ORIGINAL ARTICLE

Screening and evaluation of the glucoside hydrolaseactivity in Saccharomyces and Brettanomyces brewingyeastsL. Daenen, D. Saison, F. Sterckx, F.R. Delvaux, H. Verachtert and G. Derdelinckx

Department of Microbial and Molecular Systems, Centre for Malting and Brewing Science, Faculty of Bioscience Engineering,

Katholieke Universiteit Leuven, Leuven, Belgium

Introduction

An improvement of the organoleptic quality of beverages

and the development of new beverages can be attained

through bioflavouring. This technique relies on the

production and conversion of flavour compounds and

flavour precursors by biological methods (Vanderhaegen

et al. 2003). One method relies on the possibility to

enhance or modify the flavour profile through an

enzymatic hydrolysis of flavour precursors such as

glycosidically bound flavour compounds, which are

present in many plants, flowers and fruits. During the

processing of foods and beverages, the odourless and

nonvolatile glycosides can be extracted from the raw

materials, which lead to a mostly unused flavour potential

(Winterhalter and Skouroumounis 1997). The enzymatic

hydrolysis of glycosides requires in a first step the

cleavage of the inter-sugar bond in di- or triglycosides,

yielding b-d-glucosides (Gunata et al. 1988). The most

appropriate enzyme for hydrolysis of these b-d-glucosides

is 1,4-b-glucosidase (EC 3.2.1.21) (Sarry and Gunata

2004).

Regarding fermented beverages, a method for introduc-

ing this b-glucosidase activity into the medium is the use

of an appropriately selected yeast strain. As most fermen-

tation media of interest contain sugars exerting catabolite

Keywords

b-glucanase, b-glucosidase, bioflavouring,

brewing yeasts, co-culture,

Dekkera ⁄ Brettanomyces, hop glycosides,

Saccharomyces.

Correspondence

L. Daenen, Centre for Malting and Brewing

Science, Department of Microbial and

Molecular Systems, Katholieke Universiteit

Leuven, Kasteelpark Arenberg 22, B-3001

Leuven, Belgium.

E-mail: luk.daenen@biw.kuleuven.be

2007 ⁄ 0396: received 13 March 2007, revised

14 July 2007 and accepted 2 August 2007

doi:10.1111/j.1365-2672.2007.03566.x

Abstract

Aims: The aim of this study was to select and examine Saccharomyces and Bret-

tanomyces brewing yeasts for hydrolase activity towards glycosidically bound

volatile compounds.

Methods and Results: A screening for glucoside hydrolase activity of 58

brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was

performed. The studied Saccharomyces brewing yeasts did not show 1,4-b-glu-

cosidase activity, but a strain dependent b-glucanase activity was observed.

Some Brettanomyces species did show 1,4-b-glucosidase activity. The highest

constitutive activity was found in Brettanomyces custersii. For the most interest-

ing strains the substrate specificity was studied and their activity was evaluated

in fermentation experiments with added hop glycosides. Fermentations with

Br. custersii led to the highest release of aglycones.

Conclusions: Pronounced exo-b-glucanase activity in Saccharomyces brewing

yeasts leads to a higher release of certain aglycones. Certain Brettanomyces

brewing yeasts, however, are more interesting for hydrolysis of glycosidically

bound volatiles of hops.

Significance and Impact of the Study: The release of flavour active compounds

from hop glycosides opens perspectives for the bioflavouring and product

diversification of beverages like beer. The release can be enhanced by using Sac-

charomyces strains with high exo-b-glucanase activity. Higher activities can be

found in Brettanomyces species with b-glucosidase activity.

Journal of Applied Microbiology ISSN 1364-5072

478 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 478–488

ª 2007 The Authors

repression effects (glucose, sucrose and fructose) (Verstre-

pen et al. 2004), screening of yeasts for b-glucosidase

activity in the presence of these sugars could be very use-

ful. Most research on b-glucosidases in yeast has focused

on species indigenous to wine-making and high activities

were demonstrated, especially in nonSaccharomyces yeasts

(Rosi et al. 1994; Ferreira et al. 2001; Rodriguez et al.

2004; Villena et al. 2005). The majority of Saccharomyces

isolates does not show b-glucosidase activity on a natural

substrate like arbutin (Rosi et al. 1994; Spagna et al. 2002;

Rodriguez et al. 2004). However, Mateo and DiStefano

(1997) demonstrated the hydrolysis of grape glycosides by

crude extracts of Saccharomyces strains. Recently, this

activity was suggested to be related to the major

exo-b-glucanase EXG1 of Saccharomyces cerevisiae (Gil

et al. 2005). An interesting feature of this yeast exo-b-glu-

canase activity is a substrate nonspecificity, by attacking

besides laminarin also pustulan and small substrates such

as laminaribiose, p-nitrophenyl-b-d-glucoside (pNPG) and

4-methylumbelliferyl-b-d-glucoside (4-MUG) (Nebreda

et al. 1986; Suzuki et al. 2001). Moreover, this

exo-b-glucanase activity is produced constitutively,

independently of the carbon source (Olivero et al.

1985) and is first secreted to the periplasmic space and

then released into the culture medium (Cid et al.

1995). Over-production of this major exo-b-glucanase

in S. cerevisiae led to a moderate release of certain

volatiles from grape glycosides (Gil et al. 2005). There-

fore, screening for high exo-b-glucanase activity in

Saccharomyces strains could be an approach for obtain-

ing a flavour enhancing strain.

Until now, no data are available on the glucoside

hydrolase activities of Saccharomyces brewing yeasts or

other yeasts involved in brewing. The study of these activ-

ities is interesting for better understanding and improving

the taste and aroma of beers. In this regard, glycosides

which are extracted from hops during the production of

beer may become important (Goldstein et al. 1999). This

is certainly important for lambic and gueuze beers, where

glycosides from added sour cherries or raspberries

(Schwab et al. 1990; Pabst et al. 1992) are extracted dur-

ing a secondary fermentation. Lambic and gueuze are spe-

cial Belgian beers obtained by spontaneous fermentation

(Verachtert and Dawoud 1984). In contrast to wine mak-

ing, where Brettanomyces yeasts are mostly regarded as

spoilage agents, these yeasts are essential to obtain lambic

and gueuze beer. The following species were found during

fermentation: Brettanomyces bruxellensis, Brettanomyces

lambicus, Brettanomyces intermedius, Brettanomyces

custersii, Brettanomyces abstinens, Brettanomyces anomalus,

Brettanomyces naardenensis and Brettanomyces custersi-

anus. Brettanomyces is also found during the refermenta-

tion of other special Belgian ales (Verachtert and Dawoud

1984; Martens et al. 1997) and is involved in the biofla-

vouring of a particular Belgian trappist beer (Vanderhae-

gen et al. 2003).

The aim of this study was to select yeast strains related

to beer-making that show a pronounced glucoside hydro-

lase activity. Both b-glucosidase and exo-b-glucanase

activity were considered. Finally, the hydrolase activity of

the yeasts was validated in fermentation experiments,

using purified hop glycosides.

Materials and methods

Chemicals

The following products were supplied by Sigma Aldrich:

2-heptanol, 4-methylumbelliferyl-b-d-glucoside (4-MUG),

Amberlite XAD-2, silicone antifoam (30% in water), ar-

butin, bovine serum albumin, cellobiose (C), glucose (D),

para-nitrophenol (pNP), para-nitrophenyl-b-d-glucoside

(pNPG), octyl-b-d-glucoside and salicin (S).

Yeast strains

Saccharomyces brewing yeasts (9 lager and 27 ale strains

shown in Fig. 1) were obtained from the Centre for Malt-

ing and Brewing Science (CMBS collection, Leuven, Bel-

gium). A commercial wine yeast S. cerevisiae UVAFERM

228 (U228) with published b-glucosidase activity was

obtained from Danstar Ferment AG (Zug, Switzerland).

Four haploid S. cerevisiae wild type strains BY4741,

BY4742, W303a, W303a and a BY4742 deletion mutant

strain (EXG1D ⁄ YLR300WD) were obtained from Euro-

scarf (Frankfurt, Germany). Dekkera and Brettanomyces

yeasts (18 strains shown in Fig. 2) were from CMBS and

were isolated and identified from fermenting lambic by

Verachtert and co-workers (Vanoevelen et al. 1977; Spae-

pen and Verachtert 1982; Verachtert and Dawoud 1984;

Martens et al. 1997).

Screening for glucoside hydrolase activity

A qualitative and fast detection of glucoside hydrolase

activity in yeast was carried out by replica plating the

yeast onto agar plates containing different b-d-glucosidic

substrates like arbutin, salicin, cellobiose or 4-MUG. Solid

media (20 g l)1 agar), all adjusted to pH 5, consisted of:

(i) Arbutin-plates: 6Æ7 g l)1 yeast nitrogen base (YNB)

and 5 g l)1 arbutin, with or without 0Æ2 g l)1 ferric

ammonium citrate, which was added as described by Rosi

et al. (1994). In addition, the same agar plates were pre-

pared with yeast extract (10 g l)1) and peptone (20 g l)1)

in stead of YNB; (ii) Cellobiose-plates: 6Æ7 g l)1 YNB and

5 g l)1 cellobiose; (iii) Salicin-plates: 6Æ7 g l)1 YNB and

L. Daenen et al. Glucoside hydrolase in brewing yeasts

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 478–488 479

5 g l)1 salicin; (iv) 4-MUG-plates: 10 g l)1 yeast extract,

20 g l)1 peptone, 20 g l)1 glucose and 0Æ4 g l)1 4-MUG

(Hernandez et al. 2003).

Assay for glucoside hydrolase activity

Yeast growth

A loopful of yeast cells was inoculated from YPD plates

in 10 ml YPD or YPC at pH 5 (10 g l)1 yeast extract (Y),

20 g l)1 peptone (P), 20 g l)1 glucose (D) or cellobiose

(C)) and incubated at 25�C for 24, 48 or 72 h, depend-

ing on the growth of the yeast. Then, the yeast was

inoculated at a cell concentration of 1 · 106 cells ml)1

in Erlenmeyer flasks filled to 20% of their volume with

YPD or YPC and grown at 20�C on an orbital shaker at

150 rev min)1 until reaching the later exponential

growth phase.

Extracellular activity

Yeast cell suspension (1 ml) was centrifuged (4�C, 3000 g,

10 min) and 0Æ2 ml supernatant was kept for the determi-

nation of extracellular activity on pNPG.

Cell-associated activity

The remaining pellet was washed with 1 ml physiologi-

cal water (sterile, cold, 7 g l)1 NaCl). After centrifuga-

tion (4�C, 3000 g, 10 min) the supernatant was

removed and 0Æ2 ml McIlvaine buffer pH 5 (0Æ1 mol l)1

citric acid–0Æ2 mol l)1 Na2HPO4) was added to the

pellet.

Enzyme assay on pNPG

For determination of the extracellular and cell-associated

activity, 0Æ2 ml pNPG solution (5 mmol l)1 pNPG in

McIlvaine buffer pH 5) was added to 0Æ2 ml supernatant

and to the pellet in 0Æ2 ml buffer. After incubation at

30�C for 1 h, 0Æ8 ml carbonate buffer (pH 10Æ2;

0Æ2 mol l)1) was added and the tubes were centrifuged

(4�C, 3000 g, 10 min). The increase in absorbance by the

released pNP was measured spectrophotometrically at

400 nm. Blanks were prepared for the substrate and the

different fractions.

Figure 1 Screening of Saccharomyces yeast strains on p-nitrophenyl-b-D-glucopyranoside (pNPG). Cell-associated (left grey bars), extracellular

(middle white bars) and total enzyme (right black bars) activity are presented and expressed as mU mg)1 dry weight (DW). Yeasts were grown on

YPD pH 5. LD10–18: Saccharomyces pastorianus (nine lager yeast strains); LD20–46: Saccharomyces cerevisiae (27 ale yeast strains); BY and W: S.

cerevisiae (four haploid laboratory strains); U228: S. cerevisiae (one wine yeast strain).

Figure 2 Screening of Dekkera and Brettanomyces yeast strains on

p-nitrophenyl-b-D-glucopyranoside (pNPG). Cell-associated (left grey

bars), extracellular (middle white bars) and total enzyme (right black

bars) activity are presented and expressed as mU mg)1 dry weight

(DW). Yeasts were grown on YPD pH 5. LD70, LD86, LD87: Dekkera

bruxellensis (3); LD78-LD83: Brettanomyces bruxellensis (6); LD71,

LD73-LD77: Brettanomyces lambicus (6); LD72: Brettanomyces cust-

ersii (1); LD84: Brettanomyces anomalus (1); LD88: D. anomala (1).

Glucoside hydrolase in brewing yeasts L. Daenen et al.

480 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 478–488

ª 2007 The Authors

Yeast protein fraction

To determine the substrate specificity of the glucoside

hydrolase activity, different glucosidic substrates were

examined. To measure the released glucose, yeast protein

fractions were used in stead of whole cells or culture

medium to avoid any glucose interference. The yeast

protein fractions were obtained as described below.

After growth on YPD or YPC, 50 ml of yeast cell

suspension was centrifuged at 4�C, 1811 g for 10 min.

The extracellular fraction was obtained by concentrating

the culture fluid and performing a buffer exchange with

McIlvaine buffer (0Æ1–0Æ2 mol l)1) pH 5, by ultrafiltration

using Vivaspin 20 membranes (10 000 MWCO; Sartorius,

Vilvoorde, Belgium).

All the following steps were carried out with cooling

on ice. The yeast pellet was washed twice with sterile cold

physiological water. Then 0Æ5 ml PBS lysis buffer (pH 7Æ3)

was added to an appropriate amount of yeast cells.

The lysis buffer consisted of 10 mmol l)1 Na2HPO4, 1Æ8mmol l)1 KH2PO4, 140 mmol l)1 NaCl and 2Æ7 mmol l)1

KCl added with 150 g l)1 glycerol, 2 g l)1 Tween,

2 mmol l)1 MgCl2, 1 mmol l)1 EDTA and a protease-

inhibitor mix (Complete EDTA-free; Roche Applied Sci-

ence, Vilvoorde, Belgium). The mixture was shaken with

0Æ25 ml glass beads in a FastPrepTM instrument (FP 120,

BIO101, MP Biomedicals, Illkirch, France). Lysis was

completed by shaking for 20 s at 6 m s)1, cooling on ice

for 2 min and shaken again for 10 s. After centrifugation

(4�C, 15000 g, 5 min) the supernatant was collected in a

new centrifuge microtube and centrifuged once more.

The supernatant was used as the intracellular enzyme

activity. The pellet was used as the cell insoluble fraction.

The amount of protein was determined with Bradford

(1976) reagent (Sigma Aldrich) using bovine serum albu-

min for calibration.

Enzyme assay on different substrates

After incubation with the yeast protein fraction, the

enzyme activity and the substrate specificity were deter-

mined by the amount of glucose released from the sub-

strates presented in Fig. 4. To 0Æ85 ml McIlvaine buffer

(0Æ2–0Æ4 mol l)1 pH 5), 0Æ1 ml substrate (glucosides:

25 mmol l)1; laminarin: 25 mg ml)1) and 0Æ05 ml enzyme

fraction were added. The mixture was incubated at 30�C

for 1 h. Then, the microtubes were put on ice and

0Æ48 ml carbonate buffer (1 mol l)1 at pH 10Æ2) was

added. The released glucose was measured using a stan-

dardized reference technique (glucose oxidase, GOD-PAP;

Dialab, Wiener Neudorf, Austria).

Dry weight

Yeast dry weight was determined by filtering 20 ml of

yeast cell suspension over a preweighted 0Æ45lm filter

paper (Macherey-Nagel, Duren, Germany). The filter

papers with yeast were oven-dried to a constant weight at

100�C.

Preparation of hop glycoside extract

A mixture of glycosidic compounds extracted from hops

was used to investigate and to validate the glucoside

hydrolase activity of the selected yeast strains. Glycosides

were extracted and partially purified as described by

Goldstein et al. (1999) with some modifications. Saaz

hops (1Æ5 kg previously extracted with liquid CO2) were

shaken with 9 l of methanol ⁄ water (4 : 1 v ⁄ v) for 3 h.

The extract was filtered and concentrated using a rotary

evaporator to 1Æ1 l. Then, the glycosidic extract was

added to a column (45 · 4 cm) packed with 200 g Am-

berlite XAD-2, previously treated according to Guyot-

Declerck et al. (2000). The effluent was collected. The

column was then washed with 1Æ5 l of water and eluted

with 0Æ75 l of methanol. The column was reconditioned

and the collected effluent was brought a second time on

the column. The column was then washed and eluted as

described above. The methanol fractions were concen-

trated to dryness on a rotary evaporator and re-sus-

pended in 740 ml McIlvaine buffer pH 5. This mixture

was shaken with 25 g PVPP and three times extracted

with 145 ml of diethyl ether to remove polyphenols and

free volatiles, respectively.

Hydrolysis of hop glycoside extract

To investigate the hydrolase activity of yeast on hop gly-

cosides during an alcoholic fermentation, a loopful of

cells was incubated into 10 ml of YPD for 24–48 h at

25�C. The precultures were transferred to 50 ml YPD and

incubated on an orbital shaker for 24–48 h at 20�C. The

cells were centrifuged (4�C, 1811 g, 10 min), washed and

re-suspended in physiological water. Finally, 75 ml YPD

(10 g l)1 yeast extract, 20 g l)1 peptone, 100 g l)1 glu-

cose) supplemented with 1 ml of hop glycoside extract

was inoculated at 10 · 106 cells ml)1. Fermentations were

carried out for 6 days at a constant temperature of 20�C.

The pure culture fermentation by Br. custersii CMBS

LD72 was carried out for 12 days.

Purge-and-trap GC-MS analysis

Analysis of the released volatile aglycones was performed

as described by Vanderhaegen et al. (2004) with some

modifications. Briefly, the method was as follows. Samples

were collected in 50 ml Falcon tubes on ice and centri-

fuged (4�C, 1811 g, 10 min). Then, internal standard

2-heptanol (250 mg l)1) and 10% antifoam solution was

L. Daenen et al. Glucoside hydrolase in brewing yeasts

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 478–488 481

added to give a final concentration of 1 mg l)1 and

0Æ04%, respectively. Of this sample, 5 ml was transferred

into a Tekmar Dohrman 3000 (Emerson, Mason, OH,

USA) purge-and-trap concentrator unit in which the

sample was purged with helium carrier gas and trapped

on a Vocarb 3000 trap (Supelco, Bellefonte, PA, USA).

Then, the volatiles were transferred via a MFA815 cold-

trap ⁄ control unit (Thermofinnigan, San Jose, CA, USA)

to a Fisons GC 8000 gas chromatograph equipped with a

Chrompack CP-Wax-52-CB column (length 50 m; inter-

nal diameter 0Æ32 mm; film thickness 1Æ2 lm; Varian,

Palo Alto, CA, USA). The temperature program was

1 min at 50�C, 3�C min)1 to 150�C, 15�C min)1 to

240�C and 5 min at 240�C. Total ion mass chromato-

grams were obtained in a Fisons MD 800 quadrupole

mass spectrometer (ionization energy 70 eV; source tem-

perature 250�C) (Thermo fisher Scientific, Waltham,

MA). Quantification was performed using standard refer-

ence compounds.

Data analysis

Significance differences between experimental figures were

estimated using Student’s t-test at a significance of P £0Æ05. The values represent the means of replications;

n = 2.

Results

Screening of brewing yeasts

Methods for detecting b-glucosidase activity in yeasts are

generally based on the hydrolysis of a glucosidic substrate,

which leads to the formation of biomass or to the release

of a traceable aglycon. The b-d-glucosides which are

mostly used are arbutin, 4-MUG, pNPG, esculin, salicin

or cellobiose. These substrates with the exception of escu-

lin were used in this study. A screening for glucoside

hydrolase activity was performed on 59 yeasts of which

54 brewing yeasts belonging to the genera Saccharomyces,

Dekkera and Brettanomyces, one commercial wine yeast

S. cerevisiae U228 with b-glucosidase activity and four

haploid S. cerevisiae laboratory strains. Results for screen-

ing, presented in Table 1, show that which method is

used highly determines whether activity towards gluco-

sides is present or not. Hence, YNB agar plates containing

arbutin as sole carbon source and ferric ammonium

citrate (Rosi et al. 1994) did not show growth or colour

formation by any yeast strain. When omitting ferric

ammonium citrate from the medium, growth was

detected for some strains (Table 1). On yeast extract, pep-

tone (YP) agar plates, however, containing arbutin and

ferric ammonium citrate, growth occurred and a brown

Table 1 Qualitative detection of the glucoside hydrolase activity in brewing yeasts using screening methods based on solid media

Yeast

Solid media (2% agar)

YNB-A YP-A

YNB-C YNB-S

YPD

4-MUG⁄ Fe3+ ⁄ Fe3+

Detection by: Growth Brown color Growth Fluo-halo

No. of strains

tested� Comment

Saccharomyces

pastorianus�

9 Lager brewing

strains�

) ) ) ) ) ) + or ++

Saccharomyces

cerevisiae

27 Ale brewing

strains

) ) ) ) ) ) + or ++

S. cerevisiae 1 Wine strain ++ ) + + ++ ++ ++

S. cerevisiae 4 Haploid lab strains ) ) ) ) ) ) +

Dekkera bruxellensis 3 D. bruxellensis ) ) ) ) ) ) + or ++

Brettanomyces

bruxellensis

6 Br. bruxellensis ) ) ) ) ) ) + or ++

6 Brettanomyces

lambicus§

) ) ) ) ) ) + or ++

1 Br. custersii§ +++ ) + ++ +++ +++ +++

Dekkera anomala 1 D. anomala +++ ) + ++ +++ +++ +++

Brettanomyces

anomalus

1 Br. anomalus +++ ) + ++ +++ +++ +++

), No detectable activity; +, weak activity; ++, moderate activity; +++, strong activity; YNB, yeast nitrogen base; YP, yeast extract, peptone; A,

arbutin; C, cellobiose; S, salicin; 4-MUG, 4-methylumbelliferyl-b-D-glucopyranoside.

�The yeast strains described here are the same as presented in Figs 1 and 2.

�The S. pastorianus name is used, as proposed by Rainieri et al. (2006), for lager brewing strains which naturally occur as Saccharomyces hybrids.

§Synonyms of Dekkera Brettanomyces species according to Barnett et al. (1990).

Glucoside hydrolase in brewing yeasts L. Daenen et al.

482 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 478–488

ª 2007 The Authors

colour was formed for some Dekkera and Brettanomyces

species and with S. cerevisiae U228. For yeasts on YPD

agar plates containing 4-MUG, a fluorescent halo

appeared for all strains. The halo around the colony

increased during growth and varied in size depending on

the strain. Yeasts growth on salicin corresponded with

growth on cellobiose. This occurred with Br. intermedius

CMBS LD85, Br. custersii CMBS LD72, Br. anomalus

CMBS LD84 and Dekkera anomala CMBS LD88, but not

with any Br. bruxellensis or Br. lambicus. Neither did any

Saccharomyces strain grow on salicin nor cellobiose, ex-

cepted for the commercial strain S. cerevisiae U228.

The glucoside hydrolase activity of the strains was fur-

ther quantitatively differentiated by activity determina-

tions on pNPG. As applications of this enzyme activity

could be interesting in fermented food products contain-

ing sugars which exert catabolite repression effects, the

presence of a constitutively activity was examined by

using yeast after growth in liquid YPD and determining

the extracellular and the cell-associated activity. All strains

showed hydrolase activity, especially S. cerevisiae CMBS

LD40 and Br. custersii CMBS LD72 (Figs 1 and 2).

Exo-b-glucanase

As mentioned above, the major exo-b-1,3-glucanase

(EXG1) of S. cerevisiae is known to hydrolyse besides lami-

narin, the polymer pustulan and small substrates like lami-

naribiose, pNPG and 4-MUG (Nebreda et al. 1986; Suzuki

et al. 2001). To examine the impact of EXG1 on the results

obtained above with the screening on 4-MUG-plates and

with the screening on pNPG (Table 1 and Fig. 1), the wild

type strain BY4742 and the deletion mutant EXG1D(YLR300WD) were screened in the same way (Fig. 3). On

agar plates with 4-MUG, a fluorescent halo was observed

for the wild type strain but not for the mutant strain exg1D(Fig. 3). On pNPG, both extracellular and cell-associated

activity almost completely disappeared for the mutant

strain exg1D (Table 2). To verify this activity on a glucan

substrate like laminarin, different yeast protein fractions

were incubated both on pNPG and on laminarin

(Table 2). The hydrolase activity clearly decreased on both

substrates in case of the mutant strain exg1D.

Selection for further experiments

Based on the previous results, a selection of six yeast

strains was made for further characterization of the gluco-

side hydrolase activity. The commercial wine yeast S.

cerevisiae U228 was retained as this was the only

Saccharomyces strain demonstrating 1,4-b-glucosidase

activity in this study. Brettanomyces custersii CMBS LD72

isolated from a lambic fermentation was selected for a

relative higher b-glucosidase activity. The haploid strain

S. cerevisiae BY4742 and the mutant strain exg1D were

chosen to investigate the effect of exo-1,3-b-glucanase

(EXG1). The strains S. cerevisiae CMBS LD 25 and CMBS

LD40 were selected for a respectively low and higher glu-

coside hydrolase activity on pNPG (Fig. 1).

Figure 3 Growth of the wild type BY4742 and the exg1D(= ylr300wD) mutant strain on an YPD agar plate containing 4-methyl-

umbelliferyl-b-D-glucopyranoside (4-MUG). The fluorescent halo of the

released 4-methylumbelliferone (4-MU) under UV-radiation demon-

strates hydrolase activity.

Table 2 Specific enzyme activity of the haploid strain BY4742 and

the deletion mutant exg1D on para-nitrophenyl-b-D-glucoside (pNPG)

and laminarin

BY4742 exg1D BY4742 exg1D

pNPG

(mU ⁄ mg DW)

Extracellular activity* 0Æ54 <0Æ01

Whole cells* 0Æ24 0Æ01

pNPG

(mU ⁄ mg protein)

Laminarin

(mU ⁄ mg protein)

Extracellular activity� 49 0Æ3 149 8Æ6

Intracellular activity� 0Æ2 0Æ07 0Æ3 0Æ1

Cell insoluble fraction� 1Æ6 0Æ6 3Æ5 0Æ7

DW, dry weight.

*Specific activity is determined as in Figs 1 and 2 by measurement of

the released pNP.

�Specific activity is determined by measurement of the released pNP

or glucose after incubation of the concerning yeast protein fraction

on pNPG and laminarin, respectively.

L. Daenen et al. Glucoside hydrolase in brewing yeasts

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 478–488 483

Characterization of selected yeast strains

For the brewing yeasts, the substrate specificity of the glu-

coside hydrolase was characterized on different glucosidic

substrates. First, the b-glucosidase activity of S. cerevisiae

U228 and Br. custersii CMBS LD72 was examined. More-

over, the constitutive or inductive production of the

activity was examined by determining the activity after

growth on YP medium with respectively glucose or cello-

biose as carbon source (Fig. 4). After growth on cello-

biose, the intracellular activity of S. cerevisiae U228 and

Br. custersii CMBS LD72 was higher on all the glucosidic

substrates than after growth on glucose. The intracellular

activity of Br. custersii CMBS LD72, after growth on glu-

cose, was higher than the activity of S. cerevisiae U228

grown on glucose. Concerning substrate specificity, espe-

cially salicin, octyl-glucoside and pNPG were hydrolysed

to a great extent by S. cerevisiae U228 and Br. custersii

CMBS LD72.

Further, the glucoside hydrolase activity of the Saccha-

romyces brewing yeasts CMBS LD25 and LD40 was exam-

ined. As the results from Table 1 and Fig. 1 indicate that

this glucoside hydrolase activity is probably because of

the exo-1,3-b-glucanase, the glucan substrate laminarin

was tested as well. The results in Fig. 5 clearly demon-

strate the higher activity on the substrates pNPG, 4-MUG

and laminarin for S. cerevisiae CMBS LD40 in compari-

son with CMBS LD25.

Finally, with respect to effects on hop glycosides, fer-

mentations were started with the selected strains and also

with a mixed culture of Br. custersii CMBS LD72 and S.

cerevisiae CMBS LD25. The latter was chosen for this

mixed culture in order to minimally interfere in glucoside

hydrolysis because of a relative lower exo-b-glucanase

activity (Fig. 5). Samples for analysis of released aglycones

were taken after completion of the fermentations and

results are presented in Fig. 6. The laboratory strain

BY4742 caused a slightly higher release of methyl salicylate

and cis-3-hexen-1-ol than the mutant strain exg1D. The

strains S. cerevisiae CMBS LD40 and S. cerevisiae U228

caused a moderate but higher release of methyl salicylate

and the aliphatic alcohols 1-octen-3-ol and cis-3-hexen-1-

ol than S. cerevisiae CMBS LD25. The S. cerevisiae strains

were not very active on the linalyl-glycosides. Most inter-

esting is the high release of aglycones in fermentations

with Br. custersii CMBS LD72, in pure culture or in co-

culture with Saccharomyces. In these fermentations, linal-

ool, methyl salicylate, 1-octen-3-ol and cis-3-hexen-1-ol

were released to the highest extent.

Figure 4 Hydrolysis of different glucosidic substrates by the intracel-

lular activity of Saccharomyces cerevisiae U228 and Brettanomyces

custersii CMBS LD72 after growth on YP medium with added glucose

(U228 YPD and CMBS LD72 YPD); S. cerevisiae U228 and Br. custersii

CMBS LD72 after growth on YP medium with added cellobiose (U228

YPC and CMBS LD72 YPC). The specific enzyme activity is expressed

as mU mg)1 protein (bovine serum albumin equivalents in order of

reproduction). ( ) U228 YPD; ( ) U228 YPC; ( ) CMBS LD72 YPD;

( ) CMBS LD72 YPC.

Figure 5 Hydrolysis of different glucosidic substrates by different

yeast protein extracts [extracellular (EC), intracellular (IC) and cell

insoluble fraction (CI)] of Saccharomyces cerevisiae CMBS LD25 and S.

cerevisiae CMBS LD40 grown on YP medium with added glucose

(CMBS LD40). Specific enzyme activity is expressed as mU mg)1 pro-

tein (bovine serum albumin equivalents). ( ) CMBS LD25 EC; ( )

CMBS LD40 EC; ( ) CMBS LD25 IC; ( ) CMBS LD40 IC; ( ) CMBS

LD25 CI; ( ) CMBS LD40 CI.

Glucoside hydrolase in brewing yeasts L. Daenen et al.

484 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 478–488

ª 2007 The Authors

Discussion

The aim of this study was to select and study yeast

strains, indigenous to the brewing environment, with

hydrolase activity towards glycosidically bound volatile

compounds. This required an easy, fast and reliable detec-

tion method. The most appropriate enzyme for the

hydrolysis of glycosidically bound volatiles is b-glucosi-

dase (EC 3.2.1.21), preferably with a broad substrate spec-

ificity. Many different methods have been used to detect

b-glucosidase activity. The method with arbutin as sole

carbon source for yeast growth and addition of ferric

ammonium citrate has been proposed as reliable for

detecting b-glucosidases (Rosi et al. 1994; Ferreira et al.

2001). In our study, the brewing yeasts belonging to Sac-

charomyces or Dekkera and Brettanomyces did not grow or

develop a brown colour with that medium. This method

apparently led to false negative results with our strains, as

S. cerevisiae U228, Br. intermedius CMBS LD85, Br. cust-

ersii CMBS LD72, Br. anomalus CMBS LD84 and D. ano-

mala CMBS LD88 clearly showed the presence of a b-

glucosidase (EC 3.2.1.21) by growing on cellobiose and

salicin. By omitting ferric ammonium citrate from the

minimal medium with arbutin, these strains showed

growth. Apparently, with our strains, the combination of

arbutin and ferric ammonium citrate at the used concen-

tration inhibited growth. The assimilation of cellobiose,

salicin and arbutin by Br. intermedius, Br. custersii,

Br. anomalus and D. anomala was already reported by

Barnett et al. (1983).

Figure 6 Concentration (mg l)1) of released aglycones after fermentation of YPD with addition of purified hop glycosides by Saccharomyces ce-

revisiae BY4742 wt, S. cerevisiae exg1D, S. cerevisiae CMBS LD25, S. cerevisiae CMBS LD40, S. cerevisiae U228, Br. custersii CMBS LD72, mixed

culture of S. cerevisiae CMBS LD25 and Brettanomyces custersii CMBS LD72. Control without any enzyme activity was carried out at pH 5. (a) ( )

linalool, (b) ( ) methyl salicylate, (c) ( ) cis-3-hexen-1-ol and (d) ( ) 1-octen-3-ol.

L. Daenen et al. Glucoside hydrolase in brewing yeasts

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 478–488 485

For Saccharomyces yeasts, the presence of a sporadically

observed b-glucosidase activity remains intriguing. In our

study, none of the Saccharomyces brewing yeasts showed

such activity. On the contrary, the commercial wine yeast

S. cerevisiae U228 could grow on cellobiose, arbutin and

salicin and consequently demonstrated the presence of a

b-glucosidase. Previously, only very few S. cerevisiae iso-

lates were found which showed activity on arbutin and

thus indicating b-glucosidase activity (Rosi et al. 1994;

Spagna et al. 2002; Rodriguez et al. 2004). This is remark-

able as no gene in the genome of the haploid strain

S. cerevisiae is known for coding a 1,4-b-glucosidase (EC

3.2.1.21) (Cherry et al. 1997; http://www.yeastgenome.org;

2 January 2007). Moreover, S. cerevisiae yeasts are not

expected to assimilate cellobiose, arbutin or salicin

according to physiological identification tests proposed by

Barnett et al. (1990). However, the b-glucosidase gene of

a S. cerevisiae strain AL41 isolated on arbutin by Spagna

et al. (2002) was recently partially sequenced by Quatrini

et al. (2006). The translated amino acid sequence con-

tained one of the conserved patterns, namely FGYGLSY,

which is typical for most yeast b-glucosidases. This pat-

tern was found in the C-terminal part of the b-gluco-

sidase(s) from Saccharomycopsis fibuligera, Candida

pelliculosa and Kluyveromyces fragilis (Rojas and Romeu

1996). Apparently only some S. cerevisiae yeasts possess a

gene coding for b-glucosidase (EC 3.2.1.21) whereas the

majority does not.

On the contrary, all S. cerevisiae yeasts synthesize

b-glucanases. According to Nebreda et al. (1986), the

exo-1,3-b-glucanase EXG1 is responsible for the greatest

part of the hydrolysis of laminarin and synthetic sub-

strates like pNPG and 4-MUG, which is confirmed in this

study. The question remains whether the capacity of only

certain S. cerevisiae strains to assimilate b-glucosides like

cellobiose, salicin or arbutin is because of a b-glucanase

with an adjusted substrate specificity or to the presence of

a b-glucosidase like enzyme. Results by Ridruejo et al.

(1989) already supported the idea that yeast exo-1,3-

b-glucanases (EC 3.2.1.58) are glucosidases that also

attack laminarin, rather than glucanases capable of attack-

ing pNPG. Suzuki et al. (2001) suggested that the yeast

Exg1p may be classified as a new type of b-glucanase or

b-glucosidase that has not been described before.

Considering the regulation of the enzymes, the b-glu-

cosidase from S. cerevisiae U228 appears to be repressed

by glucose and induced by cellobiose. An inducible b-glu-

cosidase activity in S. cerevisiae was already demonstrated

by Duerksen and Halvorson (1958). The b-glucosidase of

Br. custersii CMBS LD72 also appeared to be induced by

growth on cellobiose. Possible repression effects on the

b-glucosidase production of Br. custersii CMBS LD72 will

be investigated in further research. The exo-1,3-b-glucan-

ase EXG1 of the S. cerevisiae strains is constitutively regu-

lated (Olivero et al. 1985). Results from this study show a

strain-dependent activity for this exo-1,3-b-glucanase,

which was most pronounced for S. cerevisiae CMBS

LD40.

This relative higher activity of S. cerevisiae CMBS LD40

led also to a higher release of certain aglycones from hop

glycosides during fermentation. This becomes an interest-

ing property of a brewer’s yeast when considering applica-

tions for bioflavouring. Fermentation with the haploid

mutant strain exg1D, led to a lower release of certain agly-

cones than the wild type. These results are in agreement

with those of Gil et al. (2005) who demonstrated the activ-

ity of the major exoglucanase EXG1 of S. cerevisiae towards

glycosidically bound volatile compounds. The reaction is

however somewhat specific, as linalool is almost not

released. This could be because of steric hindrance of the

reaction by the tertiary alcohol (Gunata et al. 1985; Gil

et al. 2005). However, Ugliano et al. (2006) observed

a pronounced release of linalool from grape glycosides

after fermentations with S. cerevisiae and S. bayanus wine

yeasts. The hydrolysis of glycosidically bound tertiary

alcohols like linalool apparently depends on the strain, the

fermentation conditions or on both. Further investigation

is required to clarify these observations.

The most interesting and efficient glucoside splitting

occurred with the b-glucosidase of Br. custersii CMBS

LD72. This yeast was isolated from fermenting lambic

(Verachtert and Dawoud 1984). The b-glucosidase of this

Br. custersii strain showed a broad substrate specificity

towards alkyl- and aryl-b-d-glucosides. For the hydrolysis

of hop glycosides, a co-culture with S. cerevisiae was also

very efficient. As Dekkera and Brettanomyces species are

slow fermenting yeasts, the duration of an alcoholic fer-

mentation can be reduced by a mixed fermentation in

which S. cerevisiae carries out the main fermentation and

Br. custersii takes care for the release of glycosidically

bound volatiles. Brettanomyces custersii was already recog-

nized for its interesting b-glucosidase activity as an effi-

cient cellobiose and cellotriose fermenting yeast in

ethanol production (Gonde et al. 1984; Spindler et al.

1992). Brettanomyces custersii was found to be the domi-

nant yeast species during the latest stages of lambic fer-

mentation (13th–24th month) indicating a possible

adaptation to the environment, through assimilation of

cellobiose released from the wooden fermentation casks

(Verachtert and Dawoud 1984; Vanderhaegen et al. 2003).

In conclusion, Saccharomyces strains show a strain-

dependent hydrolase activity towards certain glycosidically

bound volatile compounds. This would be mainly because

of the enzyme exo-1,3-b-glucanase. Only few Saccharomy-

ces strains appear to possess real 1,4-b-glucosidase activ-

ity. A commercial wine strain showed an inducible

Glucoside hydrolase in brewing yeasts L. Daenen et al.

486 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 478–488

ª 2007 The Authors

b-glucosidase with weak activity during fermentation.

A more pronounced b-glucosidase activity was found in

nonSaccharomyces yeasts like Br. custersii, isolated from

fermenting lambic. Fermentation carried out with a pure

culture or a co-culture of S. cerevisiae and Br. custersii led

to the highest release of volatiles from hop glycosides.

Further research will be focused on the use of Br. custersii

to introduce new flavours in beer, either through a mixed

fermentation, maturation or refermentation, either with

or without the presence of added ingredients such as

fruits, flowers or spices containing glycosides as flavour

precursors.

Acknowledgements

This research was funded by a PhD grant from the Insti-

tute for the Promotion of Innovation through Science

and Technology in Flanders (IWT-Vlaanderen, Belgium).

The authors wish to thank L. De Cooman and K. Goiris

of the Technical University KaHo in Gent for providing

the treated Saaz spent hops.

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