FEMS Microbiology Letters 235 (2004) 273–279
www.fems-microbiology.org
Characterization of bacterial diversity in Pulque, a
traditional Mexican alcoholic fermented beverage, as determined
by 16S rDNA analysis
pez-Munguıa,
Adelfo Escalante, Marıa Elena Rodrıguez, Alfredo Martınez, Agustın Lo
*
Francisco Bolıvar, Guillermo Gosset
Departamento de Ingenierıa Celular y Biocatalisis, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico, Apdo. Postal 510-3,
Cuernavaca, Morelos, 62250, Mexico
Received 12 March 2004; received in revised form 22 April 2004; accepted 23 April 2004
First published online 3 May 2004
Abstract
The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries
from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L.
acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae,
Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA
sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%).
Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new
species in pulque samples.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Pulque; Bacterial diversity; 16S rDNA sequence analysis
1. Introduction
Pulque is a traditional Mexican alcoholic fermented
beverage produced from the sap known as aguamiel,
which is extracted from several species of maguey
(Agave americana, A. atrovirens, A. ferox, A. mapisaga,
A. salmiana) [1–3]. This beverage is currently produced
and consumed mainly in the central states of Mexico.
For its production, freshly collected aguamiel is transported in wood barrels or in bags made from young goat
skins and transferred into large barrels where fermentation takes place. It has been proposed that the fermentation process starts in the maguey, where naturally
occurring microorganisms present in the aguamiel ferment part of the available carbohydrates. However, the
*
Corresponding author. Tel.: +52-7773-291601; fax: +52-7773172388.
E-mail address: [email protected] (G. Gosset).
process is accelerated by the addition of the seed (a
portion of previously produced pulque). Fermentation
time varies from a few hours to overnight, depending if
the sap is collected at daybreak or at dusk. Traditionally, development of viscosity due to exopolysaccharide
(EPS) synthesis has been the main criteria to determine
the degree of fermentation (fresh or mature pulque). The
final product is placed in wood barrels and distributed
daily for sale and consumption, without the addition of
any preservatives. The entire process is performed under
non-aseptic conditions, therefore the mixture of microorganisms involved in the fermentation process are
those naturally occurring in aguamiel and those incorporated during its collection, transport, inoculation and
manipulation [1–3].
Studies on the microbiology of pulque have focused
on the isolation and identification of microorganisms
present in the aguamiel and in fermented pulque using
traditional culture and characterization methods.
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.04.045
274
A. Escalante et al. / FEMS Microbiology Letters 235 (2004) 273–279
Microorganisms frequently identified in pulque samples
comprise several yeast and bacterial species including
homo- and hetero-fermentative lactic acid bacteria, the
alcohol producing bacteria Zymomonas mobilis and the
dextran producing bacteria Leuconostoc mesenteroides
[1–4].
Pulque production consist of three types of fermentations: acidic, alcoholic, and viscous, making this traditional beverage an interesting environment were
microorganisms or genes with potential biotechnological applications, such as those encoding sugar transporters, hydrolytic enzymes, EPS, lactic acid or ethanol
production, could be isolated.
Culture-based approaches to characterize microbial
diversity have limitations that can introduce a bias when
studying environmental samples [5]. Therefore, alternative and complementary non-culture dependent approaches have been developed and evaluated to study
microbial communities. PCR based-molecular techniques such as 16S rRNA genes (16S rDNA) amplification and sequence analysis have been widely used to
study the microbial diversity present in environmental
samples. Although it has been reported that this and
other molecular approaches may introduce some biases
[6,7], 16S rDNA analysis has been demonstrated to be a
powerful tool to investigate the biological diversity
present in environmental samples.
In this study, we analyzed 16S rDNA clone libraries
generated from total DNA of the bacterial community
present in pulque samples collected in three different
production locations in central Mexico. Clonal types
were initially screened and grouped, on the basis of
amplified 16S rDNA restriction analysis (ARDRA). 16S
rDNA sequence analysis of unique ARDRA groups led
us to identify for the first time bacterial groups not
previously detected in pulque and extend our knowledge
of the genetic diversity in this microbial community.
2. Materials and methods
2.1. Pulque origin and sampling
Home made pulque samples were collected from
central Mexico towns Aculco (19100 N, 98510 W), with
an altitude of 2320 m in a semiwarm-subwet and summer rainy region; Huitzilac (19020 N, 99160 W), with an
altitude of 2550 m in a cold weather mountainous region) and Tizayuca (19500 N, 98590 W), with an altitude
of 2260 m in a flat region with warm weather and a
summer rainy season). Average environmental temperature, sample pH and ethanol content are shown in
Table 1.
Pulque samples from Aculco and Tizayuca were
prepared from fresh aguamiel and fermented for 4–6 h,
whereas, that from Huitzilac was prepared from a same
Table 1
Sample characteristics
Sample
source
Environmental
temperature (C)a
pH
Ethanol
content (g/l)
Aculco
Huitzilac
Tizayuca
14
12.5
14
3.83
3.82
3.92
17.58 ± 0.59
45.41 ± 1.31
30.27 ± 0.59
a
Data retrieved from available averages records reported in the
Mexican National Institute for Geography, Statistics and Computer
Science home page (www.inegi.gob.mx).
day fresh aguamiel and fermented approximately for
20 h. Samples were placed in sterile plastic bags, and
transported in ice to the laboratory. Ethanol content
was analyzed by gas chromatograph using n-butanol as
internal standard (6850 Series GC System, Agilent,
Wilmington, DE).
2.2. DNA extraction and purification
Each one liter sample of pulque was used as the
substrate for indirect total bacterial DNA extraction. A
modification of the procedure previously described [8]
for the indirect DNA extraction from a fermented
dough and based on homogenization–centrifugation
rounds was performed. The integrity and the quantity of
extracted DNA were evaluated in 1.2% agarose gel
electrophoresis.
2.3. PCR amplification and cloning of 16S rDNA
16S rDNA from each sample of pulque DNA were
amplified by PCR using the pair of primers Eu530F
(50 -TGACTGGAGTGCCAGCAGCCGCGG-30 ) and
Eu1449R (50 -TGACTGACTGAGGCTACCTTGTTACGACTT-30 ), which are targeted to Eubacteria [9]. The
PCR mixture contained 2 units of Vent polymerase
(Fermentas Inc. Hanover, MD), 5 ll of 10· buffer, 200
lM each dNTP, 1.0 lM of each primer and 1 ll of
bacterial DNA extracted from each sample of pulque as
template in a final reaction volume of 50 ll. Reactions
were performed by triplicate in a Gradient 96 robocycler
(Stratagene, La Jolla, CA) as described previously [8].
Amplified 16S rDNAs from each pulque samples were
pooled and ligated into the pCR-BluntII TOPO vector
(ZeroBlunt TOPO PCR cloning kit, Invitrogen, Carlsbad, CA), according to the instructions recommended
by the supplier. Two microliters of ligation reaction
were used to electroporate competent Escherichia coli
TOP10 cells included in the ZeroBlunt TOPO kit.
Transformed cells were plated on LB-50 lg/ml kanamicin plates. Positive clones carrying 1000 bp 16S
rDNAs were identified by digestion with EcoRI of
plasmid minipreps and analysis in 1.2% agarose gel
electrophoresis of clones obtained for each library.
A. Escalante et al. / FEMS Microbiology Letters 235 (2004) 273–279
2.4. Amplified 16S rDNA restriction analysis
In order to identify unique 16S rDNA types from
pulque bacterial DNA, ARDRA profiling for each positive clone was performed. To obtain a suitable amount
of 16S rDNA for restriction analysis, 1 ll of 1/10 dilution from each plasmid miniprep was used as template
to perform PCR reactions as described above, but using
the Elongase enzyme mix, according to the supplier
recommendations (Invitrogen). Five to fifteen microliters of each PCR product were digested with restriction
enzyme HaeIII (New England Biolabs Ltd., Hertfordshire, England) at 37 C for 2 h [10]. Restricted 16S
rDNAs were analyzed by horizontal 2.5% agarose gel
electrophoresis. Restriction fragments shorter than 100
pb were not considered for restriction fragment pattern
analysis. Restriction profiles were analyzed using the
1D-Advanced program (AAB Software, Fullerton, CA)
and evaluated for the presence (1) or absence (0) of
bands. A matrix composed by ARDRA profiles from
16S rDNAs from each pulque sample was constructed
and analyzed with the TREECON v1.3b program [11].
The analysis included the calculation of similarity distance for ARDRA data, and the construction of a tree
using the unweighted pair group method with arithmetic
means (UPGMA). The resulting tree led to identify
unique ARDRA profiles corresponding to unique 16S
rDNAs present in pulque samples.
Genetic diversity obtained by ARDRA analysis was
estimated using the Shannon–Weaver diversity index (H)
previously reported to estimate bacterial diversity
[9,12]
P
and calculated from the equation H ¼ ðpi ln½pi Þ,
where pi is the proportion of each ARDRA type (based
on 100%) in relation with the total number of each
ARDRA types in sample and evenness (E), calculated
from the equation E ¼ H = lnðSÞ, where H is the Shannon–Weaver diversity index and S is the number of unique restriction types found in analyzed sample.
2.5. 16S rDNA sequencing, identification and phylogenetic analysis
Partial sequences corresponding to E. coli 16S rDNA
bases 530–1200 of unique ARDRA profiles were determined from PCR templates by the method of Taq FS
Dye Terminator Cycle Fluorescence-Based Sequencing
with a Perkin–Elmer/Applied Biosystems Model 377-18
sequencer. The obtained sequences were submitted to
the non-redundant nucleotide database at GeneBank
using the BLAST program (www.ncbi.nlm.nih.gov) in
order to determine the pulque 16S rDNA clones identity.
Nearest neighbors of pulque 16S rDNA clones matched
in the NCBI database were corroborated in the Sequence_match v 2.7 and possible chimeras were analyzed in the Check_chimera program in the ribosomal
database project (RDP) on line program. A multiple
275
alignment of pulque 16S rDNA clones and reference 16S
rRNA or rDNA sequences retrieved from GenBank
database was performed using the Clustal W program
[13]. A distance matrix calculation of nucleotide substitution rates and a phylogenetic tree was constructed
with the Jukes and Cantor algorithm and the neighborjoining (NJ) method, respectively, using the Treecon
program (v 1.3b) [11]. Bootstrap methods were used to
provide confidence estimated for tree topology in the NJ
method (100 replicates).
3. Results
3.1. Analysis of pulque bacterial diversity by ARDRA
fingerprinting
Direct cloning of PCR amplified 16S rDNA produced
three libraries composed each of 197, 187 and 189 positive clones from DNA isolated from pulque samples
obtained from Aculco, Huitzilac and Tizayuca towns,
respectively. Based on ARDRA fingerprinting analysis
of each 16S rDNA pulque library, 17, 13 and 15 unique
restriction types were detected in libraries from Aculco,
Huitzilac and Tizayuca, respectively. From them, five
groups of clones were detected: those with the same
ARDRA profile present among the three 16S rDNA
libraries (three restriction types designated as common
clones: C1, C2, C7); clones with the same ARDRA
profile but present both in libraries from Aculco and
Tizayuca (AT clones: AT4, AT11, AT13, AT57, AT95,
AT102); clones present only in library from Aculco (A
clones: A14, A18, A49, A60, A86, A191, 192, A258);
only in library from Huitzilac (H clones: H1, H3, H30,
H49, H84, H95, H121, H128, H139, H220, H270) and
clones present only in library from Tizayuca (T clones:
T17, T94, T123, T208, T226).
In order to compare the genotypic diversity based on
ARDRA, the Shannon–Weaver index (H) and evenness
(E) were used. The H value obtained from unique ARDRA types showed that the genotypic diversity in the
sample from Tizayuca (H ¼ 2:03, E ¼ 0:73) was higher
than that observed in Huitzilac (H ¼ 1:36, E ¼ 0:53)
and Aculco (H ¼ 1:23, E ¼ 0:43) samples. As evenness
increases the diversity present in an analyzed sample
increases [9].
3.2. Phylogenetic analysis of 16S rDNA clones
From the three 16S rDNA libraries, 34 unique
ARDRA types were detected and sequenced. Identities
of sequenced clones are shown in Table 2. Analyzed
sequences fell into five major lineages of the Bacteria
domain: Gram-positive bacteria from the Bacillus–Lactobacillus–Streptococcus subdivision, represented by
Lactobacillus acidophilus (126 clones); Lactobacillus
276
A. Escalante et al. / FEMS Microbiology Letters 235 (2004) 273–279
Table 2
Distribution of identified clones in pulque libraries
Clone identitya
No. (%) of clones detected in each 16S rDNA library
Bacillus–Lactobacillus–Streptococcus subdivision
Lactobacillus strain ASF360 AF157050
Aculco
Huitzilac
Tizayuca
87 (44.16)
107 (52.71)
51 (26.98)
Unique ARDRA types sequenced
1
73 (37.05)
6 (3.04)
2 (1.01)
ND
ND
16 (8.12)
8 (4.27)
ND
ND
5 (2.67)
ND
1 (0.53)
45 (23.80)
3 (1.58)
9 (4.76)
ND
1 (0.53)
47 (24.86)
C2, 2 AT4, AT13, AT57, AT102,
A86, A192, 4 H3, H30, H49, H95,
H270, 5 T244
C1, A18, A49, A191, H1
AT95, A14
A60, T17
H128
T226
C7, A258
ND
4 (2.13)
ND
H139
Arthrobacter subdivision
Microbacterium arborescens AB007421
ND
ND
3 (1.58)
T94
Flexibacter–Cytophaga–Bacteroides subdivision
Flavobacterium johnsoniae M59051
ND
ND
2 (1.05)
T208
Proteobacteria a-subdivision
Acetobacter pomorium AJ001632
Gluconobacter oxydans AF127396
Zymomonas mobilis AF281034
ND
ND
ND
4 (2.13)
59 (31.55)
2 (1.06)
ND
ND
2 (1.05)
H220
H84
H121, T123
Proteobacteria b-subdivision
Hafnial alvei Z83203
11 (5.58)
ND
26 (13.75)
3
Lactobacillus acidophilus M99740
L. kefir AB024300
L. acetotolerans M58801
L. hilgardii M58821
L. plantarum D79210
Leuconostoc mesenteroides spp mesenteroides
AB023242
Ln. pseudomesenteroides AB023237
1
2
3
AT11
4
C, common clones; AT, clones present in Aculco and Tizayuca samples; A, clones present only in Aculco; H, clones present only in Huitzilac;
T, clones present only in Tizayuca; ND, Non-detected.
a
16S rDNA or 16S rRNA sequences of organism which showed the highest percent of identity in the output result from analysis in the nonredundant nucleotide database from NCBI with blast program.
5
strain ASF360 (245 clones), L. kefir (11 clones); L.
acetotolerans (11 clones), L. hilgardii (five clones), L.
plantarum (one clone), Leuconostoc mesenteroides subsp.
mesenteroides (64 clones) and Ln. pseudomesenteroides
(1 clone). High G + C Gram positive bacteria, from the
Arthrobacter subdivision represented by Microbacterium
arborescens (three clones); Flexibacter–Cytophaga–Bacteroides subdivision, represented by Flavobacterium
johnsoniae (two clones); Proteobacteria from the a-subdivision represented by Acetobacter pomorium (four
clones), Gluconobacter oxydans (59 clones) and Z. mobilis (four clones) and Proteobacteria from the c- subdivision represented by Hafnia alvei (37 clones).
Distribution of identified clones among the three analyzed libraries is shown in Table 2. The 16S rDNA sequences and those of the identified closest neighbor
organisms in databases were used to construct a phylogenetic tree using the neighbor joining method. Tree
topology revealed that clustering of 16S rDNA clone
sequences from pulque libraries correlated with the
phylogenetic position for those 16S rDNA sequences
considered as references, however, the specific relationships between clones H121, T123 (Z. mobilis), AT11 (H.
alvei) and H84 (G. oxydans) were not resolved in this
analysis (Fig. 1).
4. Discussion
Pulque is the most important traditional non-distilled
alcoholic beverage produced in the central states of
Mexico. The use of an experimental methodology that
does not depend on microbial cultivation was explored
in this work to analyze bacterial diversity in this traditional fermented beverage. By analyzing PCR amplified
16S rDNA sequences, it was determined that several
LAB species were the most abundant in all samples. Of
this group, sequences identified as Lactobacillus strain
ASF360 and L. acidophilus were detected for the first
time in pulque and were the most abundant 16S rDNA
clones present in all samples. Lactobacillus ASF 360 and
L. acidophilus (obligately homofermentative LAB) are
microorganisms associated with the normal gut microbiota in animals and humans [14,15], however, BLAST
analysis in the NCBI server, allowed us to conclude that
Lactobacillus ASF 360 is most closely related to Lactobacillus sp. strain Y10 (data not shown), an isolate initially reported from Japanese malt whisky fermentations
[16]. L. plantarum (facultatively heterofermentative
LAB), L. hilgardii and L. kefir (obligately heterofermentative LAB), were found for first time in pulque.
These last LAB have also been reported as members of
A. Escalante et al. / FEMS Microbiology Letters 235 (2004) 273–279
277
H270 (93%)
88
93
H49 (93%)
93
H30 (95%)
H95 (97%)
A60 (96%)
H3 (96%)
83 AT4 (96%)
AT13 (96%)
C1 (95%)
H1 (96%)
A18 (96%)
T17 (96%)
100 A191 (96%)
A49 (96%)
100 A2 (95%)
A192 (95%)
Lactobacillus acidophilus M99704
Lactobacillus acetotolerans M58801
Lactobacillus acidophillus M58202
90
Lactobacillus sp. ASF360 AF157050
Lactobacillus sp. Y10 AY029223
AT57 (96%)
T244 (98%)
100
100 A102 (94%)
80
A86 (94%)
T226 (95%)
A95 (95%)
H128 (97%)
94 Lactob acillus k efir AB024300
92
Lactobacillus hilgardii M58821
86
A14 (99%)
94
Lactob acillus plantarum D79210
H139 (94%)
100
A258 (96%)
C7 (97%)
Ln. pseudomesenteroides AB023237
94
100 Leuconostoc mesenteroides AB023246
Leuconostoc mesenteroides AB023242
T123 (97%)
H21 (93%)
94
A11 (92%)
Hafnia alvei Z83203
T208 (97%)
100
Flavobacterium johnsoniae M59051
100
100 T94 (98%)
Microbacterium arborescens AB007421
100 Zymomonas mobilis AF281034
Zymomonas mobilis AF088897
Gluconobacter oxydans AF127396
100
H326 (93%)
92
Acetob acter pomorium AJ001632
H84 (93%)
Sulfolobus acidocaldarius (D14053)
Fig. 1. Phylogenetic tree of partial 16S rDNA sequences from pulque clones and partial sequences of closest neighbor 16S rRNA or 16S rDNA from
identified bacteria in the NCBI database. Percent of identity with closest reference 16S rDNA clones in the database (shown in Table 2) is indicated in
parenthesis. GenBank Accession Nos. of reference sequences are indicated. The 16S rDNA sequence of Sulfolobus acidocaldarius served as outgroup.
The percentage of 100 bootstrap resamplings that support each topological element in the neighbor-joining analysis is indicated. No values are given
for groups with bootstrap values less than 80%. The scale indicates genetic distance (0.1). C, common clones; AT, common clones for Aculco and
Tizayuca; A, clones detected only in Aculco; H, clones detected only in Huitzilac; T, clones detected only in Tizayuca. Ln, Leuconostoc.
the normal microbiota present in fermentations carried
out by a mixed yeast/bacterial microbial population
[16,17].
Homofermentative LAB such as Lactobacillus ASF
360 and L. acidophilus, produce lactate from sugars as
the main final metabolic product of the glycolytic
278
A. Escalante et al. / FEMS Microbiology Letters 235 (2004) 273–279
pathway [18]. These organisms were both detected in
fresh pulque (Aculco and Tizayuca) and in a 20 h fermented sample (Huitzilac). Therefore, it is possible to
conclude that the homofermentative lactobacilli species
detected could be the principal producers of lactic acid
in pulque during all the fermentation process. Facultatively heterofermentative LAB such as L. plantarum and
L. acetotolerans as well as obligately heterofermentative
LAB such as L. hilgardii and L. kefir, produce lactic
acid, ethanol, acetic acid and carbon dioxide as major
products, but also diacetyl, acetoin, 2-3-butanediol and
formate in minor proportions depending on the oxidizing potential of the environment [18]. Therefore, it is
possible to speculate that these organisms might have an
impact on pulque off-flavour.
Ln. mesenteroides is a LAB previously reported in
pulque and it has been identified as the microorganism
responsible for the development of viscosity, one of the
distinctive characteristics of pulque [1–4]. 16S rDNA
clones identified as Ln. mesenteroides were detected in
the three analyzed samples. Another EPS producing
bacteria, Lactobacillus kefir was detected in the sample
from Aculco. This microorganism has been also reported as a LAB producer of the capsular polysaccharide kefiran [19].
16S rDNA clones identified as G. oxydans and A.
pomorium were identified only in the pulque sample from
Huitzilac. It has been reported that G. oxidans prefers
sugary rich environments and usually dies off during
alcoholic fermentation due by its low ethanol tolerance.
On the other hand Acetobacter species prefer ethanol as
carbon source and can survive during fermentation and
the following operation of the wine maker process [20].
However, Du Toit and Lamberchts [21] reported that
growth of acetic bacteria in wine fermentations correlated with the initial pH of the must. When must pH was
relatively high (3.76), several acetic bacteria species
counts were even higher at the end of the fermentation
than at beginning, indicating that these bacteria can
grow during alcoholic fermentation. In pulque sample
from Huitzilac, measured pH value was high (3.82)
when compared with observed values reported for must
(3.4–3.76). Its possible that acetic bacteria G. oxydans
observed in pulque from Huitzilac tolerated the relatively high ethanol concentration due to high pH value
of the medium.
Clones identified as the ethanol producing bacteria Z.
mobilis, were detected in samples from Huitzilac and
Tizayuca. This organism is a facultatively anaerobic
bacteria, fermenting sugars to produce ethanol and
carbon dioxide by the Enter-Duodoroff pathway, as well
as other additional products such as lactate, acetate,
hydrogen sulfide, acetaldehyde and dimethyl sulfide
(DMS). This organism is commonly a contaminant in
yeast fermentations where molasses is used as substrate,
such as in rum or beer production [22]. Like Lactoba-
cillus sp., it has been previously proposed that Z. mobilis
is an essential microorganism in the fermentation of
pulque, responsible with yeast for ethanol production [1–
3]. Several strains of Z. mobilis have been previously
isolated from pulque samples and have been reported as
high yield ethanol producers [22].
Seven clone sequences identified tentatively as Lactobacillus strain ASF360 (A86, H49, H270, AT102), Ln.
pseudomesenteroides (H139), Z. mobilis (H21), H. alvei
(AT11) and A. pomorium (H220) exhibited less than 95%
of relatedness to NCBI database sequences. These
clones represent 78 ARDRA types (13.61% of total
clones analyzed in the three 16S rDNA libraries) which
may indicate the presence of new species in pulque
samples.
According to the results obtained in this report, obtained H and E values for genotypic diversity based on
ARDRA profile analysis of the three 16S rDNA libraries studied are in agreement with the high number of
identified 16S rDNA sequences for pulque sample from
Tizayuca town. Identity of 16S rDNA sequenced clones
showed that bacterial diversity present in pulque is
dominated by LAB (80.97% of total 16S rDNA clones).
In order to determine the precise role of this bacterial
group in the fermentation of pulque, it will be necessary
to further study the microbial composition dynamics
and the changes in physicochemical characteristics of
fresh non-inoculated aguamiel and during the fermentation process.
Acknowledgements
We thank Mercedes Enzaldo and Aurelia Ocampo
for skillful technical assistance. To Eugenio L
opez and
Paul Gaytan for primer synthesis and to Jorge Ya~
nez for
16S rDNA sequencing support. We are grateful to Patricia Lappe for assistance on bibliographic information
about pulque and Esperanza Martınez for the critical
reading of this manuscript. This work was supported by
Grants Z-003 and NC-230 from the Consejo Nacional
de Ciencia y Tecnologıa, Mexico.
References
[1] Sanchez-Marroquın, A. (1967) Estudios sobre la microbiologıa del
pulque XX. Proceso industrial para la elaboraci
on tecnica de la
bebida. Rev. Lat. Microbiol. Parasit. 9, 87–90.
[2] Sanchez-Marroquın, A., Larios, C. and y Vierna, L. (1967)
Estudios sobre la microbiologıa del pulque XIX. Elaboraci
on de
la bebida mediante cultivos puros. Rev. Lat. Microbiol. Parasit. 9,
83–85.
[3] Ulloa, M. and Herrera, T. (1976) Estado actual del conocimiento
sobre la microbiologıa de bebidas fermentadas indıgenas de
Mexico: pozol, tesg€
uino, pulque, colonche y tepache. An. Inst.
Biol. UNAM. 47–53, 145–163.
[4] Chellapandian, M., Larios, C., Sanchez-Gonzalez, M. and LopezMunguia, A. (1988) Production and properties of a dextransu-
A. Escalante et al. / FEMS Microbiology Letters 235 (2004) 273–279
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
crase from Leuconostoc mesenteroides IBT-PQ isolated from
‘pulque’, a traditional Aztec alcoholic beverage. J. Ind. Microbiol.
Biotechnol. 21, 51–56.
McDougald, D., Rice, S., Weichart, D. and Kjelleberg, S. (1998)
Nonculturability: adaptation or debilitation. FEMS Microbiol.
Ecol. 25, 1–9.
Farrelly, V., Rainey, F.A. and Stackebrandt, E. (1995) Effect of
genome size and gene copy number on PCR amplification of 16S
rRNA genes from a mixture of bacterial species. Appl. Environ.
Microbiol. 61, 2798–2801.
Suzuki, M.T. and Giovannoni, S.J. (1996) Bias caused by template
annealing in the amplification of mixtures of 16S rDNA by PCR.
Appl. Environ. Microbiol. 62, 625–630.
Escalante, A., Wacher, C. and Farres, A. (2001) Lactic acid
bacterial diversity in the traditional Mexican fermented dough
pozol as determined by 16S rDNA sequence analysis. Int. J. Food
Microbiol. 64, 21–31.
Borneman, J., Skroch, P.W, O’Sullivan, K.M., Palus, J.A.,
Rumjanek, N.G., Jansen, J.L. and Nienhhuis, J. (1996) Molecular
microbial diversity of an agricultural soil in Wisconsin. Appl.
Environ. Microbiol. 62, 1935–1943.
Urakawa, H., Kita-Tusukaoto, K. and Ohwada, K. (1999)
Microbial diversity in marine sediments from Sagami Bay and
Tokyo Bay, Japan, as determined by 16S rRNA gene analysis.
Microbiology 145, 305–3315.
Van dePeer, Y. and De Watcher, R. (1994) TREECON for
Windows: a software package for the construction and drawing of
evolutionary trees for the Microsoft Windows environment.
Comp. Appl. Biosc. 10, 569–570.
Smit, E., Leeflang, P., Sommans, S., van den Broek, J., van Mil, S.
and Wernars, K. (2001) Seasonal fluctuations of the dominant
members of the bacterial soil community in a wheat field as
determined by cultivation and molecular tools. Appl. Environ.
Microbiol. 67, 2284–2291.
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensibility of progressive ultiple sequence
alignment through sequence weighting, positions-specific gap
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
279
penalties and weight matrix chose. Nucleic Acids Res. 22, 4673–
4680.
Favier, C.F., Vaughan, E.E., De Vos, V.M. and Akkermans, D.L.
(2002) Molecular monitoring of succession of bacterial communities in human neonates. Appl. Environ. Microbiol. 68, 219–226.
Dewhirst, F.E., Chien, C.C., Paster, B.J., Ericson, R.L., Orcutt,
R.P., Schauer, D.B. and Fox, J.G. (1999) Phylogeny of the defined
murine microbiota: altered schaedler flora. Appl. Environ.
Microbiol. 65, 3287–3292.
Van Beek, S. and Priest, F.G. (2002) Evolution of the lactic acid
bacterial community during malt whisky fermentation: a polyphasic study. Appl. Environ. Microbiol. 68, 297–305.
Simpson, K.L., Pettersson, B. and Priest, F.G. (2001) Characterization of lactobacilli from Scotch malt whisky distilleries and
description of Lactobacillus ferintoshensis sp. nov., a new species
isolated from malt whisky fermentations. Microbiology 147,
1007–1016.
Liu, S.Q. (2003) Practical implications of lactate and pyruvate
metabolism by lactic acid bacteria in food and beverage fermentations. Int. J. Food Microbiol. 83, 115–131.
Yokoi, H., Watanabe, T., Fujii, Y., Mukai, T., Toba, T. and
Adachi, S. (1991) Some taxonomical characteristics of encapsulated Lactobacillus sp. KPB-167B isolated from kefir grains and
characterization of its extracellular polysaccharide. Int. J. Food
Microbiol. 13, 57–264.
Connolly, C. (1999) Bacterial contaminants and their effects on
alcohol production. In: The Alcohol Textbook. A Reference for
the Beverage, Fuel and Industrial Alcohol Industries (Jaques,
K.A., Lyons, T.P. and Kelsall, D.R., Eds.), pp. 317–334.
Nottingham University Press, Nottingham.
Du Toit, W.J. and Lambrechts, W.J.M.G. (2002) The enumeration and identification of acetic acid bacteria from South African
red wine fermentations. Int. J. Food Microbiol. 74, 57–64.
Favela, E. (1993) Producci
on de alcohol de Zymomonas mobilis.
In: Biotecnologıa Alimentaria (Garcıa-Garibay, M., QuinteroRamırez, R. and Lopez-Munguia, A., Eds.), pp. 617–636.
LIMUSA, Mexico, DF.