**3.5. Partial enzyme purification**

638 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

staining and catalase activity.

**production** 

by incubation in an alternative shaker at 37-40°C and speed of 150 oscillations per minute for 24 h. Amylases producing bacteria strains were screened on agar plate, containing (gram per liter): 10 g soluble starch, 5 g peptone, 5 g yeast extract, 0.5 g MgSO4.7H2O, 0.01g FeSO4.7H2O, 0.01 g NaCl, 15 g agar. Incubation at 37-40°C was carried out for 48 h, after which the plates were stained with lugol solution (Gram iodine solution: 0.1% I2 and 1% KI). The colonies with the largest halo forming zone were pre-selected and tested for Gram

Preliminary tests were carried out to determine the heat stability of the amylase of each isolate as we described previously [47, 48]. The gas production from glucose, growth at different temperature (10, 40, 45 °C) as well as the ability to grow in different concentration of NaCl was determined as described by Schillinger and Lucke [49] and Dykes et al [50]. The isolates which were Gram positive and catalase negative, non-motile and producing heat stable amylase and lactic acid were finally selected and identified using API 50 CH test kit (bioMerieux, France).

In order to study microbial growth, amylase and lactic acid production, the microorganism was propagated at 40°C for 70 h in 50 ml of a basal medium containing: soluble starch, 1% (w/v); yeast extract, 0.5 % (w/v) placed in 100 ml Erlenmeyer flask with shaking at 150 oscillations per minute in an alternative shaker (Kotterman, Germany). The initial pH of the medium was adjusted to 6.5 using 0.1 M HCl. After removal of cells by centrifugation (8000xg, 30 min, 4°C) in centrifugator (Heraeus, Germany), the supernatant was considered

**3.4. Optimisation of raw starch degrading thermostable amylase and lactic acid** 

The amylase and lactic acid production was optimized by studying the effect of cultural and environmental variables (carbohydrate and nitrogen sources, metal salts and surfactants) individually and simultaneously. The effect of carbohydrate sources was studied by replacing soluble starch in basal medium with different sugars, gelatinized and raw natural crude starch sources (glucose, fructose, maltose, amylose, amylopectine, cassava, corn, rice tapioca, and sorghum flours at final concentration of 1% (w/v)). Nitrogen sources were tested by replacing yeast extract with various nitrogen sources (peptone, tryptone, beef extract, soyabean meal, ammonium sulphate, and urea at final concentration of 1.5% (w/v)). The effect of metal salts was studied by adding individually various metal salts (CaCl2.2H2O, MgSO4.7H2O, FeSO4.7H2O, FeCl3, NaCl at concentration of 0.1% (w/v)). Similarly the effect of surfactants was studied by supplementing the culture medium with

All media containing gelatinized starch sources were autoclaved at 121 °C for 20 min, while for the media containing raw starch flour, starch powder was sterilized by washing in

The APILAB PLUS database identification was used to interpret the results.

as the crude enzyme solution and was also used for lactic acid evaluation.

Tween 80 and Tween 40 at concentration of 1.5% (v/v).

ethanol and added to sterile nutrient broth.

**3.3. Microbial growth, amylase and lactic acid production** 

The culture supernatant was supplemented with solid ammonium sulphate to 65% (w/v) final concentration, with mechanical stirring at 4°C. The suspension was retained for 1 h at 4 °C, and centrifuged at 8000 g for 30 min at the same temperature. The resultant supernatant was brought to 70 % w/v ammonium sulphate saturation at 4°C. 50-70% (w/v) ammonium sulphate precipitate was recovered, dissolved in 0.1 M phosphate buffer and dialysed using Spectra/PorR, VWR 2003 dialysis membrane overnight against the same buffer at 4°C and used as partial purified enzyme solution.
