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Ile–Trp (IW) and Trp–Leu (WL) are present in enzymatic hydrolysates of whey proteins and act as efficient inhibitors for angiotensin-converting enzyme (ACE) in vitro. Baseline plasma concentrations of IW and WL were analysed to 0.9 ± 0.3 n m for IW and 8.3 ± 1.3 n m for WL. Following administration of 50 mg IW or 100 mg WL to human volunteers (n = 3 for IW and n = 2 for WL, respectively), a significant increase in plasma concentrations to a maximum of 2.4 ± 0.5 n m for IW and 29–36 n m for WL after 0.5 h was observed, followed by rapid elimination. ACE activity in plasma decreased by 32% ± 8% following 50 mg IW administration. Administration of the corresponding amino acids had no effect on ACE activity. Thus, these tryptophan-containing dipeptides may serve as bioactive food additives with beneficial effects for the cardiovascular system.

Bioactive peptides from food proteins such as natural ACE (angiotensin-converting enzyme)-inhibitors have attracted particular attention for their potential to prevent hypertension. ACE-inhibiting peptides were enriched from food protein hydrolysates prepared from α-lactalbumin and lysozyme by selective adsorption on microporous activated carbons. For the eluate, it was shown by liquid chromatography that the strongest inhibitor isoleucyl-tryptophan was enriched by a factor of 11.2 compared to the initial α-lactalbumin hydrolysate. Natural inhibitors derived from lysozyme hydrolysates (e.g., alanyl-tryptophan) were successfully enriched as well. Identification of the enriched peptide fraction by mass spectroscopy revealed the hydrophobic character of the enriched peptides. The molecular weight distribution of the enriched peptide fraction can be controlled by the pore size distribution of the chosen adsorbent, which was proven by size exclusion chromatography of enriched peptide fractions derived from three different model carbons differing in their pore size. The selective enrichment of natural ACE-inhibitors from the α-lactalbumin hydrolysate lead to a 6 times stronger in vitro ACE-inhibition demonstrating the high potential as ingredients for hypotensive functional foods with reduced side effects.

Somatic angiotensin-converting enzyme (ACE) contains two active sites, the C- and N-domain, from which the C-domain is supposed to play a major role in blood pressure regulation and is therefore a promising pharmacological target to reduce blood pressure without side-effects. We report for the first time that tryptophan-containing dipeptides such as Ile-Trp or Val-Trp, which were recently found in food protein hydrolysates, are selective and competitive inhibitors for the C-domain with a selectivity factor of 40 and 70, respectively. Structure-activity studies showed that an N-terminal aliphatic amino acid and a tryptophan moiety in the P2' position are favourable structures for C-domain inhibition in dipeptides. In contrast, the lactotripeptides Ile-Pro-Pro and Val-Pro-Pro, which were widely used as ingredients for hypotensive food, showed a slight selectivity for the N-domain. Hence, tryptophan containing dipeptides are interesting ingredients for functional foods as a natural prevention for hypertension with reduced side effects due to its selective inhibition of the C-domain.

This study reports on the extraction of strongly angiotensin-converting enzyme (ACE) inhibiting dipeptides from protein hydrolysates obtained by enzymatic proteolysis. Several dipeptides with different ACE inhibitory activities and hydrophobicities were investigated regarding to their adsorption affinity on commercially available activated carbon material Norit DLC Super 50. This porous carbon exhibits extremely high adsorption capacities for the strongest ACE inhibitor Ile-Trp (IC 50 = 0.7 μM) of 726 mg/g as well as fast adsorption kinetics due to its micropore structure and small particle size. The filling of the pores was monitored by N 2 -physisorption revealing that complete pore filling occurred and Ile-Trp adsorption was only limited by the specific pore volume of Norit DLC Super 50, whereas less active peptides were adsorbed less efficient due to their higher hydrophobicity and did not impact Ile-Trp adsorption. After the adsorption, Ile-Trp was recovered by elution with ethanol. Three protein hydrolysates obtained by different enzyme combinations were mixed with activated carbon and the peptide adsorption was investigated by RP-HPLC. The amount of Trp-containing and ACE-inhibiting short chain peptides decreased selectively in contrast to more polar peptides, but the amount of adsorbed Ile-Trp is smaller than for single component adsorption.

Enzymatic hydrolysis of hen egg white lysozyme led to hydrolysates with pronounced inhibition of angiotensin I-converting enzyme (ACE). Two dipeptides, alanyl-tryptophan (AW) and tryptophanyl-tryptophan (WW), were identified for the first time in lysozyme hydrolysates by LC–ESI-TOF-MS and quantified by RP-HPLC-UV/fluorescence. Hydrolysis conditions were established for enhanced release of ACE-inhibiting tryptophan-containing dipeptides from alpha-lactalbumin and hen egg white lysozyme. The highest ACE inhibition potency and release of tryptophan-containing dipeptides were observed for the hydrolysates prepared by chymotrypsin followed by thermolysin. All identified dipeptides showed a significant and competitive inhibitory effect on ACE activity (AW: IC50 = 20 μmol/L; WW: IC50 = 68 μmol/L, IW: IC50 = 0.7 μmol/L, WL: IC50 = 10 μmol/L) in vitro and proved to be moderate resistant towards simulated gastrointestinal digestion. Protein hydrolysates containing tryptophan-containing dipeptides are interesting candidates as ingredients for functional foods with possible blood pressure-lowering effect, but also as a good source for the amino acid tryptophan.

Enzymatic hydrolysis of proteins from rice, soy, pea and wheat, with both chymotrypsin and thermolysin, resulted in hydrolysates, which are efficient inhibitors of the angiotensin-converting enzyme (ACE). IC50 values of the hydrolysates were between 27 and 39mg/l, which is comparable to enzymatically hydrolysed whey protein. A significant increase of the ACE-inhibiting effect was observed following butanol extraction due to accumulation of hydrophobic peptides (IC50 between 12 and 21mg/l). Based on the identification and quantification of individual tryptophan-, tyrosine- and phenylalanine-containing dipeptides, 50-80% of the total ACE-inhibiting potential of butanol extracts from plant protein hydrolysates could be explained. Compared to hydrolysates from whey protein, where the inhibitory effect can almost exclusively be attributed to Ile-Trp, the ACE inhibition by plant protein hydrolysates is caused by a variety of peptides, in particular tyrosine-containing peptides. Hydrolysates of plant proteins are promising ingredients for the development of functional foods.

Bioactive peptides such as Ile-Trp show great potential as natural ACE (angiotensin-converting-enzyme)-inhibitors. A continuous process for the up-scaled enrichment of ACE-inhibiting peptides based on a columnar adsorber system with an activated carbon stationary phase was developed. The particle size of the adsorbent and the flow rate was investigated as key factors affecting adsorption kinetics and separation performance. Batch and column adsorption experiments of model adsorbate systems were successfully transferred to a more complex system of an α-lactalbumin hydrolysate containing a multitude of species. Ile-Trp was successfully enriched from the hydrolysate by a factor of 4 using the optimized carbon column. The enrichment was more selective using smaller adsorbent particles due to improved adsorption kinetics.