In Vitro Generation and Stability of the Lactokinin β-Lactoglobulin Fragment (142–148)

Abstract

The objectives of this study were to investigate the generation of beta-lactoglobulin fragment (142-148) (beta-LG f(142-148) during the hydrolysis of whey proteins, and the in vitro stability of this fragment upon incubation with gastrointestinal and serum proteinases and peptidases. An enzyme immunoassay (EIA) protocol was developed for the quantification of beta-LG f(142-148) in whey protein hydrolysates and in human blood serum. The minimum detection limit was 3 ng/mL. The level of the peptide in whey protein hydrolysates was influenced by the degree of hydrolysis (DH). As expected, highest levels of this peptide were found in hydrolysates generated with trypsin. Sequential incubation of hydrolysates at different DH values with pepsin and Corolase PP, to simulate gastrointestinal digestion, generally resulted in the degradation of beta-LG f(142-148) as determined by EIA. Reversed-phase HPLC and angiotensin-I-converting enzyme (ACE) activity assays demonstrated that synthetic beta-LG f(142-148) was rapidly degraded upon incubation with human serum. Furthermore, beta-LG f(142-148) could not be detected by EIA in the sera of 2 human volunteers following its oral ingestion or in sera from these volunteers subsequently spiked with beta-LG f(142-148). These in vitro results indicate that beta-LG f(142-148) is probably not sufficiently stable to gastrointestinal and serum proteinases and peptidases to act as an hypotensive agent in humans following oral ingestion. The in vitro methodology described herein has general application in evaluating the hypotensive potential of food protein-derived ACE inhibitory peptides.

Physiological, chemical and technological aspects of milk-protein-derived peptides with antihypertensive and ACE-inhibitory activity

“…Similarly, lactokinin (ALPMHIR) was found to inhibit the release of ET-1, an endothelial peptide that evokes contractions in smooth muscle cells, an effect that might be dependent or independent of ACE inhibition (Maes et al, 2004). However, when interpreting this result, the susceptibility of ALPMHIR to degradation by gastrointestinal, brush-border and serum proteinases and the ACE inhibitory activity of its degradation products, should be taken into account (Murakami et al, 2004;Walsh et al, 2004).…”

“…Thus, the active ACE-inhibitory peptide lactokinin, ALPMHIR, that arises from tryptic hydrolysis of b-lactoglobulin (b-Lg f142-148, IC 50 ¼ 42.6 mM), was reported to be resistant to further hydrolysis by pepsin or chymotrypsin (Mullally, Meisel, & FitzGerald, 1997a). However, subsequent experiments revealed that ALPM-HIR was susceptible to degradation on incubation with gastrointestinal and blood serum proteinases and peptidases in vitro, what restricts its potential to elicit a hypotensive response in humans (Roufik et al, 2006;Walsh et al, 2004). Interestingly, the shorter fragment ALPM exerted a strong hypotensive effect in SHR despite the fact it was not an efficient ACE inhibitor in vitro (IC 50 ¼ 928 mM; Murakami et al, 2004).…”

Bioactive peptides: Production and functionality

“…Furthermore, a dose-dependent antihypertensive effect has been established in animal model studies with SHR after single oral administration of small di-and tripeptides . On the other hand, recent in vitro results by Walsh et al (2004) indicated that b-lg f(142-148), the tryptic peptide Ala-Leu-Pro-Met-His-Ile-Arg which is known to be a potent inhibitor of ACE activity in vitro (Mullally, Meisel, & FitzGerald, 1997) is probably not sufficiently stable to gastrointestinal and serum proteinases and peptidases to act as an hypotensive agent in humans following oral ingestion. This conclusion was supported by the results showing that synthetic b-lg f(142-148) was rapidly degraded upon incubation with human serum.…”

Antihypertensive Peptides from Food Proteins

“…They can be produced naturally during gastrointestinal (GI) digestion by the hydrolytic action of the proteinases pepsin, trypsin, chymotrypsin and by brush border peptidases [18]. Simulated GI digestion has been carried out on a range of protein sources to assess the effect of GI digestion on ACEinhibitory peptides [19][20][21][22][23][24]. More commonly, ACE-inhibitory peptides are produced through enzymatic hydrolysis with GI enzymes such as pepsin and trypsin or with enzyme combinations such as Alcalase™ [25].…”