Ethanol-salt based two-phase partitioning for concentration of milk clotting proteases from Sweet Indrajao (Wrightia tinctoria R. Br.) fruit

Abstract

Cheese is the naturally fermented dairy product beloved for its wide variety of textures and other nutritional qualities. Animal rennet, microbial and fermentation produced chymosin are generally considered for commercial level cheese preparation. In order to meet the growing global demand observed in cheese market, milk clotting proteases from diverse alternative sources are under exploration. Sweet Indrajao (Wrightia tinctoria R. Br.) fruit extract is known for its utility in traditional cheese preparation. Organic solvent-salt based aqueous two-phase system (ATPS) using ethanol and salt was employed to partially purify milk clotting proteases from W. tinctoria fruit. The system with ethanol-25.90% (w/w) and dipotassium phosphate-13.60% (w/w) was found favorable with yield of 96.33%, milk clotting index of 77.85, purification factor of 2.42, partition coefficient of 458.80 and selectivity of 345.58. Tricine SDS-PAGE and zymogram revealed presence of two prominent protein bands between 66 and 97.4 kDa with caseinolytic activity. Whole casein and κ- casein hydrolysis pattern was analyzed by tricine SDS-PAGE. The optimized system was scaled up to 100 g and its performance was found comparable to its 10 g counterpart. Characteristics of the cheese prepared using partitioned enzyme was comparable to that of the positive control used (Enzeco®).