Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation

Fresh low-fat milk was fermented with five mixed lactic acid bacteria for up to 30 h at 42 °C. A protease, prozyme 6, was added 5 h after the beginning of fermentation. The whey was separated from the fermented milk and freeze-dried. As the fermentation time extended to 30 h, soluble protein content increased from 30.9 to 195.9 mg g−1, free amino acid content increased from 2.8 to 192.8 mg g−1, peptide content increased from 6.4 to 402.8 mg g−1 and γ-aminobutyric acid (GABA) increased from 0 to 80.6 mg 100 g−1, while inhibition of angiotensin I-converting enzyme (ACE) increased as indicated by a decrease of IC50 from 1.18 to 0.24 mg mL−1, respectively. The amino acid sequences of two ACE inhibitory peptides were Gly–Thr–Trp and Gly–Val–Trp, of which the IC50 values were 464.4 and 240.0 μm, respectively. The systolic blood pressure and diastolic blood pressure of spontaneously hypertensive rat (SHR) were reduced 22 and 21.5 mm Hg, respectively, after 8 weeks of oral administration of diluted whey (peptide concentration 5 mg mL−1) from the 30 h fermentation.