986b. Metabolism of volatile

phenolic compounds from hydroxycinnamic

acids by Brettanomyces yeast. Arch.

Microbiol. 146:96-98

Hesford, F. K. Schneider, N.A. Porret, and J. Gafner.

2004. Identification and analysis of 4-ethyl

catechol in wine tainted by Brettanomyces off-

flavor. Avstr. Am. J. Enol. Vitic. 55:304A.

Joseph, C.M.L., G. Kumar, E. Su, and L.F. Bisson.

2007. Adhesion and biofilm production by

wine isolates of Brettanomyces bruxellensis.

Am. J. Enol. Vitic.

Kitson, B.S. 2007. The effect of amino nitrogen on

Brettanomyces/Dekkera growth and aroma

production. M.S. Thesis, University of

California, Davis.

Kurtzman, C.P., and J.W. Fell.1998. The Yeasts, A

taxonomic study, fourth edition. Elsevier

Science. Amsterdam. Pp. 450-453.

Licker, J.L., T.E. Acree, and T. Henick-Kling.

1998. What is “Brett” (Brettanomyces) flavor?

Pages 96-115 in A.L. Waterhouse and S.E.

Ebeler, eds., ACS Symposium Series vol 714,

American Chemical Society. Washington,

D.C.

Peynod, E., and S. Domercq. 1956. Sur les

Brettanomyces isolees des raisins et de vins.

Arch. Mikrobiol. 24:266-280.

Rose, A.H., and J.S. Harrison. 1971. The Yeasts.

Vol 2. Academic Press, London.

Scheffers, W.A. 1966. Stimulation of fermentation

in yeasts by acetoin and oxygen. Nature

210:533-534.

Steinke, R.D. and M.C. Paulson. 1964. The

production of steam-volatile phenols during the

cooking and alcoholic fermentation of grain. J.

Agric. Food Chem. 12:381-387.

Uscanga, M.G.A., M.L. Delia, and P. Strehaiano.

2000. Nutritional requirements of

Brettanomyces bruxellensis: Growth and

physiology in batch and chemostat cultures.

Can. J. Microbiol. 46:1046-1050.

van der Walt, J.P., and A.E. van Kerken.1958. The

wine yeasts of the Cape. Part I. A taxonomic

study of the yeasts causing turbidity in South

African table wines. Ant. Leeuwenhoek

24:239-251.

van der Walt, J.P., and A.E. van Kerken. 1960.

Wine yeasts of the Cape. Part IV: Ascospore

formation in the genus Brettanomyces. Ant.

Leeuwenhoek 26:292-296.

van der Walt, J.P., and A.E. van Kerken. 1961.

Wine yeasts of the Cape. Part V. Studies of the

occurrence of Brettanomyces intermedius and

Brettanomyces schanderlii. Ant. Leeuwenhoek

27:81-89.

Wirz, D.O. 2005. Descriptive analysis of

Brettanomyces-infected Cabernet sauvignon

wines. M.S. Thesis, University of California,

Davis.