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CHAPTER 3 MATERIALS AND METHODS
Sample preparation
Cereal and skim milk samples
Traditionally processed finger millet (Eleusine coracana) meal was purchased from rural farmers in Manicaland, a province in the Eastern Highlands in Zimbabwe. The traditional process of making finger millet meal involved roasting of the grain to loosen the peri carp and to develop flavour. The pericarp was removed by pounding with the traditional mortar and pestle. This was followed by winnowing to separate the loose pericarp from the clean grain. The clean grain was then milled using hammer milling. The finger millet meal was stored in sealed plastic bags at 10°C until required. Skim-milk powder was sourced in South Africa and stored under cool dry conditions until required.
Table 1 shows the proximate composition of the finger millet meal and the skim-milk powder.
Preparation of cereal-dairy composite gruels
Skim-milk powder was reconstituted, according to the manufacturer’s instructions. One part skim-milk powder was added to nine parts water and stirred until completely dispersed. The finger millet gruel was prepared by cooking five parts of finger millet meal in 95 parts of water at low heat for 10 min while stirring continuously. Proportions of finger millet greater than 5% gave a gruel that solidified on cooling. The reconstituted skim milk was added to the gruel to give composites containing 100%, 90010, 80%, 70%, 60%, 50% and 0% (skim milk only) finger millet gruel by volume. The unfermented gruels were sterilised by autoclaving for 15 min at 121°C.
Starter cultures
Three starter cultures were used for fermentation. YC380 (Chr. Hansen, Denmark) and Joghurt V2 (Wiesby, Denmark) were commercial yoghurt type starter cultures that were supplied by Darleon CC. in South Africa. They are often used in the dairy industry in South Africa for the manufacture of yoghurt. According to the suppliers, they contain strains of the bacteria Lactobacillus de/brueckii subsp. bu/garicus and Streptococcus thermophilus subsp. sa/ivarius. Both cultures are mixed cultures with Gram positive cocci and rods. The third starter culture, IC, was a mixed strain culture developed in our laboratory over a period of time to ferment raw cereal slurries at 25°C. Table 2 summaries the characteristics ofthe three starter cultures
Starter culture propagation
Bacterial starter cultures YC380 and V2 were each added to sterilised, reconstituted skim milk at a rate of 1 g of starter culture per 99 ml of skim milk. This was done under aseptic conditions. The mixture was thoroughly mixed and incubated at 45°C for 3 h. The fermented products were cooled to 5°C. These preliminary yoghurt cultures were subcultured into sterilised skim milk at a rate of 1 ml of culture per 99 ml of skim milk. After thorough mixing they were incubated at 45°C for 3 h and then cooled to Soc. Subculturing was repeated twice leading to batches of yoghurt culture, hereafter referred to as Starter Cultures. These were stored in the refrigerator.
The starter culture JC was prepared by mixing SO g dehulled sorghum meal and 64 ml of water at 30°C into a slurry which was then fermented at 2SoC until the pH dropped to 3.6 . The culture was maintained by inoculating a mixture of SO g of dehulled sorghum meal and 64 ml of water with S ml of the fermented slurry.
Gruel fermentation
Initially, the inoculated samples were incubated at 10°C, 2SoC, 30°C, 37°C and 4SOC for 10 h. All analyses were carried out in triplicate. In the second phase of the experiment the optimum time of incubation was determined. This was measured as the time that it took for the pH of inoculated samples to fall to 4.6 . Sterile samples were inoculated with bacterial starter cultures YC380 and V2 at an inoculation rate of 2% (v/v) under aseptic conditions. The samples were incubated at 37°C and 4SoC. At 1 h intervals the pH of the samples was determined. The control samples were not inoculated.
Gruel inoculation
Inoculation of gruel samples was carried out aseptically at room temperature and each mixed by shaking the bottle. Gruel samples that had been measured to give a volume of 98 ml were inoculated with 2 ml of starter culture to give an inoculation rate of 2%. The samples were thoroughly mixed to ensure even distribution of the inoculum.
Freeze-drying
Gruel samples that were used for the determination of crude fat, crude fibre, amino acids and energy were freeze-dried (Specht Scientific, Model no. SJ-FD-S/PC, Asslar, Germany). The freeze-dried samples were milled using a Waring blender and stored in sample bottles at 10°C away from light until required.
Physicochemical analyses
pH and titratable acidity
Acidity was detennined by the potentiometric procedure (International Dairy Federation, 1991) by weighing 10 g of the sample into small beakers, adding 10 ml of distilled water and titrating with O.IM sodium hydroxide to a pH of8.3. Acidity was expressed as % lactic acid. The pH was determined using a combined glass electrode connected to a Mettler DL-25 pH meter (Mettler-Toledo AG, Greifensee, Switzerland) by immersing the electrode directly into the gruel sample.
Syneresis
The % syneresis (vim) was detennined by a modification of the centrifugation procedure described by Harwarkar & Kalab (1983). Samples of the fermented gruel, which had been incubated at 30°C, 37°C and 45°C, were chilled to 4°C for 24 h and then centrifuged at 1000 x g for 20 min. The lower temperatures of incubation (10°C and 25°C) that were used for pH detennination were left out because the gruels fermented at these temperatures did not attain the desired pH of 4.5 or lower. The volume of serum (cm3) was expressed as a percentage of the mass (g) of the sample centrifuged.
Consistency
The consistency of the fermented gruel incubated at 37°C and 45°C was detennined as the distance flowed, in centimetres, within a time period of 30 s using a Bostwick consistometer (Gould, 1974). This instrument is used widely by the dairy industry in South Africa for quality control.
Gruel firmness
Gruel firmness was measured as the maximum force required to penetrate undisturbed gruel in glass containers using a texture analyser (TA-XT2; Stable Microsystems, Surrey, England) and a cylindrical probe which had a base diameter of 20 mm (Hess, Roberts & Ziegler, 1997). The containers were half filled and had a maximum capacity of 5 5 cm3, a base diameter of 35 mm and a height of 70 mm.
Proximate analyses
Moisture
A commonly used procedure for determining the moisture content of a food product is based on the separation of water from the solids and its measurement as the resulting loss in weight or by measurement of the amount of water lost (Joslyn, 1970). The accurate determination of moisture is difficult because of the problem of completely separating all the water from the food product without completely causing its decomposition with concomitant production of water which would be included in the determination (Joslyn, 1970). The loss of volatile constituents from the food is another factor. A modification of the oven drying method was used (AOAC, 1980a).
