Milk-clotting enzyme from marine algae: the isolation, characterisation and its action on casein from bovine milk

Abstract

The existence of proteolytic enzymes in the food industry, especially in the dairy application is substantial. The calf rennet and its substitutes still have limitation from customer and industrial perspectives, such as vegetarian preference, religious concerns, and genetically modified engineered food issue in some countries. In the meantime, marine ecosystems provide significant potential for new enzymes development which has not been well explored. In this study, the characteristic of the milk-clotting enzyme from algae was investigated.

Seven species of algae consisted of Ulva ohnoi, Sarconema filiforme, Asparagopsis taxiformis, Caulerpa lentilifera, Caulerpa racemose, Gracilaria edulis and Kappaphycus alvarezii, were extracted using 20 mM phosphate buffer pH 7. The extract of S. filiforme showed the highest protein content among all crude extract. However, of the seven species algae observed, G. edulis was the potential candidate for rennet substitute that exhibited a positive result of the milk-clotting activity and demonstrated the highest specific caseinolytic activity. The crude extract of G. edulis was subsequently partially purified by ammonium sulphate continued with dialysis as the desalting method. The analyses of purified extract indicated significant increment (P>0.05) on both caseinolytic and milk-clotting activities.

The SOS-PAGE revealed that the purified extract started to hydrolyse casein from bovine milk noticeably by 10 minutes at 37°C. Further degradation of casein by the extract was observed in longer incubation time. At 60°C, almost all casein bands were degraded in the first 10 minutes, indicating the protease expressed a stronger caseinolytic activity. The electrophoresis analysis also monitored a peptide band between 10-15 kOa generated at 37°C and 60°C, which similar to the molecular weight of para-kappa-casein produced by calf rennet. The characteristic and the action of the G. edulis protease on bovine milk casein showed a potential function as a novel milk-clotting enzyme for the dairy industry.