The Role of Brettanomyces in Wine Production

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

Department of Viticulture and Enology

University of California, Davis

Davis California, USA 95616-8749

1Professor, 2Collection curator and 3Graduate student*Corresponding author:

Phone: 1-530-752-3835FAX: 1-530-752-0382Email: lfbisson@ucdavis.edu

Key words: Brettanomyces, wine spoilage, vinyl phenol

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.

Abstract

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.

Introduction

Brettanomyces 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

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-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.

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.

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 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

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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.

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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:

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Wirz, D.O. 2005. Descriptive analysis of Brettanomyces-infected Cabernet

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

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 440µg/L1 620µg/L1

4-Ethylguaiacol Smoky, clove

25µg/L1 33µg/L2

47µg/L1110µg/L1

4-Ethylcatechol Horsy Nr3 Nr Nr

Isovlaeric acid Rancid, barnyard

Nr 33.4µg/L2 Nr

Tetrahydropyridine

s

Mousy Nr Nr Nr

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

Ethyl-2-methyl butarate

Fruity Nr 18µg/L2 Nr

1Chatonnet et al. 1993

2Ferreira et al, 2000

3Nr means not reported.

Figure Legends

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).

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

Figure 3. Principal component analysis of flavor attributes of wines inoculated with

strains of Brettanomyces from divergent geographical regions.

Figure 1:

A

B

Figure 2:

CHCH

COOH

H

OH

CHCH2

CH2

CH2

OH OH

H H

Cinnamatedecarboxylase

Vinyl phenol reductase

H OH OMe= coumaric = caffeic = ferulic

Figure 3:

Bandaid

Horsey

Earthy

Soy

PutridControl

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PC1 (41.4%)

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