www.elsevier.com/locate/ijfoodmicro

International Journal of Food Micr

Dynamics of indigenous yeast populations during spontaneous

fermentation of wines from Mendoza, Argentina

M. Combinaa,b,*, A. Elıaa, L. Mercadoa,b, C. Cataniaa, A. Gangac, C. Martinezc,d

aCentro de Estudios Enologicos-Estacion Experimental Agropecuaria Mendoza, Instituto Nacional de Tecnologıa Agropecuaria (INTA),

San Martın 3853 (5507) Lujan de Cuyo, Mendoza, ArgentinabConsejo Nacional de Investigaciones Cientıficas y Tecnologicas (CONICET), Argentina

cCentro de Estudios en Ciencia y Tecnologıa de los Alimentos (CECTA), ChiledDepartamento de Ciencia y Tecnologıa de los Alimentos, Universidad de Santiago de Chile, Alameda 3363, Estacion Central, Santiago, Chile

Received 11 May 2004; received in revised form 9 August 2004; accepted 26 August 2004

Abstract

Fermentation of wine is a complex microbial reaction, which involves the sequential development of various species of

yeasts and lactic acid bacteria. Of these, yeasts are the main group responsible for alcoholic fermentation. The aim of this work

was to study, under industrial conditions, the evolution of yeast populations and to describe the individual evolution of the most

important yeasts during three spontaneous fermentations of Malbec musts from Argentina. This work shows the significant

participation of non-Saccharomyces yeasts during spontaneous fermentation of musts, with the ubiquitous presence of three

main species: Kloeckera apiculata, Candida stellata and Metschnikowia pulcherrima.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Wine yeasts; Spontaneous fermentation; Malbec; Argentina

1. Introduction

The fermentation of grape juice into wine is a

complex microbial reaction traditionally involving the

sequential development of various species of yeast and

0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijfoodmicro.2004.08.017

* Corresponding author. Centro de Estudios Enologicos-Estacion

Experimental Agropecuaria Mendoza, Instituto Nacional de Tecno-

logıa Agropecuaria (INTA), San Martın 3853 (5507) Lujan de

Cuyo, Mendoza, Argentina. Tel./fax: +54 261 4963020/320.

E-mail address: mcombina@mendoza.inta.gov.ar

(M. Combina).

lactic acid bacteria. Yeasts are primarily responsible

for the alcoholic fermentation of the juice. Tradition-

ally, wine has been produced by the natural fermenta-

tion of grape juice by yeasts that originate from grapes

and winery equipment (Ribereau-Gayon et al., 2000).

Natural fermentation continues to be a style of

fermentation conducted in Argentina for quality wines.

Many intrinsic and extrinsic factors affect the

occurrence and growth of microorganisms on the

surface of grape berries, including rainfall, temper-

ature, berry maturity, physical damage due to bird,

insect and mould attack, and the application of

obiology 99 (2005) 237–243

Table 1

Chemical composition of grape juice

Parameter Musts

A B C

Sugar concentration (8 Brix) 22.4 22.6 22.5

pH 3.7 3.7 3.8

Total acidity (g L�1 of tartaric acid) 5.4 4.1 5.0

M. Combina et al. / International Journal of Food Microbiology 99 (2005) 237–243238

agrichemicals such as fungicides and insecticides. The

influence of winemaking technology as clarification

of grape juice, sulfur dioxide, temperature and

composition of the juice has also been reported (Fleet,

1999; Longo et al., 1991).

Wine quality is closely related to the microbial

ecology of fermentation. The various yeast species

and strains that develop during the overall fermenta-

tive process metabolize grape juice constituents,

principally the sugars, to a wide range of volatile

and non-volatile end-products, which influence and

determine the types and concentrations of many

products that contribute to the aroma and flavor

characteristics of the wine. The major volatile

products of yeast metabolism, ethanol and carbon

dioxide, make a relatively small contribution to wine

flavor. Conversely, organic acid, higher alcohols and

esters, and to a lesser extent acetaldehyde, constitute

the main group of compounds that form the

bfermentation bouquetQ (Romano et al., 2003). The

effect of non-Saccharomyces yeasts on fermentation

and wine quality is one of the principal issues

currently studied in different countries with enolog-

ical tradition (Grossmann et al., 1996; Jolly et al.,

2003).

Many ecological studies in different wine regions

of the world have identified the main species of yeast

that develop during fermentation (Jolly et al., 2003;

Povhe-Jemec et al., 2001). However, little has been

reported about the Argentinian wines. In this study,

we examined the quantitative development of indi-

vidual species of yeasts during vinification of three

Malbec wines from Mendoza, Argentina, under

industrial conditions.

2. Materials and methods

2.1. Wines

The three wines studied were produced under

commercial conditions. Two of them were sampled

during the 2000 vintage (must A and B) and the third

during the 2001 vintage (must C). The chemical

composition of the musts is shown in Table 1. Red

wines were produced from Malbec variety grapes in

two cellars located in bZona Alta del Rio MendozaQregion. The musts were sulfured to give a final

concentration of 40 mg of SO2 per liter. The sulfured

musts were transferred to large steel tanks (20,000 L)

in which fermentations were conducted at 25–26 8C.All the wines were produced by natural alcoholic

fermentation with no inoculated yeast.

2.2. Yeast enumeration and isolation

Wine samples (500 mL) were taken at frequent

intervals during vinification. Samples were taken

from the middle of the fermentation vessels once the

daily practice of pumping-over was done. Yeasts

were enumerated by spread inoculating 0.1-mL wine

(diluted in peptone 0.1%, if necessary) onto plates of

two media: Malt Extract Agar (MEA) with addition

of Rose Bengal (30 mg L�1) and other selective and

differential agents such as chloramphenicol (50 AgL�1), erythromycin (70 Ag L�1) and dichloran (2 mg

L�1 in hydroalcoholic solution); and WL nutrient

agar (Oxoid) which allows presumptive discrimina-

tion between the yeast species by colony morphol-

ogy and color (Pallmann et al., 2001). Plates were

incubated at 25 8C during 5 days for colony

development. The various colony types were

counted, and representative colonies were isolated

and subcultured onto Yeast Extract Peptone Dextrose

(YEPD): yeast extract, 5 g L�1; peptone, 5 g L�1;

dextrose, 40 g L�1; agar, 20 g L�1; for subsequent

identification.

2.3. Yeast identification

Conventional yeast identification was carried out

following the taxonomic criteria described by

Kurtzman and Fell (1998). To avoid unnecessary

testing, only the discriminating tests for wine

yeasts, based on their morphological, sexual and

physiological characteristics, were applied. The

different physiological tests were: ability to ferment

d-glucose, sucrose and raffinose; ability to use as sole

M. Combina et al. / International Journal of Food Microbiology 99 (2005) 237–243 239

source of carbon for aerobic growth, d-glucose,

galactose, l-sorbose, d-glucosamine hydrochloride,

n-acetyl-d-glucosamine, d-xylose, l-arabinose, l-

rhamnose, sucrose, melibiose, maltose, a-trehalose,

cellobiose, raffinose, melezitose, starch, glycerol,

erythritol, ribitol, xilitol, d-mannitol, inositol, dl-

lactic acid, succinic acid, citric acid and ethanol;

ability to use nitrogen compounds for aerobic growth,

nitrate, l-lysine and cadaverine; growth at different

temperatures (37 and 40 8C); cycloheximide (acti-

dione) resistance (0.01% and 0.1%); acid production

from glucose; tolerance of 1% acetic acid and growth

on 10% sodium chloride plus 5% glucose in yeast

nitrogen base. This identification was subsequently

confirmed by molecular biology.

2.4. Molecular identification of yeast

DNA extraction was carried out with maxiDNA kit

(Promega, Madison, WI, USA) according to manu-

facturer’s instructions. The region between the 18S

rRNA and 28S rRNA genes was amplified using

specific internal transcribed spacers ITS1 and ITS4

primers (White et al., 1990). This region contains the

highly conserved region of ribosomal 5.8S, and a

variable zone which is the region on the ITSs. The fact

that both zones are combined in the same fragment

makes this a useful tool for carrying out studies at

different degrees of differentiation. To achieve greater

polymorphism, the amplified genes were treated with

restriction enzymes CfoI, HinfI and HaeIII for

identification of yeasts at species level (Esteve-Zaroso

et al., 1999; Granchi et al., 1999; Guillamon et al.,

1998). The previously undescribed yeast species were

identified by sequence analyses of the PCR amplified

5.8S rRNA ITS region using the ABI Prism 3100

genetic analyzer (Valente et al., 1999). The sequence

comparisons were performed using the basic local

alignment search tool (BLAST) program within the

NCBI database (Altschul et al., 1990).

2.5. Nucleotide sequence accession number

5.8S-ITS sequences were submitted to GenBank

under accession numbers: AY679091, AY679092

(Zygosaccharomyces rouxii), AY679093 (Issatchen-

kia orientalis), AY679094 and AY679095 (Candida

bombi).

3. Results and discussion

The alcoholic fermentation of the three Malbec

musts proceeded to completion fermentation of must

sugars between 8 and 12 days after grape crushing.

Wine A had a final pH of 4.01 and a final ethanol

concentration of 13.2%; wine B had a final pH of 3.9

and a final ethanol concentration of 13.1%; and wine

C had a final pH of 3.6 and a final ethanol

concentration of 13.6%.

During fermentation, the viable population of yeast

in the grape juice increased from initial values of 104–

106 to 108–109 cfu mL�1. This growth follows a

typical pattern of microorganism grown in batch

culture; it was characterized by a lag phase, exponen-

tial phase, stationary phase and decline or death phase.

The characteristics of these phases vary with the

fermentation, being influenced by a range of factors

such as composition of grape juice and temperature of

fermentation. Although cessation of yeast growth was

determined by viable yeast counts and the cells appear

to be non-proliferating, there is still substantial

metabolic activity. Most notably, residual sugars

continue to be fermented with production of CO2

and ethanol (data not shown).

Three hundred and sixty (360) yeasts isolated

from three musts in different fermentation stages

were submitted to conventional yeast identification.

The use of the morphological, sexual and physio-

logical characteristics according to Kurtzman and

Fell (1998) proved to be adequate for clustering

similar species. ITS1-5.8S-ITS4 amplicons were

obtained from one to five isolates of each cluster.

Restriction analyses of these amplicons were highly

correlated to the conventional identification and were

able to correctly identify most of the isolates at the

genus and species level. Only one species was not

identified by this method because its 5.8S-ITS

pattern has not been previously described. Conse-

quently, we describe here a new 5.8-ITS pattern for

the species C. bombi (Table 2). The isolates which

have an ambiguous or misidentification were ana-

lysed by sequencing the ITS1-5.8S-ITS4 amplicons.

Nucleotide sequences of the 5.8-ITS gene provide

confirmation of the previous identification and were

submitted to GenBank. The majority of identification

results demonstrate a good reliability of the 5.8S-ITS

analysis as a routine technique for identification of

Table 2

Nucleotide fragment length of a new 5.8S-ITS profile described in

the study

Species AP

(bp)aFragment length(s) (bp) after restriction

endonuclease analysis with:

CfoI HaeIII HinfI

Candida

bombi

470 200+180+55+35 400+70 250+110+110

a AP, 5.8S-ITS-amplified product size.

M. Combina et al. / International Journal of Food Microbiology 99 (2005) 237–243240

wine yeast isolates. However, this analysis must be

complemented with other techniques, especially

when the isolate 5.8S-ITS pattern has not been

previously reported. It is important to point out that

the original assignation of species obtained with

classical identification techniques was confirmed in a

large number of cases.

A great population diversity of non-Saccharomy-

ces yeast species was observed in fresh must. Eleven

(11) different species belonging to nine (9) genera

were identified from fresh musts (Table 3—sampling

day 1).The most frequent species in freshly crushed

must were Kloeckera apiculata and Metschnikowia

pulcherrima, 36% to 56% and 19% to 39%, respec-

tively, followed by Candida stellata, 5% to 12%.

These yeast species are the most frequently described

in fresh musts from different places, but were found at

different isolation frequencies by Cocolin et al.

(2000), Jolly et al. (2003), and Pramateftaki et al.

(2000). I. orientalis were also isolated from the three

musts with varying percentages, 12% in must B and at

lower percentages (5% and 1%) in musts from the

Table 3

Distribution of yeast species (%) during spontaneous fermentations of thr

Yeast species Must A (2000) Must

Sampling day Samp

1 2 4 6 8 12 to 30 1

Kloeckera apiculata 56 67 67 – – – 53

Metschnikowia pulcherrima 19 11 4 – – – 20

Candida stellata 12 9 9 5 1 – 5

Zygosaccharomyces rouxii 8 6 5 – – – –

Issatchenkia orientalis 1 3 – – – – 12

Pichia membanifaciens 4 1 – – – – –

Candida vini – – – – – – 8

Candida bombi – – – – – – –

Kluyveromyces thermotolerans – – 1 – – – –

Rhodotorula sp. – – – – – – –

Saccharomyces cerevisiae – 3 14 95 99 100 2

other two wines. Z. rouxii were present only in must A

(8%) (Table 3).

Apiculate yeasts are usually one of the most

frequent yeasts in grapes and must, although there

are studies where the apiculate yeast are scarce and

other yeast species such as Cryptococcus, Rhodotor-

ula and some Candida species arise within the most

frequent yeast genera in grapes (De la Torre et al.,

1999; Rementeria et al., 2003).

The presence of other yeast species (Candida vini,

Pichia membranifaciens, Kluyveromyces thermotoler-

ans, Candida bombi and Rhodotorula species) was

less than 8% and these were not isolated from all three

musts (Table 3).

Saccharomyces cerevisiae could be isolated in 2%

of only one fresh must sample (Table 3—must B).

This observation was expected because several

researches confirm the extreme rarity of S. cerevisiae

in grapes. Although this yeast is not totally absent, its

existence cannot be proven by spreading out diluted

must on a solid medium (Martini et al., 1996;

Mortimer and Polsinelli, 1999). In spite of its low

initial incidence, this species rapidly dominated the

must a few days after the fermentation began.

Environmental conditions influence its selection

through four principal parameters: anaerobiosis, must

or grape sulfiting (SO2 addition), sugar concentration

and increasing presence of ethanol (Ribereau-Gayon

et al., 2000).

The kinetics of the main yeast species during the

fermentation process revealed that different yeast

species coexisted during the first 6 days of fermenta-

ee Malbec musts at different sampling stages

B (2000) Must C (2001)

ling day Sampling day

2 4 6 8 12 to 30 1 2 4 6 8 12 to 30

50 17 4 – – 36 63 13 – – –

18 10 – – – 39 18 13 – – –

15 13 – – – 12 16 11 1 – –

– – – – – – – – – – –

7 – – – – 5 1 0.6 – – –

– – – – – 7 1 0.6 – – –

– – – – – – – – – – –

– – – – – – – 0.6 – – –

2 – – – – – 1 1.2 – – –

– – – – – 1 – – – – –

8 60 96 100 100 – – 60 99 100 100

M. Combina et al. / International Journal of Food Microbiology 99 (2005) 237–243 241

tion. This diversity was dramatically reduced when

high concentrations of ethanol (6–7%) were reached

(Fig. 1).

Fig. 1 shows the sequential development of

dominant yeast species during the vinification of red

wines A, B and C. The behavior of the K. apiculata

population was represented by an initial proliferation

to 106–108 cfu mL�1, followed by a rapid decline and

disappearance of this species during the first week of

fermentation. M. pulcherrima and C. stellata also

reached a population of 105–106 cfu mL�1. The latter

species remained in the must up to 8–12 days of

Fig. 1. Growth of different yeast species and ethanol production

during spontaneous fermentation of three Malbec must from

Mendoza, Argentina (A, B and C).

fermentation. A rapid growth of S. cerevisiae to 108–

109cfu mL�1 to dominate the alcoholic fermentation

and the continued survival of this species to total

consumption of must sugars were observed in

accordance with many earlier studies. (Ribereau-

Gayon et al., 2000).

The inability of non-Saccharomyces yeasts to

sustain their presence in fermenting musts has been

attributed to their oxidative and weakly fermentative

metabolism and their sensitivity to increasing ethanol

concentrations produced by Saccharomyces species.

The non-Saccharomyces yeast appears to be less

tolerant to very low oxygen availability than S.

cerevisiae. Removal of residual oxygen from ferment-

ing grape juice by the vigorous growth of S.

cerevisiae could contribute to the early death of these

non-Saccharomyces species (Hansen et al., 2001).

It has been reported that K. apiculata and C.

stellata strains tolerate maximum concentrations of

ethanol ranging from 4% to 6% and from 5% to 10%,

respectively. M. pulcherrima seems to be less tolerant

to ethanol and incapable of surviving in ethanol

concentrations of 2–3% (Kunkee and Amerine, 1970).

Our results showed that K. apiculata tolerate ethanol

concentrations of up to 8%, C. stellata up to 12%, and

M. pulcherrima up to 7% (Fig. 1). Our results

evidenced a greater and longer presence of these

species during fermentation than previously reported.

These observations have also been reported by Fleet

(1990).

The ability of C. stellata to grow slowly in

fermenting must and its sustained presence almost

until high ethanol concentrations (12%) suggest that

this yeast makes a greater contribution to the ecology

of fermentation, as proposed by Fleet et al. (1984).

There are recent reports of some strains of Candida

species that may have ethanol tolerances similar to S.

cerevisiae. Strains of C. stellata fall into this category

and have been used in co-culture with S. cerevisiae to

enhance the glycerol content and other flavor charac-

teristics of wine (Soden et al., 2000; Romano et al.,

2003).

Many authors have show that K. apiculata, C.

stellata, Candida krusei and M. pulcherrima strains

have great potential to produce high concentrations of

volatile compounds such as acetic acid, acetate ethyl,

and phenol ethyl (Romano et al., 2003; Zorhe and

Erten, 2002).

M. Combina et al. / International Journal of Food Microbiology 99 (2005) 237–243242

The non-Saccharomyces yeast species not only

affect the chemical composition of wine, but could

also affect the global fermentation rate. Mortimer

(2000) shows that the rates of fermentation were

controlled by a function of the K. apiculata titers and

persistence; the higher the titers and persistence, the

slower the fermentation. It seems clear that K.

apiculata inhibits the growth of S. cerevisiae in some

way, and that this in turn decreases the rate of

consumption of sugar. The inhibition mechanism has

not been determined, but the author suggests that

Kloeckera cells can produce high concentrations of

acetic acid and acetaldehyde, both compounds being

responsible for that inhibition (Mortimer, 2000).

This work shows the significant participation of

non-Saccharomyces yeasts during spontaneous fer-

mentation of musts, with the ubiquitous presence of

three main species: K. apiculata, C. stellata and M.

pulcherrima.

Acknowledgment

This work was supported by CONICET Grant PIP

323/98.

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