Antihypertensive effect of bioactive peptides produced by protease-facilitated lactic acid fermentation of milk

Milk was fermented for up to 5 h at 43 °C with two lactic acid bacteria (Streptococcus thermophilus, Lactobacillus bulgaricus). A protease, flavourzyme, was added at the beginning of fermentation. The whey fraction was separated from the fermented milk and freeze-dried. During the 5 h of fermentation, the soluble protein content increased from 4.9 to 57.4 mg/g and peptide content increased from 2.1 to 32.8 mg/g, while inhibition of angiotensin I-converting enzyme (ACE) increased by a decrease of IC50 from 0.708 to 0.266 mg/ml, respectively. The whey was fractionated into four fractions by size exclusion chromatography on a Sephadex G-15 column. The fourth fraction of the whey showed the highest inhibitory efficiency ratio (IER) being 1329%/mg/ml. The amino acid sequence of the inhibitory peptide was Tyr-Pro-Tyr-Tyr, of which the IC50 was 90.9 μM. The whey showed mixed-type inhibition kinetics, while Captopril, the positive control showed competitive inhibition on ACE. Their Ki values were 0.188 mg/ml and 0.0067 μg/ml, respectively. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) was reduced to 15.9 and 15.6 mm Hg, respectively, in spontaneously hypertensive rat (SHR), after 8 weeks of oral administration of diluted whey (peptide concentration 4.9 mg/ml).