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 degrees C. Additionally, by investigating the effect of proteases on caseins, kappa-casein (CN) was observed to preferentially be hydrolyzed by the two proteases, followed by alpha-CN, and to a lesser extent beta-CN. These findings revealed the main milk-clotting pmteases 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.