1 23 Antonie van Leeuwenhoek Journal of Microbiology ISSN 0003-6072 Volume 100 Number 4 Antonie van Leeuwenhoek (2011) 100:497-506 DOI 10.1007/s10482-011-9605-y Yeast communities associated with artisanal mezcal fermentations from Agave salmiana A. Verdugo Valdez, L. Segura Garcia, M. Kirchmayr, P. Ramírez Rodríguez, A. González Esquinca, R. Coria & A. Gschaedler Mathis
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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
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
Antonie van Leeuwenhoek (2011) 100:497–506 501
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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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