See discussions, stats, and author profiles for this publication at:

https://www.researchgate.net/publication/10761937

Use of starter cultures of lactic acid

bacteria and yeasts in the preparation of
Togwa, a Tanzanian fermented food

ARTICLE in INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY · JUNE 2003

Impact Factor: 3.08 · DOI: 10.1016/S0168-1605(02)00386-0 · Source: PubMed

CITATIONS READS

61 341

3 AUTHORS:

Jovin K Mugula Judith A. Narvhus

Sokoine University of Agriculture (SUA) Norwegian University of Life Sciences (…
16 PUBLICATIONS 333 CITATIONS 70 PUBLICATIONS 1,429 CITATIONS

SEE PROFILE SEE PROFILE

Terje Sørhaug
Norwegian University of Life Sciences (…
55 PUBLICATIONS 1,232 CITATIONS

SEE PROFILE

All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Judith A. Narvhus
letting you access and read them immediately. Retrieved on: 03 February 2016
 International Journal of Food Microbiology 83 (2003) 307 – 318
www.elsevier.com/locate/ijfoodmicro

Use of starter cultures of lactic acid bacteria and yeasts in the

preparation of togwa, a Tanzanian fermented food
J.K. Mugula*, J.A. Narvhus, T. Sørhaug
Department of Food Science, Agricultural University of Norway, PO Box 5036, N-1432 Ås, Norway
Received 29 April 2001; received in revised form 13 July 2002; accepted 18 July 2002

Abstract

Starter cultures of lactic acid bacteria (Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus fermentum,
Lactobacillus plantarum and Pediococcus pentosaceus) and yeasts (Candida pelliculosa, Candida tropicalis, Issatchenkia
orientalis and Saccharomyes cerevisiae) isolated from native togwa were tested singly or in combination for their ability to
ferment maize – sorghum gruel to produce togwa. All species of bacteria showed an ability to ferment the gruel as judged by
lowering the pH from 5.87 to 3.24 – 3.49 and increasing the titratable acidity from 0.08% to 0.30 – 0.44% (w/w, lactic acid) in 24
h. Yeasts used singly showed little activity within 12 h, but lowered the pH to 3.57 – 4.81 and increased the acidity to 0.11 –
0.21% in 24 h. Yeasts in co-culture with lactic acid bacteria (LAB) had a modest effect on the final acidity ( P < 0.05). The
number of lactic acid bacteria and yeasts increased while the Enterobacteriaceae decreased with fermentation time. The pH was
lowered and lactic acid produced significantly ( P < 0.05) fastest in natural togwa fermentation and in samples fermented by L.
plantarum or L. plantarum in co-culture with I. orientalis. The content of fermentable sugars was reduced during fermentation.
Most volatile flavour compounds were produced in samples from fermentation by P. pentosaceus and I. orientalis in co-culture
with either L. plantarum or L. brevis.
D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Cereal fermentation; Togwa; Starter cultures; Lactic acid bacteria; Yeasts; Sugars; Organic acids; Volatile organic compounds

1. Introduction 1997; Nago et al., 1998; Kunene et al., 2000). Stable

Lactic acid bacteria (LAB) and yeasts have been
reported to be the predominant microorganisms in
most of the African indigenous fermented foods
(Nout, 1991; Halm et al., 1993; Hounhouigan et al.,
1993; Sanni, 1993; Steinkraus, 1996; Olasupo et al.,
* Corresponding author. Present address: Department of Food
Science and Technology, Sokoine University of Agriculture, PO
Box 3006, Morogoro, Tanzania. Tel./fax: +255-23-2604402.
E-mail address: jmugula@yahoo.com (J.K. Mugula).
co-metabolism between LAB and yeasts is common in
many foods, enabling the utilization of substances that
are otherwise nonfermentable (for example starch)
and thus increasing the microbial adaptability to
complex food ecosystems (Gobbetti et al., 1994; Stolz
et al., 1995; Gobbetti and Corsetti, 1997).
It has been suggested that the proliferation of
yeasts in foods is favoured by the acidic environment
created by LAB while the growth of bacteria is
stimulated by the presence of yeasts, which may pro-
vide growth factors, such as, vitamins and soluble

0168-1605/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0168-1605(02)00386-0
308 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318
nitrogen compounds (Nout, 1991). The association of
LAB and yeasts during fermentation may also con-
tribute metabolites, which could impart taste and
flavour to foods (Akinrele, 1970; Halm et al., 1993;
Brauman et al., 1996; Hansen and Hansen, 1996). The
production of acids and other antimicrobial compo-
nents in gruel during fermentation may promote or
improve the microbiological safety (Nout et al., 1989;
Svanberg et al., 1992; Kingamkono et al., 1994, 1995)
and stability of the products (Mensah et al., 1991).
According to Sanni (1993) and Kimaryo et al.
(2000), the use of starter cultures would be an
appropriate approach for the control and optimisation
of the fermentation process in order to alleviate the
problems of variations in organoleptic quality and
microbiological stability observed in African indige-
nous fermented foods. No LAB starter cultures are
commercially available yet for the small-scale pro-
cessing of traditional African foods (Holzapfel,
1997).
The development of starter cultures is one of the
prerequisites for the establishment of small-scale
industrial production of fermented foods in Africa
(Sanni, 1993). The primary consideration before intro-
ducing starter cultures for traditional small-scale fer-
mentations should be whether these would signi-
ficantly contribute to an improvement of processing
conditions and product quality with respect to: rapid
or accelerated acidification, an improved and more
predictable fermentation process, desirable sensory
attributes, improved safety and reduction of hygienic
risks (Holzapfel, 1997). Therefore, a thorough under-
standing of the fermentation process is required. The
knowledge gained with controlled starter culture may
also benefit those operating at a very small scale and
practising backslopping.
Togwa is a fermented gruel or beverage prepared
either from cassava, maize, sorghum, millet or their
combinations (Mugula et al., 2001). LAB and yeasts
are the predominant microorganisms found in togwa
(Mugula et al., 2001). Their role needs to be inves-
tigated in order to identify those functionally most
effective for the preparation of this product. This
study aimed to clarify the role of different selected
species of LAB and yeasts with respect to their
contribution to stability and safety of the product
and to the formation of flavour compounds during
fermentation in the preparation of togwa.
2. Materials and methods
2.1. Enumeration and characterization of LAB and
yeasts
Duplicate samples of togwa (10 ml) were homo-
genized with 90 ml sterile peptone physiological
saline solution (5 g peptone, 8.5 g NaCl, 1000 ml
distilled water, pH 7.0 F 0.2). The homogenate was
decimal diluted and the relevant dilutions surface
plated. M17 agar (Merck, Darmstadt, Germany)
plates containing 0.1% (w/v) glucose were incu-
bated aerobically and MRS agar (Merck) plates
containing 0.1% (w/v) natamycin (Delvocid, Delft,
The Netherlands) were incubated anaerobically
(BBL Gas Pak, H2 and CO2; Becton Dickinson,
Cockeysville, MD, USA) for 48 h at 30 jC for the
enumeration and isolation of lactic acid bacteria
(LAB). A total of 120 representative colonies were
randomly picked from higher dilution plates of
various fermentation stages and confirmed to be
Gram-positive and catalase-negative. For subsequent
purification and sub-culturing M17 and MRS agar
and broths were used. The pure bacterial cultures
were inoculated into appropriate broth, incubated
for 24 h at 30 jC, centrifuged (Kubota 2010,
Kubota, Tokyo, Japan) at 3000 rpm for 15 min
and the supernatant decanted. The cell pellets were
re-suspended either in sterile MRS or M17 broth
containing 10% (v/v) glycerol. The suspension was
aseptically transferred into sterile cryo-tubes con-
taining acid-washed glass beads and stored at 80
jC until required for identification. Aerobic meso-
philic bacteria in togwa were enumerated on plate
count agar (PCA, Merck) after incubation for 2
days at 30 jC and Enterobacteriaceae on violet red
bile glucose agar (VRBGA, Oxoid) after incubation
for 24 h at 37 jC. Yeasts were enumerated and
isolated after incubation for 3– 5 days at 25 jC on
wort agar (WA, Merck) containing 0.01% (w/v)
sterile oxytetracyline (Merck) or on Rose Bengal
Chloramphenicol agar (RBCA, Oxoid) containing
0.01% (w/v) chloramphenicol (selective supplement,
Oxoid). Purification and sub-culturing was done
using potato dextrose agar (PDA, Oxoid) and yeast
extract – malt extract (YM) broth. The purified yeast
cultures were stored on PDA slants at 4 jC until
required for identification and further use.
 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318
The bacteria were characterized by microscopic
examination and by conventional biochemical and
physiological tests. The cultures were examined for
colony and cell morphology; motility, cell arrange-
ment, Gram reaction; catalase reaction; growth in
broth at 10, 15, 40 and 45 jC; growth in presence
of 2%, 4%, and 6.5% (w/v) NaCl; production of
ammonia from arginine; production of dextran from
sucrose; and production of carbon dioxide from
glucose using Gibson’s litmus milk. These tests
were done according to procedures described by
Harrigan and McCance (1990). The production of
carbon dioxide was also determined in MRS and
M17 broth, after incubation at 30 jC for 24 h,
using an infrared gas analyzer (ADC 225 MK3,
The Analytical Development, Hertfordshire, UK)
connected to a Chromatopac (C-R3A, Shimadzu
Corporation Analytical Instruments, Kyoto, Japan)
according to Narvhus et al. (1992). Preliminary
grouping for selection of 30 isolates for API tests
was based on the above-mentioned morphological,
physiological and biochemical characteristics. The
fermentation pattern among carbohydrates was
determined by using the API 50 CH gallery with
the API 50 CHL medium (Bio Mérieux, Marcy-
l’Etoile, France). Anaerobiosis in the inoculated
tubes was obtained by overlaying with sterile par-
affin oil. The inoculated galleries were incubated at
30 jC and the observations were made after 24 and
48 h. The identification of the isolates was facili-
tated by the use of a computer programme, API-
LAB PLUS, version 3.2.2 (Bio Mérieux) and
reference to Bergey’s Manual of Systematic Bac-
teriology (Sneath et al., 1986) and Wood and
Holzapfel (1995).
The yeast isolates were identified by using the
Simplified Identification Method (SIM) described by
Deak and Beuchat (1996), with additional standard
taxonomical methods (Kurtzman and Fell, 1998), the
use of ID32C diagnostic kits (Bio Mérieux), assisted
by a computer software (API LAB PLUS version
3.2.2, Bio Mérieux). The SIM included the fermenta-
tion patterns among D-glucose, fructose, raffinose,
maltose, D -galactose (Merck), lactose, sucrose
(BDH, Poole, England); and the assimilation patterns
among xylose, melibiose, rhamnose, trehalose, man-
nitol, arabinose, citrate, soluble starch, cellobiose, D-
ribose, melezitose, DL-lactate, L-sorbose, lactose,
309
sucrose, galactose, and raffinose (Merck), erythritol,
2-ketogluconate, a-methyl-D-glucoside (Sigma, St.
Louis, MO, USA). Other tests included starch for-
mation, cycloheximide (Sigma) resistance, urease
activity, assimilation of potassium nitrate (Merck), L-
lysine and cadavarine (Sigma); growth at 37 and 40
jC; growth in 60% glucose – yeast extract agar,
growth in presence of 16% NaCl, growth in vitamin
free medium, growth in media containing 1% acetic
acid, potassium sorbate or benzoate. The formation of
mycelium and pseudohyphae was examined by micro-
scopy of Dalmau plates; ascospore formation on
Gorodkova agar, acetate agar and YM agar, and the
cell morphology on YM broth culture wet mounts.
2.2. Preparation of starter cultures
The cultures of LAB (Lactobacillus brevis, Lacto-
bacillus cellobiosus, Lactobacillus fermentum, Lacto-
bacillus plantarum and Pediococcus pentosaceus) and
yeasts (Candida pelliculosa, Candida tropicalis,
Issatchenkia orientalis and Saccharomyes cerevisiae)
isolated from native togwa as described above were
used. The cultures had been stored at –80 jC in sterile
cryo-tubes containing MRS broth with 10% (v/v)
glycerol and acid-washed glass beads until required.
LAB were cultivated by streaking on MRS agar
(Merck) and incubated anaerobically (BBL, Gas Pak,
H2 and CO2; Becton Dickinson) at 30 jC for 24 h. A
colony was picked from each pure culture plate, grown
successively in MRS broth before centrifugation at
655  g/15 min (Kubota 2010, Kubota). The pellet
was washed in peptone physiological salt solution
centrifuged again and redistributed in peptone physio-
logical salt solution. This procedure achieved a culture
preparation containing 109 colony-forming units (cfu)/
ml, checked as viable count on MRS agar. Pure cultures
of I. orientalis, S. cerevisiae, C. pelliculosa and C.
tropicalis were cultivated by streaking on Rose bengal
chloramphenicol agar (RBCA, Merck), incubated at 30
jC for 48 h and the picked colony was inoculated into
10 ml of YM broth [3 g yeast extract (Oxoid, Basing-
stoke, Hampshire, England), 3 g malt extract (Oxoid), 5
g peptone (Difco), 10 g glucose (Merck), 1000 ml
distilled water, pH 6.9] and incubated at 30 jC for 24 h.
These cultures were centrifuged and washed as
described above. This procedure achieved a culture
preparation containing 107 cfu/ml, as viable count on
310 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318
RBCA. Yeast cultures had been stored on potato
dextrose agar slants at 4 jC until required. The number
of LAB and yeasts was monitored during fermentation
by serial dilution of the samples, using the media
described above. The Enterobacteriaceae were moni-
tored in naturally fermented samples by surface spread-
ing on violet red bile glucose agar (VRBGA, Merck)
and enumeration after incubation at 37 jC for 24 h.
2.3. Preparation of samples
Maize –sorghum (1:1 w/w) flour slurry (1:9 w/v)
was boiled for 20 min to gruel and cooled to around
30 jC. Naturally fermented samples were prepared by
supplementing the gruel with either sorghum malt
alone or with sorghum malt followed by backslopping
with 1:9 (v/v) of native togwa and 100 ml quantities in
250 ml screw-capped bottles were incubated at 30 jC.
For controlled fermentation samples, the malt was
added when the gruel was at 55– 58 jC and were left
to cool for 30 min. They were then autoclaved at 121
jC for 15 min and cooled down to 30 jC prior to
inoculation.
2.4. Fermentation
In controlled fermentations, 100 ml of sterile gruel
was inoculated with 1 ml of LAB or 1 ml of yeast
suspension. Mixed fermentation by LAB and yeast
was initiated using 1 ml of each inoculum. Inoculated
samples were thoroughly mixed (Vortex Gene-2,
Model G-560E, Scientific Industries, Bohemia, NY,
USA) and incubated at 30 jC. At 0, 4, 8, 12, and 24 h
of fermentation, the samples were withdrawn for mi-
crobial counts, pH, organic acids, sugars and volatile
compounds. The experiments were replicated three
times.
2.5. Chemical analyses
The pH was determined with a pH meter (PHM61,
Radiometer, Copenhagen, Denmark) equipped with a
glass electrode (Orion 9102, Orion Research, Boston,
MA, USA). The pH meter was calibrated against
standard buffer solutions (Merck) at pH 4.0 and 7.0.
The titratable acidity was determined potentiometri-
cally according to Nout et al. (1989) by titrating 10 g
of togwa against 0.1 M NaOH to pH 8.5. The acidity
was calculated as percent (w/w) lactic acid equiva-
lent.
Organic acids were analyzed by using the high
performance liquid chromatography (HPLC) method
according to the method of Marsili et al. (1981), as
modified by Narvhus et al. (1998). Volatile com-
pounds were analyzed by using automatic static head-
space gas chromatography according to Narvhus et al.
(1998).
Total soluble sugars and reducing sugars were
determined according to the Luff-Schoorl method by
titration against 0.1 N sodium thiosulphate as
described by Egan et al. (1981). The concentrations
of glucose, fructose, and maltose were determined
during fermentation by HPLC, as for the organic acids
analysis, but using a refractive index detector (Perkin-
Elmer, Norwark, CT, USA) in series with the UV
detector and calibrated using standard sugar solutions
(Sigma).
2.6. Statistical analyses
The data obtained were subjected to analysis of
variance (SAS/Stat, 1996) and mean differences deter-
mined by Duncan’s multiple range or the least square
difference (LSD) test ( P < 0.05).
3. Results
3.1. Phenotyping of starter cultures isolated from
togwa
Among the 120 LAB isolates, rods accounted for
90%, cocci 10%, dextran producers 36%, CO2 pro-
ducers 70%, while 62% of the isolates were able to
grow at 45 jC, and 34% tolerated 6.5% NaCl. The
cocci were homofermentative, grew at 10 to 45 jC and
hydrolysed arginine. The bacteria isolates identified by
the use of a computer programme, APILAB PLUS,
version 3.2.2 (Bio Mérieux) and reference to Bergey’s
Manual of Systematic Bacteriology (Sneath et al.,
1986) and Wood and Holzapfel (1995) were tenta-
tively identified as: L. brevis, L. cellobiosus, L. fer-
mentum, L. plantarum, P. pentosaceus and W. confusa.
The species were isolated from all stages of fermenta-
tion, and L. plantarum dominated the final stages of
fermentation.
 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318 311

The yeast isolates identified by using the SIM
procedure described by Deak and Beuchat (1996),
as well as reference to the standard taxonomic key
outlined by Kurtzman and Fell (1998) and the use of
ID32C diagnostic kits (Bio Merieux), assisted by a
computer software (API LAB PLUS version 3.2.2,
Bio Mérieux, were tentatively identified as: I. orien-
talis (50%), S. cerevisiae (23%), (3) C. tropicalis
(10%) and (4) C. pelliculosa (17%). The species were
isolated from all stages of fermentation.
3.2. Starter cultures
Monocultures of the four species of LAB repre-
sented were effective in fermenting the gruel as
judged from the changes in pH and acidity (Table
1). The pH fell from 5.87 to 3.45– 3.77 in 12 h and to
pH 3.24 –3.49 in 24 h, with a simultaneous increase in
titratable acidity from 0.08% to 0.23– 0.36% and to
0.30 – 0.44% (w/w) lactic acid in 12 and 24 h of
fermentation, respectively.

Table 1
Effect of starter cultures on pH and titratable acidity of togwa*
Starter culture pH Titratable acidity
12 h 24 h 12 h 24 h
Control (sterile sample) 5.87 a** 5.87 a 0.08 j 0.08 l
Naturally fermented (Malt + BS***) 3.32 l 3.12 e 0.60 a 0.95 a
Naturally fermented (Malt) 4.20 e 3.39 ed 0.28 fcehdg 0.54 b
Issatchenkia orientalis 5.62 b 3.59 d 0.10 j 0.11 lk
Saccharomyces cerevisiae 4.89 d 4.39 c 0.16 i 0.20 jk
Candida tropicalis 5.55 b 4.81 b 0.09 j 0.11 lk
Candida pelliculosa 5.32 c 3.57 d 0.09 j 0.21 ji
Lactobacillus plantarum (LP) 3.45 jlk 3.24 ed 0.36 b 0.44 dce
LP + Issatchenkia orientalis 3.44 lk 3.24 ed 0.30 fcebd 0.41 dfce
LP + Saccharomyces cerevisiae 3.47 jilk 3.33 ed 0.30 fcebd 0.44 dce
LP + Candida tropicalis 3.45 jlk 3.30 ed 0.30 fcebd 0.46 c
LP + Candida pelliculosa 3.43 lk 3.28 ed 0.33 cb 0.45 dc
Lactobacillus brevis (LB) 3.52 jilhk 3.42 ed 0.31 cebd 0.37 gdfceh
LB + Issatchenkia orientalis 3.65 jgifhk 3.39 ed 0.30 fcebdg 0.39 gdfceh
LB + Saccharomyces cerevisiae 3.64 jgifhk 3.43 ed 0.33 cbd 0.39 gdfceh
LB + Candida tropicalis 3.67 gifh 3.40 ed 0.30 fcebdg 0.38 gdfceh
LB + Candida pelliculosa 3.64 jgifhk 3.40 ed 0.29 fcebhdg 0.39 gdfceh
Lactobacillus fermentum (LF) 3.79 gf 3.48 ed 0.26 fehdg 0.35 gdfeh
LF + Issatchenkia orientalis 3.72 gfh 3.44 ed 0.26 fehdg 0.34 gfeh
LF + Saccharomyces cerevisiae 3.71 gfh 3.48 ed 0.29 fcebhdg 0.38 gdfceh
LF + Candida tropicalis 3.72 gfh 3.46 ed 0.28 fcehdg 0.36 gdfceh
LF + Candida pelliculosa 3.67 gifh 3.44 ed 0.29 fcebhdg 0.37 gdfceh
Lactobacillus cellobiosus (LC) 3.61 jgifhk 3.42 ed 0.29 fcebhdg 0.39 gdfceh
LC + Issatchenkia orientalis 3.67 jgifh 3.41 ed 0.27 fcehdg 0.40 gdfce
LC + Saccharomyces cerevisiae 3.64 jgifhk 3.48 ed 0.30 fcebd 0.41 dfce
LC + Candida tropicalis 3.62 jgifhk 3.40 ed 0.29 fcehdg 0.37 gdfceh
LC + Candida pelliculosa 3.58 jgihk 3.39 ed 0.30 fcebd 0.38 gdfceh
Pediococcus pentosaceus (PP) 3.77 gf 3.49 ed 0.23 hg 0.30 gih
PP + Issatchenkia orientalis 3.82 f 3.51 ed 0.24 fehg 0.29 ih
PP + Saccharomyces cerevisiae 3.81 f 3.63 d 0.23 hg 0.33 gfh
PP + Candida tropicalis 3.76 gf 3.47 ed 0.22 h 0.30 gih
PP + Candida pelliculosa 3.79 gf 3.50 ed 0.23 fhg 0.32 gfh
LSD****(P < 0.05) 0.19 0.32 0.06 0.08
* Values are means of three replicates.
** Means with the same letter within the column are not significantly different ( P < 0.05).
*** BS = back-slopped with 10% (v/v) togwa.
**** LSD = least significant difference.
312 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318
Yeasts as monoculture starters, apart from S.
cerevisiae, were not very effective in lowering
the pH or increasing the acidity of the gruel in
the first 12 h. However, they were able to lower
the pH to 3.57– 4.81 and increase acidity to 0.11 –
0.21% in 24 h of fermentation (Table 1). There
was no significant difference ( P < 0.05) in pH and
acidity between the bacteria used as monocultures
or in co-culture with yeasts in their ability to
ferment the gruel.
In naturally fermented samples, the pH fell to
3.32 and 3.12 in malt-backslopped samples and, to
4.20 and 3.39 in malt-supplemented samples, with a
simultaneous increase in acidity from 0.60% to
0.95% and 0.28% to 0.54%, in 12 and 24 h of
fermentation, respectively. The combination of malt
flour and backslopping resulted into the most rapid
drop in pH and increase in acidity in comparison
with the rest of the 30 starter cultures used (Table 1).
3.3. Microbial numbers
The counts of L. cellobiosus, L. fermentum, L.
plantarum, L. brevis and P. pentosaceus increased
from about 7 to 9 log cfu/ml of togwa in 8 h, but after
this, the counts of L. brevis and P. pentosaceus de-
clined to 8 log cfu/ml (results not shown). In co-culture
with I. orientalis, the number of L. brevis reached 9 log
cfu/ml (results not shown). Yeasts increased from 5 to
7 log cfu/ml while the Enterobacteriaceae decreased
to nil within 24 h (results not shown).
3.4. Sugar content
The concentration of total soluble sugars in gruel
increased after addition of malt, from 36.5 to 39.2 mg/g
during the first 20 min, and reached 44.7 (mg/g of
gruel) after 30 min (results not shown). In the same
period, the reducing sugars increased from 1.6 to 21.4
mg/g and reached a concentration of 22.0 mg/g of gruel
after 30 min. This amount of fermentable sugar avail-
able at the beginning of the fermentation was appa-
rently sufficient to attain the acidity of 0.44%,
corresponding to a pH of about 3.24 after 24 h of
fermentation in inoculated samples (Table 1). There
was no significant difference ( P>0.05) in the levels of
fructose, glucose and maltose between malt-treated,
autoclaved and non-autoclaved gruels at the start of
fermentation except in native fermentation (data not
shown).
The concentration of fructose decreased with the
time of fermentation (0.08 – 0.09% to 0.02 –0.07% w/
w within 24 h). The most significant ( P < 0.05)
decrease was observed in samples fermented by L.
plantarum and L. plantarum in co-culture with I.
orientalis between 12 and 24 h (results not shown).
The concentration of glucose decreased with fermen-
tation time in all samples (0.52 – 0.58% to 0.04 –
0.37% w/w within 24 h) except in the naturally
fermented samples (results not shown). In the latter,
it increased radically during the first 12 h (0.52% to
1.70%) and thereafter decreased (1.37% after 24 h).
The concentration of maltose decreased with fermen-
tation time (2.38 – 2.63% to 2.06 –2.49%) (results not
shown). There was a significant ( P < 0.05) decrease
in samples fermented with monocultures of L. brevis
or L. cellobiosus and L. brevis in co-culture with I.
orientalis in 24 h. In naturally fermented samples,
maltose increased during the first 4 h from 2.38% to
3.14% and declined thereafter to 2.15% (results not
shown).
3.5. pH and organic acids
The changes in pH of togwa during fermentation
are shown in Fig. 1. The naturally fermented samples
had a significantly ( P < 0.05) lower pH at 0 h due to
backslopping. There was no significant difference
( P>0.05) in pH between the samples fermented nat-
urally and those fermented by L. plantarum or L.
plantarum in co-culture with I. orientalis after 8 and
12 h. The recommended pH for togwa (pH V 3.8) was
reached within 8 h in these samples. In samples
fermented by the rest of the cultures, this pH value
was reached within 12 h.
The changes in the concentration organic acids
during fermentation are shown in Fig. 1. Lactic acid
was produced in the largest amount, reaching about
1% (w/w) after 24 h in naturally fermented samples
rather closely followed by samples with L. plantarum
and L. plantarum co-cultured with I. orientalis within
8 to 12 h ( P < 0.05). There was no significant differ-
ence ( P < 0.05) between the naturally fermented sam-
ples and those fermented by L. plantarum after 24 h.
Citric acid was reduced to undetectable levels
within 4 h in naturally fermented samples, and
 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318 313

Fig. 1. Change in pH and organic acids in togwa during natural (NF) and controlled fermentation. LB, L. brevis; LBIO, L. brevis + I. orientalis;
LC, L. cellobiosus; LF, L. fermentum; LP, L. plantarum; LPIO, L. plantarum + I. orientalis; PP, P. pentosaceus.

within 24 h in samples fermented by L. fermentum levels of formic acid throughout fermentation, and
alone or L. plantarum and I. orientalis in co- pyro-glutamic acid after 12 h. Pyro-glutamic acid
culture. There was no significant difference between was reduced significantly ( P < 0.05) in samples
the naturally fermented and other samples in the fermented by L. cellobiosus within 24 h. Except
314 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318

for natural fermentation, there was no significant
difference between samples in the amount of uric
acid within 12 h. Succinic acid decreased signifi-
cantly ( P < 0.05) in samples fermented with L.
plantarum and P. pentosaceus after 24 h.
3.6. Volatile organic compounds (VOC)
The levels of VOC during fermentation are shown in
Fig. 2. The following malty compounds: 3-methyl-1-
butanal, 3-methyl-1-butanol, 2-methyl-1-propanal, 2-

Fig. 2. Change in volatile organic compounds in togwa during natural (NF) and controlled fermentation. LB, L. brevis; LBIO, L. brevis + I.
orientalis; LC, L. cellobiosus; LF, L. fermentum; LP, L. plantarum; LPIO, L. plantarum + I. orientalis; PP, P. pentosaceus.
 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318
methyl-1-propanol, 2-methyl-1-butanal and 2-methyl
butanol (Narvhus et al., 1998), were detected in togwa.
They were found in relatively high concentrations in
samples fermented by I. orientalis in co-culture with
either L. brevis or L. plantarum and in naturally
fermented samples (results not shown). The concen-
tration of 2-methyl butanol ranged from 0.37 to 0.39
mg/kg in samples fermented by I. orientalis in co-
culture with L. brevis; 0.10 to 0.27 mg/kg in samples
fermented by I. orientalis in co-culture with L. planta-
rum and 0.09 to 2.44 mg/kg in naturally fermented
samples between 8 and 24 h. The concentration of 2-
methyl-1-propanol ranged from 0.42 to 2.47 mg/kg in
samples fermented by I. orientalis in co-culture with L.
brevis; 0.32 to 1.41 mg/kg in samples fermented by I.
orientalis in co-culture with L. plantarum between 8
and 24 h. The concentration of 3-methyl butanal ranged
from 0.01 to 0.04 mg/kg in samples fermented by I.
orientalis in co-culture with L. brevis and 0.09 to 0.12
mg/kg in samples fermented by I. orientalis in co-
culture with L. plantarum between 8 and 24 h. It was
reduced to undetectable levels during the same period.
The concentration of 2-methyl-1-propanal ranged from
0.04 to 0.06 mg/kg in samples fermented by I. orienta-
lis in co-culture with L. brevis and decreased from 0.20
to 0.09 mg/kg in samples fermented by I. orientalis in
co-culture with L. plantarum within this period. The
concentration of 2-methyl butanal decreased (0.11 to
0.01 mg/kg) in all samples and 2-butanone remained
constant (0.02 – 0.05 mg/kg) during fermentation.
Diacetyl was produced in significantly different
amounts in samples fermented by L. plantarum
(0.39 – 0.69 mg/kg) and P. pentosaceus (0.36 – 0.47
mg/kg) within 8 to 24 h. After 24 h, acetaldehyde
was significantly higher in togwa fermented with L.
brevis or L. plantarum in co-culture with I. orientalis.
Acetoin was reduced with time in all samples. The
production of aldehydes was enhanced when L. plan-
tarum was co-cultured with yeasts ( P < 0.05).
The alcohols: 2-methyl-1-butanol, 3-methyl-1-buta-
nol and 2-methyl-1-propanol increased in naturally
fermented and in samples fermented with a combina-
tion of I. orientalis with either L. plantarum or L. brevis
within 8– 12 h ( P < 0.05). The concentration of the
latter alcohol was higher in samples fermented for 24 h.
Ethanol was the volatile compound produced in highest
concentrations. Most ethanol was found in naturally
fermented samples (0.64% w/w), followed by samples
315
fermented by I. orientalis in co-culture with L. brevis
and L. cellobiosus within 8– 12 h, in addition to L.
brevis and L. fermentum in 24 h. The production of
alcoholic flavour compounds was enhanced when the
bacteria were co-cultured with yeasts.
4. Discussion
The use of spontaneous fermentation, although a
simple way to achieve togwa fermentation, involves
a complex microbial process (Daeschel et al., 1987),
and may result in variations in quality of the gruel
(Kingamkono et al., 1995). The combination of malt
flour and backslopping is a common technique
applied to prepare togwa in Tanzania (Lorri and
Svanberg, 1995). The advantage of using a com-
bined starter method, apart from the possibility to
increase energy density (Svanberg et al., 1992;
Darling et al., 1995; Willumsen et al., 1997), is
that a desirable pH = 3.8 is quickly reached and this
may inhibit the growth and toxin production by
food-borne enteropathogens (Nout et al., 1989;
Svanberg et al., 1992; Kingamkono et al., 1994,
1998). Rapid fermentation is also desirable in order
to obtain togwa with good sensory properties (Lorri
and Svanberg, 1995).
In the present study, malt was added to gruel at
55– 58 jC and left to cool for 30 min before auto-
claving and subsequent inoculation. The concentra-
tion of reducing sugars did not increase significantly
( P < 0.05) after 20 to 30 min after addition of malt to
gruel (results not shown). The combined starters in
natural fermentation might have provided a more
diversified enzyme system from the indigenous ger-
minated cereal flour, lactic acid bacteria and yeasts.
The fermentable sugars monitored during fermenta-
tion were fructose, glucose and maltose. Mbugua et
al. (1983) reported that the fermentable sugars in
maize and sorghum during fermentation included
fructose, glucose, sucrose, maltotriose and raffinose.
They also observed that as pH is lowered during
fermentation, the production of maltose slows down
due to the inactivation of amylases. The initial ele-
vation in sugar levels during natural fermentation was
reported to be due to starch degradation by amylases
and might have an effect of increasing the microbial
count during fermentation, causing an elevation in
316 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318
acidity and a subsequent drop in pH, while subse-
quent decrease in sugar content could be due to
utilization by the fermenting microflora as a carbon
source (Mbugua et al., 1983; Odunfa and Adeyele,
1987; Umeta and Faulks, 1988; Khetarpaul and
Chauhan, 1990, 1991).
There was no significant difference after 8 and 12 h
( P < 0.05) in pH or lactic acid production between the
samples fermented naturally, by L. plantarum or by L.
plantarum co-cultured with I. orientalis. Thus in this
respect, L. plantarum could be used alone for rapid
fermentation to produce togwa. Lactic acid was the
predominant acid produced by all cultures. L. planta-
rum was comparable to natural fermentation in the
production of lactic acid after 24 h ( P < 0.05), when
the pH had reached 3.17– 3.19 (Table 1). L. plantarum
has been noted for its acid production and tolerance
(Fleming and McFeters, 1981), and for its superior
ability to utilize the substrates (Oyewole and Odunfa,
1990), including dextrins after the depletion of fer-
mentable sugars (Akinrele, 1970) and raw starch
(Giraud et al., 1994). A combined culture of yeasts
and lactobacilli has also been reported to bring about a
more significant decrease in pH and a simultaneous
increase in acidity in fermented millet than the use of
single cultures (Khetarpaul and Chauhan, 1990).
Kennes et al. (1991) reported the ability of L. planta-
rum to ferment citrate in the presence of yeast under
acid conditions. Citric acid was reduced to undetect-
able levels within 4 h in naturally fermented samples,
8 h in samples fermented by L. fermentum and, 24 h
by L. plantarum and I. orientalis.
The taste threshold value of 3-methyl-1-butanol
(1.0 mg/kg, Imhof et al., 1994) was reached in
naturally fermented togwa and in all samples fer-
mented by I. orientalis in co-culture with L. plan-
tarum or L. brevis within 12 h. The threshold value
of 2-methyl butanal (0.13 mg/kg, Sheldon et al.,
1971) was reached within 12 h in samples fer-
mented by P. pentosaceus, while that of 3-methyl
butanal (0.06 mg/kg, Sheldon et al., 1971) was
reached within 8 h in samples fermented by L.
plantarum in co-culture with I. orientalis, and that
of 2-methyl-1-propanal (0.10 mg/kg, Sheldon et al.,
1971) within 8 h by L. plantarum in co-culture with
I. orientalis and 12 h in naturally fermented sam-
ples. Thus, malty compounds should be a discern-
ible flavour aspect of togwa.
Acetaldehyde was produced in relatively large
quantities by L. plantarum in co-culture with I.
orientalis (4.19 –8.05 mg/kg within 12 –24 h) and
L. brevis in co-culture with I. orientalis (4.10 mg/kg
within 24 h). Diacetyl was produced in increasing
amounts in samples fermented by L. plantarum
(0.39 –0.69 mg/kg) followed by samples fermented
by P. pentosaceus (0.36 – 0.47 mg/kg) between 8 and
24 h. The reported threshold value is 0.03 mg/kg
(Imhof et al., 1994) and it was reduced to undetect-
able levels in samples fermented by L. brevis, L.
brevis in co-culture with I. orientalis, L. fermentum,
L. cellobiosus and naturally fermented samples
within 8 h. The threshold value of 2-butanone (60
mg/kg, Imhof et al., 1994) was not reached, as it
remained at concentrations of 0.02 – 0.05 mg/kg of
togwa. The threshold value of ethanol (100 –800 mg/
kg, Imhof et al., 1994) was reached in samples
fermented by all cultures except L. plantarum and
P. pentosaceus monocultures, within 8 h. It was still
below this value in samples fermented by P. pento-
saceus by 24 h.
The above-mentioned observations indicated that
most volatile flavour compounds were produced in
samples fermented by L. plantarum in co-culture with
I. orientalis and by P. pentosaceus. The contribution
by yeasts to flavour acceptability in lactic acid fer-
mented products has been reported by Akinrele (1970)
for Nigerian ogi.
The foregoing observations indicated that, judg-
ing from the lowering of pH value and production
of lactic acid, all LAB cultures could be used
singly to produce togwa within 8– 12 h. Their use
in co-culture with I. orientalis enhanced the pro-
duction of volatile flavour compounds in the prod-
uct. The enhancement of production of metabolites
in co-culture may be taken to be indicative of
interaction between LAB and yeasts. The ability
of various LAB cultures to produce metabolites in
different quantities might potentially be exploited to
produce different varieties of togwa.
Acknowledgements
This study was supported by grants from the
Norwegian Council of Universities’ Committee for
Development Research and Education (NUFU, Project
 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318
26/96) through the Agricultural University of Norway
and Sokoine University of Agriculture, and the
Lånekassen of Norway. We are grateful to Kari Olsen
for assistance with the GC and HPLC analyses.
References
Akinrele, I.A., 1970. Fermentation studies on maize during the
preparation of a traditional African starch-cake food. J. Sci.
Food Agric. 21, 619 – 625.
Brauman, A., Keleke, S., Malonga, M., Miambi, E., Ampe, F.,
1996. Microbiological and biochemical characterization of
cassava retting, a traditional lactic acid fermentation for foo-
foo (cassava flour) production. Appl. Environ. Microbiol. 62,
2854 – 2858.
Daeschel, M.A., Andersson, R.E., Fleming, H.P., 1987. Microbial
ecology of fermenting plant materials. FEMS Microbiol. Rev.
46, 357 – 367.
Darling, J.C., Kitundu, J.A., Kingamkono, R.R., Msengi, A.E.,
Mduma, B., Sullivan, K.R., Tomkins, A.M., 1995. Improved
energy intakes using amylase-digested weaning foods in Tanza-
nian children with acute diarrhea. J. Pediatr. Gastroenterol. Nutr.
21, 73 – 81.
Deak, T., Beuchat, L., 1996. Handbook of Food Spoilage Yeasts.
CRC Press, Boca Raton.
Egan, H., Kirk, R., Sawyer, R., 1981. Pearson’s Chemical Analysis
of Foods, 8th ed. Longman, Harlow, London, UK, pp. 152 – 153.
Fleming, H.P., McFeters, R.F., 1981. Use of microbial cultures:
vegetable products. Food Technol. 35, 84.
Giraud, E., Champailler, A., Raimbault, M., 1994. Degradation of
raw starch by a wild amylolytic strain of Lactobacillus planta-
rum. Appl. Environ. Microbiol. 60, 4319 – 4323.
Gobbetti, M., Corsetti, A., 1997. Lactobacillus sanfransisco—a key
sourdough lactic acid bacterium: a review. Food Microbiol. 14,
175 – 187.
Gobbetti, M., Corsetti, A., Rossi, J., 1994. The sourdough micro-
flora. Interactions between lactic acid bacteria and yeasts: me-
tabolism of carbohydrates. Appl. Microbiol. Biotechnol. 41,
456 – 460.
Halm, M., Lillie, A., Sørensen, A.K., Jakobsen, M., 1993. Micro-
biological and aromatic characteristics of fermented maize
doughs for kenkey production in Ghana. Int. J. Food Microbiol.
19, 135 – 143.
Hansen, A., Hansen, B., 1996. Flavour of sourdough wheat crumb.
Z. Lebensm.-Unters.-Forsch. 202, 244 – 249.
Harrigan, W.F., McCance, M.E., 1990. Laboratory Methods in
Food and Dairy Microbiology, 8th ed. Academic Press, London.
Holzapfel, W., 1997. Use of starter cultures in fermentation on a
household scale. Food Control 8, 241 – 258.
Hounhouigan, D.J., Nout, M.J.R., Nago, C.M., Houben, J.H.,
Rombouts, F.M., 1993. Microbiological changes in mawe dur-
ing natural fermentation. World J. Microbiol. Biotechnol. 10,
410 – 413.
Imhof, R., Glättli, H., Bosset, J.O., 1994. Volatile organic aroma
317
compounds produced by thermophilic mixed strain dairy starter
cultures. Lebensm.-Wiss. Technol. 27, 442 – 449.
Kennes, C., Dubourguier, H.C., Albagnac, G., Naveau, H., Veiga,
M., Nyns, E.J., 1991. Fermentation of citrate by Lactobacillus
plantarum in the presence of a yeast under acid conditions.
Appl. Microbiol. Biotechnol. 35, 369 – 372.
Khetarpaul, N., Chauhan, B.M., 1990. Effect of fermentation by
pure cultures of yeasts and lactobacilli on the available carbo-
hydrate content of pearl millet. Food Chem. 36, 287 – 293.
Khetarpaul, N., Chauhan, B.M., 1991. Sequential fermentation of
pearl millet by yeasts and lactobacilli: changes in available car-
bohydrate content. Food Chem. 40, 235 – 240.
Kimaryo, V.M., Massawe, G.A., Olasupo, N.A., Holzapfel, W.H.,
2000. The use of a starter culture in the fermentation of cassava
for the production of ‘‘kivunde’’, a traditional Tanzanian food
product. Int. J. Food Microbiol. 56, 179 – 190.
Kingamkono, R., Sjögren, E., Svanberg, U., Kaijser, B., 1994. pH
and acidity in lactic-fermenting cereal gruels: effects on viability
of enteropathogenic microorganisms. World J. Microbiol. Bio-
technol. 10, 664 – 669.
Kingamkono, R., Sjögren, E., Svanberg, U., Kaijser, B., 1995.
Inhibition of different strains of enteropathogens in a lactic-
fermenting cereal gruel. World J. Microbiol. Biotechnol. 11,
299 – 303.
Kingamkono, R.R., Sjögren, E., Svanberg, U., 1998. Inhibition of
enterotoxin production by, and growth of entropathogens in a lac-
tic acid-fermented cereal gruel. World J. Microbiol. Biotechnol.
14, 661 – 667.
Kunene, N.F., Geornaras, I., von Holy, A., Hastings, J.W., 2000.
Characterization and determination of origin of lactic acid bac-
teria from a sorghum-based fermented food by analysis of solu-
ble proteins and amplified fragment length polymorphism
fingerprinting. Appl. Environ. Microbiol. 66, 1084 – 1092.
Kurtzman, C.P., Fell, W.C. (Eds.), 1998. The Yeasts, A Taxonomic
Study, 4th ed. Elsevier, Amsterdam, pp. 77 – 100.
Lorri, W., Svanberg, U., 1995. An overview of the use of fermented
foods for child feeding in Tanzania. Ecol. Food Nutr. 34,
65 – 81.
Marsili, R.T., Ostapenko, H., Simmons, R.E., Green, D.E., 1981.
High performance liquid chromatographic determination of or-
ganic acids in dairy products. J. Food Sci. 46, 52 – 57.
Mbugua, S.K., Ledford, R.A., Steinkraus, K.H., 1983. Gas chro-
matographic determination of mono-, di- and trisaccharides in
the uji-flour ingredients and during uji fermentation. Chem.
Mikrobiol. Lebensm. 8, 40 – 45.
Mensah, P., Tomkins, A.M., Drasar, B.S., Harrison, T.J., 1991.
Antimicrobial effect of fermented Ghanaian maize dough. J.
Appl. Bacteriol. 70, 203 – 210.
Mugula, J.K., Nnko, S.A.M., Sørhaug, T., 2001. Changes in quality
attributes during storage of togwa, a lactic acid fermented gruel.
J. Food Saf. 21, 181 – 194.
Nago, C.M., Hounhouigan, D.J., Akissoe, N., Zanou, E., Mestres,
C., 1998. Characterization of the Beninese traditional ogi, a
fermented maize slurry: physiological and microbiological as-
pects. Int. J. Food Sci. Technol. 33, 307 – 315.
Narvhus, J.A., Hulbækdal, A., Thorvaldsen, K.R., Baugerød, M.,
Abrahamsen, R.K., 1992. Measurement of CO2 production and
318 J.K. Mugula et al. / International Journal of Food Microbiology 83 (2003) 307–318
O2 metabolism by pure and mixed cultures of lactic acid bacteria
growing in milk. Actes du Colloque LACTIC, vol. 91. Centre de
Publications de I’Université de Caen, Caen, France, p. 371.
Narvhus, J.A., Österaas, K., Mutukumira, T., Abrahamsen, R.K.,
1998. Production of fermented milk using a malty-compound
producing strain of Lactococcus lactis subsp. lactis biovar diac-
etylactis isolated from Zimbabwean naturally fermented milk.
Int. J. Food Microbiol. 14, 73 – 80.
Nout, M.J.R., 1991. Ecology of accelerated natural lactic fermenta-
tion of sorghum-based infant food formulas. Int. J. Food Micro-
biol. 12, 217 – 224.
Nout, M.J.R., Rombouts, F.M., Havelaar, A., 1989. Effect of natural
lactic fermentation of infant food ingredients on some patho-
genic microorganisms. Int. J. Food Microbiol. 8, 351 – 361.
Odunfa, S.A., Adeyele, S., 1987. Sugar changes in fermenting sor-
ghum during preparation of ogi-baba gruel. J. Food Agric. 1,
95 – 98.
Olasupo, N.A., Olukoya, D.K., Odunfa, S.A., 1997. Identification
of Lactobacillus species associated with selected African fer-
mented foods. Zeitsch. Naturforsch. 52, 105 – 108.
Oyewole, O.B., Odunfa, S.A., 1990. Characterization and distribu-
tion of lactic acid bacteria in cassava fermentation during fufu
production. J. Appl. Bacteriol. 68, 145 – 152.
Sanni, A.I., 1993. The need for process optimization of African
fermented foods and beverages. Int. J. Food Microbiol. 18,
85 – 95.
SAS/Stat, 1996. SAS/Stat User’s Guide. Version 6. Statistical Anal-
ysis System Institute, Cary, NC, USA.
Sheldon, R.M., Lindsay, R.C., Libbey, L.M., Morgan, M.E., 1971.
Chemical nature of malty flavour and aroma produced by Strep-
tococcus lactis var. maltigenes. Appl. Microbiol. 22, 263 – 266.
Sneath, P.H.A., Mair, N.S., Sharpe, M.E., Holt, J.G., 1986. Ber-
gey’s Manual of Systematic Bacteriology, vol. 2. Williams and
Wilkins, Baltimore.
Steinkraus, K.H., 1996. Handbook of Indigenous Fermented Foods,
2nd ed. Marcel Dekker, New York.
Stolz, P., Vogel, R.F., Hammes, W.P., 1995. Utilization of electron
acceptors by lactobacilli isolated from sourdough. Z. Lebensm.-
Unters.-Forsch. 201, 402 – 410.
Svanberg, U., Sjogren, E., Lorri, W., Svennerholm, A.M., Kaijser,
B., 1992. Inhibited growth of common enteropathogenic bacte-
ria in lactic-fermented cereal gruels. World J. Microbiol. Bio-
technol. 8, 601 – 606.
Umeta, M., Faulks, R.M., 1988. The effect of fermentation on the
carbohydrates in tef (Eragrostis tef). Food Chem. 2, 181 – 189.
Willumsen, J.F., Darling, J.C., Kitundu, J.A., Kingamkono, R.R.,
Msengi, A.E., Mduma, B., Sullivan, K.R., Tomkins, A.M.,
1997. Dietary management of acute diarrhoea in children: effect
of fermented and amylose-digested weaning foods on intestinal
permeability. J. Pediatr. Gastroenterol. Nutr. 24, 235 – 241.
Wood, B.J.B., Holzapfel, W.H., 1995. The Genera of Lactic Acid
Bacteria, vol. 2. Blackie Academic and Professional, Glasgow.