Milk clotting enzymes from marine resources and their role in cheese-making: A mini review.

With the continual increase in global cheese consumption, rennet, the traditional milk coagulant, is unable to meet the growing demand in cheese production. Although several proteases from other sources have been used for cheese-making, they suffer various shortcomings. The ocean is home to a huge and diverse range of life forms, which represent a vast potential source of proteases. Marine proteases have been isolated from a number of marine species, including sponge, jellyfish, seaweed and marine animals, and some have been shown to be suitable as milk-clotting enzymes for cheese making. This review summarizes the latest studies on rennet substitutes from marine resources and their role in cheese-making. The emphasis of the review is on the isolation and purification of the marine proteases, the biochemical characteristics of these enzymes, especially their caseinolytic and milk-clotting properties, as well as their cleavage sites on casein. Some of the marine proteases have been applied as milk-clotting agent in cheese-making, with the resultant production of cheese with comparable characteristics, including sensory characteristics, to calf rennet cheese. The review concludes by highlighting the challenges and opportunities for future research in the field.

Amino acid sequence of two new milk-clotting proteases from the macroalga Gracilaria edulis.

This study is aimed at identifying and characterising the proteases we previously extracted from the red seaweed Gracilaria edulis with the potential as milk-clotting enzymes. The protease extract was first analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Two protease bands with a molecular weight of 44 and 108 kDa were identified, and analysed using in-gel digestion and liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS). Eight peptides from the LC-MS/MS analysis matched those in existing protein databases but they were not related to any protease of the genera Gracilaria and Hydropuntia. Further analysis revealed that more than 80% of the peptide sequence of the algal proteases matched with those from members of the bacteria kingdom, including Gallaecimonas and Alteromonas. Among these, twelve matching homolog proteases were identified as metalloprotease and serine protease. The results indicated that the algal proteases have a close relationship with both algae and bacteria, and suggest that the proteases might have resulted from past bacterial colonisation of the algae and subsequent horizontal gene transfer between bacteria and algae.

Extraction, partial purification and characterization of proteases from the red seaweed Gracilaria edulis with similar cleavage sites on κ-casein as calf rennet.

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60 °C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108 kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.