Characterisation of a milk-clotting extract from Balanites aegyptiaca fruit pulp

Milk-clotting proteases were extracted and characterised from Balanites aegyptiaca fruit pulp traditionally used in sub-Saharan countries to thicken gruel. The aim was to improve the extraction conditions and milk-clotting in the context of future cheese making. B. aegyptiaca fruits were disinfected, husked and soaked in different buffers and saline solutions. The extracts obtained were diafiltered, concentrated and decoloured with active charcoal. The effects of extract concentration, milk pH and temperature on clotting-time were studied. The use of specific inhibitors showed that the B. aegyptiaca extract contains two types of proteases: an aspartic protease and a serine protease, with an optimum activity at pH 5.0 and pH 8.0, respectively, and an optimal temperature at 50 °C. The proteolytic activity is more thermostable than bovine chymosin at pH 5.0. Partial purification by anion exchange chromatography and sodium docecylsulphate polyacrylamide gel electrophoresis allowed separation of the two milk-clotting proteases.