ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 25 (2008) 616625
www.elsevier.com/locate/fm

Functional properties of selected starter cultures for sour maize bread

Mojisola O. Edemaa,, Abiodun I. Sannib
a

Department of Microbiology, College of Natural Sciences, University of Agriculture, P.M.B. 2240, Abeokuta, Nigeria
b
Department of Botany and Microbiology, University of Ibadan, Nigeria
Received 13 March 2007; received in revised form 17 December 2007; accepted 30 December 2007
Available online 29 January 2008

Abstract
This paper focuses on the functional properties of maize sour-dough microora selected and tested for their use as starter cultures for
sour maize bread. Lactic acid bacteria and yeasts isolated from spontaneously fermented maize dough were selected based on dominance
during fermentation and presence at the end of fermentation. Functional properties examined included acidication, leavening and
production of some antimicrobial compounds in the fermenting matrix. The organisms previously identied as Lactobacillus plantarum,
Lb. brevis, Lb. fermentum, Lb. acidophilus, Pediococcus acidilactici, Leuconostoc mesenteroides and Leuconostoc dextranicum and
Saccharomyces cerevisiae were used singly and as mixed cultures in the fermentation (fermentation time: 12 h at 2872 1C) of maize meal
(particle size 40.2 mm). The pH fell from an initial value of 5.623.05 in maize meals fermented with Lb. plantarum; 4.37 in
L. dextranicum+S. cerevisiae compared with the value for the control (no starter) of 4.54. Signicant differences (Pp0.05) were observed
in values obtained for the functional properties tested when starters were inoculated compared with the control (no starter) except for
leavening. Bivariate correlations at 0.01 levels (two-tailed) showed that signicant correlations existed among pH and production of
antimicrobial compounds in the fermenting meals, the highest correlation being between production of diacetyl and acid (0.694), a
positive correlation indicating that production of both antimicrobial compounds increase together with time. Antimicrobial activities of
the fermented maize dough were conrmed by their abilities to inhibit the growth of Salmonella typhi, Escherichia coli, Staphylococcus
aureus and Aspergillus flavus from an initial inoculum concentration of 7 log cfu ml1) for test bacteria and zone of inhibition of up to
1.33 cm for aatoxigenic A. flavus. The ndings of this study form a database for further studies on the development of starter cultures
for sour maize bread production as an alternative bread specialty.
r 2008 Elsevier Ltd. All rights reserved.
Keywords: Starter culture; Lactic acid bacteria; Yeasts; Fermentation; Maize meal

1. Introduction
Many foods are fermented before consumption and
lactic acid bacteria (LAB) are widely used as starter
organisms in these food fermentations because they
convert sugars into organic acids thus improving the
organoleptic and rheological properties of the products
(Konings et al., 2000; Vogel et al., 2002). Lactic and acetic
acid concentrations found in many fermented foods could
also be sufcient to impart the observed shelf stability.
Martinez-Anaya et al. (1994) reported that the efciency of
sourdough as a possible preservative agent of microbial
Corresponding author. Tel.: +234 8037119671.

E-mail addresses: moedemao@yahoo.co.uk (M.O. Edema),

abiodun_sanni@yahoo.co.uk (A.I. Sanni).
0740-0020/$ - see front matter r 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2007.12.006

spoilage of bread depends on its ability to produce acetic

acid. Pepe et al. (2003a) observed increased viscous
properties during fermentation and increased crumb
rmness in baked pizza dough leavened with LAB and
yeast. LAB are often inhibitory to other micro-organisms
and this is the basis of their ability to improve the keeping
quality of many fermented food products (Corsetti et al.,
1998a; Corsetti et al., 2000). LAB have been known to take
part in bread fermentations such as in the production of
the Swedish rye sourdough (Lonner and Preve-Akesson,
1988) and the Indian Idli (Mukherjee et al., 1965) wherein
they improve avor, texture and keeping quality through
the production of metabolites such as diacetyl, hydrogen peroxide and bacteriocins (Armero and Collar, 1996;
Arendt et al., 2007; Lacaze et al., 2007). For example, Pepe
et al. (2003b) observed an inhibition of rope-producing

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M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625

Bacillus subtilis spores for more than 15 days in breads

produced with strains of LAB isolated from sourdough. In
addition, results obtained by Budde et al. (2003) indicated
strong antilisterial activity by bacteriocin-producing
Leuconostoc carnosum without any observable undesirable
avor components.
It was customary in the beginning when cereals were
fermented by their natural ora, to put aside pieces of the
dough called sours or starters for fermenting subsequent
batches in bread making. This results in irregularities and
unpredictability that led to the development and use of
dened starter cultures of micro-organisms in modern
sourdough fermentations. To ensure products of consistent
avor, texture and shelf stability, as well as to improve
product safety, most processors have developed pure
microbial cultures to control the fermentation of their
products (Holzapfel, 2002). It is evident that with a starter
culture, the pH drops much more rapidly; hence the whole
manufacturing process is accelerated, leading to economical gains for the processor. The majority of starter cultures
are natural isolates of the desirable micro-organisms found
normally in the substrates (Holzapfel, 2002; De Vuyst and
Vancanneyt, 2007).
Starter cultures can come in fresh, frozen or freeze-dried
forms, and they can be single or mixed cultures of selected
strains of micro-organisms with denite characteristics that
are benecial in the manufacture of the desired product.
A wide variety of species of organisms have been used as
starter cultures in the food industry and many are being
investigated for their potential use as starter cultures
(De Vuyst and Neysens, 2005; Gaggiano et al., 2007). What
were probably the rst starter cultures for sourdoughs were
those developed by Kline and Sugihara (1971) for the San
Francisco sourdough. One of them was a pure culture
consisting of Lactobacillus sanfranciscensis (formerly
Lb. san Francisco) that had previously been isolated from
the San Francisco sourdough.
However, the use of sourdough starter cultures in the
baking industry is only in its infancy in Sub-Saharan Africa
and in Nigeria, is almost non-existent. Yet, there are a
number of substrates that can be exploited for use in the
development of new sourdoughs other than those existing
in Europe today. Maize is one such promising substrates
particularly as it lacks gluten which is a major source of
concern in baked goods from wheat and other cereals that
have gluten proteins, in order to avoid coeliac disease
(Di Cagno et al., 2002). Fairly successful attempts have
been made to develop sour maize bread using the
sourdough technique (Sanni et al., 1998). The sourdough
system is however a very complex one and there is a need to
study and understand the system in order to effectively
manage new products developed from novel sourdoughs
such as the sour maize meal. The composition and
dynamics of the micro-ora developing in spontaneously
fermented maize meal has therefore been studied (Edema
and Sanni, 2006). The dominating organisms in the micropopulation of the fermented maize meal were Pediococcus

617

acidilactici, Lactobacillus plantarum, Lactobacillus brevis,

Lactobacillus fermentum, Leuconostoc mesenteroides, Leuconostoc dextranicum, Lactobacillus casei, Candida albicans, Schizosaccharomyces pombe and Saccharomyces
cerevisiae. The aim of the present study was to select
starter cultures from the dominant microbial ora of sour
maize meal by investigating their functional properties with
a view to developing appropriate sour maize meal starter
for bread making.
2. Materials and methods
2.1. Sample collection and processing
A commercial our variety of white maize (Zea mays) was
obtained from Bodija market in Ibadan, southwestern
Nigeria. The grains were milled into maize meal with particle
size greater than 0.2 mm which is particularly valuable as an
ingredient for maize bread as well as meal mixes, maize
mufns and some extruded maize snack products compared
to maize our with less than 0.2 mm particle size (Okoruwa,
1995). A knife mill (Fritsch Industriestr. 8 0-55743, Idaroberstein, Germany) was used for milling. The chemical
characteristics of the maize meal were as follows: moisture
content 7.15%, fat 4.09%, protein (N  5.70) 8.96%, ber
1.48%, ash 1.33% and total carbohydrate 77.06% of dry
matter (Edema et al., 2005).
2.2. Experimental design
In the preliminary study, 34 LAB belonging to 15 species
and 13 yeasts belonging to nine species were isolated during
the spontaneous fermentation of maize meal (fermentation
time 48 h, ambient temperature 28 1C, nal pH 3.71)
(Edema and Sanni, 2006). Eight test cultures comprising
seven LAB and one yeast were chosen from these isolates
on the basis of dominance during fermentation and
presence at the end of fermentation (Holzapfel, 2002).
These were Lb. plantarum, Lb. brevis, Lb. fermentum, Lb.
acidophilus, P. acidilactici, L. mesenteroides, L. dextranicum
and S. cerevisiae.
These organisms were tested for those functional properties that are important in the sourdough technology that is
acidication and production of antimicrobial substances.
The antimicrobial properties of the test cultures in maize
dough were conrmed with inhibition of some selected
pathogens (Kingamkono et al., 1995; Holzapfel, 2002). To
eliminate bias, selected test cultures were used singly and as
mixed cultures in the fermentation of maize meal using a
completely randomized block design on a factorial basis
with three replicates. The spontaneously fermented maize
meal without added starter culture served as control.
2.3. Culture conditions and fermentation
Puried isolates of the test LAB that had been kept
as stock cultures on Hogness freezing medium were

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M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625

cultivated by transferring 2 ml of each stock culture into

8 ml of de Man, Rogosa & Sharpe (MRS) broth medium
(Oxoid, Hampshire, UK). The tubes were incubated at
30 1C for 48 h. The broth cultures were again inoculated
into fresh MRS broth medium and incubated as above.
During incubation, 1 ml each of broth culture was plated
on MRS agar plates using the pour plate technique with
routine incubation at 37 1C for 24 h. Colonies were counted
and broth cultures of corresponding plates containing
about 2  108 cfu ml1 were used as inoculums. Broth
cultures containing the required concentration of viable
cells were centrifuged (Labofuge 200, Kendro Laboratory
Products, Germany) at 6000  g for 10 min, washed in
sterile distilled water (pH 7.0) and re-centrifuged before
being suspended in sterile distilled water. The washed
harvested cells were then used as inoculum in the
fermentation of maize meals according to the method of
Lonner et al. (1986).
Yeast culture inoculum was prepared using malt extract
broth. Five ml sterile malt extract broth were added to the
yeast cultures growing on malt extract agar slants and
shaken to make a suspension. Each suspension was poured
into another 5 ml malt extract broth and incubated at 30 1C
for 24 h. Dilutions of the broth cultures were plated on
malt extract agar and incubated for another 24 h at 30 1C.
Plates of broth cultures that gave 2  108 cfu ml1 were
used as inoculum. The broth cultures having the required
counts were then centrifuged (Labofuge 200) and washed
twice in sterile distilled water before use as inoculum (Halm
et al., 1996).
Equal amounts (w/v) of maize meal to tap water were
used in all the trials. Five ml of inoculum containing
approximately the same concentration of cells each
(2  108 cfu ml1 from pour plates) was used in all cases
whether singly or mixed for 50 g maize meal. Mixing was
done manually (for up to 5 min) in glass bowls using glass
rods as stirrers. Fermentation was carried out at ambient
temperature (2872 1C) for up to 24 h. During fermentation, the number of inoculum cells was estimated by plating
1 g of fermenting maize meal on MRS agar at 30 1C
for 48 h.
2.4. Analyses of fermenting maize meal
2.4.1. pH
After fermentation, the pH values of the starterfermented maize meals were determined with a combined
glass electrode and pH meter (Mettler-Toledo, Essex
M3509 Type 340).
2.4.2. Acidity
The amount of acid produced in the fermented meals
was determined by the standard titration procedure for
total titratable acidity (TTA) according to Lonner et al.
(1986). One gram of the fermenting meal was mixed with
9 ml sterile distilled water and homogenized. The mixture
(10 ml) was titrated with 1 N NaOH using phenolphthalein

as indicator. Acid equivalent is the amount of NaOH

consumed in ml. Each ml of 1 N NaOH is equivalent to
90.08 mg of lactic acid.
2.4.3. Leavening
Leavening in the fermented meal was determined by
recording the level of the fermenting maize meal on the
graduated bottles used for fermentation at mixing and at
the end of fermentation. The difference between the initial
and the nal readings were taken as the level of leavening
in centimeters.
2.4.4. Diacetyl
Diacetyl production was determined by mixing 10 g
fermenting maize meal in 90 ml tap water. To 25 ml each of
the homogenized mixture, 7.5 ml of hydroxylamine solution (1 M) was added in two asks (one ask was for
residual titration). Both asks were titrated with 0.1 N HCl
to a greenish yellow end point using bromophenol blue as
indicator (Sanni et al., 1995). The equivalence factor of
HCl to diacetyl is 21.52 mg. The concentration of diacetyl
produced was calculated as follows:
R  S100E
,
W
where Ak (mg) is the percentage of diacetyl, R the ml of
0.1 N HCl consumed in residual titration, S the ml of 0.1 N
HCl consumed in titration of sample, E the equivalence
factor and W is the volume of sample.
Ak

2.4.5. Hydrogen peroxide

Hydrogen peroxide production was determined by
measuring 25 ml of homogenized mixture (from the same
batch used for diacetyl) into a 100-ml ask. To this was
added 25 ml of dilute H2SO4 (10%). The preparation was
then titrated with 0.1 N potassium permanganate
(KMnO4). The end point was the point at which the pale
pink color persisted for 15 s before de-colorization. Each
ml of 0.1 N KmnO4 is equivalent to 1.701 mg of H2O2
(Sanni et al., 1995). The volume of H2O2 produced was
then calculated as follows:
H2 O2 concentration

KMnO4 ml  KMnO4 N  M:E:  100

.
H2 SO4 ml  volume of sample

2.4.6. Antimicrobial activity

Antimicrobial activities of the fermenting maize meals
were determined by two procedures (Piddock, 1990). The
agar well diffusion method was used to test the antimicrobial activity of fermenting meal against aatoxigenic
mold, Aspergillus flavus previously isolated from grilled,
dry meat and conrmed to produce aatoxin by uorescence under ultraviolet radiation in Yeast Extract Sucrose
medium was used (Cotty, 1994; Onilude et al., 2005).
Spores were harvested from stock culture maintained on
agar slant by adding 10 ml sterile distilled water to dislodge

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M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625

2.4.7. Inoculum growth assessment

The growth of each inoculum was monitored during the
fermentations by plating out dilutions using the pour plate
method. Counts were also taken for the un-inoculated
sample before and after fermentation for comparison.
Total viable counts were made on MRS of de Man et al.
(1960) for the LAB and on malt extract agar for yeasts.
MRS was adjusted to pH 5.4 while streptomycin sulfate
and penicillin were added to MEA in the ratio 2:1,
respectively/liter, to inhibit the growth of unwanted
organisms. As expected, cell concentrations increased
during fermentation (Muller et al., 2001).
2.4.8. Analysis of data
All experiments were carried out in triplicate trials and
the data generated were subjected to one-way analysis of
variance (ANOVA) at 5% level of signicance and
bivariate correlations using SPSS11.0 for windows. Means
were separated by Duncans multiple range tests.
3. Results
Thirty-four LAB belonging to 15 species and 13 yeasts
belonging to nine species were isolated during the
spontaneous fermentation of maize meal (fermentation
time 48 h, ambient temperature 2872 1C, nal pH 3.71) in
a previous study by the authors (Edema and Sanni, 2006).
Counts of LAB increased steadily from 4.62 log at mixing
(0 h) to 6.45 log after 48 h fermentation while yeast counts
increased from 4.18 to 6.64 log within the same period of
fermentation (Fig. 1). Eight test cultures comprising seven
LAB and one yeast were chosen from these isolates on the
basis of dominance during fermentation and presence
at the end of fermentation. These were Lb. plantarum,
Lb. brevis, Lb. fermentum, Lb. acidophilus, P. acidilactici,
L. mesenteroides, L. dextranicum and S. cerevisiae.
Some functional properties important in soudough were
examined by inoculating the selected organisms into maize

9
8
Log of counts (cfu/g)

the spores. Molten Potato Dextrose Agar (Oxoid) in

Erlenmeyer ask was inoculated with the spore suspension
at a concentration of 5% (v/v). Incubation was 30 1C for
up to 72 h on. For test bacteria, Salmonella typhi, E. coli
and S. aureus, a modication of the method used by Rusol
et al. (1997) was adopted. From stock cultures obtained in
the Department of Microbiology, University of Agriculture, Abeokuta, one colony each of test organisms were
inoculated in MacConkey broth for E. coli, tetrathionate
broth for S. typhi and BairdParker medium for Stapholyococcus. After incubation at 37 1C for 24 h, the cells were
harvested by centrifugation at 6000  g, 15 min and washed
with sterile distilled water. Ten-fold dilution of each sample
was then plated to obtain 7 log cfu ml1. Dilutions containing the required number of cells were used as inoculum by
mixing 1 ml of the cell suspension with 9 g of fermenting
maize meal. The tubes were incubated at 37 1C for 24 h
before enumeration.

619

LAB

7
6

Yeasts

5
4

PCA
counts

3
2
1
0

12

24

36

48

Time (h)
Fig. 1. Plate counts of LAB and yeasts during spontaneous fermentation
of maize meal.

meal (fermentation conditions, 2872 1C for 12 h; inoculum

rate, 108 cfu g1; dough yield, 192). Functional properties
examined in this study included acidication and leavening
patterns as well as the production of some antimicrobial
compounds. The initial pH of the maize meal before
fermentation was 5.62. For single cultures, all LAB species
were able to lower pH during fermentation more than
yeasts with pH values ranging from 3.05 for Lb. plantarum
to 3.37 for L. mesenteroides as against 3.65 for S. cerevisiae
(Table 1). Mixed cultures also lowered the pH of the
fermenting meal to varying degrees with signicant
differences (Pp0.05) in the values obtained. All starters,
whether single or mixed were however able to lower the pH
of the fermenting meals signicantly more than that of the
control, i.e. 4.54 at the same probability level. Conversely,
production of acid, diacetyl and hydrogen peroxide in the
fermenting meals were signicantly higher in meals
fermented with starter cultures than in the spontaneous
sour. Leavening in fermented maize meals ranged from
1.20 to 1.80 cm. Signicant differences (Pp0.05) were
observed in values obtained for the functional properties
tested when starters were inoculated compared with the
control (no starter) except leavening.
Bivariate correlations at 0.01 levels (two-tailed) were
used to evaluate correlations among the functional properties examined. Signicant negative correlations existed
among pH, acid production and diacetyl production in
the fermenting meals while signicant positive correlations
were observed in the production of H2O2 and pH on one
hand, and in the production of acid, diacetyl and leavening
on the other hand (Table 2). The highest correlation was
observed between production of diacetyl and acid (0.694).
It was a positive correlation indicating that production of
both antimicrobial compounds increase together with time.
Antimicrobial activities of the fermented maize dough
were conrmed by testing their abilities to inhibit the
growth of selected indicator organisms: S. typhi, E. coli,
S. aureus and A. flavus. Numbers of inoculum were
drastically reduced from about 6 log cfu ml1 (approximate
cell concentration after inoculating 7 log cfu ml1 of

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M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625

Table 1
Functional properties of sour maize meals started with single and mixed cultures of LAB and yeasts
Starter

pH

Acid equivalent
(ml)

Diacetyl
(mg)

Hydrogen
peroxide (mM)

Leavening
(cm)

L. plantarum
L. brevis
L. fermentum
L. acidophilus
P. acidilactici
Leu. mesenteroides
Leu. dextranicum
S. cerevisiae
L. plantarum and L. brevis
L. plantarum and L. fermentum
L. plantarum and L. acidophilus
L. plantarum and P. acidilactici
L. plantarum and Leu. mesenteroides
L. plantarum and Leu. dextranicum
L. plantarum and S. cerevisiae
L. brevis and L. fermentum
L. brevis and L. acidophilus
L. brevis and P. acidilactici
L. brevis and Leu. mesenteroides
L. brevis and Leu. dexranicum
L. brevis and S. cerevisiae
L. fermentum and L. acidophilus
L. fermentum and P. acidilactici
L. fermentum and Leu. mesenteroides
L. fermentum and Leu. dextranicum
L. fermentum and S. cerevisiae
L. acidophilus and P. acidilactici
L. acidophilus and Leu. mesenteroides
L. acidophilus and Leu. dextranicum
L. acidophilus and S. cerevisiae
P. acidilactici and Leu. mesenteroides
P. acidilactici and Leu. dextranicum
P. acidilactici and S. cerevisiae
Leu. mesenteroides and Leu. dextranicum
Leu. mesenteroides and S. cerevisiae
Leu. dextranicum and S. cerevisiae
Control (no starter added)

3.05a
3.16ab
3.26ab
3.09a
3.36abc
3.37abc
3.36abc
3.65abc
3.20ab
3.12ab
3.03a
3.04a
3.15ab
3.40abc
3.35abc
3.41abc
3.48abc
3.46abc
3.39abc
3.92abc
3.55abc
3.91abc
3.88abc
3.85abc
3.85abc
3.95abc
3.71abc
4.19abc
4.08abc
4.12abc
3.93abc
3.78abc
4.08abc
3.75abc
4.04abc
4.37bc
4.54c

3.20ijkl
2.90ghij
2.80ghi
3.00hijk
2.60efgh
2.50cdefg
2.50cdefg
1.33b
3.28jkl
3.36kl
3.49l
2.80ghi
3.46l
2.92ghij
3.50l
2.15cde
2.71gh
2.63fgh
2.16cde
2.16cde
2.70gh
2.10c
2.59defgh
2.15cde
2.16cde
2.53cdefgh
2.83ghi
2.15cde
2.12cd
2.69gh
2.17cdef
2.48cdefg
2.50cdefg
2.17cdef
2.88ghij
2.50cdefg
0.80a

163.55q
180.77u
172.16s
154.94o
154.94o
129.12j
111.90d
94.69b
176.46t
172.20s
172.30s
173.07s
172.77s
185.07v
154.94o
155.07o
156.77p
154.40o
134.70m
167.88r
124.81i
122.17h
121.20gh
120.33fg
121.00gh
130.37k
119.67ef
120.13fg
120.03fg
151.40n
118.70e
118.80e
122.13h
100.27c
120.17fg
133.42l
84.47a

4.25ef
4.22def
4.25ef
4.08bcdef
4.25ef
4.29ef
4.20def
4.08bcdef
4.42f
3.17abcd
3.07ab
3.17abcd
3.35abcde
3.31abcde
4.25ef
3.11abc
3.17abcd
4.02bcdef
3.56abcdef
4.08bcdef
4.17def
4.01bcdef
4.05bcdef
3.99bcdef
3.99bcdef
4.06bcdef
3.94bcdef
3.96bcdef
3.98bcdef
4.06bcdef
4.11bcdef
4.07bcdef
4.16cdef
4.44f
4.22def
4.18def
2.89a

1.33bcd
1.33bcd
1.37cde
1.30abc
1.20a
1.43def
1.37cde
1.50efgh
1.57fghij
1.63hij
1.63hij
1.57fghij
1.67ij
1.53fghi
1.80k
1.62hij
1.59ghij
1.58ghij
1.58ghij
1.70jk
1.80k
1.57fghij
1.60ghij
1.60ghij
1.50efgh
1.63hij
1.47defg
1.50efgh
1.53fghi
1.67ij
1.47defg
1.43def
1.63hij
1.57fghij
1.67ij
1.70jk
1.23ab

Values are means of three replicates. Mean values followed by different letters within columns are signicantly different by Duncans multiple range tests
(Pp0.05).

washed cells into moistened maize meal). All starters

including the control were able to inhibit growth of the
test bacteria from an initial inoculum concentration of
approximately 6 log cfu ml1 at 35 1C; 24 h to as low as
2 log cfu ml1 for S. typhi and E. coli. S. cerevisiae recorded
the least inhibitory activity against the pathogenic bacteria
tested (Fig. 2).
For A. flavus, the agar well diffusion method was used
to determine antimicrobial activity of starter fermented
maize meals. The zone of inhibition ranged from 0.60 cm
for dough fermented with S. cerevisiae to 1.33 cm for
dough fermented with Lb. plantarum and Lb. brevis
(Fig. 3). The pattern of zone of inhibition of A. flavus
observed for maize meal fermented with mixed culture of
Lb. plantarum and Lb. brevis is shown in Fig. 4. The reason
for the central zone of inhibition is not clear but could have
resulted from production of bacteriocin-like substances

which moved in the direction of observed zone of

inhibition.
4. Discussion
The selection of starter cultures in this study involved the
investigation of some important functional properties of
strains of LAB and yeasts isolated from spontaneously
fermented maize meal. Cultures for food fermentations are
selected primarily on the basis of their stability and their
ability to produce the desired products or changes
efciently (Gobbetti, 1998; Leroy and De Vuyst, 2004).
These cultures may be established ones obtained from
other laboratories or they may be selected after testing
many numerous strains (Sanni et al., 2002; Gaggiano et al.,
2007). The latter alternative was employed in this work.
This is because organisms develop niches where they thrive

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M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625
Table 2
Correlations among functional properties of starter-generated sour maize
meals
pH
pH
Pearson correlation 1
Sig. (two-tailed)

N
111

Acid

Diacetyl Peroxide Leavening

.288 .426 .384

.002
.000
.000
111
111
111

.132
.167
111

Acid
Pearson correlation .288 1
Sig. (two-tailed)
.002

N
111
111

.694
.000
111

.025
.797
111

.256
.007
111

Diacetyl
Pearson correlation .426 .694
Sig. (two-tailed)
.000
.000
N
111
111

111

.158
.099
111

.097
.310
111

Peroxide
Pearson correlation .384
Sig. (two-tailed)
.000
N
111

.025
.797
111

.158
.099
111

111

.071
.462
111

Leavening
Pearson correlation .132
Sig. (two-tailed)
.167
N
111

.256
.007
111

.097
.310
111

.071
.462
111

111

Correlation is signicant at the 0.01 level (two-tailed).

and to transplant an organism from one natural environment to another is not a good formula for success,
particularly in sourdough fermentations. The species of
LAB and yeasts isolated from fermenting maize meal and
tested for their functional properties in this work were the
dominant micro-organisms at the end of 48 h spontaneous
fermentation of maize meal (Edema and Sanni, 2006).
Previous studies on the natural micro-biological ora of
sourdough made from different cereals report similar
organisms (Spicher, 1987; Gobbetti et al., 1995; Meroth
et al., 2004; De Vuyst and Neysens, 2005; Ehrmann and
Vogel, 2005).
Micro-organisms necessary in food fermentations may
be added as pure single or mixed cultures. Although in
some instances, no cultures may be added if the desired
micro-organisms are known to be present in sufcient
numbers in the original raw material (Lonner et al., 1986;
Randazzo et al., 2005). In the case of sour maize meal, the
presence of the desired organisms, that is LAB and yeasts,
in the raw material whether sufcient or not, is not
adequate since all the meals fermented with starter cultures
were better in acidication and other properties than the
spontaneously fermented meal used as control. This could
be because there are usually other numerous and undesirable competing micro-organisms as earlier observed
(Edema and Sanni, 2006). Although given favorable
environmental conditions, the desired organisms will
eventually act in proper succession (Wiese et al., 1996;
Gatto and Torriani, 2004), it is advantageous to use
controlled starter cultures as these will drastically reduce

621

the period of fermentation, sometimes more than half

depending on the amount of starter used (Oyewole, 1990;
Sanni et al., 1999; De Vuyst et al., 2002).
As the preparation of sourdough is very time consuming
if full leavening by LAB is to be obtained, few processes
have been developed in Germany, which ensure that the
dough is acidied by the sourdough bacteria while
leavening is achieved by bakers yeast (Ganzle, 2002).
However, bakers yeast was not used in this study because
of the short shelf life recorded in bakers yeastleavened
sour maize bread prepared in a preliminary study (Sanni
et al., 1998). Rather, a S. cerevisiae strain isolated from the
spontaneously fermented maize meal was used. It was used
singly and in combination with the selected LAB cultures
and was observed to produce inhibition of test pathogens
in addition to leavening. In sourdough, yeast is important
for good batter leavening and bread viscosity while the
LAB produce acids and other metabolites which inhibit the
growth of spoilage organisms such as molds or ropecausing bacilli (Amoa-Awua et al., 1997; Corsetti et al.,
2000). Yeasts do not produce appreciable amounts of
organic acids, their main metabolites being ethanol and
carbon dioxide (Campbell-Platt, 1987; Sanni and Lonner,
1993). The co-interaction between LAB and yeasts is
however notably common in many food and beverage
fermentations with the LAB providing the acid environment for yeast growth, while the yeasts provide vitamins
and other growth factors for the LAB (Odunfa and
Adeyele, 1985; Lonner and Preve-Akesson, 1988; Corsetti
et al., 2000). The observed antimicrobial property in maize
meal starter on yeast could therefore have been as a result
of the presence and growth of endogenous LAB in the
substrate since the our was not sterile.
Maize meals started with mixed cultures containing
Lb. plantarum produced more acid and diacetyl than the
others. Generally, it was observed that mixed cultures
appeared to produce more antimicrobial compounds than
the single cultures. Indeed maize meals started with mixed
cultures were preferred to those started with single cultures
in taste and aroma (result not shown). Lb. plantarum has
been shown to posses the ability for rapid acidication,
production of antimicrobial compounds and the most
effective antifungal effect against toxigenic strains of
Penicillium and Aspergillus (Niku-Paavola et al., 1999) as
well as the production of exopolysaccharide (Figueroa
et al., 1997).
Antimicrobial activities of the fermented maize dough
were conrmed by the inhibition of the growth of S. typhi,
E. coli, S. aureus and A. flavus. E. coli occurs normally in
human intestines and can get into foods handled without
washing hands after changing diapers or using toilets.
S. typhi is transmitted through contaminated poultry, eggs
and other foods when they are handled unhygienically.
Staphylococcus occurs almost everywhere and grows readily in foods stored at room temperature. S. aureus is found
on the skin and in the nostrils of many healthy individuals
where it causes some skin infections, but it is also an agent

L.
pl
an
L. L. taru
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L. er bre m
ac me v i
Le
u. P. ido n tu s
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L. L.
Le es ci d hil u
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p
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L.
a
a
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c n es
L. nta l ant rum an an ere i cu
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s
u n L
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an an u. . do tu
L. tar d L me ac id ph i m
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l
L. bre m a eu. sen i lac u s
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L. is ev is and L. ere ic u s
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u.
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Le e a c ci e d e x r c i
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)

Zone of inhibition (cm)

L.
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L. er bre m
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Le
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de te an nd de ter si a
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Log cfu/g

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M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625

4
S. typhi

3
E. coli

S. aureus

Starter

Fig. 2. Inhibition of three pathogenic bacteria by sour maize meals started with single and mixed cultures of LAB and yeasts (initial inoculum
concentration 7 log cfu ml1, incubation 15 1C for 24 h).

1.4

1.2

0.8

0.6

0.4

0.2

Starter

Fig. 3. Inhibition of Aspergillus flavus by sour maize meals started with single and mixed cultures of LAB and yeasts.

of food poisoning when it gets in contact with food where it

multiplies and produces its toxin (Frazier and Westhoff,
1986). A. flavus is a ubiquitous spore-former and its spores
are found everywhere in nature food surfaces inclusive. The
spores germinate in foods such as cereals whenever

prevailing environmental conditions permit. All starters

were able to inhibit growth of the tested pathogens to
varying degrees, including the maize dough started with
yeast and the spontaneously fermented maize meal. It is
believed that the growth of endogenous LAB in the maize

ARTICLE IN PRESS
M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625

Fig. 4. Inhibition of Aspergillus flavus by sour maize meal started on a

mixed culture of L. plantarum and L. brevis.

substrate was responsible for the inhibitory ability of the

spontaneously fermented maize meal. Previous studies
have also shown inhibition of similar pathogens by LAB
in related spontaneous fermentations (Svanberg et al.,
1992; Kingamkono et al., 1995; Olasupo et al., 1997; Sanni
et al., 1999).
At different pH ranges, the minimum inhibitory concentration (MIC) of the undissociated lactic acid was
different against pathogens such as Clostridium and
Enterobacter has been shown to differ (Lindgren and
Dobrogosz, 1990). In addition, the stereoisomers of lactic
acid also differ in antimicrobial activity, L-lactic acid being
more inhibitory than the D-isomer (Benthin and Villadsen,
1995). Acetic and propionic acids produced by LAB strains
through heterofermentative pathways may interact with
cell membranes, and cause intracellular acidication and
protein denaturation (Huang et al., 1986). They are usually
more antimicrobially effective than lactic acid due to their
higher pKa values (lactic acid 3.08, acetic acid 4.75, and
propionic acid 4.87), and higher percentage of undissociated acids than lactic acid at a given pH (Earnshaw,
1992). Acetic acid has been shown to be more inhibitory
than lactic acid toward Listeria monocytogenes (Ahmad
and Marth, 1989; Richards et al., 1995), and toward the
growth and germination of Bacillus cereus (Wong and
Chen, 1988). Acetic acid also acted synergistically with
lactic acid with lactic acid decreasing the pH of the
medium, thereby increasing the toxicity of acetic acid
(Adams and Hall, 1988). In addition, hydrogen peroxide
produced by LAB has been shown to produce antimicrobial effect from the oxidation of sulfhydryl groups causing
denaturing of a number of enzymes, and from the

623

peroxidation of membrane lipids thereby increasing membrane permeability. Hydrogen peroxide may also be as a
precursor for the production of bactericidal free radicals
such as superoxide and hydroxyl radicals which can
damage DNA (Yang et al., 1997). It has been reported
that the production of H2O2 by Lactobacillus and
Lactococcus strains inhibited S. aureus, Pseudomonas sp.
and various psychotropic micro-organisms in foods (Cords
and Dychdala, 1993).
Inhibition of A. flavus by sour maize meal started on a
mixed culture of Lb. plantarum and Lb. brevis revealed a
central pattern which could be as a result of production of
bacteriocin-like substance (Corsetti et al., 1998b, 2004;
Vanne et al., 2001) that moved in the direction of observed
inhibition. The inhibition is not likely to have been entirely
a result of acid production by the starters, since it has
previously been shown that Aspergillus parasiticus NRRL
2999 grew in a medium containing up to 0.75% lactic acid
at pH 3.5 (El-Gazzar et al., 1987). It can be observed
however that there is some yeast growth on the plate most
likely from endogenous culture of the maize meal. That
however did not prevent the inhibitory action of the starter,
as the starter culture was in a large quantity enough to start
off the fermentation and the action before the growth of
the endogenous organisms.
Studies on sourdough have been carried out especially in
Germany, France and Sweden where the sourdough
fermentation process has been used traditionally for rye,
rye-mixes and other ours which are difcult to bake
without souring. (Lonner et al., 1986; De Vuyst et al., 2002;
Catzeddu et al., 2006; Valcheva et al., 2006). Also, in the
United States, the San Francisco sourdough bread process
has been carried out in the San Francisco bay area for over
130 years (Sugihara et al., 1970). The use of this technique
is relatively new to Nigeria but is desirable as a way of
utilizing local substrates such as maize and cassava for
development of new food varieties. LAB are able to
produce a wide variety of compounds which give fermented
foods such as sourdough their characteristic avor and also
impart improved safety and rheology to the foods (Arendt
et al., 2007). However, the properties of the sourdough
system depend on many different concurrent factors as well
as external process conditions all of which inuence the
properties of the products. Some of these factors inuence
the properties of the system directly, while others have an
indirect inuence by affecting the activity and the
metabolism of the micro-organisms (Lacaze et al., 2007).
Consistent quality, safety and acceptability of a new
product such as the sour maize bread will be signicantly
improved by the use of well-dened starter cultures selected
on the basis of multifunctional considerations. It is
therefore imperative to properly study and regulate the
fermentation process in order to obtain a desirable
product, as in the focus on ongoing research by the
authors. Further studies are focused on the development of
fast starters with emphasis on rate of leavening, ultimate
levels of acidity produced and avor. Molecular studies,

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M.O. Edema, A.I. Sanni / Food Microbiology 25 (2008) 616625

baking trials and a standardized procedure for pilot-scale

production are also in view.
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