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Comunidades de levaduras asociadas al mezcal
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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
1 23
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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];
R. Coria
Instituto de Fisiologıa Celular, Circuito Exterior S/N
Ciudad Universitaria, Universidad Nacional Autonoma
de Mexico, Coyoacan 04510, DF, Mexico
123
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DOI 10.1007/s10482-011-9605-y
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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
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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).
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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,
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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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