Biochemical, Milk-Clotting And Cheese Making Properties of Proteases From The Marine Macroalga Gracilaria edulis

Abstract

The production of cheese involves milk coagulation as an essential step. Current milk coagulants still have various drawbacks and the search for alternatives is necessary. Marine macroalgae (seaweed) are a potential source of milk clotting enzymes that have not been well explored. This thesis investigates the extraction, characterisation and application of milk-clotting enzymes from seaweed for cheese making. Among seven species of algae studied, Gracilaria edulis was found to contain proteases with significant caseinolytic and milk-clotting activities. Optimum conditions for extracting and purifying the proteases were then determined. The proteases demonstrated optimum caseinolytic activity at 60°C and a pH range of 6–8 and were identified as serine and metalloprotease with MW of 44 and 108 kDa. The enzymes cleaved κ-casein at four main sites, one of which being the same as that of calf rennet. Amino acids sequencing showed greater than 80% matching with those from members of the bacteria kingdom, indicating that the proteases could be a novel enzyme or existing zymogen. The algal extracts were used successfully as the coagulant in making Cheddar and Feta style cheese, at a different coagulation temperature (50°C) than the usual temperature for calf rennet (37°C). The algal protease gave a higher cheese yield and moisture content but lower total solids compared to calf rennet. After aging for 6 months, proteins of the Cheddar style cheese made with algal protease were mostly digested, and the peptide profiles showed a large number of small peaks, demonstrating stronger proteolysis of the algal protease compared with calf rennet. The colour of algal cheese was lighter and less yellow, and the microstructure was similar to calf rennet cheese. Most of the textural properties of the algal cheese differed significantly (P<0.05) from the calf rennet cheese, but the hardness of both cheeses was similar (P>0.05). Sensory analysis showed that the fresh Feta algal cheese was less acidic than calf rennet cheese (P<0.05), but the two cheeses had similar texture, bitterness, and overall liking scores (P>0.05). Aged Cheddar algal cheese had similar score of texture, overall flavour, acidity (P>0.05) but higher bitterness, and lower overall liking score than aged Cheddar calf rennet cheese (P<0.05). This thesis demonstrates that the proteases from Gracilaria edulis have the potential for application in making fresh cheeses but not cheeses that require aging.