Antihypertensive peptides exert their biological activity through three main mechanisms: (i) inhibition of the Angiotensin I-Converting Enzyme (ACE) (Nakamura et al., 1995b); (ii) inhibition of the Nor-adrenaline-mediated activity of the sympathetic nervous system (Usinger et al., 2010); and (iii) modulation of vascular and endothelial function towards vasodilator response (Yamaguchi et al., 2009). The most studied mechanism is the ACE inhibition. ACE (peptidyldipeptide hydrolase, EC 188.8.131.52) converts Angiotensin-I into the potent vasoconstrictor Angiotensin-II, and cleaves the vasodilator peptide bradykinin (Petrillo and Ondetti 1982). The activity of ACE-inhibitory (ACE-I) peptides is easily measured in vitro (Holmquist et al., 1979; Nakamura et al., 1995a). It is expressed in terms of IC50, that is, the concentration (usually expressed as pmoles/L) of the peptide needed to inhibit 50% of enzyme activity. The lower the value of IC50, the higher is the inhibitory potency of the peptide.
The ACE-I activity of peptides is strongly affected by their primary structure, especially by the three last C-terminal residues (Ondetti and Cushman, 1982). In detail, high ACE-I activity was reported for peptides containing aromatic amino acids (especially tryptophan, phenylalanine, and tyrosine), or proline (P) at their C-terminus (Li et al., 2004; Wu et al., 2006). The presence of proline would make the digestion of peptides more difficult (Walker et al., 2001). The residues at the N-terminus also affect the capacity of peptides to bind to the active sites of ACE. Most potent ACE-I peptides contain branched chain amino acids (valine, leucine, isoleucine) at the N-terminus (Li et al., 2004; Wu et al., 2006). Regardless of the primary structure, ACE-I peptides exerting in vivo effect should be resistant to hydrolysis by gastrointestinal and serum peptidases (Foltz et al., 2008) and transportable through intestinal cells (Aito-Inoue et al., 2007). For this reason, the in vitro activity of a given peptide (expressed as IC50) cannot be directly linked to the in vivo antihypertensive effect.
ACE-I peptides of various structure and molecular mass (from 2 to 20 amino acid residues) have been found in various fermented milk beverages and cheeses (Korhonen, 2009). IPP (isoleucine-proline-proline) and VPP (valine-proline-proline) are two lac- totripeptides that show high ACE-I activity (IC50 values of 5 and 9 pM, respectively). Because of their peculiar features (presence of proline at the C-terminus, low molecular mass), these peptides have been reported to decrease the level of blood pressure (Seppo et al., 2003). IPP and VPP are released from p-casein (and, only for IPP, also from к-casein) during milk fermentation by selected strains of L. helveticus. Nevertheless they may be obtained using other lactic acid bacteria (L. acidophilus, L. rhamnosus, L. casei) as starters for milk fermentation (Muguerza et al., 2006; Solieri et al., 2015). Calpis and Evolus, two commercial L. helveticus-fermented milk beverages, contain both lactotripeptides (Nakamura et al., 1995b; Kitamura and Ueyama, 2010). A meta-analysis based on IPP- and VPP-based dietary interventions restricted to European countries showed that although Japanese populations show better response, the combination of lactotripep- tides, at daily doses ranging from 2 to 6 mg, moderately lowers the level of systolic blood pressure (Cicero et al., 2013).
In addition to lifestyle changes (moderate alcohol consumption or dietary sodium restriction), consumers with high blood pressure could benefit by including milk beverages containing IPP/VPP in their diet. As shown by meta-regression approaches and subgroup meta-analyses, the effect of IPP/VPP seems stronger in younger subjects, suggesting that the two lactotripeptides could be used in slightly hypertensive middle-aged adults as a means for preventing hypertension (Cicero et al., 2013). However, based on the available evidences, the European Food Safety Authority (EFSA) concluded that further studies are necessary in order to establish a cause/effect relationship between the dietary intake of the two lactotripeptides and the decreased level of blood pressure (EFSA Panel on Dietetic Products Nutrition and Allergies, 2009).
Besides those lactotripeptides, RYLGY and AYFYPEL, obtained upon hydrolysis of casein with pepsin, showed ACE-I activity and are contained in the commercial product named Lowpept, which showed antihypertensive effect in humans (Recio et al., 2011). FFVAPFPEVFGK is another ACE-I dodecapeptide released from as1-casein upon hydrolysis with trypsin and contained in the antihypertensive formulate marketed with the name Peptide C12 (Cadee et al., 2007). However, unlike IPP and VPP, the antihypertensive activity of the purified form of this dodecapeptide and of those peptides contained in Lowpept has not been ascertained.