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

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