Enzyme and Microbial Technology 40 (2007) 420–425

␤-Glucosidase activity in wine yeasts:

Application in enology
Marı́a Arévalo Villena ∗ , Juan Francisco Úbeda Iranzo, Ana Isabel Briones Pérez
Departamento de Quı́mica Analı́tica y Tecnologı́a de Alimentos, Universidad de Castilla la Mancha, Spain

Abstract
A method for quantifying ␤-glucosidase activity in wine yeasts is reported. After selecting the appropriate strain, conditions for enzyme synthesis
were optimized: synthesis of extracellular enzyme was induced by the presence of cellobiose in the medium under aerobic conditions, with peak
production at 42 h growth. The enzyme was partially purified and biochemically characterized. At the same time, aroma precursors were measured
in various musts from grapes grown in Castilla la Mancha (Spain) in order to quantify potential aroma, using a laboratory-developed method based
on glycoside retention in reversed-phase C18 columns, recovery by elution with appropriate solvents and acid hydrolysis. This provided information
on the most suitable substrate for wine yeast ␤-glucosidase enzymes. Finally, wines were made at laboratory scale using a selection of white grape
varieties ranging from highly aromatic to neutral (Muscat, Riesling and Airén), adding yeast enzyme to the wine and comparing the results to
those obtained with a widely used commercial enzyme preparation of fungal origin. Release of glycoside terpenes was measured by the method
indicated above and by gas chromatography/mass spectrometry. Results were satisfactory and displayed good correlation, showing that enzymes
obtained from wine yeasts could be used in place of the commercial fungal enzyme preparations currently used in winemaking.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Wine aroma; Non-Saccharomyces yeasts; ␤-Glucosidase enzymatic activity; Terpenes

1. Introduction directly influence the fermentation medium; their effect may be

In traditional winemaking, must fermentation takes place nat-
urally and spontaneously through the action of yeasts present in
the raw material, which convert glucose to ethanol, CO2 and
other metabolites. Although yeasts are the main agents in the
fermentation process, certain bacteria are also involved [1].
Both Saccharomyces and non-Saccharomyces yeasts are uti-
lized for this purpose. The most important non-Saccharomyces
genera are Debaryomyces, Hanseniaspora, Brettanomyces,
Candida, Metschnikowia, Pichia and Zygosaccharomyces [2,3].
These yeasts are always present during the early stages of
fermentation, but due to extreme conditions in the must (low
pH, high initial sugar concentrations gradually converted into
ethanol, addition of sulphur dioxide as an antioxidant, anaerobic
conditions), only the best-adapted yeasts survive to the end of the
winemaking process; Saccharomyces gradually displaces other
yeasts [4].
Nevertheless, all yeasts are important in the winemaking pro-
cess because during development they synthesize enzymes that
∗ Corresponding author. Tel.: +34 926 295300x3424; fax: +34 926 295318.
E-mail address: Maria.Arevalo@uclm.es (M.A. Villena).
harmful or beneficial, depending on the nature of the enzyme
and on the environmental conditions.
The major enzyme groups in winemaking are oxidoreduc-
tase, pectinase, protease and finally ␤-glucosidase, the main
object of the present study. Any study of ␤-glucosidase enzymes
must address the terpene compounds that contribute to the vari-
etal character of wines. Over recent years, increasing attention
has been paid to complex, non-volatile glycosides, known as
aroma precursors, which occur in high concentrations in musts
[5–15]. Reports indicate that not all glycosides are present in all
grape varieties, and that concentrations vary according to variety
[16], ranging from 500 to 1700 ␮g/L of must [17]. Major pre-
cursors include structures such as ␤-d-glucopyranoside, 6-O-␣-
l-arabinosil-␤-d-glucopyranoside, 6-O-␣-l-ramnopyranosyl-
␤-d-glucopyranoside and 6-O-␤-d-apiofuranosyl-␤-d-gluco-
pyranoside apiosylglycosides [7,18,19].
Aglycon chemical structure (volatile when free) may vary,
taking the form of a terpenol (linalol, geraniol, nerol, cit-
ronelol, ho-trienol or ␣-terpienol), linalol oxide, linear or cyclic
alcohol (hexanol, phenylethanol, benzyl alcohol), C13 noriso-
prenoid, phenolic acid and/or volatile phenol. It is the mixture
of these compounds, rather than the influence of any individual
compound, that defines the varietal characteristics of a wine;

0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2006.07.013
 M.A. Villena et al. / Enzyme and Microbial Technology 40 (2007) 420–425
the olfactory threshold of the mixture is lower than that of
any individual component [20]. These compounds are released
through the action of ␤-glucosidase enzymes, which break the
terpene–sugar bond, contributing to increased intensity of wine
flavour.
␤-Glucosidase may be obtained from humans [21], insects
[22], plants [23–25], or microorganisms. The first three sources
are of little value in winemaking, since they are inhibited by
processing conditions (low pH, high glucose and ethanol con-
centrations); the only viable enzyme source is thus microorgan-
isms.
At present, aroma release is often enhanced using commer-
cial enzyme preparations of fungal origin, mainly Aspergillus
spp. The composition of these preparations varies, and they
are in reality a mixture of non-specific glucanases. One of the
preparations most widely used in winemaking displays exo-
␤-glucanase, endo-␤-1,3-glucanase, exo-␤-1,6-glucanase and
␤-glucosidase activity [26]. A drawback to the use of such prepa-
rations is that since glucanases are non-specific, they may trigger
collateral hydrolysis reactions detrimental to the product [27].
An alternative to employing commercial fungal enzyme
preparations in winemaking could be the use of specific enzymes
contained in yeasts forming part of the wine ecosystem and thus
adapted to their ecological environment. These enzymes would
release conjugated terpenes without affecting other components,
enhancing the varietal characteristics increasingly prized by con-
sumers [4].
This paper reports on the search for wine yeasts with ␤-
glucosidase activity, for use in winemaking.
2. Materials and methods
2.1. Screening for β-glucosidase activity in wine yeasts
A total of 102 pure yeast cultures isolated from wine ecosystems were stud-
ied; 82 from wine cellars in Castilla la Mancha (Spain) and the remaining 20
from Stellenbosch (South Africa). ␤-Glucosidase activity in all strains was tested
using cellobiose as substrate, since the glycoside bond linking the two glucoses
is ␤-(1–4), characteristic of the aroma precursors found in grape musts [28].
Testing comprised qualitative and quantitative assays.
2.1.1. Qualitative assay
The aim was to select yeasts displaying enzyme activity. Yeasts were grown
in YPD broth and transferred to carbon-free YNB broth in order to fully consume
residual sugars. Strains were subsequently streaked onto YP agar plates contain-
ing cellobiose as single carbon source. Strains showing good development on
agar were considered ␤-glucosidase positive.
2.1.2. Quantitative assay
Having selected the best yeasts, their ␤-glucosidase activity was quantified
in all cell-culture fractions using a protocol developed by Arévalo Villena et
al. [12], which enabled evaluation of activity in supernatant, whole cells, cell
wall, cytosol and membrane. Enzyme activity was quantified by measuring the
glucose released from the cellobiose substrate using a specific enzyme kit.
2.2. Optimization of growing conditions to ensure maximum
activity
Using the yeast considered most suitable for ␤-glucosidase enzyme synthe-
sis, culture conditions were optimized using some different culture media (YPD,
YPC, YPD + C, YNB-A, YNB-C, YNB-G, YNB-A + G) and incubation times
421
(24, 48, 72 h), in both strictly anaerobic and diverse aerobic conditions (aeration
using Erlenmeyer flasks versus test tubes) [12,13].
2.3. Enzyme characterization
Supernatant from the selected yeast strain was partially purified by cold
acetone precipitation of the protein fraction and subsequent differentiation by
filtration through size-exclusion filters. The ␤-glucosidase enzymes obtained
were characterized biochemically (in terms of KM and Vmax values, optimal
temperature and pH and thermal stability). A study was made of the influence of
various metabolites on enzyme activity (glycerol, EDTA, acetic acid, SDS, Triton
X100, glucose, ethanol, Ca, Co, Mg, K, Na, Zn), and of substrate specificity using
molecules with different types of bonds (cellobiose, pNPG, laminarine, maltose,
mandelonitryl beta-d-glucoside, N-octyl-beta-d-glucoside, lactose and arbutin)
[13].
2.4. Rapid method for quantifying aroma precursor content in
musts and wines
Having obtained the sought-after activity “in vitro”, it was necessary to
establish an intermediate stage between the laboratory and real winemaking
processes. A quick, simple method was developed for reliably quantifying aroma
precursors in grape extract, musts and wines of different grape varieties, in order
to determine baseline aroma potential and the degree of hydrolysis achieved by
treatment using the enzymes obtained earlier [14,15].
The protocol developed by Williams et al. [29] was modified at various
stages, following careful analysis of column recovery capacity, flow rate, degree
of recovery and interference of other (phenol) compounds.
The resulting protocol was used to isolate and quantify aroma precur-
sors in a range of grape extracts (Airén, Chardonnay, Chenin, Garnacha,
Muscat, Rosanne, Sauvignon Blanc and Verdejo), musts (Airén, Chardonnay,
Gewürztraminer, Macabeo, Muscat, Saugvignon Blanc and Riesling) and wines
elaborated at laboratory level using a commercial Saccharomyces cerevisiae
strain (UCLM 325) [30]. Precursor content was measured in all samples, and
tests were performed to check for possible declines in glycosylated terpene lev-
els following fermentation. In the light of the results obtained, aroma precursors
were isolated and quantified from musts (Airén, Macabeo and Muscat), wines
made from these musts after fermentation with the same strain, and the same
wines treated with a commercial enzyme preparation of fungal origin widely
used in winemaking [31].
2.5. Application of Debaryomyces pseudopolymorphus enzymes
in winemaking processes
Vinification was performed in a multitube fermenter at 18 ◦ C, thus repro-
ducing wine cellar fermentation conditions. Musts of varying aroma precursor
content were used (Airén, Riesling and Muscat).
Batches of wines were treated with either a commercial enzyme prepara-
tion of fungal origin, widely used in winemaking (positive control), or with
the purified yeast extract showing ␤-glucosidase activity. Wines with no added
enzyme extract were used as negative control. Release of volatiles was quanti-
fied by the aroma precursor retention method and by gas chromatography/mass
spectrometry [32].
A sensory analysis was carried out using a triangular test designed to detect
differences between wines treated with purified yeast extract, those treated with
the commercial enzyme preparation and untreated wines.
3. Results and discussion
3.1. Screening for β-glucosidase activity in wine yeasts
3.1.1. Qualitative assay
Of the 102 yeasts grown on YP cellobiose plates, only 25%
displayed good growth; growth by the remainder was moderate,
weak or non-existent. All yeasts displaying good growth were
422 M.A. Villena et al. / Enzyme and Microbial Technology 40 (2007) 420–425

Fig. 1. Kinetics of growth and ␤-glucosidase biosynthesis in D. pseudopolymor-

phus. Enzyme activity is expressed as nmol glucose produced per ml of super-
natant fluid h−1 . Growth in YPD () and in YPC (); enzyme activity in super-
natant YPD (䊉) and YPC (). Standard deviations were between 5% and 10%.

non-Saccharomyces species; only one Saccharomyces strain
grew well on cellobiose [12].
3.1.2. Quantitative assay
␤-Glucosidase activity in the selected yeasts was quantified
in all cell-culture fractions. Most strains displayed marked intra-
cellular, but weak extracellular, activity. However, five yeast
species excreted the enzyme at the growth medium: Debary-
omyces pseudopolymorphus, Hanseniaspora uvarum, Candida
oleophila, Debaryomyces polymorphus and Brettanomyces spp.
[10,12].
Fig. 2. SDS-PAGE electrophoresis of the various steps of ␤-glucosidase iso-
lation. Lane 1, supernatant fraction; lane 2, after acetone precipitation; lane 3,
after ultrafiltration, fraction with greatest activity (≥100 kDa). The numbers to
the right of the figure indicate the position and molecular weight in KDa of the
marker (SigmaMarkerTM high molecular weight range).
column (pretreatment of cartridges with methanol and water,
sample loading and glycoside elution with appropriate solvents),
acid hydrolysis of precursors obtained and colorimetric mea-
surement of released glucose using a specific enzyme kit.

3.2. Optimization of growing conditions to ensure

maximum activity

Of these five pre-selected yeasts, D. pseudopolymorphus was

identified as the best producer of ␤-glucosidase enzymes. Max-
imum synthesis was recorded with the following conditions:
growth in YP cellobiose broth for 42 h at 30 ◦ C in Erlenmeyer
flasks 1/5 full, shaken at 150 rpm, showing that synthesis was
induced. In Fig. 1 can be observed that enzymatic synthesis was
induced, so in YPD broth it was weak.

3.3. Enzyme characterization

Partial purification showed that ␤-glucosidase enzymes

were monomers with a molecular weight of around 100 kDa
(Fig. 2). Vmax and KM values were 70.92 ␮mol/min mg prot
and 11.92 ␮mol/ml, respectively. Values for optimum temper-
ature, pH and heat stability are shown in Figs. 3–4. The various
metabolites tested (glycerol, EDTA, acetic acid, SDS, Triton
X100, glucose, ethanol, Ca, Co, Mg, K, Na, Zn) had no signifi-
cant influence on enzyme activity. Tests for substrate specificity
showed that the highest activity of the enzyme was on mono-␤-
d-glycosides [13].

3.4. Rapid method for quantifying aroma precursor content

in musts and wines
The method used here comprised the following stages: sam-
ple preparation, glycoside retention on a C18 reversed-phase
Fig. 3. Relative enzyme activity at different temperatures (A) and pH (B) values
for the purified ␤-glucosidase. The scale of relative activity (%) indicates the
percentage of the experimental value at various temperatures and pH’s relative
to the maximum. The values shown here are means from quadruplicated assays
±5% standard deviation.
 M.A. Villena et al. / Enzyme and Microbial Technology 40 (2007) 420–425 423

3.5. Application of D. pseudopolymorphus enzymes to

finished wine

Vinification and enzyme treatments were as described in Sec-

tion 2. The two analytical methods used to measure released
volatiles provided similar results; both treatments (commercial

Fig. 4. Thermostability of ␤-glucosidase. The scale of residual activity (%)

indicates the percentage of experimental value to the maximum. The sta-
bility over time is of purified ␤-glucosidase incubated at 40 ◦ C and pH 4.
The values shown here are means from quadruplicated assays ±5% standard
deviation.

With regard to the variables considered when developing the

protocol, the following were noted: column recovery capac-
ity was found to be satisfactory (linear correlation coeffi-
cient = 0.997); flow rates of 2, 3 or 4 ml/min may be used with
equal efficacy; each column may be used up to three times with
no significant loss of glycosides; and phenol compounds – at
the concentrations found in the varieties for which the protocol
is tested (i.e. white) – did not interfere with aroma precursor
recovery [14].
The protocol was applied in the first instance for iso-
lation and quantification of glycosylated terpenes in grape
extract using some different varieties (showed in Section 2).
Aroma precursor content was found to vary among varieties;
Muscat displayed the greatest potential (Fig. 5A). Precursors
were subsequently isolated from musts (Airén, Chardonnay,
Gewürztraminer, Macabeo, Muscat, Sauvignon Blanc and Ries-
ling) and from wines made from these musts at laboratory scale
using a commercial S. cerevisiae strain (UCLM 325). Precur-
sor concentrations were measured in all cases, and it was found
that glycosylated terpene levels declined during fermentation,
perhaps due to the residual glucanase activity characteristic
of this strain. The problem would appear to be that released
volatiles were drawn off by the CO2 produced during the pro-
cess, and lost due to the metabolite exchange taking place
between the fermentation medium and the atmosphere. This
loss, however, is not a source of concern for future enzyme
treatments, since – as Fig. 5B shows – unhydrolyzed precur-
sors still remained in the finished wine. Finally, in the light of
the results obtained, aroma precursors were isolated and quan-
tified from musts (Airén, Macabeo and Muscat), wines made
from these musts after fermentation with the same strain, and
the same wines treated with a commercial enzyme preparation Fig. 5. Aroma precursor concentrations (expressed as nmol glucose g−1 grape).
of fungal origin widely used in winemaking. As the results in (5A) In grapes of different varieties: Airén (1), Chardonnay (2), Chenı́n (3),
Fig. 5C show, there was again some release during fermentation, Garnacha (4), Muscat (5), Rosanne (6), Sauvignon Blanc (7) and Verdejo (8).
(5B) In musts and wines of different varieties: Airén (1), Chardonnay (2),
although a decline in precursor levels following enzyme treat- Gewürztraminer (3), Macabeo (4), Muscat (5), Sauvignon Blanc (6) and Ries-
ment suggested that released volatiles, in this occasion, remained ling (7). (5C) In musts, untreated wines and enzyme-treated wines of different
in the wine. varieties: Airén, Macabeo and Muscat.
424 M.A. Villena et al. / Enzyme and Microbial Technology 40 (2007) 420–425

Table 1
Aroma precursors (nmol glucose/ml wine) at 6 and 12 days in must, control wine and wines treated with the enzyme extracts
Variety Must Control Treated wine

CEP P

6 12 6 12

Airén 88.3 72.5 a 48.9 48.9 b 59.9* 26.8* c

Riesling 163.9 124.5 a,a 115.1* a 97.7* b 66.2* b 47.3* c
Muscat 214.3 146.6 a,a 129.2* b 110.3* b 78.8* c 60.0* c

P: purified yeast extract; CEP: commercial enzyme preparation. Different letters indicate significant differences (95 % confidence) between wines treated with
different extracts for 6 (normal letters) and 12 days (letters with prime) in each variety for the different enzymatic treatments.
* Significant differences between treatment times for the same enzymatic preparation.

Table 2
Percentage agreement between wines treated with the two enzyme preparations
C–P P–CEP

Airén 90 ND
Riesling 99 99
Muscat 99.9 95

C: untreated wine; P: wine treated with purified yeast extract; CEP: wine treated
with commercial enzyme preparation of fungal origin.

mould-derived preparation and purified yeast enzyme extract)

were effective, prompting a gradual decline in precursor content
and thus increasing the volatile fraction of wines with respect to
controls (Table 1; Fig. 6A–C).
Hydrolysis using yeast extract was found to be more con-
trolled. Fig. 6C shows that Muscat wine treated with the com-
mercial enzyme preparation displayed higher levels of ethyl
octanoate, a volatile not desirable in wines. The sensory analysis
distinguished between the two treatments with different confi-
dence levels depending on the type of enzyme and the grape
variety (Table 2).

References

Fig. 6. Release of volatile compounds (% hydrolysis with respect to controls)
in control wines and in wines treated with purified yeast extract (P) and a com-
mercial enzyme preparation (CEP), after 12-day treatment for all varieties (A:
Airén, B: Riesling and C: Muscat). (A) 1: limonene, 2: 1-hexanol, 3: 3-hexen-
1-ol, 4: citronelol, 5: 2-phenylethanol, 6: ethyl acetate, 7: ethyl butyrate, 8:
isoamyl acetate, 9: ethyl hexanoate, 10: hexyl acetate, 11: ethyl octanoate, 12:
2-phenylethyl acetate. (B) 1: 1-Hexanol, 2: 3-hexen-1-ol, 3: linalol, 4: citronelol,
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isoamyl acetate, 12: ethyl hexanoate, 13: hexyl acetate, 14: ethyl octanoate, 15:
2-phenylethyl acetate.
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