Screening and typing of Patagonian wine yeasts forglycosidase activities

M.E. Rodrıguez1*, C.A. Lopes1*, M. van Broock2*, S. Valles3, D. Ramon3,4 and A.C. Caballero1

1Laboratorio de Microbiologıa y Biotecnologıa, Departamento de Quımica, Facultad de Ingenierıa, Universidad Nacional del Comahue,

Neuquen, Argentina, 2Laboratorio de Microbiologıa y Biotecnologıa, Departamento de Biologıa, Centro Regional Universitario Bariloche,

Universidad Nacional del Comahue, Bariloche, Rıo Negro, Argentina, 3Departamento de Biotecnologıa, Instituto de Agroquımica y

Tecnologıa de Alimentos (C.S.I.C.), Paterna, Valencia, Spain, and 4Departamento de Medicina Preventiva y Salud Publica,

Bromatologıa, Toxicologıa y Medicina Legal, Universitat de Valencia, Valencia, Spain

2003/0457: received 2 June 2003, revised 7 August 2003 and accepted 9 August 2003

ABSTRACT

M.E . RODR IGUEZ, C .A . LOPES, M. VAN BROOCK, S . VALLES, D. RAMON AND A.C . CABALLERO. 2003.

Aims: The purpose of this study was to select autochthonous glycosidase producer yeasts with potential use in

industrial production of Patagonian red wines.

Methods and Results: The study was carried out in oenological autochthonous yeasts from Comahue region

(Argentinean North Patagonia). A set of screenable yeast phenotypic characteristics indicative of their potential

usefulness in more aromatic red wine production was defined and tested in both, Saccharomyces and non-

Saccharomyces populations. Twelve isolates showing six different glycosidase phenotypes were selected and they

were characterized at species and strain levels using molecular methods. A close correlation between molecular and

phenotypic characteristics was observed. Five strains belonging to Candida guilliermondii, C. pulcherrima and

Kloeckera apiculata with highest constitutive b-glucosidase activity levels without anthocyanase activity were

discriminated. Some of them also showed constitutive b-xylosidase and inductive a-rhamnosidase activities.

Conclusions: The extension of the selection of oenological yeast to non-Saccharomyces species provided strains

possessing novel and interesting oenological characteristics which could have significant implications in the

production of more aromatic young red wine.

Significance and Impact of the Study: As these non-Saccharomyces are indigenous to wine, they can be used in

mixed starters at the beginning or as pure cultures at the end fermentation to contribute in enhancing the wine

nuance that is typical of this specific area.

Keywords: anthocyanase activity, glycosidase activities, killer behaviour, non-Saccharomyces diversity, Patagonian

oenological yeast.

INTRODUCTION

Flavour (aroma and taste) is the most important distin-

guishing characteristic of wine (Lambrechts and Pretorius

2000). Wine flavour is classified according to the sources of

the different compounds contributing to it, this includes

varietal, prefermentative, fermentative and postfermentative

flavours (Schreier 1979; Rapp 1998). It is well known that

several secondary metabolites accumulated in grapes are

responsible for providing the basis of the �varietal character�of the wine (Rapp and Versini 1991). These compounds can

be found in grapes and musts as free, volatile and odorous

forms as well as in flavourless, nonvolatile forms b-glyco-

sidically bound to disaccharide molecules such as 6-O-a,

*Consejo Nacional de Investigaciones Cientıficas y Tecnicas de la Republica

Argentina (CONICET).

Correspondence to: Adriana C. Caballero, Laboratorio de Microbiologıa y

Biotecnologıa, Departamento de Quımica, Facultad de Ingenierıa, Universidad

Nacional del Comahue, Neuquen, Argentina (e-mail: ecastro@neunet.

com.ar).

ª 2003 The Society for Applied Microbiology

Journal of Applied Microbiology 2004, 96, 84–95 doi:10.1046/j.1365-2672.2003.02032.x

LL-arabinofuranosyl-, 6-O-a, LL-rhamnopyranosyl- and 6-O-

b-DD-apiofuranosil-b-DD-glucose (Vasserot et al. 1995; Win-

terhalter and Skouroumounis 1997). These flavourless

glycosidic complexes are generally more abundant than free

odorous forms and they represent an important potential

source of the fragrant compounds in wine requiring

enzymatic or acidic hydrolysis for the liberation of their

fragrances (Gunata et al. 1985; Williams et al. 1989). The

enzymatic hydrolysis of these glycosidic complexes is a two-

step sequential reaction: first, an a-LL-arabinofuranosidase,

an a-LL-rhamnosidase or a b-DD-apiofuranosidase activity

cleaves the (1 fi 6) osidic linkage, and secondly, the flavour

compounds are liberated from the monoglucosides by the

action of a b-DD-glucosidase activity (Gunata et al. 1988).

Unlike acidic hydrolysis, enzymatic hydrolysis does not

result in modifications of the aromatic character (Gunata

et al. 1990a). Because of their characteristics (glucose

inhibition, poor stability at the low pH), grape glycosidase

activities have a minimal effect on enhancing varietal aroma

during winemaking (Canal-Llauberes 1993). As an alternat-

ive to the inefficient grape glycosidases, the presence of these

activities has been investigated in several fungal and yeast

species and it has been found that some of them possess

interesting properties for practical uses (Gunata et al. 1985,

1990a,b; Aryan et al. 1987; Vasserot et al. 1991; Rosi et al.

1994; Rogerson et al. 1995; Riou et al. 1998; Riccio et al.

1999; Spagna et al. 2002).

In this work, we investigated the presence of several

glycosidase activities in oenological indigenous yeasts from

Comahue region (north-western Patagonia, Argentina) in

order to use these yeasts themselves, or their enzymes, to

increase the varietal character of the regional red wines. To

do this, a large number of Saccharomyces and non-

Saccharomyces yeasts isolated from grape surfaces and

fermentation musts were screened twice to determine the

presence of b-glucosidase, b-xylosidase, a-LL-arabinofura-

nosidase and a-LL-rhamnosidase activities and their antho-

cyanase capabilities. The taxonomic identity of 12

indigenous yeast isolates with highest b-glucosidase activity

was confirmed by molecular methods at species and strain

levels, and their killer phenotype was characterized. On the

basis of the relationships between their phenotypic and

genotypic characteristics, these indigenous yeast isolates

were clustered in six different strains, some of which showed

interesting characteristics for winemaking.

MATERIALS AND METHODS

Yeasts

A total of 180 indigenous yeast isolates belonging to the

genera Candida (74 isolates), Torulaspora (six isolates),

Kloeckera (25 isolates) and Saccharomyces (75 isolates) were

analysed. The yeast isolates were obtained in our laboratory

from Merlot, Malbec and Trousseau type grapes (van

Broock et al. 1996; Lopes 1999) and from three different

fermentation stages of their musts spontaneously fermented

at laboratory and industrial scales (Rodrıguez 1999). Yeasts

have already been identified at genus and species level

according to conventional physiological and morphological

tests and keys proposed by Kreger-van Rij (1984) and

Kurtzman and Fell (1998) (van Broock et al. 1996; Lopes

1999; Rodrıguez 1999).

Glycosidase screening methods

Screening for detecting b-glucosidase activity was carried

out on agar plates with arbutin as substrate. The medium,

containing 6Æ7 g yeast nitrogen base (YNB; Difco), 5 g

arbutin, 20 g agar per litre and pH 5Æ0, was autoclaved at

120�C for 15 min. Immediately after sterilization 2 ml of a

sterile 1% (w/v) ferric ammonium citrate solution was

added to 100 ml melted medium (Rosi et al. 1994). Each

plate was inoculated in radial streaks with 24-h-old yeast

cultures, incubated at 25�C and examined daily during a

week. An uninoculated plate served as the control. Yeast

isolates with b-glucosidase activity hydrolyse the substrate

and a dark brown halo develops in the agar medium.

Screening for detecting all b-glucosidase, b-xylosidase,

a-rhamnosidase and a-arabinofuranosidase activities was

carried out on agar plates containing 1Æ7 g YNB (without

amino acids and ammonium sulphate; Difco), 5 g ammo-

nium sulphate, 5 g source carbon and 20 g agar per litre.

The pH was adjusted to 5Æ5. The carbon sources assayed

were either arabinose, cellobiose, glucose, rhamnose and

xylose (Sigma). Glycosidase activities were determined by

using the appropriate 4-methylumbelliferyl glycoside (Sig-

ma) as substrate, as described by Manzanares et al. (1999).

The presence of the enzymatic activity was visualized as a

fluorescent halo surrounding yeast growth by plate expo-

sition to u.v. light.

b-Glucosidase activity

Liquid culture conditions. A medium containing 1Æ7 g

YNB, 5Æ0 g (NH4)2SO4, 5Æ0 g yeast extract, 5Æ0 g peptone,

5Æ0 g glucose per litre and pH 5Æ0 was used to examine

yeasts showing b-glucosidase activity and to localize the

enzymatic activity. Inocula were prepared by growing a

loopful of cells from GPY plate (5Æ0 g yeast extract, 5Æ0 g

peptone, 40Æ0 g dextrose, 20 g agar per litre; pH 5Æ0) in

10 ml of liquid medium. After 24 h, a 2% volume of the

inoculum was added to 100 ml Erlenmeyer flasks filled to

20% of their volume and shaken at 180 rev min)1 in a

Vicking shaker at 25�C for 24 h (aerobic conditions). In

studies on the influence of anaerobic growth conditions, the

GLYCOSIDASE ACTIVIT IES IN PATAGONIAN YEASTS 85

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 84–95, doi:10.1046/j.1365-2672.2003.02032.x

yeasts were grown in 20 ml screw-capped tubes totally filled

and incubated without shaking at 25�C for 3 days. Growth

control was checked by monitoring the culture absorbance at

600 nm and tubes or Erlenmeyer flasks containing uninoc-

ulated media were used as the blank. Once the assays were

finished, cultures were centrifuged (5000 g, 10 min, 4�C)

and the cells and culture supernatant were assayed for

enzymatic activity and the dry weight was determined.

Whole cell preparation. Cells harvested from 1 ml of

culture as indicated above, were washed twice with cold

sterile distilled water, centrifuged and the pellet was

resuspended in 0Æ5 ml of 100 mmol l)1 citrate–phosphate

buffer, pH 5Æ0, and used for enzymatic assay.

Permeabilized cell preparation. The procedure of

Salmon (1984) with some modifications was used: 5 ml of

liquid culture were centrifuged and washed with 5 ml of cold

sterile distilled water. The pellet was resuspended in 1 ml of

75 mmol l)1 imidazole buffer pH 7Æ5, and added quickly to

50 ll of 0Æ3 mmol l)1 glutathion, 10 ll of 10% Triton X-100

and 50 ll of toluene/ethanol (1 : 4 v/v). The suspension was

shaken vigorously for 5 min and then centrifuged. The yeast

pellet was suspended in 5 ml cold sterile water: 1 ml of

this suspension was centrifuged, washed with cold sterile

distilled water and the pellet was resuspended in 0Æ5 ml

citrate–phosphate buffer and used for enzymatic assay.

Enzyme assay. b-Glucosidase activity was assayed by

measuring the amount of p-nitrophenol (pNP) released from

p-nitrophenyl-b-DD-glucoside (pNPG) used as substrate.

Enzyme solution (0Æ1 ml) was mixed with 0Æ4 ml of a

2 mmol l)1 solution of pNPG in 100 mmol l)1 citrate–

phosphate buffer, pH 5Æ0. The reaction mixture was incu-

bated at 30�C for 5–15 min and subsequently, 2Æ0 ml of

250 mmol l)1 Na2CO3 was added to stop the reaction. The

released pNP in this mixture was measured spectrophoto-

metrically at 405 nm in a Shimadzu UV–V spectrophoto-

meter (Shimadzu Corporation, Kyoto, Japan). The pNP molar

extinction coefficient used was � ¼ 18 300 mol)1 l cm)1.

One unit (U) of enzyme activity was defined as the quantity

of enzyme that released 1 lmol of pNP per minute under the

above experimental conditions. The values of intracellular

activity were obtained subtracting the whole cell enzymatic

activity values from the permeabilized cell values. All assays

were performed in duplicate.

Dry weight. Ten millilitres of culture samples were

centrifuged at 10 000 g 5 min. The pellets were washed

twice with 5 ml of cold sterile distilled water and resus-

pended in sterile distilled water. These cell suspensions were

placed on preweighed dishes and dried at 105�C until

constant weight.

PCR–RFLP analysis of rDNA

The 5Æ8S-ITS region was amplified in a Progene thermo-

cycler (Techne, Cambridge, UK) using ITS1 (5¢-TCCGT

AGGTGAACCTGCGG-3¢) and ITS4 (5¢-TCCTCCGCT

TATTGATATGC-3¢) primers already described (White

et al. 1990). PCR conditions were identical to those

described by Esteve-Zarzoso et al. (1999). The amplified

DNAs (0Æ5–10 lg) were digested without further purifica-

tion with the restriction endonucleases CfoI, HaeIII, HinfI

and DdeI (Roche Molecular Biochemicals, Mannheim,

Germany) according to the supplier’s instructions. PCR

products and their restriction fragments were separated on

1Æ4% (w/v)and 3% (w/v) agarose gels, respectively, with 1x

TAE buffer (40 mmol l)1 Tris–acetate, 1 mmol l)1 EDTA,

pH 8). After electrophoresis, gels were stained with ethi-

dium bromide (5 lg ml)1) and visualized under u.v. light. A

100-bp DNA ladder marker (Gibco BRL, Gaithersburg,

MD, USA) served as size standard.

The taxonomic identity of indigenous S. cerevisiae isolates

tested in this study has already been confirmed by us using

the same molecular method (Lopes et al. 2002).

Karyotype analysis

DNA was prepared in agarose plugs as described by Carle

and Olson (1985). Chromosomal profiles were determined

using the CHEF technique with a CHEF-DRIII (Bio-Rad,

Richmond CA, USA). The yeast chromosomes were

separated on 1% (w/v) chromosomal grade agarose gels

(Bio-Rad) under the following running conditions: electric

field, 6 V cm)1; angle, 120�; temperature, 14�C; block 1,

60 s pulse time for 15 h; block 2, 120 s pulse time for 13 h;

buffer 0Æ5 · TBE (45 mmol l)1 Tris–borate pH 7Æ5,

1 mmol l)1 EDTA pH 8Æ0). After electrophoresis, gels were

stained with ethidium bromide (5 lg ml)1) and visualized

under u.v. light.

mtDNA-RFLP analysis

Total DNA extraction and mtDNA restriction analysis were

performed by the method of Querol et al. (1992) modified

by Lopez et al. (2001). Yeast DNA was digested with HinfI

restriction enzyme (Roche Molecular Biochemicals, Mann-

heim, Germany) and the fragments were separated in TAE

buffer containing 1% (w/v) agarose gels (Fernandez-

Espinar et al. 2000).

Killer behaviour

Killer behaviour was assayed using the seeded agar-plate

technique as described by Sangorrın et al. (2002). The killer

sensitivity of the indigenous yeasts were determined

86 M.E. RODRIGUEZ ET AL.

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 84–95, doi:10.1046/j.1365-2672.2003.02032.x

contrasting them to reference panel of the 10 collection well-

known killer strains and their killer character was evaluated

against two collection sensitive strains. The collection killer

strains used were S. cerevisiae YAT 679 (K1 type), S.

cerevisiae NCYC 738 (K2 type), S. capensis NCYC 671 (K3

type), C. glabrata NCYC 388 (K4 type), Hansenula anomala

NCYC 434 (K5 type), Kluyveromyces fragilis NCYC 587 (K6

type), C. valida NCYC 327 (K7 type), H. anomala NCYC

435 (K8 type) and H. mrakii NCYC 500 (K9 type), K.

drosophilarum NCYC 575 (K10 type), provided by Professor

Isato Kono (Industrial Technology Center of Okayama

Prefecture, Japan), and S. cerevisiae P352 (K1 type) from

PROIMI yeast collection from Tucuman (Argentina). The

collection-sensitive strains used were S. cerevisiae P351

(PROIMI yeast collection) and C. glabrata NCYC 388 above

cited.

Anthocyanase activity

Anthocyanase activity was measured using red wine as

substrate. Aerobically grown YNB-cellobiose or rhamnose

cells of 1.5 ml was collected by centrifugation and resus-

pended in 1Æ5 ml of red wine and anaerobically incubated at

4�C during a week. Wine decolourization was monitored

measuring the absorbance at 520 nm in 2 mm light path

cuvettes. Glycosidase activity was also assayed using the

appropriate pNP glycoside (Sigma) as substrate.

Chemicals

Arbutin, pNPG, pNP, MUG, MUX, MUA, MUR,

glutathion and Triton X-100 were supplied by Sigma, and

culture medium constituents by Difco. All other chemicals

used were of reagent grade.

Data analysis

Significant differences between experimental figures were

estimated using Student’s test. Dendrograms were gener-

ated using an unweighed pair group with arithmetic average

(UPGMA) algorithm and the concordance between indi-

viduals was (1-SM) matching.

RESULTS

Glycosidase screenings and characterization

Patagonian Saccharomyces and non-Saccharomyces yeast

isolates, all of oenological origin, were screened for

b-glucosidase activity on agar plates containing arbutin as

substrate. Forty-five of 180 analysed isolates showed enzy-

matic activity and they were characterized as strong (11

isolates), moderate (eight isolates) or weak (26 isolates)

according to the colour intensity (dark, fair or light brown)

of the halo (Table 1). Among these producer isolates, 44

belonged to C. guilliermondii, C. pulcherrima and Kloeckeraapiculata species and only one belonged to S. cerevisiae; this

isolate came from grape berry surface and it was a weak

producer (Table 1).

All isolates characterized as enzyme producers on plate

assay were grown in liquid media under aerobic conditions

and they were tested for b-glucosidase activity using

p-nitrophenyl-b-DD-glucoside as substrate (in vitro assays).

In these assays, whole cells and culture media were used as

enzyme source. Under assay conditions, none of the yeast

isolates showed any capacity to excrete the enzyme into the

medium. Analysis of whole cell activity data, using the box-

and-whiskers statistic method, revealed that within

C. guilliermondii, C. pulcherrima and K. apiculata producer

species, the activity levels found in must isolates (median

values of 13Æ00, 1Æ00 and 0Æ07 U g)1 dry weight, respect-

ively) were greater than in grape isolates (median values of

2Æ1, 0Æ40 and 0Æ05 U g)1 dry weight, respectively). These

results also evidence that C. guilliermondii isolates from must

(M229, 231 and 425) were the greatest b-glucosidase

producers. However, some single grape isolates of this

species (A135, M102 and M119) as well as some single

isolates of C. pulcherrima (T152 and M227) and K. apiculata

(M321, 325 and 328) also showed very high enzyme activity

levels (outlier values), between four to 40 times higher than

the medians of their populations. These 11 best b-glucosi-

dase producers, characterized as strong on plate assay, were

selected to continue the study. The moderate to weak

producer K. apiculata M326 was also included in order to

continue checking the screening and selection method and

the 12 isolates were renamed as V1–V12 (see Table 2).

Because yeast strains may have exocellular, parietal (cell

wall bound) and intracellular b-glycosidase activity, this

activity was assayed on the culture supernatant medium

(extracellular), on whole cells (parietal) and on permeabi-

lized cells; intracellular activity was estimated from the latter

(see �Methods�). Taking into account that winemaking is an

anaerobic process, yeast cells were grown under aerobic and

partially anaerobic conditions, and total b-glucosidase

activity was estimated as the sum of the three activities.

As a whole, the results from these assays showed two

different behaviour patterns among indigenous isolates.

C. pulcherrima isolates and four of the six C. guilliermondii

isolates (V3, V5, V7 and V8) yielded the highest levels of

enzyme activity and neither b-glucosidase production nor

cellular location were significantly affected by culture

aeration conditions (Table 2). Additionally, whereas in the

first isolates almost all the activity was detected in perme-

abilized cells, indicating the presence of an intracellular

enzyme, the last isolates showed important levels in whole

cells and permeabilized cells, indicating the presence of

GLYCOSIDASE ACTIVIT IES IN PATAGONIAN YEASTS 87

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 84–95, doi:10.1046/j.1365-2672.2003.02032.x

both parietal and intracellular enzymes (Table 2). However,

under anaerobic conditions, the other two C. guilliermondii

isolates (V1 and V2) and K. apiculata isolates showed the

highest levels of activity on whole cells (‡89Æ5 and ‡96Æ9% of

the total activity, respectively), evidencing the presence of

parietal enzyme. Aerobic growth conditions greatly stimu-

lated b-glycosidase activity in these isolates (Table 2).

Candida guilliermondii V2 shows fivefold more enzymatic

activity under this condition than under anaerobic condi-

tions, with a significant increase of both intracellular and

parietal activities. Although their activity values are lower,

the same trend may be observed in C. guilliermondii V1 and

all K. apiculata isolates. Additionally, certain activity was

observed in supernatant media, exocellular enzyme, but its

level was small and, except for V8 isolate, it was only

observed under anaerobic conditions (Table 2).

A second plate screening for detecting other glycosidase

activities were realized on the yeast isolates. In this assay,

yeast cells were grown on agar plates containing different

carbon source and b-DD-xylosidase, a-LL-rhamnosidase and

a-LL-arabinofuranosidase as well as b-DD-glucosidase were

detected using the appropriate 4-methylumbelliferyl-

glycoside as substrate. The results showed in Table 3 reveal

that all C. guilliermondii and C. pulcherrima isolates were

capable to produce both b-glucosidase and b-xylosidase

activities on all carbon source where they can grow,

indicating that the enzymes could be synthesized constitu-

tively, whilst K. apiculata isolates only produced b-glucosi-

dase. Candida guilliermondii V2 also showed capability to

hydrolyse MUR (a-rhamnosidase activity), but this enzy-

matic activity was only detected using rhamnose as substrate

(inducible activity) (Table 3). None of the 12 yeast strains

produced a-arabinofuranosidase activity.

Killer behaviour

Under assay conditions, neither C. pulcherrima, K. apiculatanor C. guilliermondii isolates were capable of producing killer

toxin. However, whereas the isolates belonging to the two

former species were immune against all assayed killer toxins

Table 1 Indigenous yeast isolates tested for

b-glucosidase activity

Species Source

Tested

isolate

number Activity* Isolate identity�

Saccharomyces cerevisiae Grape 1 +(1) A102w

Must 72 – –

Saccharomyces kluyveri Must 2 –

Kloeckera apiculata Grape 4 +(3) A117w and 119w; T124m

Must 21 +(15) M201m, 204m, 313w, 315w, 321s,

325s, 326m, 327s; 402w, 407w, 411w,

412w and 414w T202w and 206w

Torulaspora delbrueckii Must 4 – –

Torulaspora pretoriensis Must 2 – –

Candida colliculosa Must 7 – –

Candida dattila Must 16 – –

Candida guilliermondii Grape 13 +(13) M102s and 119s; A108w,

109w, 110w, 111w,

114w, 131w, 132m, 135s;

142w, 146w and 150w

Must 3 +(3) M229s, 231s and 425s

Candida parapsilosis Grape 2 – –

Candida pulcherrima Grape 10 +(6) T140w, 149w and 152s A123w,

128w and 162m

Must 17 +(4) M227s T205m; 218m and 220w

Candida stellata Must 6 – –

Total 180 +(45)

*Activity is expressed as hydrolysis of arbutin. The figures between brackets indicate the number

of yeast isolates with b-glucosidase activity.

�The capital letters indicate the grape variety: A, Merlot; M, Malbec; T, Trousseau. The first

digit indicates the source: 1, grape; 2, must completely processed in laboratory; 3, must partially

processed in laboratory; 4, must completely processed in winery and the last two digits represent

the isolate number within each series. The superscript letters s, m and w indicate strong, moderate

or weak b-glucosidase production, respectively.

88 M.E. RODRIGUEZ ET AL.

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 84–95, doi:10.1046/j.1365-2672.2003.02032.x

Table 3 Glycosidase activity in indigenous yeasts grown on different carbon source

Strain

Carbon source

DD-glucose Cellobiose DD-xilose LL-arabinose LL-rhamnose

1 2 1 2 1 2 1 2 1 2

C. guilliermondii

V1, V3, V5, V7 and V8 (+) BGL (+) BGL (+) BGL (+) BGL (+) BGL

BXL BXL BXL BXL BXL

V2 (+) BGL (+) BGL (+) BGL (+) BGL (+) BGL

BXL

BXL BXL BXL BXL ARH

C. pulcherrima

V4 and V6 (+) BGL (+) BGL (+) BGL ()) ND ()) ND

BXL BXL BXL

K. apiculata

V9, V10, V11 and V12 (+) BGL (+) BGL ()) ND ()) ND ()) ND

1, cell growth; 2, enzymatic activity. The activity was detected on plate using MUG (b-DD-glucosidase activity, BGL); MUX (b-DD-xylosidase activity,

BXL); MUA (a-LL-arabinofuranosidase activity, AAF) and MUR (a-LL-rhamnosidase activity, ARH) as substrates. ND, not detected.

Table 2 b-Glucosidase activity in indigenous yeasts under anaerobic and aerobic conditions

Source Species

Isolate identity

(original name)

Enzymatic activity�

Total Exocellular Parietal Intracellular

Grapes C. guilliermondii V1 (A135) 9Æ90 ± 0Æ30

(33Æ7 ± 3Æ10)*

0Æ34 ± 0Æ150

(ND)

9Æ56 ± 0Æ15

(24Æ12 ± 1Æ24)*

ND

(9Æ57 ± 1Æ86)

V2 (M102) 12Æ90 ± 1Æ52

(60Æ00 ± 1Æ70)*

0Æ35 ± 0Æ09

(ND)

11Æ54 ± 2Æ51

(38Æ30 ± 3Æ92)*

1Æ01 ± 0Æ90

(21Æ24 ± 2Æ12)**

V3 (M103) 41Æ75 ± 3Æ00

(33Æ33 ± 5Æ15)

0Æ29 ± 0Æ00

(ND)

24Æ87 ± 5Æ14

(20Æ47 ± 0Æ91)

16Æ63 ± 2Æ14

(12Æ82 ± 6Æ06)

C. pulcherrima V4 (T152) 27Æ67 ± 2Æ75

(30Æ98 ± 4Æ00)

0Æ22 ± 0Æ12

(ND)

1Æ63 ± 0Æ06

(6Æ20 ± 2Æ98)

25Æ89 ± 2Æ93

(24Æ80 ± 1Æ02)

Must C. guilliermondii V5 (M425) 27Æ50 ± 3Æ81

(35Æ83 ± 8Æ21)

0Æ14 ± 0Æ06

(ND)

17Æ11 ± 2Æ11

(18Æ87 ± 4Æ36)

10Æ25 ± 5Æ98

(16Æ93 ± 3Æ85)

V7 (M229) 36Æ60 ± 6Æ53

(51Æ86 ± 8Æ93)

0Æ44 ± 0Æ12

(ND)

20Æ06 ± 9Æ12

(33Æ22 ± 6Æ42)

16Æ10 ± 2Æ47

(18Æ64 ± 2Æ51)

V8 (M231) 43Æ4 ± 1Æ88

(37Æ9 ± 3Æ27)

0Æ34 ± 0Æ00

(0Æ45 ± 0Æ01)

27Æ04 ± 3Æ89

(25Æ01 ± 0Æ78)

16Æ02 ± 2Æ01.

(12Æ43 ± 2Æ48)

C. pulcherrima V6 (M227) 25Æ15 ± 4Æ50

(33Æ7 ± 5Æ13)

ND

(ND)

5Æ23 ± 1Æ63

(4Æ71 ± 1Æ72)

19Æ92 ± 6Æ13

(28Æ98 ± 6Æ85)

K. apiculata V9 (M321) 5Æ01 ± 0Æ28

(22Æ08 ± 1Æ61)*

0Æ15 ± 0Æ01

(ND)

4Æ85 ± 0Æ29

(16Æ70 ± 1Æ42)*

ND

(5Æ39 ± 3Æ03)

V10 (M325) 3Æ42 ± 0Æ25

(15Æ56 ± 0Æ42)*

0Æ09 ± 0Æ07

(ND)

3Æ31 ± 0Æ18

(13Æ59 ± 0Æ72)*

ND

(2Æ01 ± 0Æ30)

V11 (M326) 1Æ4 ± 0Æ08

(9Æ1 ± 0Æ32)*

0Æ01 ± 0Æ02 (ND) 1Æ39 ± 0Æ05

(7Æ06 ± 0Æ61)*

ND

(2Æ03 ± 0Æ29)

V12 (M328) 3Æ96 ± 0Æ30

(12Æ82 ± 0Æ80)*

0Æ08 ± 0Æ11 (ND) 3Æ92 ± 0Æ19

(11Æ28 ± 1Æ00)*

ND

(1Æ52 ± 1Æ80)

�Enzymatic activity is expressed as U g)1 dry weight. Values without brackets indicate the enzymatic activity after 72 h under anaerobic conditions

and values in italics and within brackets indicate the enzymatic activity after 24 h under aerobic conditions. ND, not detected.

*P < 0Æ01, **P < 0Æ001 Student’s test, n ¼ 2.

GLYCOSIDASE ACTIVIT IES IN PATAGONIAN YEASTS 89

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 84–95, doi:10.1046/j.1365-2672.2003.02032.x

displaying a neutral phenotype (S)K)), C. guilliermondii

isolates showed a sensitive phenotype to K8, K9 and K10

isolates (S+K)).

Antocyanase activity

In this assay yeast cells grown on cellobiose (cell bound b-

glucosidase activities ‡10Æ00 ± 1Æ50 U g)1 dry weight) or

rhamnose (cell bound V2a-rhamnosidase activity ¼5Æ84 U g)1 dry weight) were used as enzyme source and

the progress of decolourization of a commercial red wine was

studied during a week. No significant differences in colour

figures were observed between control (uninoculated red

wine, A520 ¼ 0Æ731 ± 0Æ010, n ¼ 2) and experimental sam-

ples (V2, V5, V6, V9-cellobiose and V2-rhamnose inoculated

red wine, A520 ¼ 0Æ669± 0Æ039, 0Æ671 ± 0Æ051, 0Æ780 ±

0Æ021, 0Æ702 ± 0Æ060 and 0Æ739 ± 0Æ014, respectively;

n ¼ 2). However, low but significant residual b-glycosidase

(‡100 ± 9 mU g)1 dry weight) and a-rhamnosidase activ-

ities (700 ± 23 mU g)1 dry weight) were observed at the

end of the assay.

Molecular analysis and typing of the yeast isolates

The correct taxonomic identification of an isolate at species

level is a previous essential step to its definitive character-

ization at enzymatic level.

The taxonomic species identity assigned to 12 indigenous

yeast isolates by means of conventional methods was

confirmed by PCR–RFLP analysis of the ribosomal internal

transcribed spacers including the 5Æ8S rRNA gene region.

Figure 1 and Table 4 show that the indigenous isolates,

characterized as belonging to C. guilliermondii, C. pulcherr-

ima and K. apiculata species present the same species-

specific patterns reported in literature for type strains

belonging to their teleomorph forms Pichia guilliermondii,

Metschnikowia pulcherrima and Hanseniaspora uvarum

(Esteve-Zarzoso et al. 1999). As the anamorph and teleo-

morph forms yielded the same pattern by using this method,

we concluded that all selected isolates had been correctly

characterized at species level.

The population homogeneity within each species was

analysed using karyotype analysis and mtDNA RFLP, two

molecular methods widely used to characterize indigenous

wine yeasts at strains level (Querol et al. 1992; Schutz and

Gafner 1993; Nadal et al. 1996; Fernandez et al. 2000).

Figure 2a shows the CHEF separation of DNA from the 12

isolates. Candida guilliermondii and K. apiculata isolates

displayed chromosomal-length polymorphism. Four of six

isolates of C. guilliermondii, V3, V5, V7 and V8, showed a

similar banding pattern, with seven bands ranging in size

from ca 2200 to 590 kb, that differed from the V1 and V2

karyotype in one additional band of ca 1700 kb (Fig. 2a

300

Cfo

IH

aeIII

MW

MW

MW

600

MW

(a)

(b)

(c)

bpbp

Hin

fID

deI

V1

V2

V3

V4

V5

V6

V7

V8

V9

V10

V11

V12

V 9to

V 12V 9

toV 1

2V

1V

2V

3V

4V

5V

6V

7V

8V

9V

10V 1

1V 1

2V 1

V 2V 3

V 4V 5

V 6

Fig

.1

PC

R–

RF

LP

ofin

dig

enou

sye

ast

ITS

regi

ons.

(a)

Am

pli

fied

pro

du

cts.

(b,

c)R

estr

icti

onfr

agm

ents

.M

W:

100-

bp

DN

Ala

dd

er.

Th

ere

stri

ctio

nen

don

ucl

ease

su

sed

inea

chca

sear

e

ind

icat

edat

the

top

ofth

ep

ictu

res

and

ind

igen

ous

isol

ate

iden

tity

atth

eb

otto

m

90 M.E. RODRIGUEZ ET AL.

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 84–95, doi:10.1046/j.1365-2672.2003.02032.x

marked with arrow). However, two karyotype patterns were

observed within K. apiculata isolates. Both patterns consis-

ted of five chromosomal bands ranging in size from ca 2200

to 670 kb and they could be differentiated by the

electrophoretic mobility of their smallest band (Fig. 2a).

Under assay conditions, the chromosomes of C. pulcherrima

could no be separated appropriately. Additionally, using

mtDNA RFLP method we could distinguish C. guillier-mondii V1 from V2 but no difference, neither between

C. pulcherrima V4 and V6 isolates nor between others, could

be detected (Fig. 2b).

Taking the molecular patterns as variables, we performed

a cluster analysis (Fig. 3b). The isolates found with linkage

distance zero show identical patterns in all used molecular

methods and they can be considered as belonging to the

same strain (Vezinhet et al. 1992; Fernandez et al. 2000).

The resulting dendrogram reveals three different strain

subpopulations within C. guilliermondii species, two within

K. apiculata and only one within C. pulcherrima species

(Fig. 3b). Additionally, when this dendrogram was com-

pared with that obtained from biochemical, killer and

glycosidase phenotypes (Fig. 3a) a close correlation between

clusters was observed. That is, from genetic and phenotypic

data sets, we can discriminate exactly the same six strains

among indigenous isolates.

DISCUSSION

Volatile compounds derived from grape glycosidic com-

plexes make an important contribution to varietal flavour of

Table 4 PCR–RFLP profiles of indigenous yeast isolates and collection type strains

Strain

AP

(bp)�

Restriction frgment (bp)�

Cfo HaeIII HinfI DdeI

V1, V2, V3, V5, V7 and V8 625 300 + 265 + 60 400 + 115 + 90 320 + 300 ND

V4 and V6 400 205 + 100 + 95 280 + 100 200 + 190 ND

V9, V10, V11 and V12 750 320 + 310 + 105 750 350 + 200 + 180 300 + 180 + 95 + 90 + 85

Pichia guilliermondii 1019) 1021) 1438* 625 300 + 265 + 60 400 + 115 + 90 320 + 300 –

Metschnikowia pulcherrima 1691) 10408) 10546* 400 205 + 100 + 95 280 + 100 200 + 190 –

Hanseniaspora uvarum 1444) 10389* 750 320 + 310 + 105 750 350 + 200 + 180 300 + 180 + 95 + 90 + 85

*Type strain: from the Spanish Type Culture Collection (CECT). PCR–RFLP profiles were extracted from Esteve-Zarzoso et al. 1999.

�PCR amplified product size.

�Size of restriction fragments.

1020

2200

1125

1600

MW MWV 1 V 2 V 3 V 4 V 5 V 6 V 7 V 8 V 9 V 10 V 11 V 12

CI CI CII CII CII CII AI AI AII AI

(a)V 1 V 2 V 3 V 4 V5 V 6 V 7 V 8MW

(b)

680

Fig. 2 Molecular analysis of indigenous yeast isolates. (a) Electrophoretic karyotypes. MW: chromosomes of Saccharomyces cerevisiae YNN295

used as the size standard. (b) HinfI mtDNA restriction patterns. MW: weight marker-lambda DNA digest with HindIII. The codes at the tops

of both figures indicate the identity of the indigenous isolates and the codes at the bottom indicate the karyotype patterns

GLYCOSIDASE ACTIVIT IES IN PATAGONIAN YEASTS 91

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 84–95, doi:10.1046/j.1365-2672.2003.02032.x

the wine (Francis et al. 1992, 1999; Williams and Francis

1996; Gueguen et al. 1997). b-Glucosidase plays a key role

in this aroma enhancing process and this activity has been

extensively researched in wine yeast. Although some

activity in different strains of S. cerevisiae has been

described (Delcroix et al. 1994; Mateo and Di Stefano

1997; Hernandez et al. 2002), most studies demonstrate

that higher b-glucosidase producers are non-Saccharomycesspecies (Rosi et al. 1994; Miklosy and Polos 1995;

Charoenchai et al. 1997; Fernandez et al. 2000; Mendes

Ferreira et al. 2001; Strauss et al. 2001). Our screening

and preliminary in vitro assay results confirm these find-

ings. The best b-glucosidase activity producer were all

non-Saccharomyces yeasts belonging to C. guilliermondii,

C. pulcherrima and K. apiculata species (Table 1). This

yeast isolates were preselected and they were characterized

according to a set of selectable phenotypic characteristics

indicative of their potential usefulness in more aromatic

red wine production.

The first criterion to discriminate among strains included

the levels of specific b-glucosidase activity, its cellular

location and the effect of aeration conditions on them.

b-Glucosidase, by releasing aglicon from monoglycosidic

complexes, enhances the varietal aroma of wines. The

second criterion was the presence of the other glycosidases

activities involved in the first step of glycosidic complex

Linkage distance

V11

V12

V10

V9

V6

V4

V8

V7

V5

V3

V2

V1

0·00 0·05 0·10 0·15 0·20 0·25 0·30 0·35 0·40 0.45 0.50

C. guilliermondii

C. guilliermondii

C. guilliermondii

C. guilliermondii

C. guilliermondii

C. guilliermondii

C. pulcherrima

C. pulcherrima

K. apiculata

K. apiculata

K. apiculata

K. apiculata

(a)

Linkage distance

V11

V12

V10

V9

V6

V4

V8

V7

V5

V3

V2

V1

0·0 0·2 0·4 0·6 0·8 1·0 1·2

C. guilliermondii

C. guilliermondii

C. guilliermondii

C. guilliermondii

C. guilliermondii

C. guilliermondii

C. pulcherrima

C. pulcherrima

K. apiculata

K. apiculata

K. apiculata

K. apiculata

StrainI

StrainII

Strains III

Strains IV

Strains V

Strain VI

(b)

––

–

Fig. 3 UPGMA cluster analysis of selected yeast isolates based on phenotype (a) and molecular (b) characteristics

92 M.E. RODRIGUEZ ET AL.

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 96, 84–95, doi:10.1046/j.1365-2672.2003.02032.x

sequential hydrolysis as well as b-xylosidase activity. There

are no data available about the potential of this activity to

improve aroma and flavour properties of wine. However,

b-xylosidase contribution to the formation of flavour and

colour in other foods obtained from fermentation industries

has been reported (Ohta et al. 1991; Kimura et al. 1999),

so we decided to include it. The third criterion was a

technological property, the killer behaviour of the isolates.

Non-Saccharomyces yeasts can be employed at the begin of

winemaking as mixed starter cultures in conjunction with

more ethanol-tolerant S. cerevisiae strains (Rainieri and

Pretorius 2000). To determine the killer behaviour is very

important because different killer interactions between

starters and between starters and wild yeast strains could

be established during fermentation affecting its normal

evolution. These interactions become relevant in areas

where killer yeasts are widespread such as the north

Patagonian region (Sangorrın et al. 2001). The fourth and

last, unfavourable criterion, was the presence of anthocy-

anase activity. Anthocyanins are phenolic molecules com-

posed of a glycosilated flavylium ion and they are

responsible for wine colour. Some b-glucosidase from

fungis and yeasts break the linkage between the glucose

and the anthocyanidin moieties inducing loss of wine

colour (Wightman et al. 1997; Sanchez-Torres et al. 1998;

Manzanares et al. 2000).

On the basis of these results, we characterized six

phenotypically different subpopulation among the selected

isolates including the weak isolate used as control

(Fig. 3a). Then, using molecular methods we confirmed

their taxonomical identity at species level (Fig. 1 and

Table 4) and their intraspecific variability (Figs 2 and 3b).

From the ecological point of view, these results showed a

great diversity of Patagonian indigenous glycosidase

producer strains (Tables 2 and 3), with significantly

different b-glucosidase profiles. These different glycosi-

dase profiles seem to be more closely related to strains

than to species. However, as far as is known, our study is

the first one that shows a correlation between molecular

polymorphism and specific phenotypic traits in wild

indigenous strains. In order to simplify the selection

process, the use of molecular methods before the appli-

cation of oenological criteria have been proposed to

discriminate among strains (Esteve-Zarzoso et al. 2000).

However, more than one molecular method could be used

so as not to miss any strain. With these considerations,

molecular polymorphism analysis may be very useful to

identify strains with specific phenotypic properties in

selection protocols.

From the technological point of view, some characteristics

of these Patagonian indigenous strains could justify their

utilization in industrial production of more aromatic red

wine. One of them is the high b-glucosidase activity levels

showed by these strains under anaerobic conditions, much

higher than those reported by other authors for yeast strains

belonging to these same species (Rosi et al. 1994; Fernandez

et al. 2000; Manzanares et al. 2000; Mendes Ferreira et al.

2001). Another characteristic is the lack of killer character

and their immunity to S. cerevisiae killer toxins. Although

S. cerevisiae killer toxins are only active against a few yeast

species including S. cerevisiae and C. glabrata, non-Sac-

charomyces killer toxins have a broader anti-yeast spectrum

including S. cerevisiae species. We have observed that

indigenous non-Saccharomyces killer yeasts resident in

Patagonian winery surfaces and initial musts could hinder

the implantation of commercial S. cerevisiae starters in

guided fermentation, inducing stuck fermentation

(I. Zajonskovsky, T.L. Lavalle, M.E. Rodrıguez, Ch. Lopes,

M. Sangorrın and A.C. Caballero 2002, personal commu-

nication). The last remarkable trait of these strains is the

presence of b-glucosidase activities lacking anthocyanase

activity and maintaining low but significant residual levels

after exposition to finished wine conditions during a

significant period of time.

Because some non-Saccharomyces yeasts produce undes-

irable concentrations of acetic acid and ethyl acetate from

sugars, they had always been regarded as unsuitable for

winemaking. The potential application of non-Saccharomy-

ces yeast strains in this process has been explored only fairly

recently. Due to their sensitivity to ethanol, non-Saccharo-myces yeast could be employed at the beginning of

winemaking as mixed cultures together with Saccharomyces

strains or in sequential non-Saccharomyces–Saccharomyces

inoculation protocols (Rainieri and Pretorius 2000; Soden

et al. 2000). However, the results obtained in this study

allow us to think in an additional and more advantageous

way of using these non-Saccharomyces strains towards the

end of fermentation, on the young wine.

ACKNOWLEDGEMENTS

This work was supported by the Comahue University Grant

(B091). The authors wish to thank S. Genoves and E. Ibanez

for help during the work in IATA and Ph.D. S. Bramardi

(U.N. Comahue) for his statistical assistance.

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