Sour orange Citrus aurantium L. flowers: A new vegetable source of milk-clotting proteases

- Citrus flower represent a new source of proteases for milk-clotting.
- Proteolytic activity over a broad pH range indicates several types of proteases.
- Milk-clotting activity was temperature dependent with a maximum between 65 and 70 °C.
- Milk-clotting times were similar to those used in commercial cheese making process.
- Citrus flower proteases holds a potential use for biotechnological application.

The need to find suitable calf rennet substitutes for cheese-making has led to an increased interest in screening for new proteases to be used as natural coagulants. The aim of this work was to detect and characterize proteases in citrus flower extracts (CFE) by evaluating milk-clotting and proteolytic activity. Proteolytic enzymes in raw CFE showed milk-clotting activity over a broad temperature range (35-70 °C) and were able to hydrolyze substrates such as casein, hemoglobin and bovine serum albumin. The protease activity of the raw extract was adequate to clot milk (0.33 milk clotting units mL−1) in clotting times similar to commercial rennet. Inhibition studies suggested that some enzymatic activity in the crude CFE was due to the action of aspartic proteases since most of the activity at pH 3 was inhibited by pepstatin A. Proteolytic activity at pH 7 was sensitive to dithiothreitol (DTT), indicating that possibly disulfide bridges contribute to stabilizing the native enzyme structure. CFE contains different types of proteases and holds potential as a new source of enzymes for biotechnological applications (e.g., cheese production).