We describe a clinical trial to study the efficacy of a casein hydrolysate, prepared using an Aspergillus oryzae protease, containing the major angiotensin-I-converting enzyme inhibitory peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) in a single-blind, placebo-controlled study. A total of 131 volunteers with high-normal blood pressure and mild hypertension were randomly divided into four groups (n 32 or 33 in each group). Each volunteer was given two tablets containing four different dosages of VPP and IPP (VPP þ IPP: 0, 1·8, 2·5 and 3·6 mg), daily for 6 weeks. A significant decrease in systolic blood pressure was observed at 6 weeks in the active group receiving 1·8 mg (P,0·01) VPP and IPP; in the active groups receiving either 2·5 mg or 3·6 mg, systolic blood pressure was decreased at both 3 weeks (P,0·05 and P,0·05) and 6 weeks (P,0·001 and P,0·0001) compared with systolic blood pressure measured before treatment. Changes in the systolic blood pressure after 6 weeks of treatment in the four groups were 21·7, 26·3, 26·7 and 210·1 mmHg, and these effects were dose dependent. In addition, a significant difference in systolic blood pressure between the placebo group and the VPP and IPP group receiving 3·6 mg was observed (P,0·001) by two-way ANOVA. The antihypertensive effect was greater in mildly hypertensive subjects (n 20 or 21 in each group) than in any of the other subjects. No significant change of diastolic blood pressure was observed for all the test groups, and no differences in diastolic blood pressure in the test sample groups compared with the placebo group were observed during the test period.

Objective: To examine the effects of the degree of body mass index and weight gain as the risks for hypertension, hypercholesterolemia and diabetes in Japanese men, and to compare that to the corresponding effects in a Caucasian population. Design: Prospective cohort study. Setting: Cohort of all male employees at a company. Subjects: A total of 4737 male employees followed until retirement or for 4 y (longest term 4 y). Results: Increase of body mass index beyond 22 kg=m 2 related to an increased risk for hypertension even after being adjusted for possible confounding factors such as age, smoking status, alcohol intake, family history and baseline value of systolic blood pressure. The risks greatly increased in subjects with a body mass index above 27 kg=m 2 for hypertension, and 29 kg=m 2 for diabetes and hypercholesterolemia. Weight gain (more than 2 kg) was strongly related to an increased risk for hypertension and hypercholesterolemia after being adjusted for possible confounding factors. However, weight gain was not related to diabetes, and weight loss did not decrease the risks for any of the three diseases. Conclusions: Among Japanese, the degrees of body mass index associated with risks for hypertension, diabetes and hypercholesterolemia were lower than those in Caucasians. The risks for hypertension and hypercholesterolemia were strongly associated with weight gain in a Japanese male population who showed a low prevalence of severe obesity, and the risks were similar to or somewhat higher than those in a Caucasian population with a high prevalence of severe obesity.

Angiotensin-I converting enzyme inhibitory activities were measured after hydrolysis of casein by 9 different commercially available proteolytic enzymes. Among these enzymes, a protease isolated from Aspergillus oryzae showed the highest angiotensin-I converting enzyme inhibitory activity per peptide. The A. oryzae peptide also showed the highest antihypertensive effect in spontaneously hypertensive rats when the systolic blood pressure was measured 5 h after oral administration of 32 mg/kg of various enzymatic hydrolysates. Significant antihypertensive effects were observed with dosages of 9.6, 32, and 96 mg of the A. oryzae peptide/kg of body weight (BW), and the effects were dependent on these peptide dosages. Analysis of peptide length showed the A. oryzae hydrolysate was the shortest of all tested casein hydrolysates; the peptide mixture had an average value of 1.4 amino acids (AA) in the sequence. To further characterize the A. oryzae hydrolysate, we analyzed the AA sequence of the whole peptide mixture. Various AA were detected at the first AA position, however, an increased number of Pro residues were observed at the second and third position of the A. oryzae hydrolysate. No strong signals were detected after the fourth AA position of the A. oryzae hydrolysate. These results suggest that the casein hydrolysate of A. oryzae, which expressed potent antihypertensive effects in spontaneously hypertensive rats, mainly contain short peptides of X-Pro and X-Pro-Pro sequences.

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