Quantitative analysis of the antihypertensive peptides Val-Pro-Pro and lie-Pro-Pro in casein hydrolyzate using an Aspergillus oryzae protease: an LC-MS method

摘要

Potent antihypertensive peptides, Val-Pro-Pro and lie-Pro-Pro, previously isolated from milk fermented with Lactoba-cillus helveticus, were analyzed from the proteolytic digest of casein with an Aspergillus oryzae protease. A quantitative method for Val-Pro-Pro and lie-Pro-Pro in the casein hydrolyzate was tested by a HPLC method. However, it was difficult to detect specific peaks corresponding to these two peptides. These peaks were easily detected, if an LC-MS method was applied, using Ala-Pro-Propeptide as internal standard. Validation studies of the measurements for Val-Pro-Pro and lle-Pro-Pro in A. oryzae using the LC-MS method showed sufficient precision, linearity, recovery and reproducibility. Recoveries of Val-Pro-Pro were 103.5, 101.3,105,7, 103.0, 99.3 and 99,1%, and of Ile-Pro-Pro: 108.3,102.5,101.1,103..5, 102,4 and 97.5%, if both synthetic peptides were added at 3,6,7,2,12,0, 24.0, 36.0 and 48.0 mu g/ml, respectively. Analysis was repeated 10 times in order to measure reproducibilityof the LC-MS analysis: the variation for Val-Pro-Pro and Ile-Pro-Pro was 3.9 and 41%, respectively. Our results suggest that the LC-MS method is an appropriate analytical method to specifically quantify the antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, in A. oryzae-digest.