International Journal of Food Microbiology 124 (2008) 126-134

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Analysis of bacterial community during the fermentation of pulque, a traditional

Mexican alcoholic beverage, using a polyphasic approach
Adelfo Escalante a,, Martha Giles-Gmez b, Georgina Hernndez a, Mara Soledad Crdova-Aguilar a,
Agustn Lpez-Mungua a, Guillermo Gosset a, Francisco Bolvar a
a
Departamento de Ingeniera Celular y Biocatlisis, Instituto de Biotecnologa, Universidad Nacional Autnoma de Mxico (UNAM), Av. Universidad 2001,
Col Chamilpa, 62210, Cuernavaca, Morelos, Mxico
b
Departamento de Biologa, Facultad de Qumica, UNAM, Ciudad Universitaria, Coyoacn, 04510, Mxico D.F. Mxico

A R T I C L E I N F O A B S T R A C T

Article history: In this study, the characterization of the bacterial community present during the fermentation of pulque, a
Received 2 June 2007 traditional Mexican alcoholic beverage from maguey (Agave), was determined for the rst time by a
Received in revised form 14 November 2007 polyphasic approach in which both culture and non-culture dependent methods were utilized. The work
Accepted 3 March 2008
included the isolation of lactic acid bacteria (LAB), aerobic mesophiles, and 16S rDNA clone libraries from
total DNA extracted from the maguey sap (aguamiel) used as substrate, after inoculation with a sample of
Keywords:
Pulque
previously produced pulque and followed by 6-h fermentation. Microbiological diversity results were
Traditional alcoholic fermentation correlated with fermentation process parameters such as sucrose, glucose, fructose and fermentation product
Microbial diversity concentrations. In addition, medium rheological behavior analysis and scanning electron microscopy in
Lactic acid bacteria aguamiel and during pulque fermentation were also performed. Our results showed that both culture and
non-culture dependent approaches allowed the detection of several new and previously reported species
within the -, -Proteobacteria and Firmicutes. Bacteria diversity in aguamiel was composed by the
heterofermentative Leuconostoc citreum, L. mesenteroides, L. kimchi, the -Proteobacteria Erwinia rhapontici,
Enterobacter spp. and Acinetobacter radioresistens. Inoculation with previously fermented pulque incorpo-
rated to the system microbiota, homofermentative lactobacilli related to Lactobacillus acidophilus, several -
Proteobacteria such as Zymomonas mobilis and Acetobacter malorum, other -Proteobacteria and an
important amount of yeasts, creating a starting metabolic diversity composed by homofermentative and
heterofermentative LAB, acetic and ethanol producing microorganisms. At the end of the fermentation
process, the bacterial diversity was mainly composed by the homofermentative Lactobacillus acidophilus, the
heterofermentative L. mesenteroides, Lactococcus lactis subsp. lactis and the -Proteobacteria A. malorum.
After a 6-h fermentation, 83.27% of total sugars detected after inoculation were consumed (228.4 mM hexose
equivalents) and a carbon (C) recovery of 66.18% in fermentation products was estimated. They were
produced 284.4 mM C as ethanol, 71.5 mM C as acetic acid and 19 mM C as lactic acid, demonstrating the
presence of homo- and heterofermentative, acetic and alcoholic metabolisms in the nal product. It was also
found, after hydrolysis, that the exopolysaccharide produced during the fermentation was mainly composed
by fructose residues, probably inulin or levan.
2008 Elsevier B.V. All rights reserved.

1. Introduction
Pulque is a non-distilled traditional alcoholic beverage produced
by the fermentation of the sap known as aguamiel (AM), which is
extracted from several species of maguey such as Agave atrovensis and
A. americana. This beverage is currently produced and consumed
mainly in the central states of Mexico. For its production, freshly
collected AM is transported to large open barrels where fermentation
takes place. The process is accelerated by the addition of a portion of
Corresponding author. Tel./fax: +52 55+5622 7648.
E-mail address: adelfo@ibt.unam.mx (A. Escalante).
previously produced pulque and the fermentation time varies from
few hours to overnight, depending on whether the sap is collected at
daybreak or dusk. The development of viscosity due to exopolysac-
charide (EPS) synthesis and the alcohol content are the main
parameters used to determine the degree of fermentation. The
process is static and performed under non-aseptic conditions; there-
fore, the populations of microorganisms involved in the fermentation
are those naturally occurring during AM accumulation in the maguey
and those incorporated during collection, transport, inoculation and
manipulation (Snchez-Marroqun and Hope, 1953; Godoy, 1987;
Garca-Garibay and Lpez-Mungua, 1993; Escalante et al., 2004).
Several studies have been performed to characterize the microbial
diversity of AM and pulque samples using traditional culture dependent

0168-1605/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2008.03.003
 A. Escalante et al. / International Journal of Food Microbiology 124 (2008) 126-134
and identication methods. Important industrial microorganisms
isolated from various pulque samples, comprise several yeasts, such as
the ethanol producing Saccharomyces cerevisiae and species of Kluyver-
omyces producers of inulinase (Snchez-Marroqun and Hope, 1953;
Estrada-Godina et al., 2001; Cruz-Guerrero et al., 2006). Isolated
bacterial species include the alcohol producing Zymomonas mobilis
(Snchez-Marroqun and Hope, 1953; Godoy, 1987) and the dextran
producing lactic acid bacteria (LAB) Leuconostoc mesenteroides (Chella-
pandian et al., 1988). Among them, S. cerevisiae, L. mesenteroides and Z.
mobilis have been proposed as essential microorganisms for the pulque
fermentation process (Snchez-Marroqun and Hope, 1953; Godoy,
1987; Favela, 1993; Garca-Garibay and Lpez-Mungua, 1993).
We recently reported a study of the bacterial diversity present in
three samples of pulque from different geographical origins, based on
16S rDNA sequence analysis amplied from total bacterial DNA. Our
results indicated the presence of a microbial community composed of
previously reported microorganisms such as L. mesenteroides and Z.
mobilis, but several Proteobacteria, homo- and heterofermentative
LAB were detected and reported for the rst time in pulque samples.
This study revealed the presence of common microbial species in this
traditional fermented beverage, independently of the sample origin
(Escalante et al., 2004). However, to date, no information regarding
the microbial dynamics during the fermentation process of pulque is
available.
Microbiological characterization of traditional fermented foods
and beverages produced domestically and at semi-industrial level
represent the rst stage in the process for industrialization and scale-
up of this type of products. Results from this initial characterization
should allow the identication of microorganisms that are essential in
these processes (Blandino et al., 2003). This strategy is not only
restricted to local fermented foods and beverages; several studies
have been performed to characterize the microbial communities
involved in fermentations in order to improve important alcoholic
distilled beverages, such as Scotch malt whisky (Simpson et al., 2001;
van Beek and Priest, 2002).
In Mexico, interest in alcoholic beverages obtained from Agave has
increased in recent years due to their acceptance both in local and
international markets. Among these beverages, tequila and in a minor
proportion mezcal are of particular importance due to their
economical impact and the complex microbial process involved in
their production; therefore, it is possible to foresee that some of these
traditional fermentation processes could become industrialized. In the
present study, we report the characterization of the bacterial
community present in AM, and its dynamics during the fermentation
of pulque using cultured and non-cultured dependent methods. The
microbiological study was correlated with the sucrose, glucose,
fructose and fermentation products concentration, as well as medium
rheological behavior and scanning electron microscopy analyses in
AM and during pulque fermentation.
2. Materials and methods
2.1. Pulque sampling and fermentation analysis
Samples of freshly collected AM and overnight fermented pulque
to be used as inoculum were obtained from Huitzilac, a town in
Morelos State, Mexico (altitude of 2550 m in a cold weather
mountainous region), placed in sterile plastic recipients and trans-
ported to the laboratory.
One fermentation was carried out in a 4 L sterile glass container
and was started by addition of previously fermented pulque and fresh
AM (2:3 v/v) (as recommended by local supplier). Temperature and pH
were monitored in AM, after inoculation (T0), 3 (T3) and 6 (T6) h of
fermentation. Sucrose, glucose, fructose, ethanol, lactic- and acetic
acids concentrations were determined in all samples using a Waters
HPLC system, equipped with an Aminex column for fermentation
127
analysis. One ml of each sample was centrifuged at 10,000 g/5 min;
supernatants were then ltered through a 0.45 m membrane and a
20 l aliquot was injected to the column.
2.2. Microbiological determinations
Ten ml of each sample were diluted in 90 ml of sterile 0.1% Peptone
(DIFCO) water and serial dilutions (101 to 106) were made. Growing
aerobic mesophilic bacteria and LAB were determined by plating 1 ml of
each serial dilution on Nutritive-agar (DIFCO) and on APT-agar (DIFCO),
respectively. In order to obtain a general view of the interactions of the
different microbial groups involved in the process, we decided to include
the yeasts counts in our analysis. One ml of each serial dilution was
plated by duplicate on Malt extract-agar (DIFCO) supplemented with
10% w/v tartaric acid as antibacterial agent. Microbial determinations
were performed by triplicate. All plates were incubated at 30 C and
monitored for colony appearance from 1 to 3 days. Representative plates
of Nutritive-agar and APT-agar were used to isolate and purify an
average of 100 CFU, from AM and pulque samples, respectively. Gram
staining and light microscopy observations were used as criteria to
determine the purity of each isolate.
2.3. Amplied rDNA restriction analysis (ARDRA) of isolated cultures and
microbial identication
ARDRA was performed to Gram-positive and Gram-negative isolated
bacteria. Total DNA was released from each cultured isolate by a previously
used method that ensures an effective lysis of diverse bacterial groups
(Smit et al., 2001). One l of each lysate was used as template for 16S rDNA
fragment amplication as previously reported (Escalante et al., 2004)
using the primers Eu530F (5'-TGACTGGAGTGCCAGCAGCCGCGG-3') and
Eu1449R (5'-TGACTGACTGAGGCTACCTTGTTACGACTT-3'), which target to
Eubacteria. These primers had already been applied to determine the
bacterial diversity present in soil (Borneman et al., 1996) and pulque
samples (Escalante et al., 2004). Amplied 16S rDNA fragments were
digested with Hae III, for 2 h at 37 C. Restriction proles were visualized
using 2.5% agarose gel electrophoresis and analyzed with the molecular
weight analysis mode of the Eagle Eye Image Capture and Analysis
Software (Stratagene). Cultured isolates were grouped based on their
ARDRA patterns after identication of unique common restriction proles.
16S rDNA fragments corresponding to unique restriction types were
sequenced by the Taq FS Dye Terminator Cycle Fluorescence-Based
Sequencing method, with a Perkin Elmer/Applied Biosystems Model 377-
18 sequencer. The resulting DNA sequences were submitted to the non-
redundant nucleotide database at GeneBank using the BLAST program to
determine their identity.
2.4. Total bacterial DNA extraction from AM and pulque samples, PCR
amplication and cloning of 16S rDNAs
Total bacterial DNA was extracted from AM and pulque samples
using the microbial genomic DNA isolation kit of MoBio (12224-250).
This kit was selected due to its lytic efciency reported for several
Gram-positive and Gram-negative bacteria and EPS producing species
(www.mobio.com). Five independent extractions were performed for
each sample and pooled. One l of each pooled sample was used as
template for 16S rDNA fragment amplication using the Eu530F/
Eu1449R primer set. Three independent PCR reactions were per-
formed and pooled for AM and pulque samples. A 16S rDNA clone
library was created for each sample by cloning 4 l of amplied 16S
rDNA into TOPO Blunt vector (Invitrogen) and used to transform E. coli
TOP10 electrocompetent cells (Invitrogen). Positive clones were
screened by digestion of 2 l alkaline plasmid minipreps from each
16S rDNA clone library with EcoRI and visualized using 1% agarose gel
electrophoresis in order to identify those clones with the 16S rDNA
insert. An average of 100 positive TOPO Blunt 16S rDNA clones from
128 A. Escalante et al. / International Journal of Food Microbiology 124 (2008) 126-134

each AM, T0, T3 and T6 samples were selected, ARDRA was performed
to the positive ones and data analyzed as described in Section 2.3.
2.5. Non-culture dependent diversity analysis in 16S rDNA clone libraries
from AM and pulque samples
Unique ARDRA types identied from AM and pulque 16S rDNA
libraries were sequenced and identied as described in Section 2.3.
The EstimateS program (version 8.0.0; Colwell, 2006) was used to
calculate the nonparametric richness estimator Chao 1 (Hughes et al.,
2001; Buckley et al., 2006), which species the extent of diversity
richness based on 100 clones sampled. In order to determine whether
the size of the libraries was statistically different from each other or
whether one or more was a subset of others, a LIBSHUFF analysis was
performed (Schloss et al., 2004). Multiple alignment of AM and pulque
16S rDNA clones and reference 16S rDNA sequences retrieved from
GenBank database was performed using the Clustal W program.
Distance matrix calculation of nucleotide substitution rates and a
phylogenetic tree was constructed with the Jukes and Cantor
algorithm and the Neighbor-Joining (NJ) method, respectively, using
the Phylip (v 1.3b) program. Bootstrap methods were utilized to
provide condence estimated for tree topology in the NJ method
for both microbial groups after inoculation was 1.2 107 and
1.5 108 CFU/ml, respectively, and remained relatively constant until
the end of the process (3.5 107 and 1.5 108 CFU/ml at the end of the
fermentation, respectively). Yeasts concentration detected in AM was
3.1 10 4 CFU/ml. Inoculation with previous fermented pulque
increased the log number of yeasts to 8.8 106 CFU/ml and increased
further to reach 1.4 107 CFU/ml after 3-h culture to remain constant
for the rest of the fermentation process.
An average of 100 colonies of aerobic mesophiles and LAB from
representative plates of AM and each pulque sample were isolated.
DNA from each colony from the different samples was isolated and
subjected to ARDRA by digesting the 16S rDNA fragment with Hae III.
Using restriction proles generated with this enzyme, microbial
Table 1
Identity of cultivated and non-cultivated microorganisms by 16S rDNA analysis from
AM and during pulque fermentation
Lineagea Identityb Isolate and/or 16S rDNAd
clone detected by
Cultivated 16S rDNA library
analysis

(1000 replicates). -Proteobacteria/ Acetobacterium malorum T3332

Rhodospirillales Acetobacter orientales T638
-Proteobacteria/ Zymomonas mobilis subsp. T321
2.6. Scanning electron microscopy (SEM) analysis
Sphingomonadales pomaceae
-Proteobacteria/ Citrobacter spp. AM116
One ml of AM and pulque samples were xed with 1 volume of Enterobacteriales Enterobacter agglomerans T0100
4% v/v glutaraldehyde and 0.01% w/v OsO4. Fixed samples were Enterobacter spp. AM119
successively dehydrated in 30%, 50%, 70%, 100% ethanol, super critical Erwinia rhapontici AM230
Kluyvera ascorbata T0330 T3344
CO2 dried in a Sandri-780 apparatus (Tousimis) and coated with gold Kluyvera cochleae AM143
particles in a JFC-110 ion sputter device (JEOL). Samples were observed Providencia spp. T6126
in a JEOL scanning electron microscope model JSM 5410LV. Serratia grimensii AM14
-Proteobacteria/ Acinetobacter radioresistens AM83
Psedomonadales
2.7. Hydrolysis of EPS produced in pulque fermentation and rheological
-Proteobacteria/ Sterotrophomonas spp. T040
analysis Xhantomonadales
Flavobacterias Chryseobacterium spp. T6342
Acid and enzymatic hydrolysis were performed to determine EPS Firmicutes/Bacillales Bacillus sp. T621
components produced at the end of the fermentation. For the acid Bacillus licheniformis T023
Firmicutes/ Lactobacillus spp. AC07c T33
hydrolysis, 1 ml aliquot of pulque sample T6 was centrifuged for Lactobacillales Lactobacillus spp. ASF360c T01
10 min at 12,000 g and the supernatant sterilized under UV Lactobacillus spp. Y10c T3239
exposition during 4 h. The sample was then hydrolyzed by addition Lactobacillus acidophilus T3160
of 0.3 N HCl, incubated for 1 h at 105 C and neutralized with 0.1 N Lactobacillus hilgardii T0225
Lactobacillus paracollinoides T3150
NaOH. Reducing sugars were determined by the DNS method before
Lactobacillus sanfranciscencis T0218
and after hydrolysis. For the enzymatic hydrolysis, to a 1 ml aliquot of Lactococcus spp. AM123 AM96
UV sterilized sample adjusted to pH 4.6 was added 1 ml of the enzyme Lactococcus lactis T3239
Fructozyme (1% v/v nal concentration). Reaction was heated to 60 C Lactococcus lactis subsp. cremoris T011
and incubated 2 h. Finally, the resulting concentrations of reducing Leuconostoc kimchi AM146 AM235
Leuconostoc citreum AM140 AM132
sugars were measured (DNS method) and analyzed by HPLC with a Leuconostoc gasicomitatum T0121
Waters HPLC system. Leuconostoc mesenteroides AM212 AM118
Rheological determinations were performed using a rotational AR Leuconostoc pseudomesenteroides T3245 T358
1000 TA Instruments rheometer (New Castle, DE, USA) tted with a Pediococcus urinaeequi T325
Streptococcus devriesei AM446
stainless steel cone/plate geometry (60 mm of diameter, 1 cone
Uncultured bacterial Uncultured bacterial clone AM312
angle). AM and pulque samples were transferred to the peltier plate, clones BANW647c
removing the excess material with lter paper. All experiments were Uncultured bacterial clone T3135
conducted at room temperature. Data in the laminar ow regime P-2988-9F2c
(Re b 10) was tted to the power law model (t = Kgn). From this model, Uncultured bacterial clone T365
rRNA342c
polymer consistency index, K (Pa sn) and ow index, n (dimensionless)
Eukaryotic Saccharomyces cerevisiae T0323
were determined. a
Based on information retrieved from the NCBI taxonomy homepage. b16S rDNA or 16S
rRNA sequences of organisms which showed the highest percent of identity in the
3. Results output result from analysis in the non-redundant nucleotide database from NCBI using
the BLAST program. cStrain or clone designation of closest 16S rDNA sequences in
3.1. Culture-dependent analysis of bacterial diversity in AM and during database are indicated only for Lactobacuillus spp. and uncultured bacterial clones.
pulque fermentation Previously reported microorganisms or 16S rDNA clone sequences for AM and pulque
are highlighted in bold letters (Snchez-Marroqun and Hope, 1953; Godoy, 1987;
Garca-Garibay and Lpez Mungua, 1993; Escalante et al., 2004). dAguamiel (AM), after
The log number of CFU/ml of aerobic mesophiles and LAB present inoculation (T0), 3 (T3) and 6 (T6) h indicate the sample source of isolates or 16S rDNA
in AM was 1.3 107 and 3.2 108 CFU/ml, respectively. The log number clones detected.
 A. Escalante et al. / International Journal of Food Microbiology 124 (2008) 126-134 129

isolates with similar patterns were grouped and the 16S rDNA of one
member of each group was sequenced and identied. From total
isolates, 21 different species and 14 different genera were found,
51.44% were identied as Firmicutes (Bacillales and Lactobacillales)
and 48.55% distributed among -Proteobacteria, -Proteobacteria and
Flavobacteria. Identities of isolated bacteria in AM and pulque samples
are presented in Table 1 and its distribution during the fermentation is
given in Fig. 1.

3.2. Non-culture dependent diversity analysis in 16S rDNA clone libraries

from AM and pulque samples

Total microbial DNA was extracted from AM and pulque samples.

Amplication of the 16S rDNA fragment was performed indepen-
dently from each DNA extraction and cloned into the TOPOBlunt
system. An average of 100 clones from each 16S rDNA library was then
digested with Hae III and unique restriction types from each library
were detected and selected for sequence analysis. Retrieved results
from GenBank analysis revealed that 79.1% of analyzed clones were
identied as cultivated Firmicutes and 8.2% as members of the
cultivated -Proteobacteria, -Proteobacteria and Enterobacteriales.
These included some of the identied microorganisms by the culture-
dependent approach (Table 1). Interestingly, 7.2% of the analyzed
sequences were identied as uncultured bacterial clones. Finally, 5.4%

Fig. 2. Rank abundance curves of unique 16S rDNA clones from AM and pulque samples.
A. AM, B. T0, C. T3, D. T6. Nick names for identied isolates are the same as in Fig. 1.
Additional abbreviations for AM: Lki, Leuconostoc kimchi; Ara, Acinetobacter radio-
resistens; Unb, Uncultured bacterial clones; Sde, Streptocccus devriesei. For T0: Zmo,
Zymomonas mobilis; Lhi, Lactobacillus hilgardii, Lga, Leuconostoc gasicomitatum. For T3:
Lac, Lactobacillus acidophilus; Lsa, Lactobacillus sanfranciscencis. For T6: Aor, Acetobacter
orientalis. Sequences identied as Saccharomyces cerevisiae (abbreviated Sce) are also
included.

of analyzed clones were identied as S. cerevisiae, the most abundant

Fig. 1. Rank abundance curves of both total aerobic mesophiles and LAB isolated from AM
and pulque samples. A. AM, B. T0, C. T3, D. T6. Abbreviations for identied isolates in AM are:
Erh, Erwinia rhapontici; Lci, Leuconostoc citreum; Lme, L. mesenteroides; Ent, Enterobacter
spp.; Cit, Citrobacter sp.; Lla, Lactococcus lactis; Sgr, Serratia grimensii; Kch, Kluyvera cochleae.
For T0: Eag, Enterobacter aglomerans; Kas, Klyuvera ascorbata; Ama, Acetobacter malorum;
Ste, Sterotrophomonas spp.; Lps, Leuconostoc pseudomesenteroides; Bli, Bacillus licheniformis;
Llc, Lactococcus lactis spp. cremoris. For T3: Lbs, Lactobacillus spp.; Lcs, Lactococcus spp., Pur,
Pediococcus urinaeequi. For T6: Pro, Providencia spp.; Chr, Chryseobacterium sp; Bac, Bacillus
spp.
yeast present in pulque fermentation. As it was observed for the
culture-dependent approach, several clones were identied as
microorganisms non-previously reported in pulque (Table 1).
LIBSHUFF analysis was used to compare the phylogenetic
composition of 16S rDNA clone libraries of all samples in order to
nd out whether the average of the analyzed clones was statistically
different. Results revealed that the AM library was statistically
different to that of sample T0, whereas samples T3 and T6 were not
statistically different as a consequence of the presence of common
species in T0 and T3, and T3 and T6 libraries, that disappeared, were
maintained without relative changes or were enriched during the
fermentation (Fig. 2). Calculation of the Chao 1 estimator revealed a
low difference with respect to the expected number of unique 16S
rDNA clones for AM, T0 and T3 samples. These results indicated that
the number of analyzed clones in these libraries adequately described
the extent of diversity richness present, except for T6 library, in which
the estimator showed a higher difference with respect to the
expected diversity. Phylogenetic analysis of 16S rDNA sequences
demonstrated a high similarity to isolates or environmental clones
previously obtained from diverse sources and deposited in GenBank
database (Fig. 3). However, other clones showed less than 95%
relatedness to NCBI database sequences, which may indicate the
presence of new species.
130 A. Escalante et al. / International Journal of Food Microbiology 124 (2008) 126-134

Fig. 3. Phylogenetic tree of unique 16S rDNA sequences detected in AM and pulque samples and sequences of closest neighbor 16S rDNA from identied bacteria or environmental
clones in the NCBI database. GenBank accession number of closest 16S rDNA sequences in database are indicated. The 16S rDNA sequence of Sulfolobus acidocaldarius served as
outgroup. The percentage of 1000 bootstrap samplings supporting each topological element in the neighbor-joining analysis is indicated. No values are given for groups with
bootstrap values less than 80%. Identities of unique 16S rDNA sequences presented in the tree are shown in Table 1. Identity percentage with closest reference to 16S rDNA clones in
the database is indicated in parenthesis.

3.3. pH, temperature, sugar content and fermentation products during 66.2% of total C in products were recovered. The concentration of
AM and pulque fermentation ethanol reached a nal value of 142.17 mM; meanwhile nal
concentrations of acetic and lactic acids were 35.73 mM and
AM had an initial pH of 6.0, which after inoculation with previous 6.32 mM, respectively (Table 2).
fermented pulque (pH = 4.0) decreased to 4.5 as a consequence of the
addition of fermentation products. The nal pH of pulque after 6-h
fermentation was 4.3. The traditional process took place at room Table 2
temperature: The AM sample had an in situ temperature of 17 C, Sugar content, fermentation product concentrations and physicochemical parameters
which after inoculation increased to 20 C and then gradually up to in aguamiel (AM) and pulque samples
22 C after 3 h and 24 C after 6 h. This was the result of important Sample Sugar content Product concentrations Viscosity
microbial activity during fermentation, in spite of the relatively (mM hexose equivalents) (mM C)
constant total bacterial counts observed all through the process. To
Sucrose Glucose Fructose Ethanol Lactic Acetic n ()a K (Pa sn)
asses the overall metabolic activity of the microbial community in AM
acid acid
and pulque samples, the concentrations of sucrose, glucose, fructose
AM 410.16 83.53 533.35 4.34 46.95 30.48 1.060 0.0012
and fermentation products were determined (Table 2). Total sugars
T0 334.79 30.86 188.05 655.10 87.25 36.58 1.030 0.0017
consumed after 6 h of culture were 228.4 mM hexose equivalents; this T3 210.69 28.19 71.21 749.31 95.91 94.84 0.842 0.0071
is equivalent to 1370.5 mM C, which was found as CO2. It was assumed T6 180.19 3.82 12.43 939.46 106.24 108.05 0.754 0.0142
that in molar terms, the amount of CO2 produced is equal to the Changes during the fermentation process: after inoculation (T0), 3 (T3) and 6 (T6) h of
amount of acetic acid and ethanol. When 176.5 mM C corresponding fermentation.
to 5.3 g/L of fructose in EPS were also included (see Section 3.4), then Values are the mean of triplicate determinations. aDimensionless.
 A. Escalante et al. / International Journal of Food Microbiology 124 (2008) 126-134 131

Fig. 4. Scanning electron microscopy micrographs of AM and pulque samples. A. AM; B. T0; C. T3; D. T6.
132 A. Escalante et al. / International Journal of Food Microbiology 124 (2008) 126-134
3.4. Analysis of EPS produced in pulque fermentation and rheology
EPS production is an important distinctive characteristic of fermented
pulque. HPLC analysis of sugars generated from EPS by acid hydrolysis
revealed no changes in glucose concentration while a signicant increase
in fructose residues was observed. Calculations derived from HPLC and
reducing sugars analysis indicated that 5.3 g/L of fructose were present in
the polymeric structure. Based on these observations, the EPS produced
after 6-h fermentation must be a polymer composed by fructose residues
that could be either inulin or levan.
Two rheological properties were determined, the ow index (n)
and the consistence index (K). Results in Table 2 indicate that AM and
pulque polymer behaved as non-Newtonians uids. As fermentation
developed, n decreased indicating the change from a uid lightly
dilatant (AM) to a pseudoplastic product after 6 h. Based on the
evolution of K values, which increased from AM to T6, it is possible to
propose the presence in AM of one type of EPS which is degraded
during fermentation and the synthesis of another type of polymer
with higher viscosity (probably inulin or levan).
3.5. Scanning electron microscopy of AM and pulque samples
SEM observations of AM revealed the presence of bacteria with
several morphologies including short bold and large thin rods, cocci
organized in pairs and short chains with smooth surface, cocci in large
chains with rough surface, and few yeasts, probably Saccahromyces
cerevisiae (Fig. 4A). After inoculation (Fig. 4B), rough cocci in large
chains, probably EPS producing Leuconcostoc species, were observed
as the most abundant cell morphology followed by budding yeasts.
Samples corresponding to T3 (Fig. 4C) and T6 (Fig. 4D) showed cocci in
short chains and rods, an important increase in yeast counts, as well as
the enrichment of cocci in large chains, which was dened as the most
abundant bacterial morphology at the end of fermentation.
4. Discussion
The bacterial diversity present in AM and during the fermentation
of pulque, a Mexican traditional alcoholic beverage, was studied for
the rst time to determine microbial community changes involved in
the process. In addition, we determined several physicochemical
parameters during the fermentation in order to obtain a more detailed
description of the process. Bacterial diversity was explored using both
culture and non-culture dependent methods in order to minimize
biases currently associated with either of these methodologies.
Although both approaches allowed the detection of common species
among the -, -Proteobacteria and Firmicutes, it was observed that
each method revealed a poor diversity for some species, highlighting
the limitations of using only one approach to study bacterial diversity.
Rank abundance curves were elaborated to integrate the results
obtained with both approaches. Besides, important microbial diversity
changes were determined during the fermentation from AM to the
nal fermented product. Identities obtained from 16S rDNA clone
sequence analysis and strain isolation revealed that species of Leuco-
nostoc mesenteroides, L. kimchi and L. citreum were the most abundant
LAB in AM (36.6% of total cultured organisms and 51.9% of total clones
analyzed for AM). Among them, L. mesenteroides had been previously
reported in pulque (Snchez-Marroqun and Hope, 1953; Godoy, 1987;
Garca-Garibay and Lpez Mungua, 1993), whereas L. kimchi, L.
citreum and Lactococcus lactis (in minor proportions) were detected
for the rst time in both AM and pulque. Furthermore, it was observed
that the -Proteobacteria identied by culturing as Erwinia rhapontici
and Enterobacter spp. (47.9% of total isolates) and 16S rDNA clones
identied as Acinetobacter radioresistens (18.3% of total clones
analyzed) were the second dominant group in sap (Figs. 1A and 2A).
Members of the genus Leuconostoc are heterofermentative LAB
occuring in pairs and chain aggregates; they are commonly found in
fruits and vegetables. They play important roles in the fermentation of
dairy products, vegetables and meats given their ability to ferment a
wide diversity of sugars as those present in pulque (sucrose, glucose
and fructose), producing CO2, D() lactic acid, glucans and fructans
from sucrose (Carr et al., 2002). SEM of AM sample revealed the
presence of abundant cocci organized in pairs and short chains with
smooth surface and cocci in large chains, probably Leuconcostoc
species.
Although it has been reported that some -Proteobacteria detected
in AM are naturally distributed in several environments including
fresh water, soil, plant and vegetable surfaces, some of them are also
considered opportunistic human pathogens (Ashraf et al., 1997;
Janssen et al., 1997; Holt et al., 2000; Waleron et al., 2002). It is
possible that some of the -Proteobacteria found in AM are members
of the natural bacterial diversity of the sap that were incorporated
from the environment during its accumulation in the maguey,
whereas others could be incorporated during its extraction, handling
and storage under non-aseptic conditions.
Inoculation of AM with previously fermented pulque added new
bacterial species to the system and an important content of yeasts;
however, abundant clones in sap such as those identied as L. kimchi
and A. radioresistens, were diluted and therefore detected in low
proportions (5.6% of total clones), whereas culturable L. mesenteroides,
L. citreum (33% of total isolates in T0) stayed relatively constant with
respect to AM. After inoculation, the most abundant microorganisms
detected by clone analysis were Lactobacillus spp. (75.3% of total
clones), a LAB closely related to homofermentative L. acidophilus, and
the -Proteobacteria Enterobacter agglomerans (21.2% of total isolates);
both species were not detected in AM. Furthermore, -Proteobacteria,
other -Proteobacteria and Gram-positive bacteria not detected in AM
were observed after inoculation (Figs. 1B and 2B). The -Proteobac-
teria Zymomonas mobilis and Acetobacter malorum were found after
inoculation.
Z. mobilis, previously reported in pulque, has been traditionally
considered in association with yeasts responsible for ethanol produc-
tion (Snchez-Marroqun and Hope, 1953; Godoy, 1987, Favela, 1993;
Garca-Garibay and Lpez-Munga, 1993). Several strains of Z. mobilis
have been previously isolated from pulque samples and reported as
high ethanol yield producers (Favela, 1993). Although in low
proportions, A. malorum was also detected for the rst time in pulque.
Species of this microorganism occur naturally in sugar rich environ-
ments such as alcoholic fermentation musts of sake, tequila (another
fermented beverage from Agave), palm wine, grape wine, among
others (Holt et al., 2000, Du Toit and Lambrechts, 2002). SEM after
inoculation revealed the addition of yeasts to the system, while
previously observed cocci in pairs and chains with smooth and rough
surface remained relatively abundant. Important physicochemical
changes were observed in T0, pH detected in AM changed from 6.0 to
4.5 as a consequence of the addition of fermented pulque, whereas
total C in fermentation products increased from 116.6 mM to
1470.6 mM.
At T0, starting bacterial diversity for pulque fermentation was
composed by those species present in AM and those incorporated
by inoculation; integrating a metabolic diversity of homofermenta-
tive and heterofermentative LAB, acetic and ethanol producing
bacteria and yeasts. After 3-h fermentation important changes in
bacterial diversity were observed. Lactobacillus spp. (49.5% of total
clones), L. mesenteroides and Enterococcus agglomerans (61.8% of
total isolates) became the dominant bacterial species. Other bacteria
such as Z. mobilis, A. malorum and A. radioresistens were found
relatively constant since inoculation; L. citreum was detected in
minor proportion and some others disappeared (Figs. 1C and 2C).
16S rDNA clones identied for the rst time the homofermentative
LAB L. acidophilus found in the fermentation as a minor group; also
clones identied as Lactococcus lactis subsp. cremoris increased
after inoculation. During the rst 3-h fermentation, yeasts increased
 A. Escalante et al. / International Journal of Food Microbiology 124 (2008) 126-134
from 8.8 106 CFU/ml to 1.4 107 CFU/ml. Fields of this sample
analyzed by SEM demonstrated abundant presence of yeasts,
smooth cocci in pairs, rough cocci aggregated in chains and some
rods.
At the end of the fermentation process, the bacterial diversity
observed by clone analysis was mainly composed of the homo-
fermentative Lactobacillus acidophilus (88.1% of total clones), mean-
while major culture diversity consisted of the heterofermentative LAB
L. mesentreoides (50%), L. lactis subsp. lactis (12.5%), a homofermenta-
tive Lactobacillus spp. (14.8%) and the -Proteobacteria A. malorum
(13.6% of total isolates). Yeasts counts were maintained relatively
constant with respect to T3. Analyzed elds by SEM showed abundant
yeast and the same bacterial morphologies observed previously. As a
consequence of the microbial activity, after 6-h fermentation 83.27%
of total sugars detected after inoculation were consumed (228.4 mM
hexose equivalents). It was estimated that 18% of C was incorporated
into fermentation products in the following manner: 284.4 mM C as
ethanol, 71.5 mM C as acetic acid and 19 mM C as lactic acid. Thus, the
presence of homo- and heterofermentative, acetic and alcoholic
metabolisms was demonstrated during the whole process.
Traditionally, studies on the microbiology of pulque have reported that
L. mesenteroides and Z. mobilis are the most important bacteria involved in
the fermentation process. However, although Lactobacillus species have
been previously detected as well, no detailed information about the
identity and the role of this LAB group is available (Godoy, 1987; Escalante
et al., 2004). In this work, it was shown that homofermentative lactobacilli
species could probably play an important role during the fermentation
process, given the high number of L. acidophilus identied clones and
closely related Lactobacillus spp. in all pulque samples. Lactobacillus
acidophilus has been reported to ferment glucose, fructose, sucrose and
produce DL lactic acid (Carr et al., 2002). Other lactobacilli detected in
minor proportions were L. hilgardii, Lactobacillus spp. ACO7 (related to L.
hilgardii) and L. sanfranciscensis (heterofermentative LAB). They also
ferment glucose and fructose but not sucrose, and produce DL lactic acid.
It has been reported that some species are able to grow in environments
with 20% ethanol (Carr et al., 2002.). Therefore, lactic acid bacteria
resistance to high ethanol concentrations and their capacity to grow in
acid environments (pHb 4.5) could explain their relative abundance
during the fermentation process.
It is possible that bacterial species that apparently disappeared
during fermentation were not able to resist the acidication of the
environment, increased ethanol concentration, possible antimicrobial
effect of LAB such as H2O2 production, or other antibacterial activities.
The liquid consistence of pulque avoids the formation of microenvir-
onments or gradients observed in solid traditional fermentations that
allow survival of large populations of Enterobacteriaceae despite the
high acid production (Ampe et al., 1999).
The use of universal primers Eu530F and Eu1449R for amplication
of 16S rDNA fragments demonstrated the amplication of yeast S.
cerevisiae and uncultured bacterial species ribosomal genes. Detection
of eukaryotic genes using universal prokaryotic primers has been
previously observed (Barns et al., 1994; Rheims et al., 1996; Escalante
et al., 2001). These results could be explained as a bias associated with
the following circumstances: the methodology used for extraction of
total bacterial DNA could have lyzed non-bacterial cells, low primer
annealing temperature used during amplication, and the relative
abundance of S. cerevisiae in pulque. Uncultured bacterial clones
detected in this study were related to known sequences of Acineto-
bacter spp. and Lactobacillus spp. (Fig. 3).
One of the main distinctive properties of pulque is its viscous
consistence, which is dependent on EPS production. The main
microorganism associated with this property has been L. mesenteroides,
widely studied for its dextran producing capacity (Snchez-Marroqun
and Hope, 1953; Garca-Garibay and Lpez-Mungua, 1993; Chellapan-
dian et al., 1998). However, in this study we reported for the rst time in
pulque the presence of an EPS composed by fructose (levan or inulin
133
type) observed after 6-h fermentation. It has been published that
L. citreum CW28 and L. mesenteroides (NRRL B512F and ATCC8293
strains) produce an EPS composed by fructose as a consequence of
fructosyltranferase activitiy which transforms fructose from sucrose to a
growing fructan polymer (Olivares-Illana et al., 2003; Ortiz-Soto et al.,
2004). Characterization of this fructose-EPS is currently in progress.
The bacterial diversity in AM and during the fermentation of
traditional pulque was studied by using a polyphasic approach. The
microbial diversity detected in AM and T0 was composed by homo-,
heterofermentative, acetic, ethanol and EPS producer species. The
process started in an acid and sugar rich environment, with a complex
bacterial diversity composed by both Gram-positive and Gram-
negative bacteria from which the heterofermentative L. citreum,
L. mesenteroides and L. kimchi were the most abundant species during
the rst hours. Homofermentative L. acidophilus was the most
abundant species at the end of fermentation. Z. mobilis subsp. poma-
ceae and Citrobacter species contributed to the production of lactic
acid (DL), CO2, ethanol and small amounts of acetic acid which are
important for the sensorial characteristics of fermented pulque. All
these microorganisms, their proportions and properties must be
considered in order to dene a well known bacterial starter to perform
the fermentation of pulque under controlled conditions. However, it is
still necessary to organize additional studies to gain further informa-
tion on the interactions between the bacterial diversity present in
pulque with yeasts, the most important microbial group responsible
for alcohol production.
Acknowledgements
We thank Fernando Gonzlez, Mercedes Enzaldo and Rodrigo
Conca for technical assistance; Eugenio Lpez, Paul Gaytan, Jorge
Yaz and Soledad Jurez for primer synthesis and sequencing
support; Araceli Patrn and Rosa Mara Picasso for SEM support, and
Mr. Alfonso Dvila for pulque sampling. This work was supported by
grants CONACYT P46052-Z, D43243-Z, PAPIIT/UNAM IN-205005-2
and PAPIME/UNAM PE205606.
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