Identification, structure, and caseinolytic properties of milk-clotting proteases from Moringa oleifera flowers

The protein extract of Moringa oleifera flowers is reported to have milk-clotting activity (MCA), but information regarding its protease is unclear. In this study, two milk-clotting proteases (MoFP 12 and MoFP 13) with molecular weights of 42.304 kDa and 31.741 kDa, respectively, were isolated and identified from M. oleifera flowers using liquid chromatography-mass spectrometry (LC-MS/MS). Bioinformatics analysis showed that these two milk-clotting proteases were primarily involved in hydrolase activity and catabolic process, and exhibited hydrophilicity. The secondary structure of MoFP 12 consisted of 43.65% helix, 13.23% strand, and 43.12% coil, and MoFP 13 consisted of 26.51% helix, 20.14% strand, and 53.35% coil. The proteases were stable in the pH range of 5.0 to 8.0 and showed their maximum MCA at 70℃. Additionally, by investigating the effect of proteases on caseins, κ-casein (CN) was observed to preferentially be hydrolyzed by the two proteases, followed by α-CN, and to a lesser extent β-CN. These findings revealed the main milk-clotting proteases in M. oleifera flowers and its milk-clotting properties, indicating its potential for application in the dairy and food sectors, especially in the cheese-making industry.