(khamir) produced from sorghum in Gizan region, Saudi Arabia

Journal of Applied Microbiology 1999, 86, 221–225
Study of the micro-organisms associated with the fermented
bread (khamir) produced from sorghum in Gizan region, Saudi
Arabia
INTRODUCTION
M.A.A. Gassem
Department of Food Science and Nutrition, College of Agriculture, King Saud University, Riyadh, Saudi Arabia
6707/04/98: received 22 April 1998, revised 21 August 1998 and accepted 26 August 1998
M.A.A. GASSEM. 1999. Traditional bread (khamir) was made from sorghum flour of two
local varieties, Bayadh and Hamra. The bread was prepared by mixing the
sorghum flour with water and spices (onion, garlic, lemon juice and fenugreek) in a
1:0·8 (w/w) ratio and fermented for 24 h at 30 °C. Two other fermentations were carried out
using an inoculum from the previous fermentation. The micro-organisms were isolated
from different plates and identified using different characterization systems. Both total
bacterial populations and lactic acid bacteria increased with fermentation time and
reached the highest number at 16 h (first fermentation) and at 8 h (second and third
fermentation). The content of lactic acid was increased with time to reach 1·2%, but
the increase was higher for the second and third fermentations (1·6% each). The
pH dropped with time from 6·77 to 4·35 in the first fermentation and from 6·65 to
4·18, and 6·57–3·93, in the second and third fermentations, respectively. The microorganisms,
which were isolated and characterized during the 24 h fermentation,
included: bacteria (Pediococcus pentosaceus, Lactobacillus brevis, Lact. lactis subsp.
lactis, Lact. cellobiosus, Klebsiella oxytoca, Kl. pneumoniae, Enterobacter aerogenes, Ent.
sakazakii, Serratia marcescens and Ser. odourifera), moulds (Penicillium sp.,
Rhizopus sp., Aspergillus niger, Alternaria sp., Fusarium sp. and Mucor sp.) and yeasts
(Candida parapsilosis, C. orvegnsis and Rhodotorula glutinis).
Sorghum (Sorghum bicolor L. Moench) is a staple food for
the people of the arid and semi-arid tropical regions of Africa
and Asia (Chavan et al. 1988). Graham et al. (1986) reported
that millions of people in the semi-arid regions of the world,
and especially in Africa and the Asian subcontinent, depend
on sorghum as a major source of dietary protein and energy.
Sorghum is cultivated in different parts of the world; grain
sorghum is considered to be the fifth most extensively grown
crop in the world and the third largest crop harvest in the
USA (Kazanas and Fields 1981). In Saudi Arabia, sorghum
is grown in different regions, but it is the main cereal crop in
the south-western region (Chaudhary 1989).
Different traditional fermented foods and beverages are
Correspondence to: Dr M.A.A. Gassem, Department of Food Science and
Nutrition, College of Agriculture, King Saud University, PO Box 2460, Riyadh
11451, Saudi Arabia (e-mail: MAGASSEM@KSU.EDU.SA).
© 1999 The Society for Applied Microbiology
prepared from the grain sorghum in different parts of the
world, for example, kisar, asida, merrissa and nasha in Sudan
(Dirar 1978; El-Tinay et al. 1985; Graham et al. 1986;
Mohammed et al. 1991), ogi and obiolar in Nigeria (Adeyemi
1988; Acki 1990), feni in India (Chavan and Kadam 1989)
and khamir and lahoh in Saudi Arabia.
Fermentation of the sorghum was found to enhance its
digestibility (Au and Fields 1981; Eggum et al. 1983;
MacLean et al. 1983; Graham et al. 1986). Grain sorghum is
used in the Gizan region (south-western Saudi Arabia) for
making fermented bread called khamir. Traditionally, the
bread is made by utilizing the natural microflora in sorghum
flour. No information is available regarding khamir making
and its fermentation in Saudi Arabia. Therefore, it is necessary
to understand the fermentation process and to identify the
micro-organisms involved. The aim of this study was to
isolate and identify micro-organisms associated with sorghum
fermentation in order to obtain the necessary information

222 M.A.A. GASSEM
for improving khamir production, and to study the changes
occurring in pH and titratable acidity which might have a
major role in microbial distribution during fermentation.
MATERIALS AND METHODS
Two local varieties of sorghum grains (Baydah and Hamra)
were obtained from a grain market in Abu-Arish (south-west
Saudi Arabia). The sorghum was milled at the local grain
market to fine flour using a Diamant mill, model 500-mm
(Denmark). The flour was then transferred to the laboratory
in Riyadh and stored at 25 °C until used. Sorghum fermentation
was carried out in the traditional way by mixing
sorghum flour with sterile deionized water and spices (onion,
garlic, lemon juice and fenugreek) in a 1:0·8 (w/w) ratio to
make a dough (traditionally called ‘ajeen’). The mixture was
incubated at 30 °C for 24 h in a sterile covered flask; usually,
at this time, the dough will have a good consistency and sour
taste. Two consecutive fermentations were carried out using
3% inocula (traditionally called ‘shetiah’) from the previous
fermentation to start each subsequent batch. Each fermentation
was performed in duplicate and sampled every 4 h
for 24 h.
Sampling
At each sampling time, a 50 g sample was placed in a sterile
stomacher bag and mixed with 450 ml sterile 0·1% peptone
water (Oxoid) using a Stomacher Lab-Blender 400 (Seward
Medical, London, UK). Appropriate serial dilutions in 0·1%
peptone water were prepared and samples were plated on the
different agar media.
Microbiological analysis
Total plate count was determined on Plate Count Agar (PCA,
Oxoid). The plates were incubated at 32 °C for 24 h.
Coliforms were enumerated on Violet Red Bile Agar
(VRBA, Oxoid) and incubated at 37 °C. Counts were taken
at 24 and 48 h. Coliforms were isolated from VRBA plates,
purified on PCA and identified using the API 20 system
(BioMérieux, Marcy l’Etoile, France).
Lactic acid bacteria were enumerated on de Man, Rogosa
and Sharpe (MRS, Oxoid) agar. The plates were incubated
at 37 °C for 48 h under anaerobic conditions using GasPak
(H 2 and CO 2) anaerobic systems (BBL Microbiology Systems,
Cockeysville, MD, USA). Pure colonies from countable
plates were Gram-stained, maintained in MRS broth,
and fur-ther characterized using the API 50 CHL system
(BioMérieux).
Yeasts and moulds were enumerated on acidified potato
dextrose agar (APDA, Oxoid). The plates were incubated at
25 °C for 3, 5 and 7 d. Isolated yeasts were purified on Sabou-
raud medium (Difco) and characterized using the API AUX
system (BioMérieux). Moulds were identified using 10 dayold
cultures on potato dextrose agar (Oxoid). Cultural and
microscopic characteristics were examined and moulds were
classified according to Frazier and Westhoff (1988) and Barnett
and Hunter (1972).
Microbial identification
Representatives from each colony type were selected from
plates used for viable counts at each sampling time according
to shape and/or colour. Isolates were purified by repeated
streaking on appropriate media and identified using appropriate
identification systems.
Measurement of pH and titratable acidity
The pH of the fermented dough was measured using a pH
meter (Jenway PHM10, Felsted, UK). Titratable acidity,
expressed as percentage lactic acid, was determined by
titrating the samples with 0·1 mol l −1 NaOH to the phenolphthalein
end point.
RESULTS
Microbial counts
Microbial counts of the initial fermentation of sorghum (Bayadh
variety) are presented in Fig. 1. The microflora of the
naturally fermented sorghum flour consisted of different
micro-organisms, such as lactic acid bacteria, coliforms,
moulds and yeasts. Both the total bacteria and lactic acid
bacteria counts increased with increasing fermentation time
until they reached stationary phase at 16 h (Fig. 1a). The yeast
and mould counts remained constant during the fermentation.
The coliform counts decreased until 12 h, then
increased, and thereafter decreased.
When sorghum was fermented using an inoculum from
the previously fermented sorghum, the microbial counts also
increased with time. However, the total bacterial populations
and lactic acid bacteria reached maximal growth in half the
time of the initial fermentation (Fig. 1c). No changes in mould
and yeast counts were noticed during fermentation. The coliforms
decreased and were not detected after 12 h of fermentation.
Data for the third fermentation (not shown) which was
carried out using an incoulum from the previous fermentation
were similar to those obtained for the second fermentation
except for the coliforms, which were not detected after 8 h.
For the Hamra variety, similar results were obtained for
total bacterial counts, lactic acid bacteria, and yeasts and
moulds in the initial, second and third fermentations.
However, for coliforms, initial fermentation counts increased
© 1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 221–225

Log cfu g –1
Log cfu g –1
12
10
8
6
4
2
0
12
10
8
6
4
2
0
0
0
(a)
(c)
5 10 15
Time (h)
20 25
5 10 15
Time (h)
20 25
30
30
MICRO-ORGANISMS IN FERMENTED SORGHUM BREAD 223
% Lactic acid
% Lactic acid
1·8
1·5
1·2
0·9
0·6
0·3
0·0 0
1·8
1·5
1·2
0·9
0·6
0·3
0·0 0
(b)
(d)
5 10 15
Time (h)
20 25
5 10 15
Time (h)
20 25
Fig. 1 Microbial counts, percentage lactic acid, and pH during the fermentation of khamir bread using the Bayadh variety. (a), (b) 24 h
of initial fermentation (natural); (c), (d) 24 h of the second fermentation with the addition of inoculum. ( ), Total plate count; (ž),
lactic acid bacteria; (t), coliforms; (T), yeasts and moulds; ( ), pH; (Ž), percentage lactic acid
until 8 h, then decreased, and persisted thereafter (data not
shown). For the second and third fermentations, the results
were similar to those for the Bayadh variety (Fig. 1c).
Acidity and pH
In the initial fermentation, pH decreased with a concomitant
increase in acidity as lactic acid accumulated. Lactic acid
content (Fig. 1b) increased with fermentation time from 0·19
to 1·22%, and from 0·21 to 1·13%, during 24 h for Bayadh
and Hamra varieties, respectively. This resulted in a pH drop
© 1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 221–225
7
6
5
4
3
30
7
6
5
4
3
30
from 6·77 to 4·35 (Bayadh), and from 6·73 to 4·29 (Hamra).
When adding an inoculum, the amount of lactic acid in the
Bayadh variety increased from 0·27 to 1·40% in 16 h and by
the end of 24 h, the amount of the acid was 1·60% (Fig. 1d).
In the Hamra variety, the amount of acid was lower than in
the Bayadh, increased from 0·26 to 1·2% in 16 h, and reached
1·36% at 24 h (data not shown). In the third fermentation
(data not shown), the pH decreased from 6·57 to 4·18 in 12 h
and reached 3·92 in 24 h for the Bayadh variety, while for the
Hamra variety, the pH dropped from 6·57 to 4·06 in 12 h and
at the end of 24 h, was 3·85. The amount of lactic acid
pH
pH

224 M.A.A. GASSEM
(Bayadh) increased from 0·32 to 1·2% in 12 h and at the end
of 24 h fermentation, it was 1·60%. Similar results were
obtained for the Hamra variety.
Classification of micro-organisms
Different micro-organisms were isolated at different fermentation
times and are presented in Table 1. Two species
of lactic acid bacteria (Lactobacillus and Pediococcus) were
identified. Three strains of Lactobacillus were isolated, Lact.
lactis subsp. lactis, Lact. brevis and Lact. cellobiosus. Lactobacillus
lactis subsp. lactis was isolated and identified from
both sorghum varieties (Bayadh and Hamra), while Lact.
brevis and Lact. cellobiosus were isolated and identified only
from the Hamra variety. The Pediococcus isolates were identified
as Ped. pentosaceus and were found in both sorghum
varieties. Pediococcus pentosaceus was dominant at the end of
the fermentation and maintained a high number.
The coliforms are listed in Table 1. Klebsiella pneumoniae,
Kl. oxytoca, Enterobacter aerogenes, Ent. sakazakii and Serratia
marcescens were found in both sorghum varieties. Serratia
odorifera was found in the Bayadh variety only.
Yeasts (Table 1) were identified as Candida parapsilosis, C.
norvegnsis and Rhotorula glutinis. Candida norvegnsis was
found only in the Bayadh variety. Moulds (Table 1) were
identified as Rhizopus sp., Penicillium sp., Alternaria sp., Apergillus
niger, Mucor sp. and Fusarium sp.
DISCUSSION
There were several micro-organisms that are found naturally
in sorghum flour. These micro-organisms included coliforms,
lactic acid bacteria, yeasts and moulds. Chavan and Kadam
(1989) have indicated that the micro-organisms involved in
Table 1 Micro-organisms isolated during khamir fermentation
—––––––––––––––––––––––––––––––––––––––––––––––––––– ––
Bacteria Yeasts and moulds
—––––––––––––––––––––––––––––––––––––––––––––––––––– ––
Lactic acid bacteria n Yeasts n
Pediococcus pentosaceus (8) Candida parapsilosis (10)
Lactobacillus brevis (2) C. norvegnsis (1)
Lact. lactis subsp. lactis (4) Rhodotorula glutinis (2)
Lact. cellobiosus (3)
Coliforms n Moulds n
Klebsiella oxytoca (1) Penicillium sp. (4)
Kl. pneumoniae (3) Rhizopus sp. (1)
Enterobacter aerogenes (1) Aspergillus niger (1)
Ent. sakazakii (4) Alternaria sp. (1)
Serratia marcescens (2) Fusarium sp. (2)
Ser. odorifera (1) Mucor sp. (1)
—––––––––––––––––––––––––––––––––––––––––––––––––––– ––
the natural fermentation of cereals are essentially the surface
flora of seeds. The Enterobacteriaceae are common on plant
material. Members of the Enterobacteriaceae were found in
natural fermentations of different products such as sorghum
(Nout 1991; Mohammed et al. 1991) and soybeans (Mulyowidarso
et al. 1989). In the first sorghum fermentation, numbers
of coliforms remained constant. However, in the second
and third fermentations, coliforms were not detected after
12 h and 8 h, respectively. Similar results were reported by
Mica (1955), Nout et al. (1989), Nout (1991) and Mohammed
et al. (1991). Mohammed et al. (1991) suggested that Enterobacter
may be inhibited by the growth of lactic acid bacteria
which outnumber the Enterobacter and result in faster acid
production.
The numbers of lactic acid bacteria increased with time and
dominated the fermentation (Fig. 1a,c). Chavan and Kadam
(1989) indicated that lactic acid bacteria constitute the predominant
flora in the natural fermentation of water/cereal
meal mixture. Some of the lactic acid bacteria that were
isolated and identified in this study were isolated from different
fermented foods. Lactobacillus brevis has been isolated
from ‘tef’ (Gashe 1987), ‘fufu and ogi’ (Adegoke and Babalola
1988) and kisra (Mohammed et al. 1991). Lactobacillus cellobiosus
was isolated from fermented corn meal (Fields et al.
1981), and Ped. pentosaccus was isolated from kisra (Mohammed
et al. 1991). The increase in lactic acid bacteria resulted
in increased amounts of lactic acid produced and lower pH.
Similarly, in the natural fermentation, there was an increase
in the amount of lactic acid produced and a decrease in pH.
However, in the second and third fermentations, the amount
of acid produced was higher and the drop in pH was faster.
The faster drop in pH was probably due to the higher number
of lactic acid bacteria in the inoculum than the initial fermentation.
Similar results were obtained by Nout (1991) and
Mohammed et al. (1991). Therefore, the presence of lactic
acid bacteria is important in khamir production.
Numbers of yeasts and moulds remained constant during
the fermentation for both varieties, except for a slight decrease
during the third fermentation. Candida parapsilosis and
Rhodotorula glutinis were isolated from both Bayadh and
Hamra. Candida norvegnsis was isolated from Bayadh only.
Yeasts were isolated from different indigenous fermented
foods such as kisra, ogi, fufu, tef, burukutu and merissa
(Steinkraus 1983; Gashe 1987; Adegoke and Babalola 1988;
Chavan and Kadam 1989; Mohammed et al. 1991).
In conclusion, this study has shown that there are some
micro-organisms that are naturally present in sorghum flour.
During khamir fermentation, these micro-organisms result
in some microbial and biochemical changes. Changes in pH
and acidity were due to the outgrowth of lactic acid bacteria
which become the predominant organisms. Additional work
is needed on the use of isolated micro-organisms for the
development of starter cultures for khamir production.
© 1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 221–225

ACKNOWLEDGEMENTS
The author gratefully acknowledges the valuable technical
assistance of Mr Ibrahim Al-Zain throughout this work.
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