FOOD SCIENCE ›› 2021, Vol. 42 ›› Issue (10): 104-110.doi: 10.7506/spkx1002-6630-20200322-322

• Bioengineering • Previous Articles     Next Articles

Hydrolysis Site of Casein by Milk-Clotting Protease from Moringa oleifera Lam. and Preparation and Evaluation of Casein Phosphopeptide and Casein Glycomacropeptide

SHI Yanan, ZHANG Jiayan, HUANG Aixiang   

  1. (College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China)
  • Online:2021-05-25 Published:2021-06-02

Abstract: In this study, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to explore the cleavage site and hydrolysis characteristics of casein by a milk-clotting protease from Moringa oleifera Lam. Then, casein phosphopeptide and casein glycomacropeptide in the resulting hydrolysate were analyzed. The results showed that the protease could specifically cleave κ-casein at the Arg93-His94 site leading to milk clotting, which is distinct from other reported chymosins. The Michaelis constant (Km) and maximum reaction rate (Vm) of the milk-clotting protease were 0.49 mg/ml and 45.2 U/min, respectively, and it could promote the aggregation and coagulation of casein micelles. The enzyme could hydrolyze α, β and κ-casein to different extents and exhibit the fastest hydrolysis rate toward α-casein, indicating that it was suitable for the processing of soft cheese. Casein phosphopeptide (CPP) was obtained by barium-ethanol precipitation method with casein as the substrate, with a yield of 15.87%. CPP could delay the precipitation of calcium by 23 min, which was beneficial to the intestinal absorption of calcium. When buffalo milk was used as the substrate, the concentration of casein glycomacropeptide (CGMP) was 6.61 mg/mL in whey, and the degree of glycosylation was 477.527 μg/mg. This study provides an important theoretical basis for the development and application of new plant chymosins in cheese and casein-derived bioactive peptides.

Key words: Moringa oleifera Lam. milk-clotting protease; enzyme cleavage site; κ-casein; casein phosphopeptide; casein glycomacropeptide

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