Pulque

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
Adelfo Escalante, Mar Elena Rodr
ıa
ıguez, Alfredo Mart
ınez, Agust Lopez-Mungu
ın 
ıa,
*
Francisco Bol
ıvar, Guillermo Gosset
Departamento de Ingenier Celular yBiocatlisis, Instituto de Biotecnolog Universidad Nacional Autnoma de Mxico, Apdo. Postal 510-3,
ıa
a
ıa,
o
e
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 rDNAclone 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 rDNAsequenced 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.
Ó 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
*

Correspondingauthor. Tel.: +52-7773-291601; fax: +52-7773172388.
E-mail address: gosset@ibt.unam.mx (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 hasbeen 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 occurringin 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 Ó 2004 Federation of European Microbiological Societies.Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.04.045

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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...