Identification of antihypertensive tripeptides in the culture medium of Lactobacillus helveticus D75 and D76 strains

The aim of the current work was to search for bioactive peptides Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP) with antihypertensive action in the culture medium (CM) of probiotic Lactobacillus helveticus D75 and D76 strains when cultured in milk. The CM composition was analyzed using HPLC-UV, HPLC-MS, and MALDI Fourier transformation ion-cyclotron resonance mass spectrometry (MALDI FT-ICR MS, Varian). Direct analysis of CM using FT-ICR failed to detect peptides in the original CM due to the high concentration of concomitant components, but after preliminary fractionation of CM by HPLC, both peptides were detected. The masses (m/z) of characteristic ions were 312.19 (VPP) and 326.21 (IPP). Peptides were quantified using a single reference method with HPLC and time-of-flight mass spectrometry. The concentrations of VPP and IPP in the CM of the D75 strain, respectively, reached 18.0 µg/mL and 25.4 µg/mL, in the CM of the D76 strain they were lower (12.7 µg/mL and 16.2 µg/mL), and in the mixed CM (both strains) the concentrations reached intermediate values of 15.7 µg/mL and 24.0 µg/mL. The data obtained indicate the existence of an active complex of extracellular enzymes in L. helveticus D75 and D76, which provide the production of IPP and VPP peptides with antihypertensive effect. The results also confirm the symbiotic properties of D75 and D76 strains (synergism and syntrophy). Functional foods based on milk fermented by probiotic L. helveticus D75 and D76 strains, which contains IPP and VPP peptides in effective doses (>3.0 mg/day), can be considered a safe and effective non-pharmacological intervention in patients with mild hypertension.