Sorghum malt preparation
SESO 3, a red seeded 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 then washed using pressurized water. Ten kilograms of grain were soaked in 15 liters of potable water containing 0.3% sodium hydroxide (Merck, Germany) and allowed to steep 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 water was drained and the grain transferred to germination beds at 25°C. Germination was halted after two days when the rootlets of the grain reached 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 room temperature (25-27°C) till further use.
Propagation of microbial strains
Lactobacillus plantarum MNC 21 and Saccharomyces cerevisiae MNC21Y isolated from Obushera  were used in the study. Lb. plantarum MNC21 was grown in 100 ml of MRS broth (CONDA, Madrid, Spain) at 30°C for 24 h. S. cerevisiae MNC21Y was grown in 100 ml of Yeast Mold Broth (CONDA, Madrid, Spain) at 30°C for 24 h. The cells were separately centrifuged at 7500 x g for 10 min (Centrofriger – BL II, JP Selecta, Barcelona, Spain) and the pellets washed thrice using sterile quarter strength Ringer’s solution (Oxoid Limited, Basingstroke, Hampshire, England). The pellets were then separately suspended in 10 ml of sterile quarter strength Ringer’s solution.
Preparation and inoculation of sorghum malt 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 sorghum malt was mixed with potable water to make a slurry of 12.5 % total solids. The slurry was heated with continuous stirring to 90°C and held at that temperature for 15 min. The hot slurry (200 ml) was then aseptically transferred to sterile 250 ml glass bottles and allowed to cool to 30°C. The porridge was inoculated with 6.0 log cfu.ml -1 of Lb. plantarum MNC 21 and S. cerevisiae MNC 21Y cultures and incubated at 30°C for 24 h. Samples were drawn at 0, 4, 8, 12 and 24 h to measure cell counts, pH and titratable acidity.
Storage of starter cultures in fermented sorghum malt slurries
Starter cultures can be stored in a refrigerator at 2 – 5°C or frozen with cryoprotectant at −20°C to −40°C, −80°C or −196°C (Parente, Cogan and Powell 2017). Other authors have reported storing starters at −20°C to 10°C (Kringelum and Kragelund 2010). In this study 5°C and −18°C were used because they are the common chiller and freezer cabinet temperatures, respectively observed for refrigerators in Uganda (Makumbi et al, 2015). Therefore, most processors of Obushera with access to refrigerators are likely to store the starters at either 5°C or −18°C. To store the starter cultures, 50 ml of the fermented sorghum malt slurries were distributed in sterile 100 ml plastic bottles. Some of the bottles containing the fermented sorghum malt slurries were stored in a refrigerator and the rest in a freezer.
Determining the fermentation ability of the stored starter cultures
Samples were drawn periodically to determine cell counts and fermentation ability of the stored cultures. Fermentation ability of the stored cultures was determined by inoculating 500 ml of freshly prepared and sterile sorghum malt slurries (12.5% total solids) with 1% (v/v) of the stored starter cultures. The inoculated slurries were incubated at 30°C for 24 h. Samples were drawn periodically to determine pH and titratable acidity (0, 4, 6, 12 and 24 h), and flavor development (24 h).
Acidity and pH
The pH was measured using a pH meter (Mettler-Toledo AG model, Mettler-Toledo Group, Schwerzenbach, Switzerland). Titratable acidity was determined by titrating 10 ml of the sample against a 0.1M solution of sodium hydroxide using phenolphthalein as the indicator (Horwitz 2000).
Flavor development by the stored starter cultures
The starter culture combination of Saccharomyces cerevisiae MNC21Y and Lactobacillus plantarum MNC21 is known to produce acceptable sensory attributes (aroma, taste, texture and color) which are similar to those of the traditional product (Mukisa 2012; Mukisa et al. 2017). Therefore, beside evaluating viability and acidification potential of the stored culture, this study assessed the production of the typical flavor of Obushera. Flavor development was determined by the researchers (n = 3) who were all familiar with Obushera. The products were sniffed to detect for the characteristic flavor of Obushera. Samples were scored on consensus as follows: +++ = strong flavor development; ++ = mild flavor development; + = weak flavor development; ND = not detected.
Serial dilutions were prepared using ¼ strength ringer’s solution (Oxoid Limited, Basingstroke, Hampshire, England). Enumeration of Lactobacillus plantarum MNC21 was carried out by pour plating selected serial dilutions in MRS agar (CONDA, Madrid, Spain) and incubating at 30°C for 48 h. Enumeration of Saccharomyces cerevisiae MNC21Y was carried out by surface plating selected serial dilutions of the culture in Potato Dextrose Agar (CONDA, Madrid, Spain) with chloramphenical supplement and incubating at 30°C for 72 h. Microbial counts were determined at days 0, 5, 10, 20, 30, 40, 50, 60, 70, 80 and 90 during storage of starters at 5°C and only up to day 30 for starters stored at -18°C.
Means were subjected to one way analysis of variance (ANOVA) to test for significant differences at a 5% level of significance. The least significant difference test (Fisher’s LSD) was used to determine means that were significantly different from one another after the ANOVA test. All statistical analyses were performed by XLSTAT (2010, Addinsoft, Paris, France). Experiments were carried out in triplicate.