Research Article

Received: 20 March 2019 Revised: 27 June 2019 Accepted article published: 1 July 2019 Published online in Wiley Online Library: 27 July 2019

(wileyonlinelibrary.com) DOI 10.1002/jsfa.9907

Influence of fermentation by different

microflora consortia on pulque and pulque
bread properties
Maciej Kuligowski,a* Ricardo López Otero,b Katarzyna Polanowska,a,c
Didier Montet,c Iwona Jasińska-Kuligowskad and Jacek Nowaka

Abstract
BACKGROUND: Pulque bread is a traditional Mexican product obtained by fermentation using microflora present only in
pulque. In this study, the possibility of creating a pulque microbial consortium under laboratory conditions and its applications
were evaluated. A laboratory-made consortium was compared with a consortium originating in Mexico in bread and pulque
production. They were tested in various growth medium systems: pulque made from agave sap and malt extract, Mexican wheat
and rye pulque bread, and European wheat and rye bread.

RESULTS: Depending on the growth medium, consortiums showed differing influence on many factors, such as specific volume,
weight loss after baking, soluble proteins, and crust and crumb color. Indigenous starters increased sensorial acceptance of
pulque and Mexican rye bread, decreased pH, and increased titratable acidity of the breads at the highest level whereas
laboratory consortia improved sensory acceptance of wheat breads. The laboratory-prepared starter in some cases improved
antiradical activity. All pulques received similar consumer evaluations. However, malt pulque was the least appreciated
beverage.

CONCLUSION: The results show the possibility of creating a pulque microbial consortium under laboratory conditions. Depend-
ing on the flour type and the breadmaking technique, the use of a particular microbial consortium allowed modification of
certain physicochemical parameters. In conclusion, it is feasible to modify bread parameters to obtain features corresponding
to consumer demands by using an appropriate microflora, pulque, or flour type. Moreover, this research describes, for the first
time, the use of rye malt for pulque and rye flour for pulque bread preparation as raw materials.
© 2019 Society of Chemical Industry

Keywords: pulque; pulque bread; antioxidant activity; sensory evaluation

INTRODUCTION The other low alcoholic fermented beverage popular in East-

Bread is one of the main cereal foods consumed all over the
world. However, the production methods and ingredients vary
markedly, depending on the region and on tradition. The recipes
have changed over centuries with culture transfer. In terms of the
unique microflora used in the bread preparation, pulque bread
is an interesting example, although it is not popular in European
countries. This kind of bread is a traditional Mexican product
obtained by fermentation using microflora present in the Mexican
drink, pulque, or its sediment, called xaxtle.1
Pulque is a traditional fermented beverage originating in Mex-
ico. The saps of several agave species are used for its preparation.2
Agave sap is a rich source of sugars, especially fructans, which
exhibit prebiotic activity.3 Pulque consumption was proved to
have many health benefits, such as reduced risk of low ferritin and
hemoglobin values in Mexican women. Moreover, it was described
as a good source of ascorbic acid, thiamin, riboflavin, iron,4 and
of anti-infective3 and anti-inflammatory probiotics.5 The probiotic
effect is ensured by significant lactic acid bacteria content, which
ranges from 7.7 to 11.3 log CFU mL−1 . Other microorganisms
ern European countries is kvass. It has a golden-brown color, a
pleasant aroma of rye bread, and appropriate sweetness. The kvass
microflora differ slightly from those used for pulque production
and consists of yeast and lactic acid bacteria. They are tradition-
ally made from rye malt, rye flour, and stale rye bread. However,
∗ Correspondence to: M Kuligowski, Faculty of Food Science and Nutrition,
Poznań University of Life Sciences, Poznań, Poland.
E-mail: maciej.kuligowski@up.poznan.pl
a Faculty of Food Science and Nutrition, Poznań University of Life Sciences,
Poznań, Poland
b La Universidad Autónoma del Estado de México, Instituto Literario 100, Colonia
Centro, Toluca de Lerdo, Mexico
c Qualisud, Univ Montpellier, CIRAD, Montpellier SupAgro, Univ d’Avignon, Univ
de La Réunion, Montpellier, France
d Department of Food Commodity, Faculty of Commodity Sciences, Poznań
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found in pulque were Saccharomyces and Zymomonas mobilis.1,2,6 University of Economics and Business, Poznań, Poland

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nowadays it is becoming more common to use rye malt or extracts
from other cereals.7
Beside the sources from indigenous cultures for fermentation,
mentioned above, pulque bread was described as a mixture of
wheat flour, milk, eggs, margarine sugar, and salt.8 In central
Europe, wheat bread is produced with flour, water and yeast
by a one-phase or two-phase method.9 Rye bread production
involves even more phases of sourdough fermentation from 3
to 5.10 Rye bread is receiving more attention due to its high
nutritional value – especially the fiber content and longer shelf life
compared to wheat bread. Incorporation of rye flour into different
kinds of bread may positively influence the nutritional and sensory
aspects.11
There is little information in the scientific literature about typical
pulque bread but the available evidence proves its strong poten-
tial as a functional food. For example, Torres-Maravilla et al. used
xaxtle as an inoculum for dough fermentation and reported that
the baked products had a lower glycemic index.1 Other authors
found a high concentration of several prebiotic compounds in
this type of bread.8 The fermentation of traditional pulque is per-
formed by indigenous microorganisms associated with the sap of
the agave plant. There are no reports describing attempts to cre-
ate such a microbial mixture in industrial or laboratory conditions.
The aim of the study was therefore to create the microbial starter in
laboratory conditions similar to the indigenous Mexican microbial
consortium used for pulque production. In our study this microbial
starter was obtained by mixing the microorganisms characteristic
of pulque in defined proportions. To verify its suitability for pulque
and pulque bread production some physicochemical aspects were
evaluated. The influence of the microbial consortia was tested in
several research systems using two types of pulque and four dif-
ferent methods of breadmaking. The suitability of using rye malt
for pulque production and rye flour for traditional Mexican pulque
bread preparation were also tested.
MATERIALS AND METHODS
Microflora
The indigenous starter (IS) was pulque sediment obtained from a
pulquería located in Toluca City, Mexico.
The laboratory-prepared microbial consortium (LP) was made
from yeast Saccharomyces cerevisiae (Babuni, Wołczyn, Poland), Z.
mobilis (CD-BB-603, Mexican Culture Collection), and Lactobacillus
casei Shirota (Poznań University of Life Sciences Culture Collec-
tion).
Materials
The following bread ingredients were used: wholemeal rye flour
(type 2000), wheat flour (type 750) (Komplexmłyn, Wa̧growiec,
Poland), sucrose (Diamant, Poland), Saccharomyces cerevisiae
(Babuni, Poland), butter (Gostyńskie, Poland), salt (Kłodawa,
Poland), rye malt extract (Uldo, Poland), and agave syrup (Allos,
Mexico).
Pulque of the El Tlaquiche brand (TL) was obtained from Ocoyoa-
cac, Mexico, and the Llanos de Apan brand (LL) from Puebla, Mex-
ico.
Chemicals and reagents
2,2′ -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammo-
6308
nium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carbo-
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M Kuligowski et al.
xylic acid (Trolox), chloramphenicol, and cycloheximide were from
Sigma-Aldrich (St Louis, MO, USA).
De Man, Rogosa and Sharpe (MRS) broth and potato dextrose
agar (PDA) were from BTL (Poland).
METHODS
Pulque preparation
Pulque was prepared by dissolving rye malt extract (malt
pulque – MP) or agave syrup (agave pulque – AP) in water to
achieve 120 g of sugar in 1 kg of liquid and fermenting at 25 ∘ C
for 13 days using either the indigenous starter (IS) or laboratory-
prepared microbial consortium (LP).
Bread preparation
Recipe 1 (R1) consisted of flour (500 g) (wheat – WR1, rye – RR1),
sucrose (65 g), salt (5 g), butter (65 g), yeast (20 g) and pulque
(200 mL). The dough was kneaded and incubated at 30 ∘ C for
70 min. The dough was then portioned and held at 30 ∘ C for
35 min, before being baked at 180 ∘ C for 8 min. Different types
of pulque were added to the breads: agave pulque and malt
pulque fermented by indigenous starter (IS-AP and IS-MP), and
agave pulque and malt pulque fermented by laboratory-prepared
microbial consortium (LP-AP and LP-MP).
Recipe 2 consisted of wheat flour (WR2) (350 g), salt (5 g), yeast
(10.4 g), and pulque (200 mL). After kneading, the dough was
held at 26 ∘ C for 90 min, portioned, and incubated for 35 min at
30 ∘ C, before baking for 20 min at 180 ∘ C. Different pulques were
added to the breads: agave pulque and malt pulque fermented
by indigenous starter (IS-AP and IS-MP), agave pulque and malt
pulque fermented by laboratory-prepared microbial consortium
(LP-AP and LP-MP).
Recipe 3 (RR3) was performed using a three-stage method.
First, rye flour (120 g), pulque (200 mL), and yeast (20 g) were
mixed, kneaded, and held at 20 ∘ C for 16 h. Next, 480 g of rye
flour and 450 mL of pulque were added, mixed well, and left at
30 ∘ C for 3 h. Then rye flour (350 g) and pulque (260 mL) were
added, kneaded, portioned, and incubated at 35 ∘ C for 30 min.
The bread was baked in the oven at 200 ∘ C for 35 min. Different
pulques were added to the breads: agave pulque and malt pulque
fermented by indigenous starter (IS-AP and IS-MP), agave pulque,
and malt pulque fermented by a laboratory-prepared microbial
starter (LP-AP and LP-MP).
Recipe 1 (R1) and recipe 2 (R2) are based on semi-methods, and
recipe 3 (R3) on an indirect method of bread making. The blank
samples were prepared with water replacing the pulque addition
(recipe 1: WR1-B with wheat flour, RR1-B with rye flour; recipe 2:
WR2-B, recipe 3: RR3-B). The procedures of the bread making are
explained in Table 1.1
Microbiological analysis
Zymomonas mobilis and L. casei Shirota were separately cultured in
MRS broth.
The MRS medium was mixed with cycloheximide, and agar was
applied to determine changes in the bacterial population. Potato
dextrose agar with chloramphenicol was used to determine the
quantity of yeast.
Bacteria and yeast in the pulque sediment underwent sequence
analysis using polymerase chain reaction denaturing gradient gel
electrophoresis (PCR-DGGE).12
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Influence of fermentation by different microflora consortia on pulque
Table 1. The procedures of the bread making
With water With malt pulque
Type of flour Control sample Indigenous starter
Mexican recipea
wheat WR1-B IS-MP-WR1
rye RR1-B IS-MP-RR1
European recipea
wheat WR2-B IS-MP-WR2
rye RR3-B IS-MP-RR3
a The breads were made in three independent replications.
Abbreviations: IS, indigenous starter; LP, laboratory-prepared microbial consortium; MP, malt pulque; AP, agave pulque; WR1, recipe 1, wheat flour;
RR1, recipe 1, rye flour; WR2, recipe 2, wheat flour; RR3, recipe 3, rye flour; B, blank sample, water replacing the pulque addition.
pH and titratable acidity of bread
Within 24 h after baking, 5 g of each bread was crushed, placed
into Erlenmeyer flasks, and shaken for 50 min with 100 mL of
distilled water. The suspensions were filtered and supernatants
were collected. The pH was measured using a pH meter, and
the extracts were titrated with a 0.1 mol L−1 NaOH solution until
pH 7.10
Specific volume and weight loss of bread
The weight difference was used to determine the weight loss dur-
ing baking. The portioned bread loaves were weighed immedi-
ately before being placed inside the oven, after baking, and 1 h
later. The specific volume was determined using the rapeseed
replacement method.13 The specific volume (cm3 g−1 ) was calcu-
lated as the ratio of the volume (cm3 ) to the mass of the bread (g).14
Bread color analysis
The colors of the crust and crumb of the breads were measured
using a Konica Minolta CM-3600d spectrophotometer (Konica
Minolta Holdings, Japan). The results were expressed using CIE L*,
a*, b* color system coordinates. Measurements were performed
using a D65 illuminant, a 25.4 mm diameter measurement hole,
ten standard observers, and an specular component excluded
measurement model. The results represent the means of six mea-
surements at different points of the bread crust and crumb. The
color difference between the samples was calculated according to
Eqn (1):
√
ΔE = (L1 − L2 )2 + (a1 − a2 )2 + (b1 − b2 )2
Protein content
One gram of lyophilized and powdered sample of bread or pulque
was mixed on a roller–shaker for 30 min in test tubes with 10 mL of
0.5 mol L−1 NaCl. The mixture was then filtered using filter papers
(𝜙 320 mm) into a volumetric flask and topped up to 10 mL. The
protein content of the extracts was determined using the Bradford
method15 with thaumatin as the protein standard.
Antiradical activity
Antiradical activity was measured using the ABTS method16 in
extracts prepared in the same way as for protein analysis and
expressed as Trolox equivalents.
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With agave pulque
Laboratory consortium Indigenous starter Laboratory consortium
LP-MP-WR1 IS-AP-WR1 LP-AP-WR1
LP-MP-RR1 IS-AP-RR1 LP-AP-RR1
LP-MP-WR2 IS-AP-WR2 LP-AP-WR2
LP-MP-RR3 IS-AP-RR3 LP-AP-RR3
Sensory evaluation
A hedonic test was used to determine the color, taste, aroma,
and texture of breads based on the degree of liking or disliking,
according to a ten-point hedonic scale (1: dislike extremely, 10:
like extremely).17 For the pulque evaluation, the participants were
asked to use the preference method and ten-point hedonic scale.
The consumers group consisted of 120 people (20–40 years old).
Statistical analysis
All the experiments were performed independently in at least
triplicate. Statistical analysis was carried out using Statistica 12
(StatSoft software). Tukey’s multiple means comparison test was
used to verify differences between the samples. P < 0.05 was set
as the criterion for significance.
RESULTS
Microbiological analysis
The yeast concentration at the beginning of fermentation using
the indigenous starter (IS) was 4.67 log CFU mL−1 , whereas the
bacteria numbered 3.64 log CFU mL−1 . The maximum number of
microorganisms was obtained on the third day of incubation in the
malt extract medium (Table 2). Growth on the agave medium was
weaker than on the malt extract for the 13 days of fermentation. On
day 13, the laboratory microbial consortium (LP) had a yeast con-
centration similar to that of the pulque fermented with the IS, but
the number of bacteria was lower by 3.88 and 1.68 log CFU mL−1 in
the agave syrup and rye malt extract, respectively. In all cases the
yeast outnumbered the bacteria.
(1) Analysis of the 16S rRNA and 26S rRNA profiles of bacteria
and yeast communities from the indigenous starter showed that
the closest matching sequences corresponded to Bacillus subtilis
(100% similarity), Lactobacillus spp. (buchneri, kefiri, otakinesis:
97%), Leuconostoc pseudomesenteroides (95%), Saccharomyces
spp. (paradoxus, cariocanus, mikatae, cerevisiae: 95%), and Z.
mobilis (94%).
pH and titratable acidity of bread
The use of different starters in the same bread recipe did not result
in any differences in the pH of the breads in the case of MP-WR1,
AP-RR1, AP-WR2 and AP-RR3. However, the indigenous starter (IS)
led to a significant pH reduction in the breads made according to
the AP-WR1, MP-RR1, MP-WR2, and MP-RR3 recipes.
The breads prepared without pulque – WR1-B with wheat flour,
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RR1-B with rye flour; recipe 2: WR2-B, recipe 3: RR3-B (henceforth
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Table 2. Viable cell counts of microorganisms, variation of amount of proteins, antiradical activity in pulque, and the consumers’ preferences

Log CFU mL−1a

Productb Fermentation time Bacteria Yeast Protein mg L−1a TROLOX mg L−1a Consumers preference

IS-AP 0 3.6 ± 0.3E 4.7 ± 0.3D 37 ± 5E 534 ± 12D -

IS-MP 0 3.6 ± 0.3E 4.7 ± 0.3D 1492 ± 28C 1093 ± 32B -
IS-AP 3 5.1 ± 0.4C 7.3 ± 0.7B 47 ± 7E 544 ± 15D -
IS-MP 3 9.2 ± 0.3A 9.7 ± 0.5A 1572 ± 50B 1091 ± 16B -
IS-AP 7 6.2 ± 0.6C 7.5 ± 0.6B 71 ± 9E 542 ± 15D -
IS-MP 7 8.4 ± 0.2B 8.6 ± 0.1B 1439 ± 46C 1094 ± 9B -
IS-AP 13 8.1 ± 0.6B 7.6 ± 0.6B 68 ± 12E 542 ± 13D 6.9
IS-MP 13 8.2 ± 0.6B 8.6 ± 0.3B 1329 ± 48D 1258 ± 49A 4
LP-AP 13 4.2 ± 0.3D 6.5 ± 0.5B 50 ± 8E 534 ± 10D 6.5
LP-MP 13 6.5 ± 0.1C 7.2 ± 0.6B 1649 ± 34A 913 ± 26C 3.9
LL - 0.0 0.0 187 ± 20E 591 ± 10E 6.4
TL - 0.0 0.0 221 ± 15E 463 ± 10E 5.6
a Values (means ± SDs) bearing the same superscripts in each column are not statistically significantly different (P < 0.05). The tests were done in
triplicates.
SDs, standard deviations; b IS, indigenous starter; LP, laboratory-prepared microbial consortium; MP, malt pulque; AP, agave pulque; LL, commercial
pulque Llanos de Apan brand from Puebla, Mexico; TL, commercial pulque El Tlaquiche brand from Ocoyoacac, Mexico.

the ‘blank samples’) – showed a higher pH and the lowest titrat-
able acidity values (Table 3).
The type of microflora used for fermentation had no specific
effect on the titratable acid value, but in the case of AP-WR1,
MP-WR2, and MP-RR3, the indigenous starter led to a significant
increase in this parameter.
Volume and weight loss of bread
The effect of the different starters on the specific volumes was
observed only in breads made with recipe 1 (WR1), whereas the
use of LP decreased this parameter (Table 3). A similar dependence
was observed for RR1, although the differences in this case were
not statistically significant. The effect of the fermented pulque
starter on bread volume ranged from 2.4% (AP-WR2) to 17.8%
(MP-WR1).
The addition of the laboratory-prepared microbial consortium
resulted in a weight loss reduction after baking in the case of WR1
and MP-RR3, and in a weight loss increase in the case of WR2
(Table 3). The changes in weight loss after baking caused by the
LP starter (using the same recipe in each case) ranged from 0.03%
(AP-RR3) to 1.83% (MP-RR3).
The indigenous starter led to a reduction in the loss of bread
mass, as evaluated 1 h after baking (MP-RR1, WR2, RR3), although
in the case of recipe WR1 this effect was observed for the
laboratory-prepared microbial consortium. One hour after baking,
the percentage differences in weight loss in the breads ranged
from 0.23% (MP-WR1) to 2.68% (MP-RR3).
Bread color analysis
The results in Table 4 show that type of microbial starter did not
affect the crusts’ L*, a*, b* color system coordinates. However, color
differences between the samples (ΔE) caused by the starter were
observed. In the case of samples made using the AP-WR1, WR2,
and R3 recipes, the laboratory-prepared microbial consortium led
to greater differences as compared with the blank samples in the
crusts. However, in the case of the MP-WR1 and RR1 recipes, this
6310
In the crumbs made according to the same recipe, the L* param-
eter was predominantly higher in bread prepared with indigenous
starter (Table 4), although the difference was in some cases not sta-
tistically significant.
The indigenous starter significantly increased the intensity of red
(parameter a) and yellow (parameter b) of MP-WR2 and MP-RR3.
In turn, the laboratory-prepared microbial consortium caused a
significant increase in the red intensity of MP-WR1 and in the
yellow of AP-WR1 and AP-RR1.
Greater color differences (ΔE) occurred in breads made with
the laboratory-prepared microbial consortium using the MP-RR1,
WR2, and MP-RR3 recipes. When considering the applied starters,
WR1 showed the smallest changes in bread color.
Soluble protein content
The soluble protein content of the pulque was the highest on
the 13th day of fermentation in the case of the rye malt extract
(Table 2). The effect of the starter on protein levels depended
on the growth medium. Considering the rye malt extract, the
highest amounts of soluble protein was obtained by the use of
the laboratory-prepared microbial consortium. The opposite effect
was observed in pulque from agave syrup. Pulque made from
rye malt extract had at least six times greater protein content
than commercial pulque or pulque made from fermented agave
syrup.
Generally, the use of the LP decreased soluble protein in the
breads, although sometimes these differences were not statis-
tically significant. However, in two cases (AP-RR1 and MP-RR3),
the use of IS resulted in a decrease in protein level. Differences
in the soluble protein amount in breads with the addition of a
pulque fermented by both microbial consortiums ranged from
5.2% (MP-WR1) to 56.4% (AP-WR2) (Table 3).
Antiradical activity
During pulque fermentation, no effect of microflora growth on
antiradical activity in the agave syrup medium was observed

happened when the indigenous starter was used. (Table 2). The influence of microbial starter on antiradical activity

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Table 3. The properties of prepared breads (pH, titratable acidity, specific volume, loss weight after baking, amount of protein, antiradical activity)

Titratable Specific volume Weight loss Baking weight loss Protein TROLOX
Product pHa aciditya (cm3 g−1 )a after baking (%)a after 1 h (%)a (g kg−1 DM)a (g kg−1 DM)a

WR1-B 5.7 ± 0.1AB 0.4 ± 0.1F 4.85 ± 0.35A 5.8 ± 0.1BC 7.5 ± 0.2AB 1.66 ± 0.08C 5.44 ± 0.05B
IS-MP-WR1 5.2 ± 0.1CD 0.8 ± 0.1D 5.21 ± 0.50A 5.7 ± 0.1BC 7.0 ± 0.1AB 1.73 ± 0.05C 4.63 ± 0.03D
LP-MP-WR1 5.0 ± 0.1D 1.0 ± 0.1D 4.27 ± 0.45BC 5.2 ± 0.2B 6.7 ± 0.2A 1.64 ± 0.10C 4.63 ± 0.06D
IS-AP-WR1 5.1 ± 0.1D 0.9 ± 0.1D 4.70 ± 0.81AB 6.9 ± 0.2D 8.1 ± 0.2B 2.02 ± 0.17AB 5.40 ± 0.06B
LP-AP-WR1 5.5 ± 0.1B 0.6 ± 0.1E 4.08 ± 0.63C 5.6 ± 0.9C 6.7 ± 0.1A 1.90 ± 0.06AB 5.40 ± 0.03B
RR1-B 6.0 ± 0.1A 0.9 ± 0.1D 1.48 ± 0.13E 9.0 ± 0.3F 11.9 ± 0.5C 1.61 ± 0.04C 4.61 ± 0.08D
IS-MP-RR1 5.3 ± 0.1C 1.9 ± 0.2C 1.51 ± 0.15E 9.3 ± 0.3F 11.7 ± 0.1C 1.81 ± 0.09B 3.98 ± 0.08F
LP-MP-RR1 5.6 ± 0.1B 1.6 ± 0.1C 1.43 ± 0.14E 10.2 ± 0.3F 12.9 ± 0.6C 1.61 ± 0.06C 4.03 ± 0.06F
IS-AP-RR1 5.8 ± 0.1A 1.0 ± 0.1D 1.55 ± 0.13E 9.9 ± 0.3F 12.2 ± 0.3C 1.29 ± 0.11D 4.33 ± 0.08E
LP-AP-RR1 5.8 ± 0.1A 0.9 ± 0.1D 1.39 ± 0.16E 9.1 ± 0.4F 11.7 ± 0.3C 1.52 ± 0.12C 4.63 ± 0.05D
WR2-B 5.8 ± 0.1A 0.4 ± 0.1F 2.62 ± 0.04D 4.3 ± 0.2A 6.9 ± 0.1AB 1.18 ± 0.12D 5.54 ± 0.05A
IS-MP-WR2 4.7 ± 0.2E 1.8 ± 0.1C 2.52 ± 0.04D 8.1 ± 0.4E 9.5 ± 0.8B 1.18 ± 0.10D 4.84 ± 0.10CD
LP-MP-WR2 5.3 ± 0.1C 0.9 ± 0.1D 2.74 ± 0.12D 9.0 ± 0.2F 10.3 ± 0.5B 1.03 ± 0.11DE 4.63 ± 0.11D
IS-AP-WR2 5.3 ± 0.1C 0.6 ± 0.1E 2.72 ± 0.04D 7.2 ± 0.2D 8.8 ± 0.6B 199.20 ± 15.30AB 5.40 ± 0.07B
LP-AP-WR2 5.3 ± 0.1C 0.6 ± 0.1E 2.73 ± 0.07D 8.8 ± 0.3F 10.4 ± 0.2B 0.87 ± 0.19E 5.32 ± 0.10B
RR3-B 5.8 ± 0.1A 0.9 ± 0.1D 1.39 ± 0.08E 14.9 ± 0.5I 15.7 ± 0.3DE 0.84 ± 0.11E 5.04 ± 0.09C
IS-MP-RR3 4.8 ± 0.2E 4.0 ± 0.2A 1.55 ± 0.06E 13.4 ± 0.4H 13.7 ± 0.6D 0.80 ± 0.11E 4.47 ± 0.07DE
LP-MP-RR3 5.1 ± 0.1D 2.9 ± 0.1B 1.62 ± 0.10E 11.6 ± 0.5G 16.4 ± 1.0E 0.88 ± 0.06E 4.26 ± 0.09E
IS-AP-RR3 5.2 ± 0.1CD 1.9 ± 0.2C 1.27 ± 0.09E 14.8 ± 0.5I 15.8 ± 1.0DE 1.17 ± 0.08D 4.61 ± 0.05D
LP-AP-RR3 5.2 ± 0.1CD 1.7 ± 0.2C 1.38 ± 0.04E 14.8 ± 0.5I 17.9 ± 1.1E 0.76 ± 0.09E 4.92 ± 0.11C
a Values (means ± SDs) bearing the same superscripts in each column are not statistically significantly different (P < 0.05). The tests were done in
triplicates.
Abbreviations: SDs, standard deviations; DM, dry mass.

was observed when the growth medium was prepared from
malt extract. On the 13th fermentation day, the indigenous
starter increased the antiradical activity by 27% compared to
laboratory-prepared microbial consortium.
The antiradical properties of MP were twice as high as agave
pulque. The agave pulque prepared showed a similar level of
antiradical activity to commercial pulque (LL and TL). No effect
on this parameter was observed when considering the kind of
microbial starter used for fermentation of pulque, which was then
added to breads (Table 3).
The antiradical activity slightly increased in the case of MP-WR2
and MP-RR3 pulque with the indigenous starter. However, the LP
significantly increased the antiradical activity by 6% in AP-RR1 and
AP-RR3.
Consumer sensory evaluation
The parameters evaluated by sensory evaluation were aroma,
color, taste, and texture. The use of the laboratory-prepared micro-
bial consortium for pulque fermentation, which was in turn used as
a bread additive, resulted in statistically higher consumer assess-
ment results in the case of the WR1 recipe (Table 5). The oppo-
site situation – though often without statistical significance – was
observed in the case of the RR1 recipe, where the products made
with the indigenous starter were rated higher. Partially statistically
significant increases in the color and smell scores were noted with
the use of the laboratory-prepared microbial consortium in RR2
and AP-RR3.
There was a similar perception of commercial pulque brands
(TL and LL) and pulque made from agave (Table 2). The malt
indigenous starters were given higher evaluations for the same
type of pulque.
DISCUSSION
Pulque microflora have been described as containing mainly Lac-
tobacillus spp., S. cerevisiae, Z. mobilis, and L. mesenteroides.2,5
Some other specific strains have also been isolated from this
medium, such as Kluyveromyces marxianus,18 L. mesenteroides,3
and Leuconostoc kimchi.19 Valadez-Blanco et al.6 reported the pres-
ence of lactic acid bacteria in quantities ranging from 6 × 107 to
2 × 1011 CFU mL−1 . The pulques produced with IS were thus typi-
cal in terms of the quantity of bacteria and the type of microflora.
Interestingly, in our study we reported B. subtilis for the first time
alongside the typical microflora in the Mexican pulque sediment.
Pulque is described as a thick drink with a greater than usual
density. This is usually attributed to L. mesenteroides, a creator of
exopolysaccharides. However, it could also be caused by B. subtilis
activity. Up to this time, in the pulques only Bacillus licheniformis
was described from Bacillus sp.20
The microflora of our laboratory-prepared microbial consortium
was similar to typical pulque – for example, it contained S. cere-
visiae, Z. mobilis and L. casei Shirota. Both starters were used to
ferment pulque, which was then added to bread. Recipe 1 was a
typical Mexican recipe for pulque bread, for which wheat was used
(WR1). In the RR1 variant, wheat was replaced by rye flour, which
has not been described so far. WR2 and RR3 are typical breads of
central Europe with wheat and rye flours, respectively.
The longer fermentation time of European rye bread caused a
lowering of the pH level and an increase in buffering capacity.
6311

pulque was the least appreciated beverage. Pulques obtained with The acidifying bacteria could multiply for longer but the effect

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www.soci.org M Kuligowski et al.

Table 4. Results of the bread color analysis

Crumb Crust
Product La aa ba ΔEb La aa ba ΔEb

WR1-B 74.3 ± 0.4B 1.4 ± 0.1J 22.2 ± 1.1BC 64.6 ± 4.7AB 10.7 ± 1.7ABCD 30.7 ± 1.8A
IS-MP-WR1 43.3 ± 0.4EF 10.3 ± 0.3C 23.2 ± 0.9AB 32.3 43.9 ± 1.5CD 11.8 ± 1.0ABC 22.4 ± 0.8C 22.3
LP-MP-WR1 42.9 ± 0.6EF 10.9 ± 0.2B 23.4 ± 0.5A 32.7 47.1 ± 1.6CD 12.8 ± 0.7AB 24.4 ± 0.6BC 18.7
IS-AP-WR1 76.1 ± 0.6A 1.3 ± 0.1J 20.1 ± 0.6E 2.8 66.7 ± 5.9AB 8.9 ± 1.3BCDE 30.8 ± 2.0A 2.8
LP-AP-WR1 76.2 ± 0.8A 1.4 ± 0.1J 21.4 ± 0.3CD 2.1 69.1 ± 5.1A 8.4 ± 3.1BCDE 30.3 ± 2.5A 5.1
RR1-B 42.4 ± 1.0EFG 12.3 ± 0.8A 14.8 ± 0.4HI 45.0 ± 4.1CD 15.4 ± 1.0A 18.2 ± 2.4D
IS-MP-RR1 29.5 ± 0.6I 7.4 ± 0.1F 9.9 ± 0.1J 14.7 38.3 ± 3.2D 6.8 ± 0.1CDE 13.5 ± 0.2DEFG 11.8
LP-MP-RR1 23.5 ± 0.6K 7.7 ± 0.3F 9.2 ± 0.3J 20.3 37.8 ± 2.0D 7.9 ± 1.1BCDE 14.1 ± 0.6DEFG 11.2
IS-AP-RR1 46.2 ± 0.8D 5.3 ± 0.1I 15.5 ± 0.3H 8.0 55.4 ± 1.1 BC 4.4 ± 0.4F 15.5 ± 2.7DEF 15.4
LP-AP-RR1 42.7 ± 0.4EF 5.4 ± 0.1I 17.1 ± 0.4FG 7.2 48.2 ± 2.2CD 5.4 ± 0.8EF 15.2 ± 0.3DEF 10.9
WR2-B 71.1 ± 1.8C −0.2 ± 0.0K 20.5 ± 0.1DE 67.5 ± 1.7AB 5.7 ± 1.2DEF 28.1 ± 1.8AB
IS-MP-WR2 37.6 ± 0.6H 9.7 ± 0.1D 21.1 ± 0.2CDE 34.9 40.1 ± 4.6CD 10.2 ± 2.0BCDE 15.9 ± 1.0DE 30.3
LP-MP-WR2 35.7 ± 0.8H 8.9 ± 0.2E 17.8 ± 0.6F 36.7 38.4 ± 1.9CD 9.9 ± 0.8BCDE 15.4 ± 2.0DEF 32.0
IS-AP-WR2 72.0 ± 0.3C −0.6 ± 0.1K 20.4 ± 0.3DE 1.00 48.8 ± 2.8CD 13.5 ± 2.4AB 25.1 ± 4.6BC 20.5
LP-AP-WR2 74.4 ± 0.4B −0.7 ± 0.0K 20.3 ± 0.2DE 3.3 48.5 ± 2.8CD 13.5 ± 4.2AB 24.4 ± 4.2BC 20.9
RR3-B 40.6 ± 0.8G 5.2 ± 0.2I 13.7 ± 0.8I 47.0 ± 9.8CD 8.2 ± 3.4BCDE 16.7 ± 3.0DE
IS-MP-RR3 25.6 ± 1.7J 7.0 ± 0.6G 9.0 ± 0.6J 15.8 35.0 ± 5.9D 5.9 ± 0.4DE 9.6 ± 3.4EF 14.1
LP-MP-RR3 19.5 ± 0.9L 6.3 ± 0.2H 7.0 ± 0.7K 22.2 30.8 ± 1.0D 6.1 ± 1.2DE 8.9 ± 2.5F 18.1
IS-AP-RR3 43.7 ± 1.4E 6.0 ± 0.2H 15.7 ± 0.5GH 3.8 40.5 ± 7.1CD 6.6 ± 0.9DE 12.6 ± 3.4DEF 7.9
LP-AP-RR3 41.7 ± 0.1FG 6.2 ± 0.0H 16.2 ± 0.2FGH 2.9 33.8 ± 4.3D 8.7 ± 2.8BCDE 12.2 ± 2.2DEF 14.0
a Values (means ± SDs) bearing the same superscripts in each column are not statistically significantly different (P < 0.05). The tests were done in ten
repetitions.
Abbreviations: SDs, standard deviations; L*, lightness of the sample (0 = black. 100 = white); a*, redness when positive or greenness when negative;
b*, yellowness when positive or blueness when negative; b ΔE, the difference between colors.

of their activity was not greater. However, the samples without
pulque (blank samples) were always characterized by higher pH
and lower titratable acidity. The lowest pH was observed when
using pulque with the indigenous starter in AP-WR1, MP-RR1,
MP-WR2, and MP-RR3. This might be due to the higher quantity of
bacteria in the IS pulque. That the lowest pH and the highest TTA
were found in breads with the addition of pulque – especially with
the IS-MP addition – suggests that these products might have a
longer shelf life. The higher concentrations of organic acids exhibit
antimicrobial activity and are effective against rope spoilage; they
may thus be responsible for the extended shelf life of sourdough
breads.21
The pH of prepared wheat breads was similar to that described
by Zhang et al.22 in bread supplemented with Lactobacillus plan-
tarum. In the case of traditional Italian wheat bread technology,
sourdough was propagated for 11 days to obtain a pH of 4.24–4.31
and a total titratable acidity of 8.4–9.2 mL.23 The total titratable
acidity for modern Polish rye bread is on the level of 5.2,10 compa-
rable to the data presented in Table 3. The microflora from pulque
usually altered the pH of rye breads in a way similar to central Euro-
pean rye sourdoughs.
Vernon-Carter et al.24 found that incorporation of pulque led to
a significant decrease (55%) in the bread’s moisture diffusivity,
resulting in less weight loss during baking. We did not observe
this effect in all the breads. A similar level of loss (Table 3) in wheat
bread was described by Romankiewicz et al.9 and for rye bread by
Ostasiewicz et al.10 On the basis of these results it seems that the
type of flour is the determining factor.
When xaxtle was used as a leavening agent in Italian straight
dough,8 the resulting bread had smaller volume than the control
6312
samples showed statistically higher specific volume than the
pulque breads. In our studies, we combined the pulque microflora
with commercial yeast, and this way of achieving the desired
bread growth with the addition of pulque seems to be useful.
The effects of pulque on bread described in the literature
(moisture diffusivity and less weight loss during baking) could
be related to the presence of exopolysaccharides produced by
microflora. Nevertheless, even the presence of exopolysaccharides
synthesized by bacteria is not capable of affecting these param-
eters if the dough is acidified,25 which may explain our results. In
our study, the products with the highest specific volume (WR1
recipe) also had the best texture rating. Moreover, products made
with the laboratory-prepared consortium of microorganisms were
rated decidedly higher, which indicates a significant influence
of microflora type on the texture. However, the influence of the
microflora differed depending on the recipe used, namely the
type of flour and the method of kneading, and to a lesser extent
the pulque type.
The pulque breads that obtained the worst scores for taste
(IS-MP-RR3 and LP-MP-RR3) were characterized by low pH and
high titratable acidity. A similar effect has been described by other
authors as conducive to the low sensory evaluation of products
obtained from sourdough.26 The blank samples of each bread
type obtained lower scores of some tested parameters in the sen-
sory evaluation than breads prepared with pulque (Table 5). Sen-
sory analyses are difficult to compare, not only because of dif-
fering hedonic scales17,27 but also due to consumer habits. This
may have been the main reason why the wheat breads produced
with the laboratory-prepared microbial consortium obtained the
highest scores. Generally, Europeans are more familiar with prod-

sample. In our study, only one bread variant among the blank ucts fermented by S. cerevisiae and L. casei than with B. subtilis

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Influence of fermentation by different microflora consortia on pulque www.soci.org

on the flour and the breadmaking technique. This study shows the
Table 5. Results of the bread consumers sensory analysis
possibility of modifying bread parameter to obtain features corre-
Product Colora Smella Tastea Texturea sponding to consumers’ demands by using appropriate microflora,
pulque or flour type.
WR1-B 7.2 ± 1.7C 7.1 ± 1.8B 6.9 ± 2.8BC 7.9 ± 1.4AB
IS-MP-WR1 7.2 ± 1.7C 5.8 ± 2.5C 7.3 ± 2.2B 8.1 ± 1.3AB
LP-MP-WR1 9.3 ± 0.6A 8.1 ± 1.3A 9.2 ± 2.7A 9.8 ± 0.4A REFERENCES
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