Food Microbiology 76 (2018) 267–278

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Occurrence of lactic acid bacteria and yeasts at species and strain level T
during spontaneous fermentation of mawè, a cereal dough produced in West
Africa
Marcel Houngbédjia,1, Pernille Johansenb,1, Sègla Wilfrid Padonoua, Noël Akissoéa,
Nils Arneborgb, Dennis S. Nielsenb, D. Joseph Hounhouigana, Lene Jespersenb,∗
a
Laboratoire de Sciences des Aliments, Faculté des Sciences Agronomiques, Université d’Abomey-Calavi, 03 BP 2819, Jéricho, Cotonou, Benin
b
Department of Food Science, University of Copenhagen, Rolighedsvej 26, 1958, Frederiksberg C, Denmark

A R T I C LE I N FO A B S T R A C T

Keywords: Mawè is a West African spontaneous fermented cereal-based dough. Different types of mawè exist varying in
Mawè type of cereal and/or production condition, with fermentations lasting 24–48 h. With the aim of obtaining a
Cereal doughs comprehensive understanding of the microbial ecology of mawè processing, a microbiological characterisation
Spontaneous fermentation was performed for four mawè types, produced at eight sites in Benin. At the onset of the fermentations lactic acid
Lactic acid bacteria
bacteria (LAB) and yeast counts were on average 7.5 ± 1.03 and 4.8 ± 0.79 Log10 cfu/g, which increased to
Yeast
9.2 ± 0.38 and 7.4 ± 0.42 Log10 cfu/g, respectively, at the end of the fermentations.
LAB (n = 321) and yeasts (n = 298), isolated during the fermentations, were identified. The predominant
LAB and yeast species were Lactobacillus fermentum and Pichia kudriavzevii, respectively, followed by
Kluyveromyces marxianus, all present throughout the mawè fermentations. Further, microbial successions took
place with Weissella confusa occurring mostly at the onset, while Pediococcus acidilactici and Saccharomyces
cerevisiae were mainly associated with the end of the fermentations. Species diversity was influenced both by
type of cereal and production condition. The dominating strain clusters of L. fermentum and P. kudriavzevii were
ubiquitous and strain diversities were influenced by type of cereal and production site.

1. Introduction
Spontaneous fermentation is a very common way of processing
cereal grains in West African countries. In Benin and Togo, mawè is one
of the most popular spontaneous fermented uncooked cereal doughs
and constitutes an important part of the daily food intake. Made from
dehulled or undehulled cereals (maize, sorghum, millet or rice), mawè
is used for the preparation of a variety of West African traditional
cooked dishes, including paste (makumè, akassa and come), porridge
(koko and aklui) and beverage (akpan). At the early stage of fermen-
tation, the dough can moreover be used to prepare other dishes like
steam-cooked bread (ablo), fritters (massa, pâté) and couscous (yèkè-
yèkè) (Hounhouigan et al., 1993a). Several types of mawè are being
produced in both urban and rural areas of Benin and the duration of the
fermentation may vary from 24 -48 h, depending on the rate of acid-
ification and the preference of the producer.
In previous studies focus has been on two types of mawè, i.e.
commercial- and homemade maize mawè (Greppi et al., 2013a, 2013b;
Hounhouigan et al., 1993b, 1994). Hence, other types of mawè such as
commercial sorghum mawè and undehulled maize mawè remain spar-
sely characterised or not characterised at all from a technological and
microbiological point of view. Variations in the type of cereal, pro-
duction conditions and in the geographical and agro-climatic char-
acteristics could lead to variations in the microbiota (Jespersen et al.,
1994, 2005). Consequently, it is relevant to investigate different types
of mawè, which are based on different types of cereals as e.g. maize and
sorghum, under different production conditions and produced in both
urban and rural areas.
In previous studies, lactic acid bacteria (LAB) and yeasts were re-
ported as the dominating microorganisms during spontaneous fermen-
tation of commercial maize mawè and homemade maize mawè
(Hounhouigan et al., 1993b, 1994). In these studies, Lactobacillus fer-
mentum, Lactobacillus brevis and Lactobacillus reuteri were identified as
the dominant LAB species and Pichia kudriavzevii (formerly named Is-
satchenkia orientalis, anamorph Candida krusei) as the dominant yeast
species, although Kluyveromyces marxianus (anamorph Candida kefyr),

∗
Corresponding author.
E-mail address: lj@food.ku.dk (L. Jespersen).
1
Authors contributed equally.

https://doi.org/10.1016/j.fm.2018.06.005
Received 9 March 2018; Received in revised form 6 June 2018; Accepted 8 June 2018
Available online 09 June 2018
0740-0020/ © 2018 Elsevier Ltd. All rights reserved.
M. Houngbédji et al.
Candida glabrata and Saccharomyces cerevisiae were also present. In
these previous studies, only methods with low discriminative power,
such as morphological, physiological and biochemical identification
methods were applied to determine the microbial diversity and suc-
cession in mawè. Further, due to changes in the taxonomical status of
some microbial species and the discovery of many new LAB and yeast
species during recent years (Guyot, 2010), reinvestigation of some of
the traditional fermented foods, using molecular tools for the un-
ambiguous determination of microbial diversity, would be valuable.
Recently, Greppi et al. (2013a, 2013b) investigated the yeast dynamics
in commercial maize mawè by culture dependent and culture in-
dependent molecular methods and reported that P. kudriavzevii was the
predominant yeast species followed by C. glabrata, K. marxianus, Cla-
vispora lusitaniae and S. cerevisiae. Even so, there is a need for a com-
bined analysis of LAB and yeasts involved in the spontaneous fermen-
tation of different types of mawè using molecular techniques, to obtain
a more comprehensive understanding of the microbial ecology and the
microbial successions taking place in mawè processing. Besides, the
diversity at the strain level has never been investigated for either LAB
or yeasts.
The aim of the present study was to provide a comprehensive in-
vestigation of the microorganisms taking part in the spontaneous fer-
mentation of four different types of mawè from Benin (commercial
sorghum mawè, commercial maize mawè, homemade maize mawè and
undehulled maize mawè). LAB and yeasts were clustered by (GTG)5-
based repetitive-PCR (rep-PCR) followed by identification by sequen-
cing of 16S rRNA gene for LAB and the D1/D2 domain of 26S rRNA
gene for yeasts. For the predominant species, strain variations were
investigated based on rep-PCR profiles of the isolates. A deeper un-
derstanding of the detailed microbial ecology of mawè processing could
add to enhance the quality of the final mawè products in terms of or-
ganoleptic properties, nutritional value, shelf life and safety.
2. Materials and methods
2.1. Mawè production and sampling
Four types of mawè varying in either type of cereal and/or pro-
duction conditions were each studied at two separate production sites,
i.e. commercial sorghum mawè (site A1 and A2), commercial maize
mawè (site B1 and B2), homemade maize mawè (site C1 and C2) and
undehulled maize mawè (site D1 and D2), of which the latter is directly
cooked into the street food named “come”. All production sites were
located in Southern Benin and the mawè processing followed the flow
diagram in Fig. 1. In Table 1 information about the type of cereal, lo-
cation of production sites, soaking conditions during the production
and moisture content after kneading is reported. The four mawè types
were processed by the local producers at the production sites until the
fermentation step, using 10 kg of maize or sorghum purchased at the
nearest market. For the four types of mawè, the onset of the fermen-
tations (0 h) was set after the step where the milled grits (for the de-
hulled mawè types, i.e. commercial sorghum mawè, commercial maize
mawè and homemade maize mawè) or hot-soaked milled maize (for
undehulled maize mawè) were kneaded with water and left for spon-
taneous fermentation (Fig. 1). Samples were taken in parallel from two
separate production sites for each of the four mawè types as follows. At
0 h, 200 g of mawè was transferred to a sterile stomacher bag (Seward,
West Sussex, UK). Simultaneously, 4 × 1000 g of mawè was transferred
into four separate clean plastic containers with lids (similar to the ones
used by the producers), representing the fermentation time points 6 h,
12 h, 24 h or 36 h, respectively. Sampling was performed aseptically in
duplicate at the eight production sites. Immediately after sampling at
the production site, all samples were transported from the production
sites to the laboratory, of which the 0 h samples were transported on
ice. Fermentation took place in the separate plastic containers on the
laboratory bench at ambient temperature (28–32 °C) to reproduce the
268
Food Microbiology 76 (2018) 267–278
fermentation conditions at the production sites. When a certain con-
tainer reached the fermentation time point (6 h, 12 h, 24 h or 36 h), the
surface (app. 2–4 cm) of the mawè dough was removed using a ster-
ilized spatula and 25 g was taken from the centre of the dough. For all
fermentation time points, microbiological and physiochemical analyses
were performed using 5 g for moisture content determination, 10 g for
pH determination and 10 g for microbial enumerations.
2.2. Determination of moisture content and pH
The moisture content was determined for 5 g of mawè calculated
based on the weight before and after 72 h of oven drying according to
the American Association of Cereal Chemists method 44-15A (AACC,
2000). Determination of pH was carried out by homogenizing 10 g of
mawè in 20 mL of distilled water, using an InoLab digital pH-meter
(3505 pH meter, JENWAY).
2.3. Enumeration and isolation of microorganisms
For each mawè dough sample, 10 g were resuspended in 90 mL of
sterile diluent [0.1% (w/v) bactopeptone (Oxoid, Hampshire, UK),
0.85% (w/v) of NaCl (Merck, Darmstadt, Germany), pH 7.0] and
homogenised for 30 s at 230 rpm with a Stomacher (Lab Blender, Model
400, Seward Medical, London, England). Microbial enumeration was
done from appropriate 10-fold dilutions. According to Amoa-Awua and
Jakobsen (1995), lactic acid bacteria (LAB) was enumerated on de Man,
Rogosa and Sharp (MRS) (CM0361 Oxoid), pH 6.2 agar plates, in-
cubated under anaerobic conditions (Anaerocult® A, Merck KGaA,
Darmstadt, Germany) at 30 °C for 3 days. Yeasts were enumerated ac-
cording to Greppi et al. (2013a) on MYGP agar [3 g of yeast extract
(Oxoid), 3 g of malt extract (Oxoid), 5 g of bactopeptone (Oxoid), 10 g
of glucose (Sigma, Milan, Italy) and 20 g of agar (Oxoid) per liter of
distilled water, pH 5.6, and added chloramphenicol (Fisher chemicals,
Milan, Italy) and chlortetracycline (Sigma) to concentrations of 50 mg/
L and 25 mg/L, respectively], and incubated aerobically at 25 °C for 3
days. Plates with 30–200 colony forming units (CFU) were counted and
results expressed as Log10 (CFU/g). Aiming at 20 isolates, all colonies
from a random segment (> 15% of the area) of the highest dilutions or
suitable plates were picked and purified by repeated streaking on MRS
and MYGP for LAB and yeasts, respectively (Amoa-Awua and Jakobsen,
1995).
2.4. Identification of LAB and yeasts
2.4.1. Phenotypic and biochemical characterisation
Prior to genotypic identification, presumptive LAB isolates on MRS
agar were examined for Gram reaction, catalase activity as well as cell
morphology and motility. Gram reaction was performed by dissolving a
loop full of freshly grown colony material in a drop of 3% KOH (Merck)
on a microscope slide, observing whether ropy strings occurred
(Gregersen, 1978). Catalase activity was determined by adding a drop
of H2O2 (Merck) solution (30%) to a bit of colony mass on a glass slide,
observing whether air bubbles were generated (Taylor and Achanzar,
1972). After genotypic identification, further complementary bio-
chemical tests were performed, when required, in order to identify
certain isolates, as described below. Pediococcus acidilactici and Pedio-
coccus lolii were differentiated based on ribose fermentation and growth
at 45 °C and 48 °C (Doi et al., 2009; Franz et al., 2014). Weissella con-
fusa, Weissella cibaria and Weissella koreensis were differentiated based
on the production of acids from carbon compounds (ribose, galactose,
arabinose, cellobiose and maltose (Sigma)), and growth at 45 °C
(Björkroth et al., 2002; Collins et al., 1993).
Prior to genotypic identification, presumptive yeast colonies grown
on MYGP agar were examined for cell morphology (Olympus BX 40) by
description of micro- and macro morphological characteristics. Further,
fermentation of carbon compounds (glucose, maltose, lactose, raffinose
M. Houngbédji et al. Food Microbiology 76 (2018) 267–278

Fig. 1. Flow diagram for the processing of the four types of mawè each investigated at two separate production sites; commercial sorghum mawè, commercial maize
mawè, homemade maize mawè and undehulled maize mawè. Samples were withdrawn for analysis at 0 h, 6 h, 12 h, 24 h and 36 h during the spontaneous
fermentation. *At sites A1 (commercial sorghum mawè) and B1 (commercial maize mawè), part of the soaking water was reused from a previous mawè production.

and galactose (Sigma)) was performed when appropriate, according to amplified using the universal primers NL1 and NL4 (Kurtzman and
Kurtzman et al. (2011). Robnett, 1998) under the following conditions: initial denaturation at
95 °C for 5 min, 30 cycles at 95 °C for 90 s, 53 °C for 30 s, 72 °C for 90 s
followed by a final extension step at 72 °C for 7 min. PCR products were
2.4.2. Genotypic identification
sent to a commercial sequencing facility (Macrogen, South Korea). Se-
DNA was extracted using InstaGene™ Matrix (Bio-Rad Laboratories,
quences were manually corrected and assembled using CLC Genomics
Hercules, CA 94547, USA) according to the manufacturer's instructions.
Workbench 8.0 (Aarhus, Denmark). Subsequently, the corrected se-
(GTG)5-based rep-PCR, agarose gel electrophoresis and fingerprint
quences were aligned to 16S rRNA gene sequences in the EzBioCloud
clustering were performed as described by Gevers et al. (2001) and
database (Yoon et al., 2017) for LAB and 26S rRNA gene sequences in
Nielsen et al. (2007), with slight modifications. First, the extracted DNA
the GenBank for yeasts, using the BLAST algorithm (Zhang et al., 2000).
was PCR-amplified on a SureCycler 8800 (Agilent Technologes, MY
The nucleotide sequences obtained in this study have been assigned
25110007) under the following thermocycling conditions: 5 min of in-
GenBank Accession Nos. MG245791-MG245859, as indicated in Figs. 2
itial denaturation at 94 °C, 30 cycles of 95 °C for 30 s, 45 °C for 60 s,
and 3.
65 °C for 8 min followed by a final elongation step of 60 °C for 16 min.
Lactobacillus plantarum and Lactobacillus pentosus were differentiated
Next, the PCR products were separated by 1.5% agarose gel electro-
by sequencing of the recA gene, according to Torriani et al. (2001).
phoresis in 0.5 × TBE (5 h, 120 V) using a Generuler 1 kb DNA ladder
Kluyveromyces marxianus and Kluyveromyces lactis were differentiated
(Thermo Scientific, Lithuania) as reference. The electrophoresis gels
by sequencing of the internal transcribed spacer (ITS) region, using the
were stained with ethidium bromide, documented using a digital
primers ITS1 and ITS4, according to Nielsen et al. (2007), under the
camera (Computar® TV Zoom LENS, H6Z0812M, Japan) and cluster
following conditions: 3 min of initial denaturation at 94 °C; 30 cycles of
analysis were carried out using Bionumerics 7.1 (Applied Maths, 9830
94 °C for 2 min, 60 °C for 1 min, 72 °C for 2.5 min followed by a final
Saint- Martens-Latem, Belgium) based on the Dice's coefficient of si-
elongation step of 72 °C for 10 min.
milarity with the unweighted pair group method with arithmetic
averages clustering algorithm (UPGMA). Based on the clustering, re-
presentatives of each cluster (at least the square root of the number of 2.5. Statistical analysis
isolates within the cluster) were chosen for sequencing. For the selected
LAB, the 16S rRNA gene was amplified using the universal primers 27F Microbial enumerations, pH measurements and moisture content
and 1540R (Jensen et al., 2009) under the following conditions: initial analyses were conducted in duplicate and means and standard devia-
denaturation at 95 °C for 5 min, 35 cycles at 95 °C for 30 s, 60 °C for 30 s tions were calculated. Data were subjected to one-way analysis of
and 72 °C for 120 s, followed by a final extension at 72 °C for 10 min. variance (ANOVA) and means were separated by least significant dif-
For the selected yeasts the D1/D2-region of the 26S rRNA gene was ference test of Fisher (P < 0.05) using the MINITAB 18 statistical

269
M. Houngbédji et al. Food Microbiology 76 (2018) 267–278

software package (MINITAB Inc. Release 17 for windows, 2015).

Soaking water partly reused
from previous production 3. Results

3.1. pH and microbial counts during spontaneous fermentations of mawè

Throughout the spontaneous mawè fermentations, pH decreased in

all types of mawè from an average pH of 5.4 ± 0.53 at the onset (0 h)
to 4.1 ± 0.33 at the end (36 h) of the fermentations (Table 2). The
yes

yes
no

no

no

no
–

–
acidification of the cereal grains initiated while they were soaking
(4–10 h), which gave rise to the relatively low pH observed at the onset
mawè (w/w) after kneading

of the mawè fermentations. The type of cereal used for mawè produc-
Moisture content of fresh

tion affected the pH during the fermentation. Especially at the end of

the fermentation (36 h), significantly (P ≤ 0.05) higher pH values were
observed for mawè based on sorghum (commercial sorghum mawè) and
lower pH values were in general observed from 6 h to the end of the
fermentation for the mawè types based on maize.
43.4%

49.7%
44.1%

53.2%

55.1%

42.3%

41.3%

The type of cereal used for mawè production only affected the LAB
ND
Type of cereal, location of production site, production capacity, soaking conditions and moisture content after kneading of the four different types of mawè.

counts at the onset of fermentation (0 h), where significantly different

(P ≤ 0.05) LAB counts were obtained for mawè based on sorghum and
maize. From 6 h and to the end of the fermentations (36 h) no sig-
beginning
°C at the

nificant difference in LAB counts between mawè based on sorghum and

29.0 °C

30.0 °C
30.0 °C

29.5 °C
29.5 °C

30.0 °C

95.0 °C

97.0 °C
Soaking

maize were observed. Production conditions were found to affect the

LAB counts. Similar LAB counts were obtained throughout the fer-
Duration

mentations for the three types of mawè based on dehulled cereals

(commercial sorghum mawè, commercial maize mawè and homemade
10 h

10 h

10 h
4h
9h

6h

8h

9h

maize mawè) (Table 2). Contrary, mawè produced by using undehulled

maize that was soaked in boiled water showed significantly (P = 0.027)
Production capacity

lower LAB counts, at the onset of the fermentation (0 h). However, at

the end of the fermentation (36 h) the LAB counts had reached the same
level (P = 0.229) as those of the other types of mawè. Further, pro-
-; not known, ND: not determined; *: “come” is a fermented, cooked and slightly salted paste sold as a street food.
(kg/week)

900–1000

900–1200
500–600

700–900

duction of mawè by reusing part of the soaking water from a previous

mawè production as performed at site A1 (commercial sorghum mawè)
and B1 (commercial maize mawè) led to higher LAB counts, as com-
–

pared to the production sites where fresh soaking water was used
Street vendor in Zogbadjè, Abomey-Calavi,
consumption in Dogbo, Couffo, rural area
consumption in Comè, Mono, rural area

(commercial sorghum mawè at site A2 and commercial maize mawè at

St Michel market in Cotonou; Littoral,

Pahou market; Atlantique, urban area

Godomey market in Abomey-Calavi;

site B2). The type of cereal used for mawè production did not affect
Comè market in Mono, rural area

Comè market in Mono, rural area

Inside a household for domestic

Inside a household for domestic

yeast counts, since no significant difference in yeast counts were ob-

served throughout the fermentation between the three mawè types
based on either sorghum or maize (commercial sorghum mawè, com-
Atlantique, urban area

Atlantique, urban area

mercial maize mawè and undehulled maize mawè). However, produc-

tion conditions had some effect on yeast counts since homemade maize
mawè, produced at household for domestic consumption, had sig-
urban area

nificantly lower counts of yeast throughout the fermentation

Location

(P ≤ 0.05), except for the last sampling point (36 h) where the yeast
counts were similar to those of the other types of mawè (P = 0.983).
Further, the production site significantly influenced both pH and mi-
Site

D1

D2
A1

A2

C1

C2
B1

B2

crobial counts of both LAB and yeasts for all types of mawè at almost all
fermentation time points (P ≤ 0.05) (Table 2).
Cereal type

Sorghum

Maize

Maize

Maize

3.2. LAB species identified during spontaneous fermentations of mawè

A total of 321 Gram-positive, catalase negative rod or coccoid

shaped isolates obtained from MRS were presumptively considered as
Production scale

Processed into

LAB. The presumptive LAB isolates were clustered by (GTG)5-based rep-

For domestic
consumption
Commercial

Commercial

PCR fingerprinting, dividing the isolates into five clusters.

“come”*

Representatives of each cluster were identified based on their 16S rRNA

gene sequence followed by BLAST search at EzBioCloud (Fig. 2). The
predominant part of the isolates was in cluster IV (85% of the total LAB
isolates), which was identified as Lactobacillus fermentum (97.8–99.8%
Commercial sorghum

Commercial maize

similarity to EzBioCloud sequences). Isolates of cluster II (5% of the

Undehulled maize
Homemade maize

total LAB isolates) had high similarity to EzBioCloud sequences of

Type of mawè

Pediococcus acidilactici (99.6–100%) and Pediococcus lolii (99.8%–100%

mawè

mawè

mawè

mawè

similarity to EzBioCloud sequences). The cluster II isolates were un-

Table 1

ambiguously identified as P. acidilactici based on their ability to pro-

duce acid from ribose as well as to grow at 45 °C and 48 °C (Doi et al.,

270
M. Houngbédji et al.
Fig. 2. Species identification of LAB isolated during spontaneous fermentations of the four different types of mawè at two separate production sites. Dendrogram
obtained by cluster analysis of (GTG)5-based rep-PCR fingerprints of representative LAB, based on Dice's coefficient of similarity with the unweighted pair group
method with arithmetic average clustering algorithm (UPGMA). LAB species were identified by sequencing of 16S rRNA gene and EzBioCloud searches.
2009; Franz et al., 2014). Isolates of cluster V (4% of the total LAB
isolates), had similarity to EzBioCloud sequences of Lactobacillus plan-
tarum (99.8–99.9%) and Lactobacillus pentosus (99.7–100% similarity to
EzBioCloud sequences). Subsequently, sequencing of the recA gene
identified the isolates in cluster V as L. plantarum. Isolates of cluster I
(4% of the total LAB isolates) had high similarity to EzBioCloud se-
quences of Weissella confusa (99.2–100%) or Weissella cibaria
(98.5–99.3% similarity to EzBioCloud sequences). All cluster I isolates
were identified as W. confusa as they produced acid from cellobiose,
galactose and maltose, but not arabinose (Collins et al., 1993) and were
not able to grow at 45 °C (Björkroth et al., 2002). Isolates of cluster III
representing a minor part (1% of the total LAB isolates) were identified
as Pediococcus pentosaceus (100% similarity to EzBioCloud sequences).
3.3. Yeast species identified during spontaneous fermentations of mawè
A total of 298 isolates growing on MYGP agar were, after micro- and
macro morphological examination, presumptively considered as yeasts.
The presumptive yeast isolates were clustered by (GTG)5-based rep-PCR
fingerprinting, dividing the isolates into six clusters. Representatives of
each cluster were identified by sequencing of the D1/D2 region of the
26S rRNA gene followed by BLAST search at GenBank (Fig. 3). The
majority of the yeast isolates was in cluster V (62% of the total yeast
isolates), which was identified as Pichia kudriavzevii (100% similarity to
271
Food Microbiology 76 (2018) 267–278
GenBank sequences). All isolates were able to ferment glucose, but did
not ferment maltose, lactose, raffinose and galactose. Isolates of cluster
IV (28% of the total yeast isolates) had similarity to GenBank sequences
of Kluyveromyces marxianus (99.8–100%) and Kluyveromyces lactis
(99.1% similarity to GenBank sequences). The cluster IV isolates were
unambiguously identified as K. marxianus by sequencing of the ITS
regions, resulting in a fragment of approx. 680 bp., with 100% simi-
larity to GenBank sequences of K. marxianus. The identified K. marx-
ianus isolates were able to ferment glucose and raffinose, but did not
ferment maltose and lactose. Isolates of cluster VI (6% of the total yeast
isolates) were identified as Saccharomyces cerevisiae (99.6–99.8% simi-
larity to GenBank sequences). The S. cerevisiae isolates were able to
ferment glucose and maltose but did not ferment lactose and galactose.
Isolates of clusters I, II and III together comprising about 4% of the total
yeast isolates, were identified as Candida glabrata, Wickerhamomyces
anomalus and Ogataea polymorpha, respectively, each with 100% simi-
larity to GenBank sequences.
3.4. LAB and yeast species succession during mawè fermentations and their
distribution in the four different types of mawè
The occurrence of the identified LAB and yeast species during the
fermentations was determined for the four different types of mawè each
investigated at two separate production sites. For LAB, L. fermentum was
M. Houngbédji et al. Food Microbiology 76 (2018) 267–278

rep-PCR Sequence database

Similarity to GenBank

100
Isolate Identities GenBank (%) accession no. Cluster Identity
60

80
C2-24-Y3 572/572 100 MG245841
I C. glabrata
C2-6-Y2 597/597 100 MG245821
H1-0-Y6 563/563 100 MG245842 II W. anomalus
Cm1-12-Y2a 578/578 100 MG245822
III O. polymorpha
H1-12-Y7 558/558 100 MG245843
C1-0-Y4a 532/532 100 MG245844
C1-36-Y2 535/535 100 MG245845
S1-0-Y2 563/564 99.8 MG245823
S1-6-Y4a 561/562 99.8 MG245824
S2-24-Y5 562/563 99.8 MG245825
IV K. marxianus
C1-0-Y6 564/565 99.8 MG245826
S2-6-Y6 560/560 100 MG245827
C1-0-Y1 562/563 99.8 MG245828
H2-0-Y2 531/531 100 MG245846
H2-6-Y1 523/523 100 MG245847
C2-0-Y6 581/581 100 MG245829
S2-6-Y4 585/585 100 MG245830
S2-6-Y10 558/558 100 MG245848
H2-12-Y1 585/585 100 MG245831
S1-12-Y2a 581/581 100 MG245832
S2-24-Y11 575/575 100 MG245833
S2-6-Y3 556/556 100 MG245849
Cm1-24-Y3 550/550 100 MG245850
H2-0-Y15 557/557 100 MG245851
H1-0-Y5 581/581 100 MG245834
V P. kudriavzevii
Cm2-24-Y4 550/550 100 MG245852
H2-0-Y13 581/581 100 MG245835
H2-6-Y5 548/548 100 MG245853
Cm2-6-Y4 556/556 100 MG245854
H2-0-Y3 559/559 100 MG245855
C2-0-Y2a 586/586 100 MG245836
C2-36-Y3 541/541 100 MG245856
S1-24-Y6 583/583 100 MG245837
Cm1-6-Y9 557/557 100 MG245857
Cm2-24-Y9 582/582 100 MG245838
Cm1-36-Y6 562/562 100 MG245858
Cm2-36-Y11 560/560 100 MG245859
VI S. cerevisiae
C2-0-Y2 589/590 99.8 MG245839
C4-24-Y4 548/550 99.6 MG245840

Fig. 3. Species identification of yeasts isolated during spontaneous fermentations of the four different types of mawè at two separate production sites. Dendrogram
obtained by cluster analysis of (GTG)5-based rep-PCR fingerprints of representative yeasts, based on Dice's coefficient of similarity with the unweighted pair group
method with arithmetic average clustering algorithm (UPGMA). Yeast species were identified by sequencing of 26S rRNA gene and GenBank searches.

the dominating species in all types of mawè and was present throughout
the fermentations (Fig. 4). The highest relative abundance was ob-
served in undehulled maize mawè followed by commercial maize
mawè. At lower abundancies, W. confusa and P. acidilactici were de-
tected in all types of mawè, however, not at all production sites.
Weissella confusa was mostly associated with the onset (0 h) of the
fermentation, whereas P. acidilactici was mostly observed at the end
stages of the fermentation (24 h and 36 h). Two LAB species, comprising
a minor fraction of the total LAB, only occurred in some of the in-
vestigated types of mawè, i.e. L. plantarum, which was identified in only
the three dehulled types of mawè and did not occur in undehulled
maize mawè and P. pentosaceus, which was only identified in two types
of mawè (commercial sorghum mawè and homemade maize mawè).
Among the yeasts isolated from mawè, P. kudriavzevii was the
dominating species in all types of mawè, being present throughout the
mawè fermentations. Further, K. marxianus occurred in all types of
mawè and was isolated throughout the mawè fermentations, however
at lower abundancies. Kluyveromyces marxianus was the predominant
yeast species at one of the production sites investigated (site B1, com-
mercial maize mawè). At lower abundance, S. cerevisiae occurred in two
types of mawè (commercial maize mawè and undehulled maize mawè)
and was mostly associated with the end stages of these mawè fermen-
tations (24 h and 36 h). Three yeast species, comprising a minor frac-
tion of the total yeasts, only occurred in some of the investigated mawè
types, i.e. C. glabrata in commercial maize mawè, W. anomalus in
homemade maize mawè as well as O. polymorpha in homemade maize

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Table 2
pH, LAB and yeast counts [Log10 (cfu/g)] during spontaneous fermentation of the four different types of mawè in Benin.

Product Prod. site pH LAB

0h 6h 12 h 24 h 36 h 0h 6h

e bc b b b a
Commercial A1 5.1 ± 0.02 4.7 ± 0.03 4.6 ± 0.07 4.5 ± 0.01 4.4 ± 0.02 9.1 ± 0.03 9.9 ± 0.02a
sorghum A2 6.1 ± 0.04a 5.1 ± 0.01a 5.0 ± 0.02a 4.9 ± 0.01a 4.8 ± 0.01a 6.7 ± 0.02f 9.9 ± 0.01a
mawè

Commercial B1 5.3 ± 0.07d 4.7 ± 0.01b 4.2 ± 0.01de 4.0 ± 0.02af 3.9 ± 0.01de 8.7 ± 0.03b 10.0 ± 0.02a
maize B2 4.5 ± 0.01f 4.2 ± 0.02f 4.1 ± 0.01f 4.0 ± 0.03f 3.9 ± 0.02e 7.9 ± 0.04c 8.9 ± 0.03d
mawè

Homemade C1 5.6 ± 0.03c 4.4 ± 0.02e 4.2 ± 0.01d 4.1 ± 0.01ce 4.0 ± 0.02d 7.4 ± 0.02d 9.6 ± 0.01b
maize C2 5.7 ± 0.03c 4.7 ± 0.03b 4.4 ± 0.02c 4.3 ± 0.04c 4.2 ± 0.03c 7.5 ± 0.01d 9.1 ± 0.10c
mawè

Undehulled D1 6.0 ± 0.01b 4.5 ± 0.04d 4.2 ± 0.03d 4.1 ± 0.03d 3.9 ± 0.03de 6.0 ± 0.01g 10.0 ± 0.04a
maize D2 5.1 ± 0.03de 4.6 ± 0.01c 4.1 ± 0.01e 3.9 ± 0.02f 3.9 ± 0.02e 6.9 ± 0.08e 9.0 ± 0.06cd
mawè

Product LAB Yeasts

12 h 24 h 36 h 0h 6h 12 h 24 h 36 h

a a c cd c de c
Commercial 9.9 ± 0.03 10.0 ± 0.09 9.2 ± 0.01 4.7 ± 0.02 5.5 ± 0.04 5.8 ± 0.11 6.9 ± 0.08 7.0 ± 0.02e
sorghum 10.0 ± 0.02a 9.8 ± 0.08a 9.8 ± 0.05a 6.5 ± 0.03a 8.0 ± 0.07a 7.8 ± 0.02a 7.6 ± 0.08b 7.7 ± 0.08b
mawè

Commercial 9.8 ± 0.04a 9.9 ± 0.05a 9.6 ± 0.02b 4.8 ± 0.02d 6.6 ± 0.04b 6.7 ± 0.04c 8.1 ± 0.01a 7.5 ± 0.01c
maize 9.2 ± 0.04c 9.2 ± 0.02c 8.6 ± 0.02e 5.0 ± 0.02b 5.1 ± 0.01c 5.6 ± 0.11e 6.5 ± 0.02d 7.2 ± 0.03d
mawè

Homemade 9.5 ± 0.02b 9.5 ± 0.06b 9.0 ± 0.05cd 3.7 ± 0.06f 4.5 ± 0.07e 5.6 ± 0.04e 6.5 ± 0.04d 7.6 ± 0.08b
maize 9.3 ± 0.10c 10.0 ± 0.05a 9.0 ± 0.06d 4.5 ± 0.01e 4.6 ± 0.04e 5.7 ± 0.05de 6.1 ± 0.03e 7.2 ± 0.02d
mawè

Undehulled 10.0 ± 0.03a 8.9 ± 0.02d 9.0 ± 0.02d 4.5 ± 0.04e 6.7 ± 0.04b 7.0 ± 0.02b 7.5 ± 0.05b 8.0 ± 0.02a
maize 9.0 ± 0.03d 9.0 ± 0.02cd 9.0 ± 0.04d 4.6 ± 0.07de 5.1 ± 0.03d 6.0 ± 0.03d 6.1 ± 0.07e 6.7 ± 0.02f
mawè

Means that do not share superscripts (a,b,c,d,e,f,g)

are significantly different (P ≤ 0.05).

mawè and undehulled maize mawè.
The type of cereal used for mawè production was found to influence
both the LAB and yeast species diversity in the mawè dough. Mawè
based on sorghum (commercial sorghum mawè) showed a higher LAB
species diversity compared to the three mawè types based on maize.
Contrary for yeasts, where lower yeast species diversity occurred in
mawè based on sorghum. Production conditions were also found to
influence the LAB and yeast species diversity. Production of mawè from
undehulled maize that was soaked in boiled water (undehulled maize
mawè) resulted in the lowest LAB species diversity compared to the
three other mawè types, which was contrary to yeasts where more di-
verse yeast species distributions were observed in undehulled maize
mawè. The production site only had a minor influence on the LAB and
yeast species diversity. LAB species diversities were similar at the two
production sites for all four types of mawè. For yeasts, similar species
diversities were observed at the two production sites investigated for
three of the mawè types and only at the two separate production sites
for commercial maize mawè (site B1 and B2) more different species
distributions were observed.
3.5. L. fermentum and P. kudriavzevii strain diversity
As shown in Fig. 4, L. fermentum and P. kudriavzevii were found to be
the predominant LAB and yeast species, respectively, during the dif-
ferent mawè fermentations. Based on the rep-PCR profiles, the L. fer-
mentum isolates from all types of mawè could be divided into 10 clusters
representing different strains or groups of closely related strains
(Fig. 5). The most abundant L. fermentum strain clusters (I and IV) re-
presented 29% and 54% of the total L. fermentum isolates, respectively,
and were interestingly isolated from the four types of mawè at all
production sites. Of the 10 L. fermentum strain clusters, three were only
identified at a single production site, hence termed single-site clusters.
Of these, two belonged to production sites located in urban areas, i.e.
cluster VIII at site A2 (commercial sorghum mawè) and cluster II at site
B1 (commercial maize mawè), whereas the third single-site cluster (X)
were from a production site located in rural area (site D2 undehulled
maize mawè). The isolates of P. kudriavzevii from all mawè types could
be divided into 10 strain clusters based on their rep-PCR profiles
(Fig. 6). For P. kudriavzevii the most abundant strain cluster (VI) re-
presented 56% of the total P. kudriavzevii isolates and was identified in
the four types of mawè at all production sites. Further, three strain
clusters (V, VII and IX) occurred in all of the mawè types, at varying
abundancies and were detected at one or both of the production sites
investigated. Four single-site clusters were identified for P. kudriavzevii.
These were cluster I at site B2 (commercial maize mawè), cluster II at
site C2 (homemade maize mawè) as well as clusters III and IV at site D2
(undehulled maize mawè), all of which were located in rural areas.
The type of cereal used for mawè production had an influence on
both the L. fermentum and P. kudriavzevii strain diversity, respectively.
In mawè based on sorghum (commercial sorghum mawè) one L. fer-
mentum strain cluster (VII) occurred at both production sites (site A1
and A2), which was not detected in any of the other mawè types based

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Not detected
with part of the soaking water being reused from a previous production

80-100%
(site A1 commercial sorghum mawè and site B1 commercial maize

60-80%

40-60%

20-40%

<20%
mawè) two single-site clusters (VIII and II) for L. fermentum were ob-
served, respectively, which could indicate the establishment of a house
hold microbiota. Further, production of mawè by using undehulled
maize that was soaked in boiled water (undehulled maize mawè) re-
0 6 12 24 36

sulted in P. kudriavzevii strain diversities that were different from the

Undehulled maize mawè

Site D2

three other mawè types, however, at the same time being highly in-
fluenced by the production site. Even though opposite to the observa-
tions at species level, the production site had a high influence on strain
diversities for both L. fermentum and P. kudriavzevii. For three of the
0 6 12 24 36

mawè types, L. fermentum strain diversities differed at the two pro-

Site D1

duction sites and only similar L. fermentum strain diversities occurred at

the two production sites for homemade maize mawè (site C1 and C2).
For each of the four mawè types, P. kudriavzevii strain diversities dif-
fered at the two production sites which could indicate that a specific
0 6 12 24 36

house hold microbiota had been established. Further, the location of the
Homemade maize mawè

Site C2

production site seemed to have an influence on the P. kudriavzevii strain

diversity, since higher strain diversities were observed at the produc-
tion sites located in rural areas (site B2 commercial maize mawè, site
C1 and C2 homemade maize mawè as well as site D2 undehulled maize
0 6 12 24 36

mawè) compared to the production sites located in urban areas (site A1

Site C1

and A2 commercial sorghum mawè, site B1 commercial maize mawè

and site D1 undehulled maize mawè).
Mawè type

4. Discussion
0 6 12 24 36
Commercial maize mawè

Site B2

For all types of mawè, pH was relatively low at the onset of the
spontaneous fermentation step (on average 5.4 ± 0.53), presumably
due to acidification during soaking, as previously observed by Halm
et al. (1993) for “kenkey” a fermented maize dough from Ghana. Fur-
0 6 12 24 36

ther, the LAB (7.5 ± 0.80 Log10 cfu/g) and yeast (4.8 ± 0.62
Site B1

Log10 cfu/g) counts were, at the onset of the fermentations, relatively

high compared to previous findings, which could possibly be explained
by the shorter soaking step applied in the previously studied mawè
types (Hounhouigan et al., 1993b, 1994). At two production sites, site
0 6 12 24 36
Commercial sorghum mawè

A1 (commercial sorghum mawè) and site B1 (commercial maize

Site A2

mawè), part of the soaking water was reused from a previous mawè
production, resulting in significantly higher LAB counts at 0 h of fer-
mentation (P < 0.05), compared to the remaining mawè types. The
LAB counts of mawè at the end of the fermentation were similar to
Fermentation time 0 6 12 24 36

those obtained on sourdoughs using MRS media, however in sourdough

Site A1

a slightly higher LAB count has been obtained by using maltose MRS,
indicating that the use of MRS for colony isolation may guarantee an
accurate description of LAB diversity in mawè doughs (Vera et al.,
Production site

2009). The decrease in pH and increase of LAB and yeasts counts during
Ogatea polymorpha
Pichia kudriavzevii
Lactobacillus fermentum

Klyuveromyces marxianus
Lactobacillus plantarum

Candida glabrata
Weissella confusa

Pediococcus pentosaceus

Saccharomyces cerevisiae
Pediococcus acidilactici

Wickerhamomyces anomalus

the fermentation were in accordance with previous findings (Akabanda

et al., 2013; Greppi et al., 2013b; Hounhouigan et al., 1993a; Jespersen
et al., 1994; Jespersen, 2003; Owusu-Kwarteng et al., 2012).
In the present study, five different LAB species were identified in the
different mawè types, namely L. fermentum, L. plantarum, P. acidilactici,
W. confusa and P. pentosaceus. The most prevalent LAB species in all
four mawè types was L. fermentum, which confirms the findings re-
ported by Hounhouigan et al. (1993b). The predominance of L. fer-
mentum has also been observed in many other West African cereal-
b)
a)

based foods such as “ogi” from Nigeria and Benin, “koko” from Ghana,
Fig. 4. Relative abundance of a) LAB and b) yeast species, isolated at 0 h, 6 h,
“dolo” from Burkina Faso and “kenkey” from Ghana (Halm et al., 1993;
12 h, 24 h, and 36 h of spontaneous fermentation from the four different types
of mawè at two separate production sites; commercial sorghum mawè (site A1 Hayford et al., 1999; Lei and Jakobsen, 2004; Nago et al., 1998;
and A2), commercial maize mawè (site B1 and B2), homemade maize mawè Sawadogo-Lingani et al., 2007). The dominance of L. fermentum in
(site C1 and C2) and undehulled maize mawè (site D1 and D2). mawè and in many other cereal dough fermentations may partly be
explained by its tolerance to low pH, as reported by Santoyo et al.
(2003). Thus, L. fermentum would play a predominant role in the
on maize. For P. kudriavzevii isolates the overall lowest strain diversity
acidification of mawè and could help in meeting the consumer's pre-
was observed when mawè was based on sorghum (commercial sorghum
ference, as acidic taste is one important quality criteria of mawè. In the
mawè). Production conditions also had an influence on L. fermentum
present study, L. plantarum was isolated from commercial sorghum
and P. kudriavzevii strain diversity, respectively. Production of mawè
mawè and homemade maize mawè on average comprising 12% and

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M. Houngbédji et al. Food Microbiology 76 (2018) 267–278

Fig. 5. Strain diversity of L. fermentum isolates from the four different mawè types at two separate production sites; commercial sorghum mawè (site A1 and A2),
commercial maize mawè (site B1 and B2), homemade maize mawè (site C1 and C2) and undehulled maize mawè (site D1 and D2). a) The dendrogram is based on
Dice's coefficient of similarity with the unweighted pair group method with arithmetic average clustering algorithm (UPGMA). b) Relative distributions of the
different L. fermentum strain clusters in the four mawè types from the two separate production sites. Numbers in brackets indicate the number of isolates included.

14% of the LAB isolated from these mawè types, respectively. This is
contrary to previous findings in commercial maize mawè and home-
made maize mawè, where no L. plantarum was identified (Hounhouigan
et al., 1993b). However, in accordance with the findings of
Hounhouigan et al. (1993b), the present study identified P. acidilactici,
P. pentosaceus and W. confusa (formerly named Lactobacillus confosus) to
be involved in spontaneous fermentations of the different types of
mawè.
Microbial successions are common during spontaneous fermenta-
tion of cereal-based foods (Greppi et al., 2013b; Hounhouigan et al.,
1993b; Jespersen et al., 1994; Jespersen, 2003). For mawè, no studies
using molecular biology based methods for investigating LAB

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M. Houngbédji et al. Food Microbiology 76 (2018) 267–278

Fig. 6. Strain diversity of P. kudriavzevii isolates from the four different mawè types at two separate production sites; commercial sorghum mawè (site A1 and A2),
commercial maize mawè (site B1 and B2), homemade maize mawè (site C1 and C2) and undehulled maize mawè (site D1 and D2). a) The dendrogram is based on
Dice's coefficient of similarity with the unweighted pair group method with arithmetic average clustering algorithm (UPGMA). b) Relative distributions of the
different P. kudriavzevii strain clusters in the four mawè types from the two separate production sites. Numbers in brackets indicate the number of isolates included.

succession during fermentation have previously been performed. In
contrast to the findings of Hounhouigan et al. (1993b), the present
study demonstrated a rather low LAB species diversity during mawè
fermentations despite of a great diversity in geographical situations,
agro-climatic conditions, type of cereal used and traditional preparation
practices applied in the investigated mawè types. Hounhouigan et al.
(1993b) identified Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus
curvatus, Lactobacillus buchneri, Lactobacillus salivarius, Lactococcus
lactis, and Leuconostoc mesenteroides in commercial maize mawè and
homemade maize mawè, using the commercial API system combined
with phenotypic and biochemical characterisations. The high diversity
obtained in this previous study might be overestimated due to mis-
identifications resulting from low discriminatory power of phenotypic
and biochemical characterisations as well as poor reproducibility of the
API system (De Vuyst and Vancanneyt, 2007).
Yeasts are generally reported to be associated with LAB during West
African cereal-based food fermentations (Greppi et al., 2013a;
Hounhouigan et al., 1994; Jespersen, 2003; Nago et al., 1998). The
present study confirmed that yeasts also are involved in the sponta-
neous fermentations of the different types of mawè. Pichia kudriavzevii
and K. marxianus dominated the mawè fermentations, while S. cerevi-
siae, O. polymorpha, C. glabrata and W. anomalus were detected but in
lower abundance. Hounhouigan et al. (1994) also demonstrated the
predominant involvement of P. kudriavzevii in commercial maize mawè
using the API system method. Recently, Greppi et al. (2013b) used
Denaturing Gradient Gel Electrophoresis (DGGE) and rep-PCR typing
and reported P. kudriavzevii as the predominant yeast during the
spontaneous fermentation of commercial maize mawè. As observed in
the present study, Greppi et al. (2013b) also reported the occurrence of
S. cerevisiae. In the present study, C. glabrata was only detected at one
site (site B2, commercial maize mawè) with low abundance. This is
contrary to the findings of Greppi et al. (2013b) where a high

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involvement of C. glabrata was reported which may be related to the
sanitary level at the production sites or the microbiota associated with
the producers (Fidel et al., 1999). Candida glabrata has been reported as
an opportunistic pathogen that accounts for an increasingly large po-
pulation of nosocomial fungal infections (Fidel et al., 1999).
Strain diversity has so far not been studied before in mawè and only
to a limited extent in other fermented foods (Petersen et al., 2002;
Siezen et al., 2010). In the present study, rep-PCR profiles were used to
study the strain cluster diversity within the predominant LAB and
yeasts, i.e. L. fermentum and P. kudriavzevii, and further to correlate the
strain diversity to mawè type and production site. Interestingly, it was
observed that the two predominant strain clusters of L. fermentum and
P. kudriavzevii were ubiquitous, occurring at all production sites, which
indicates that these strain clusters could be broadly distributed in the
environment of mawè processing. Further, a few strain clusters were
detected only at a single production site, of which the majority were
located in rural areas of Benin. Additionally, especially the type of
cereal affected the strain cluster diversity for both L. fermentum and P.
kudriavzevii. The occurrence of dominating strain clusters of both L.
fermentum and P. kudriavzevii, which were universally present at all
production sites indicate a strong link between particular strains and
mawè processing. Likewise, other studies have determined that specific
strains have been linked to a specific food product where the dominant
strain was found to be better adapted to the environmental conditions
(Petersen et al., 2002). Moreover, the occurrence of single-site clusters,
where a unique strain cluster occurs, indicates a strong selection pres-
sure at the individual production sites and point to the importance of a
house hold specific microbiota. The fact that single-site clusters were
observed does likewise indicate that the mawè food matrix may act as a
reservoir for adaptation and evolution at subspecies level.
5. Conclusion
In conclusion, the present study confirmed that the spontaneous
mawè fermentations are dominated by adventitious LAB and yeasts.
Lactobacillus fermentum and P. kudriavzevii were identified as the pre-
dominant LAB and yeast species, respectively, and were present
throughout the mawè fermentations. Further, LAB and yeast succes-
sions took place with W. confusa occurring mostly at the onset, while P.
acidilactici and S. cerevisiae were mainly associated with the end of the
fermentations. The species diversity was influenced both by the type of
cereal and the production conditions of the four mawè types. The
dominating strain clusters of L. fermentum and P. kudriavzevii were
ubiquitous, whereas few strain clusters were detected only at a single
production site, which were mainly located in rural areas of Benin.
Additionally, L. fermentum and P. kudriavzevii strain diversities were
influenced by the type of cereal and production site. The deeper un-
derstanding of the microbial ecology of mawè processing obtained in
this study could add in upgrading the mawè processing to enhance
quality and food safety of mawè and derived products. Thus, there is a
need to standardise the different mawè processing methods and to turn
the mawè processing from spontaneous fermentation to controlled
fermentation at semi-industrial scale.
Acknowledgments
The authors would like to acknowledge Danida for funding through
the project Preserving African food microorganisms for Green Growth
(project number DFC No. 13-04KU).
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