artículo avl verdugo et al.2011

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Antonie van LeeuwenhoekJournal of Microbiology ISSN 0003-6072Volume 100Number 4 Antonie van Leeuwenhoek (2011)100:497-506DOI 10.1007/s10482-011-9605-y

Yeast communities associated withartisanal mezcal fermentations from Agavesalmiana

A. Verdugo Valdez, L. Segura Garcia,M. Kirchmayr, P. Ramírez Rodríguez,A. González Esquinca, R. Coria &A. Gschaedler Mathis

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ORIGINAL PAPER

Yeast communities associated with artisanal mezcalfermentations from Agave salmiana

A. Verdugo Valdez • L. Segura Garcia • M. Kirchmayr •

P. Ramırez Rodrıguez • A. Gonzalez Esquinca •

R. Coria • A. Gschaedler Mathis

Received: 29 January 2011 / Accepted: 3 June 2011 / Published online: 17 June 2011

� Springer Science+Business Media B.V. 2011

Abstract The aims of this work were to characterize

the fermentation process of mezcal from San Luis

Potosi, Mexico and identify the yeasts present in the

fermentation using molecular culture-dependent meth-

ods (RFLP of the 5.8S-ITS and sequencing of the D1/

D2 domain) and also by using a culture-independent

method (DGGE). The alcoholic fermentations of two

separate musts obtained from Agave salmiana were

analyzed. Sugar, ethanol and major volatile com-

pounds concentrations were higher in the first fermen-

tation, which shows the importance of having a quality

standard for raw materials, particularly in the concen-

tration of fructans, in order to produce fermented

Agave salmiana must with similar characteristics. One

hundred ninety-two (192) different yeast colonies were

identified, from those present on WL agar plates, by

RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA

gene, with restriction endonucleases, HhaI, HaeIII and

HinfI. The identified yeasts were: Saccharomyces

cerevisiae, Kluyveromyces marxianus, Pichia kluy-

veri, Zygosaccharomyces bailii, Clavispora lusitaniae,

Torulaspora delbrueckii, Candida ethanolica and

Saccharomyces exiguus. These identifications were

confirmed by sequencing the D1-D2 region of the 26S

rRNA gene. With the PCR-DGGE method, bands

corresponding to S. cerevisiae, K. marxianus and

T. delbrueckii were clearly detected, confirming the

results obtained with classic techniques.

Keywords Mezcal � Yeast diversity � Fermentation

process � RFLP � DGGE

Introduction

Mezcal is a traditional Mexican distilled beverage

produced by fermenting the juices of cooked agave

plant core (‘pina’ in Spanish). Mezcal is produced

from various species of Agave in a denomination of

origin region, which includes the states of Durango,

Guerrero, Oaxaca, San Luis Potosi, Zacatecas and

some districts of Guanajuato and Tamaulipas

(Mexican Ministry of Commerce and Industry

1994; Garcıa Mendoza 1998; Lappe-Oliveiras et al.

2008). Agave salmiana is used mainly in Mexico’s

A. Verdugo Valdez � A. Gonzalez Esquinca

Facultad de Ciencias Biologicas, Universidad de Ciencias

y Artes de Chiapas, Libramiento Norte 1150, colonia

Lajas Maciel, Tuxtla Gutierrez, CHIS, Mexico

L. Segura Garcia � M. Kirchmayr �P. Ramırez Rodrıguez � A. Gschaedler Mathis (&)

Centro de Investigacion y Asistencia en Tecnologıa y

Diseno del Estado de Jalisco A.C, Av. Normalistas # 800,

colonia Colinas de la Normal, 44270 Guadalajara,

JAL, Mexico

e-mail: [email protected];

[email protected]

R. Coria

Instituto de Fisiologıa Celular, Circuito Exterior S/N

Ciudad Universitaria, Universidad Nacional Autonoma

de Mexico, Coyoacan 04510, DF, Mexico

123

Antonie van Leeuwenhoek (2011) 100:497–506

DOI 10.1007/s10482-011-9605-y

Author's personal copy

Altiplano region (De Leon-Rodrıguez et al. 2006,

2008). The general steps in the production process

are: harvesting the raw material, cooking, milling,

fermentation, distillation, and in some cases, matu-

ration. In San Luis Potosi cooking is done in rustic

ovens where the heat is provided by steam injection,

in this step the fructans contained in the agave are

hydrolyzed into simple sugars, mainly fructose. The

juice of cooked agave is obtained in a rudimentary

mill (named ‘‘tahona’’), which has a circular stone of

about 1.5 m in diameter rotating in a circular pit over

the cooked agave. During the milling process, water

is added, and the resulting juice is fermented through

a spontaneous process and then distilled. The taste

and the aroma of the mezcal are provided by the

composition of a complex mixture of compounds

produced during the process (cooking, fermentation

and distillation), and other elements that come

directly from the agave plant (De Leon-Rodrıguez

et al. 2006, 2008).

Many studies have demonstrated that several

species of non-Saccharomyces yeasts are predomi-

nant at the beginning of spontaneous wine fermen-

tations (Fleet 2003), and these contribute significantly

to sensory characteristics of the beverage (Romano

et al. 2003).

In spite of the significance of the process and the

sensorial characteristics of the end product, few works

have addressed the identification and characterization

of the yeasts involved in the fermentation process of

the different agave spirits. The first study to address

this goal was conducted on traditional tequila fer-

mentation by Lachance (1995). He identified, using

classic microbiological techniques, the yeast commu-

nities and showed the great diversity in these

processes. Samples from the early fermentation

process contained a rich mixture of yeast species.

However, as fermentation progressed, the number of

species present tended to diminish, and finally only

one biotype of Saccharomyces cerevisiae became

dominant. Another study carried out in mezcal

fermentation from Agave salmiana (Escalante-Min-

akata et al. 2008), revealed lower biodiversity of

yeasts through molecular identification methods. In

the last decade, it has been shown that neither classic

microbiological methods nor culture depended molec-

ular methods accurately detect complex microbial

diversities in artisanal fermentations (Ben Omar and

Ampe 2000; Tu et al. 2010). The most widely used

culture-independent method for the study of microbial

communities is analysis by PCR-DGGE (denaturing

gradient gel electrophoresis). This method has been

used to study microbial communities, for example, in

wine (Cocolin et al. 2001; Renouf et al. 2007), in

sourdough (Meroth et al. 2003; De Vuyst et al. 2009;

Moroni et al. 2011), in cocoa bean (Nielsen et al.

2005; Lefeber et al. 2011), in sausages (Rantsiou and

Cocolin 2006), in soybean paste (Kim et al. 2009) and

in several other fermented products.

The goal of this work is to characterize the

fermentation process of mezcal of San Luis Potosi

and to identify the yeasts present during the fermen-

tation using molecular methods, by RFLP analysis of

the 5.8S-ITS region (Esteve-Zarzoso et al. 1999), and

sequencing of the D1/D2 domain (Kurtzman and

Robnett 1998). Additionally, and for the first time in

an agave distilled spirit, a culture-independent

method (PCR-DGGE) was used to study the succes-

sion of the different species of the yeast during the

fermentation process.

Materials and methods

Mezcal fermentations and sampling procedures

Sampling was carried out at the distillery of Laguna

Seca in San Luis Potosi (Mexico). After milling, the

crushed cooked agave is transferred in a special vat

(called a ‘‘lavadero’’) where water is added and the

agave is washed in order to extract the sugars. Then,

the juice (without the fibers) is transferred to

fermentation tanks. A smaller tank is used to

propagate a starter ferment in agave juice at 10�Bx

supplemented with (NH4)2PO4 (1 g/l). The yeasts

used in this starter culture are the autochthonous

yeasts of the factory, which were preserved at 4�C

from a previous fermentation. Fermentation is carried

out in 7000 l of agave juice supplemented with

(NH4)2PO4 (1 g/l). The sugar concentration varies,

depending on the raw material.

Multiple samples were taken from two separate

fermentations (fermentation I and II): from the

‘‘lavadero’’; the starter culture; from the initial juice

before inoculation; and at various points along the

fermentation process. After sampling, yeast cultures

were performed in the distillery, and aliquots of the

same samples were frozen immediately until they

498 Antonie van Leeuwenhoek (2011) 100:497–506

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were processed in the laboratory (analytical methods

and PCR-DGGE).

Yeast isolation

For all fresh samples, decimal dilutions in saline

physiological solution (9 g/l NaCl) were prepared

and used to inoculate WL nutrient agar (Fluka) plates,

supplemented with 0,01% chloramphenicol. The

plates were incubated at 29�C for 3–5 days for

colony development. The various colony types were

counted, and representative colonies of each type

were isolated and subcultured in YPD (yeast extract

10 g/l; peptone 20 g/l; dextrose 20 g/l; agar 20 g/l)

for subsequent identification.

Analytical methods

For the determination of major volatile compounds

(ethanol, methanol, amyl alcohols, acetaldehyde,

isobutanol, 1-propanol and ethyl acetate), the must

samples were volatilized in a Hewlett Packard head-

space sampler HP 7694E and analyzed in a Hewlett

Packard 6890 series gas chromatograph equipped

with a flame ionization detector (FID), and a

60 m 9 320 9 0.25 lm film thickness HP-Innowax

capillary column. The chromatographic conditions

were 45�C for 7 min, increased at 10–160�C,

20–220�C/min, and maintained at this temperature

for 8 min. Helium was used as carrier gas at a flow

rate of 1.8 ml/min, and the injector and detector

temperature were at 250�C.

Reducing sugar concentration was determined by

the dinitro-salicylic acid (DNS) method (Miller 1959)

using fructose as the standard.

Molecular identification

Yeasts were directly collected from the colonies and

suspended in a PCR reaction mixture. For amplifica-

tion of the ITS-5.8S rRNA region, the primers ITS1

(50-TCC GTA GGT GAA CCT GCG-30) and ITS4

(50-TCC TCC GCT TAT TGA TAT GC-30) were

used. PCR conditions described by Esteve-Zarzoso

et al. (1999) were followed and the enzymatic

digestions were carried out using restriction enzymes

HhaI, HaeIII and HinfI. Restriction fragments were

analyzed by electrophoresis in 3%(w/v) agarose gels

(Invitrogen, Carlsbad, CA, USA). The migration was

conducted at 100 V for 1 h in TAE 1X buffer

(Sigma–Aldrich). The gels were stained with ethi-

dium bromide (Sigma–Aldrich, Steinheim, Ger-

many), visualized under UV light using an GelDoc

system (Bio-Rad, Hercules, CA, USA), the size of the

fragments was estimated by comparison with TrackIt

100 bp DNA ladder (Invitrogen), and analyzed using

the Quantity one software (Bio-Rad).

Identity of the yeasts was confirmed by sequencing

the variable domain D1/D2 of the large (26S)

ribosome subunit (Kurtzman and Robnett 1998).

The PCR products were sequenced by Macrogen

(Rockville, MD, USA). The resultant sequences were

aligned in GenBank using the BLAST program for

identification.

PCR-DGGE analysis

For direct DNA extraction from musts samples, the

MasterPure Yeast Purification Kit (Epicentre Bio-

tecnologies, Madison, WI) was used. The D1 region

of the 26S rRNA gene was amplified by PCR, using

the primers NL1GC (50-GCG GGC CGC GCG

ACC GCC GGG ACG CGC GAG CCG GCG GCG

GGC CAT ATC AAT AAG CGG AGG AAA

AG-30) (the GC clamp is underlined) and LS2 (50-

ATT CCC AAA CAA CTC GAC TC–30), following

the conditions described by Cocolin et al. (2001). In

case of direct amplification from isolated yeasts, an

initial step of denaturalization was added (25 min at

95�C). Amplified products were analyzed on 3%

ultrapure agarose (Invitrogen) gels containing

0.5 mg/ml ethidium bromide, visualized under UV

light and analyzed with the Quantity one software

(BioRad).

The DcodeTM Universal Mutation Detection

System (BioRad) was used for DGGE analysis.

Electrophoreses were performed in a 1.0 mm poly-

acrylamide gel [8% (w/v) acrylamide:bisacrylamide

37.5:1] using a denaturant gradient increasing in

the direction of the electrophoretic run. Amplicons

produced by PCR were analyzed in a denaturant

gradient from 45 to 75%. Electrophoretic runs were

carried out at a constant temperature of 60�C in 1X

TAE at 100 V for 16 h. After electrophoresis, the

gels were stained for 10 min in 1X TAE containing

ethidium bromide 0.1 ll/ml, visualized under UV

light, and analyzed with the Quantity One software

(BioRad).

Antonie van Leeuwenhoek (2011) 100:497–506 499

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Results

Fermentation kinetics and general yeast counts

Figure 1 shows the time course of the two alcoholic

fermentations sampled. Generally, fermentation times

are short; the process is completed in 24–48 h. The

sugar and ethanol concentrations are higher in

fermentation I than in fermentation II. However, the

conversion yields of sugar to ethanol are still

identical: 0.39 and 0.38 g/g for fermentation I and

II, respectively. An important characteristic of this

type of fermentation is the high temperature of the

must, which is maintained between 33 and 35�C. The

yeast population in the inoculums reached 4 9 107

cells/ml. The maximum concentration during fer-

mentation was reached after 10 h and decreased

slightly until the end of the process. Figure 1 also

shows the concentration of the major volatile

compounds found in the must. In general, fermenta-

tion I (A) generated greater concentrations of these

compounds than fermentation II (B).The amyl alco-

hols are the predominant compounds, followed by

acetaldehyde, isobutanol, 1-propanol and ethyl ace-

tate. The concentration of higher alcohols (sum of

concentrations of amyl alcohols, isobutanol and

1-propanol) was 54.8 and 27.5 mg/l in fermentation

I and II respectively.

Molecular identification and enumeration

of yeasts

One hundred ninety-two (192) different colonies were

isolated from WL plates and identified by RFLP

analysis of the ITS1-5.8S-ITS2 of rRNA gene.

Twenty-six (26) different morphologies were distin-

guished, resulting in eight different restriction profiles

of RFLP. Table 1 shows the nucleotide fragment size

Fig. 1 Evolution of alcoholic fermentation and production of volatile compounds in Fermentation I (a) and II (b) in distillery

‘‘Laguna Seca’’ in San Luis Potosı, Mexico


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(pb) of the ITS-5.8S region amplified by PCR and

digested with restriction endonucleases, which were

compared to the data previously described (Esteve-

Zarzoso et al. 1999). The identified yeasts were:

Saccharomyces cerevisiae, Kluyveromyces marxi-

anus, Pichia kluyveri, Zygosaccharomyces bailii,

Clavispora lusitaniae, Torulaspora delbrueckii, Can-

dida ethanolica and Saccharomyces exiguus. These

identifications were confirmed by sequencing the D1-

D2 region of 26S rRNA gene for each RFLP profile.

Table 2 shows the species found in different areas

of the distillery, the ‘‘lavadero’’ where the cooked and

crushed agave is washed; in the agave juice before

the inoculation; and in the tank of starter culture. S.

cerevisiae, K. marxianus, S. exiguus and T. del-

brueckii were detected at every site. C. ethanolica

was only detected in the starter culture, and

P. kluyveri and Z. bailii only in the agave juice

before inoculation. During fermentation (Table 3),

S. cerevisiae was the predominant yeast. Large

differences in the populations of non-Saccharomyces

were observed between the two fermentations. At the

beginning, fermentation I exhibited high diversity,

while in fermentation II less non-Saccharomyces

species were detected. At the end of fermentation I

only K. marxianus and T. delbrueckii were detected

whereas, in fermentation II, K. marxianus, P. kluy-

veri, Z. bailii, C. lusitaniae, T. delbrueckii, and

S. exiguus were found together with a significant

population of Z. bailii.

PCR-DGGE analysis

The PCR-DGGE profiles obtained from the extracted

musts samples are shown in Fig. 2 and compared with

profiles from the different isolated strains. Bands

corresponding to S. cerevisiae, K. marxianus and

T. delbrueckii are clearly detected and confirmed the

results obtained with the traditional techniques. A few

bands were observed which didn’t correspond to any of

the isolated yeast. However, these bands were a result

of either secondary structures of the isolated species or

belonged to fungi genomes (Penicillium spp.).

Discussion

The fermentation process of Agave salmiana was

analyzed, including a general characterization of the

fermentation process; the generation of some volatile

Table 1 Yeast identified and sizes of ITS-5.8S region amplified by PCR (AP) and the fragments obtained after digestion with

restriction endonucleases HhaI, HaeIII and HinfI

Yeast identified AP (pb) Restriction fragments

HhaI HaeIII HinfI

Saccharomyces cerevisiae 875 373–347–138 321–241–180–133 374–127

Kluyveromyces marxianus 784 298–196–171–90 644–85 256–190–118–87

Clavispora lusitaniae 380 165–99 379 257–217

Saccharomyces exiguus 658 361–297 496–246 354–248–137

Torulaspora delbrueckii 800 325–220–153-100 795 410–378

Zygosaccharomyces bailii 723 321–284–94–94 700 339–226–158

Pichia kluyveri 465 168–108–86 316–95 257–207

Candida ethanolica 450 152–101 301 250–201

Table 2 Yeast

identification from different

sites and fermentations

process from the factory

Laguna Seca in the State of

San Luis Potosı, Mexico

Isolation site Lavadero Starter culture Fermentation tank

(before inoculation)

Isolated yeast S. cerevisiae S. cerevisiae S. cerevisiae

K. marxianus K. marxianus S. exiguus

S. exiguus S. exiguus K. marxianus

T. delbrueckii T. delbrueckii T. delbrueckii

C. ethanolica P. kluyveri Z. bailii


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compounds; and a detailed study of the yeast

populations using culture-dependent and -indepen-

dent methods.

Fermentation process and generation of volatile

compounds

The Agave salmiana used for the elaboration of this

mezcal is not cultivated but is collected in the Mexican

Altiplano. The characteristics of the agave depend

where it is collected. In particular, the concentration of

fructans, which are the stored sugar of the plant, may

vary. In fact, the agave plants used for each fermentation

arrived from different sites and had large difference in

the initial sugar concentrations. Since it had less sugar,

fermentation II produced less ethanol than fermentation

I. A similar effect was observed with the volatile

compounds: amyl alcohols, isobutanol and 1-propanol,

since sugar concentration affects this process as well. In

tequila, synthesis of isobutanol and amyl alcohols is

increased when the C/N ratio is increased (Arrizon and

Gschaedler 2007). In our case, initial nitrogen concen-

tration was similar in the two fermentations (addition of

(NH4)2PO4, 1 g/l) so the differences in the volatile

compounds profiles could be due to different sugar

contents in the raw material. Although only a few

compounds could be directly measured in the must,

these reflect the overall behavior of the volatile com-

pounds. De Leon-Rodrıguez et al. (2006) analyzed

sixteen mezcal brands from San Luis Potosi and

identified thirty-seven compounds; nine of them were

classified as major compounds. Five of the compounds

determined in this study belonged to this group, and had

an impact on the organoleptic properties and the bouquet

of the final product. The first conclusion of this work is

that it is essential to have quality standards for the raw

material, particularly in the sugar concentration, in order

to generate a fermented must with similar concentra-

tions of ethanol and volatile compounds.

Yeast identification succession and generation

of volatile compounds

Like numerous previous studies (Zott et al. 2008;

Tofalo et al. 2009; Csoma et al. 2010; Li et al. 2010;

Table 3 Occurrence of yeast populations in fermentation tank

at the beginning and at the end of the fermentation

Species Fermentation ages

Initial (%) End (%)

Fermentation I

S. cerevisiae 94.00 96.22

S. exiguus 0.90

K. marxianus 2.20 2.00

T. delbrueckii 0.50 1.78

P. kluyveri 0.50

Z. bailii 1.40

C. lusitaniae 0.50

Fermentation II

S. cerevisiae 97.63 77.26

S. exiguus 0.18

K. marxianus 2.19 1.68

T. delbrueckii 1.43

P. kluyveri 0.67

Z. bailii 13.68

C. lusitaniae 5.28

Fig. 2 Migration profile of PCR-DGGE from fermentation I

(a) and II (b). Line M corresponds to mixture of pure strains

isolated on WL medium and identified by RFLP; lines 1–9DGGE profiles of the DNA amplicons obtained directly from

musts corresponding to 7.5, 10, 15, 23, 25.5, 27, 30, 32 and

47 h in (a); lines 1–7 corresponding to 0, 3, 6, 9, 11, 13 and

24 h in (b). Abbreviations: C. sake (Cs), T. delbrueckii (Td),

K. marxianus (Km), Z. bailii (Zb), S. cerevisiae (S.c.),

R. mucilaginosa (Rm), S. exiguus (Se), C. ethanolica (Ce)

and P. membranifaciens (Pm)


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Cordero-Bueso et al. 2011), PCR–RFLP analysis was

successfully used to identify yeast species. Sequenc-

ing was used to confirm the identities obtained, by

comparing the RFLP patterns with similar published

data. Escalante-Minakata et al. (2008) reported

K. marxianus, P. fermentans and C. lusitaniae, in

another Agave salmiana fermentation in the same

region. However, the use of only one restriction

enzyme, Hae III, and different solid mediums for

yeast isolation, may explain the dissimilarities in

diversity and species encountered. The use of WL

medium in this study was very useful for the

detection of yeast diversity (Pallmann et al. 2001;

Cocolin et al. 2006; Urso et al. 2008; Li et al. 2010);

based on not only morphological characteristics, but

also on differences in color of the colonies. In

addition to the latter, we used a medium supple-

mented with agave juice, and we found the same

yeasts (data not shown).

In tequila, another agave distilled spirit, Lachance

(1995) found S. cerevisiae, Z. bailii, Candida milleri and

Brettanomyces anomala, as dominant yeasts and

B. bruxellensis, Hanseniaspora guilliermondii, H. vi-

nae, P. membranaefaciens, T. delbrueckii and

K. marxianus as secondary yeasts in the fermentation

process. On the fly drosophila, which is a vector for

yeast, Lachance found P. kluyveri. However, it wasn’t

detected in the fermentation. So, five of the eight yeasts

detected in these mezcal fermentations were also found

in the tequila fermentation. Regarding this study, tequila

fermentations present more diversity of yeasts than the

Agave salmiana fermentations. One reason could be the

characteristics of the raw material. Agave salmiana

contains a high level of saponins (Zamora et al. 2010),

and these compounds are known to be inhibitors of

yeasts growth (Miyakoshi et al. 2000). In another work,

Cira et al. (2008) showed that the heterologous expres-

sion of Fusarium oxysporum tomatinase (which detox-

ifies steroidal saponins) in Saccharomyces cerevisiae

increases its resistance to saponins and improves

ethanol production during the fermentation of Agave

must. Finally, another probable reason could be the

geographical location of the distillery: the Mexican

altiplano is an arid semi-desert region with a low

population of insects, which are the possible vectors

of yeasts in this kind of fermentation as demonstrated

by Lachance (1995).

In wine must, and recently in vineyards and

wineries, various studies have been carried out in

order to characterize the current yeast populations,

emphasizing non-Saccharomyces yeasts. In a study of

yeasts from grape berries, Clavijo et al. (2010) found

that 84% of the total yeast population was non-

Saccharomyces species, and that Kluyveromyces

thermotolerans, H. guilliermondii, H. uvarum and

Issatchenkia orientalis represented the 42.7%. Ocon

et al. (2010) studied the yeasts present in the facilities

and cellars of four wineries from the D.O.Ca. Rioja

Region. Pichia and Cryptococcus genera and the

Pichia anomala species were found in all four

wineries; T. delbrueckii and P. membranifaciens

were detected in four wineries; and Aerobasidium

pullulans, Kloeckera apiculata and Debaryomyces

hansenii were isolated in two wineries. Zott et al.

(2008) found 19 yeasts species in the wine elabora-

tion process in France, which includes a cold

maceration prior to fermentation. Hanseniaspora

uvarum and Candida zemplinina were the predomi-

nant non-Saccharomyces yeasts. Gonzalez et al.

(2006) in Spain found 27 species with a high number

of Candida and Pichia. In Argentina, 11 species were

isolated by Combina et al. (2005). In general, the

diversity of species is higher in wine fermentations

than in the studied mezcal process. Here, the raw

material (agave) is first cooked, which eliminates all

microorganisms present in the raw material. In wine,

in contrast, the principal source of non-Saccharomy-

ces yeasts are the grapes which are only crushed, so

any microorganisms that are present remain alive and

inoculate the fresh wine must. Kluyveromyces spp,

Zygosaccharomyces spp and Torulaspora spp, the

principal non-Saccharomyces yeasts found in this

study, have been also detected in wine fermentations;

however, only as minor species. K. marxianus has

been isolated from a great variety of habitats and has

great potential in biotechnological applications, par-

ticularly in the production of enzymes (Fonseca et al.

2008). K. marxianus seems to be closely related to

fermentations carried out with Agave as raw material.

Perez-Brito et al. (2007) reported K. marxianus in

plants and must of henequen (Agave fourcroydes);

Lachance (1995) found it in the tequila fermentation;

Lappe and Ulloa (1993) in pulque, which results in

the spontaneous fermentation of the sap or aguamiel

of different Agave species.

The behavior of the different yeasts populations is

quite different between the two fermentations. For

fermentation II, the dominant yeast is S. cerevisiae,


123


the population of non-Saccharomyces is higher than

in fermentation I: at the end of the process, five

different species are detected (Table 3). It’s well

known that non-Saccharomyces has low ethanol

tolerance, so with lower ethanol content then in

fermentation I, the diversity of non-Saccharomyces is

still high at the end of fermentation II. These strains,

particularly Z. bailii and C. lusitaniae are able to

grow with ethanol concentration of 12 g/l whereas in

fermentation I with 24 g/l of ethanol they didn’t

survive until the end of the fermentation. Zott et al.

(2008), like other authors (Nissen et al. 2003; Perez-

Nevado et al. 2006), proposed that there is some

negative interaction between the S. cerevisae and the

non-Saccharomyces. In our case, the quantity of

Saccharomyces is higher in fermentation I than in

fermentation II so that could be another reason of a

lower non-Saccharomyces population. However, it

will be important to study more fermentations and the

behavior of the isolated yeasts in controlled labora-

tory conditions in order to understand this specific

point. However, these changes in the yeast population

probably have a great impact on generation of

volatile compounds, as demonstrated in wine fer-

mentation (Romano et al. 2003). Few studies have

dealt with the behavior of specific yeasts isolated

from agave fermentations and their role in the

generation of volatile compounds. Arrizon et al.

(2006) demonstrated great differences between agave

and grape yeasts, particularly in the production of

volatile compounds in must prepared with agave and

grape juice. Although the non-Saccharomyces spe-

cies, e.g. K. marxianus, are well known to produce

high amounts of volatile compounds, particularly

esters (Fonseca et al. 2008), it is barely possible to

associate the levels of volatiles with the succession of

the global or specific yeast populations in the studied

fermentation.

Even though in this work the bacterial community

wasn’t studied, we detected considerable amounts of

bacteria during the process which may be an another

important factor in the generation of volatile com-

pounds. Previous studies demonstrated the presence

of lactic and acetic bacteria as well as Zymomonas

mobilis in these kinds of fermentations (Escalante-

Minakata et al. 2008; Narvaez-Zapata et al. 2010).

The real contribution of these microorganisms is still

unknown and needs further research in order to

elucidate its role.

PCR-DGGE

Recently, numerous authors have employed a combi-

nation of culture-dependent and culture-independent

methods, in order to study the behavior of the

microbiota that participates in the elaboration of

fermented products (Cocolin et al. 2002; Prakitchai-

wattana et al. 2004; Nielsen et al. 2005; Rantsiou et al.

2005; Cocolin et al. 2006; Rantsiou and Cocolin 2006;

Obodai and Dodd 2006; Dolci et al. 2008; Oelofse et al.

2009; Kim et al. 2009; Andorra et al. 2010; Lacerda

Ramos et al. 2010) and to understand the ecological

relationship between the microorganisms and the

influence of this diversity on the characteristics of the

end product. As in wine, PCR-DGGE has been shown

to be a reliable method for direct qualitative assessment

of the yeast populations present in mezcal fermenta-

tions. According to Cocolin et al. (2001), PCR–DGGE

avoids the problems often associated with microbial

enrichments. Moreover, it can be performed in a

reasonably rapid fashion (one day) and with minimal

sample volume. In this study, the PCR-DGGE detected

a microbial consortium composed of S. cerevisiae,

T. delbrueckii and K. marxianus throughout the

fermentation process. In complex mixed yeast popu-

lations, this method detected species present at 10–100

fold less than other species, but not when the ratio

exceeded 100 fold (Prakitchaiwattana et al. 2004).

When yeast populations fell below 104 CFU/ml, the

corresponding DGGE bands faded or disappeared.

This threshold is likely the result of a larger quantity of

Saccharomyces DNA in these samples competing with

the smaller amounts of template from the non-

Saccharomyces yeasts for amplification of the rDNA

(Mills et al. 2002). This can explain the fact that in the

case of the minority yeasts, S. exiguus, P. kluyvery and

Z. bailii, the detected bands were very weak.

Acknowledgments This study was developed within the PhD

research program (Ciencias Biologicas) from Universidad

Nacional Autonoma de Mexico, and supported by the SEP-

CONACYT # 24556 project. The authors thank Consejo

Nacional de Ciencia y Tecnologıa (CONACyT) for economic

support (grant for the PhD to Verdugo Valdez A.) and the

distillery ‘‘Real de Magueyes’’ for their interest and help.

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