2.1 Material and methods
2.1.1 Microbial cultures
L. plantarum MNC 21 and S. cerevisiae MNC 21Y isolated from Obushera (Mukisa 2012) were used. L. plantarum MNC 21 and S. cerevisiae MNC 21Y were separately grown in MRS broth and Yeast Mold Broth, respectively. The broths were supplied by CONDA, Madrid, Spain. Cultures were incubated at 30 °C for 24 h. The cells were centrifuged at 7500xg for 10 min and the pellets washed thrice using sterile quarter strength Ringer’ solution (Oxoid Limited, Hampshire, England). The pellets were suspended in sterile quarter strength Ringer’s solution.
2.1.2 Sorghum malt
SESO 3, a red sorghum variety obtained from the National Semi-arid Resources Research Center in Serere, Uganda was used to prepare sorghum malt. The sorghum grain was placed on a suspended wire mesh and sorted to remove chaff. The grain was washed using pressurized water. Ten kilograms of grain were soaked in 15 L of potable water containing 0.3% NaOH then steeped for 6 h. The water was drained and the steep vessel refilled with fresh water. The grains were steeped for a further 10 h after which the grain was transferred to germination beds at 25 °C. After two days when the rootlets were 1 cm long, the grain was spread out in a 2 cm thick layer in a drying chamber at 65 °C. The grain was considered dry if it broke ‘cleanly.’ The dried sorghum malt was stored in moisture proof bags at ambient temperature till further use.
2.1.3 Preparation and inoculation of slurries
Dried sorghum malt was milled using a Wondermill (Grote Molen Inc., Pocatello, USA) at the bread texture control setting. At this setting, 99% of the resulting flour passed through a 1000 mm mesh and 85% through a 500 mm mesh. The flour was mixed with potable water to make slurry (12.5% total solids) which was then heated with continuous stirring to 90 °C and held at that temperature for 15 min. The hot slurry was aseptically transferred to a sterile glass bottle and cooled to 30 °C. It was then inoculated with 6 log cfu/mL of L. plantarum MNC 21 + S. cerevisiae MNC 21Y mixture and incubated at 30 °C for 24 h. Samples were drawn at 0, 4, 8, 12 and 24 h to determine cell counts, pH and titratable acidity. Experiment was done in triplicate.
2.1.4 Storage of starter cultures
The fermented slurries were distributed in sterile bottles and some stored at -18 °C while others at 5 °C. Samples were drawn periodically to determine cell counts and fermentation ability. Fermentation ability was determined by inoculating 100 mL of sterile freshly prepared sorghum malt slurries (12.5% total solids) with 1% (v/v) of cultures and incubating at 30 °C for 24 h. Samples were drawn periodically to determine pH, titratable acidity and flavor development.
2.1.5 Analyses
Acidity and pH
The pH was measured using a pH meter (Mettler-Toledo AG model, Schwerzenbach, Switzerland). Titratable acidity was determined by titrating 10 mL of Obushera against a 0.1M solution of NaOH using phenolphthalein as the indicator (Horwitz, 2000).
Flavor development
Flavor development was determined by sniffing the products to detect the characteristic flavor of Obushera. The intensity of flavor was scored as follows: +++ (strong), ++ (mild) and + (weak).
Microbiological analyses
L. plantarum MNC 21 was enumerated by pour plating selected serial dilutions of the culture in MRS agar then incubated at 30 °C for 48 h. The yeast was enumerated by surface plating selected serial dilutions of the culture on Potato Dextrose Agar supplemented with chloramphenicol then incubated at 30 °C for 72 h. Counts were determined on days; 0, 5, 10, 20, 30, 40, 50, 60, 70, 80 and 90 for cultures stored at 5 °C and 0, 5, 10, 20 and 30 for those kept at -18 °C.
Statistical analyses
Means were subjected to one way analysis of variance to test for significant differences at α = 0.05. The Fisher’s LSD was used to separate the means. Analyses were done using XLSTAT software version 2010 (Addinsoft, Paris, France).