Pea and whey in the control of high blood pressure

Hypertension or high blood pressure is a major risk factor for the development of cardiovascular diseases, which are the leading causes of morbidity and mortality in western society. It is estimated that 20% of the world’s adult population suffers from hypertension. Recently, some functional foods have received considerable attention for their effectiveness in both the prevention and the treatment of hypertension. This is among others due to the presence of food derived bioactive peptides with potential antihypertensive properties. Compared to antihypertensive drugs, these peptides, as part of a (functional) food or as nutraceutical, have no side effects and are less expensive. Moreover, as a more natural product, they appeal to the consumer.

Angiotensin I converting enzyme (ACE) inhibitory peptides exert an antihypertensive effect by inhibition of the angiotensin I converting enzyme in the cardiovascular system, hence preventing the formation of the potent vasoconstrictor angiotensin II and the degradation of the vasodilator bradykinin. In order to reduce the blood pressure after oral administration, ACE inhibitory peptides have to reach the bloodstream in an active form. Therefore, stability against human proteases and intestinal transport of active peptides is required. In the current field of ACE inhibitory peptide research, the maintenance of the activity in the oral delivery route is often not taken into account. ACE inhibitory peptides have mainly been isolated from milk proteins and have not yet been studied from pea protein.

The aim of this work was to investigate the release of ACE inhibitory activity from pea and whey protein. Moreover, whey protein, from which certain bioactive peptides already have been characterised, was compared to the unknown pea protein. Emphasis was placed on the in vitro study of the activity of ACE inhibitory peptides after oral administration in the human body, from the gastrointestinal tract to their site of action, the cardiovascular system. The maintenance of bioactivity in the oral delivery route is necessary to exert an antihypertensive effect.

A diagnostic assay to measure ACE activity was transformed into an ACE inhibition assay and subsequently optimised to obtain a more sensitive and less expensive test. This spectrophotometric method, where ACE inhibition was measured with the substrate FAPGG and rabbit lung acetone extract as ACE source, was validated by the antihypertensive drugs captopril, enalapril and its active derivative enalaprilat, and by the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg. In addition, it indicated the release of ACE inhibitory activity after digestion of pea and whey protein. A more delicate and standardised assay was obtained by applying pure ACE from porcine kidney as ACE source. The ACE inhibitory activity of captopril and Ala-Leu-Pro-Met-His-Ile-Arg was also verified in this assay. The ACE inhibition assay presented a relatively simple and reliable tool to screen for ACE inhibitory peptides from food proteins.

The activity of the potent ACE inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, originally derived from a tryptic digest of b-lactoglobulin, was investigated in vitro in the oral delivery route. During in vitro gastrointestinal digestion, the heptapeptide was partially degraded by chymotrypsin, likely to Ala-Leu-Pro-Met and His-Ile-Arg, the latter also possessing ACE inhibitory activity. Nevertheless, after hydrolysis by stomach and pancreatic proteases, half of the initial peptide concentration and 70% of the initial ACE inhibitory activity could still be recovered. The lactokinin was rapidly broken down by rat intestinal tissue peptidases, while almost no degradation was observed after incubation with Caco-2 homogenates. The intestinal transport of 1 mM of the lactokinin was investigated in a Caco-2 Bbe cell monolayer mounted in an Ussing chamber. After 10 min of incubation at 37°C, substantial ACE inhibitory activity was found in the serosal compartment after threefold concentration of the samples. Concomitantly, MALDI-TOF spectrometry detected the heptapeptide in the serosal compartment. Under the observed experimental conditions, the ACE inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg was transported intact through the Caco-2 Bbe cell monolayer, although at low concentrations. It seems that the degradation by intestinal peptidases constitutes the major barrier in the oral delivery route of this lactokinin.

Fermentation by different lactic acid bacteria was studied as a means to release ACE inhibitory activity from pea and whey protein. Fermentation of pea protein by Lactobacillus helveticus yielded the highest ACE inhibitory activity and was therefore selected for subsequent experimentation. Next, pea and whey protein were fermented by Lactobacillus helveticus and the yeast Saccharomyces cerevisiae in monoculture and in combination at 28 and 37°C. Fermentation was always followed by in vitro gastrointestinal digestion and the digests of non-fermented protein solutions served as controls. After fermentation, the ACE inhibitory activity (%) of a 2.73 mg/ml sample increased by 18 to 30% for all treatments, except for the fermentations of whey protein by Saccharomyces cerevisiae at 28°C, where no significant change was observed. After digestion, however, both fermented and non-fermented samples reached maximal ACE inhibitory activity (%). The degree of proteolysis showed only a minor increase after fermentation and augmented sharply after digestion. The whey (fermented) digests tended to have lower 50% inhibitory concentrations (IC50) (0.148-0.072 mg/ml), hence higher ACE inhibitory activity, than the pea (fermented) digests (0.183-0.093 mg/ml). The non-fermented digested samples were at least as ACE inhibitory active as the fermented ones. The non-fermented whey protein digest showed the lowest IC50 value and hence the highest ACE inhibitory activity of all. For pea protein, the non-fermented sample had the one but lowest IC50 value. These results suggest that in vitro gastrointestinal digestion was the predominant factor controlling the formation of ACE inhibitory activity, hence indicating its importance in the bioavailability of ACE inhibitory peptides.

Therefore, the formation of ACE inhibitory activity during in vitro gastrointestinal digestion of pea and whey protein was investigated more profoundly.

Firstly, the conditions during in vitro gastrointestinal digestion of pea and whey protein leading to maximal release of ACE inhibitory activity were characterised. In batch experiments, three in vitro gastrointestinal digestions varying in pH and incubation time in the stomach and the small intestine phase, were compared for pea and whey protein. The digestion simulating the physiological conditions of protein hydrolysis in the human body sufficed to achieve the highest ACE inhibitory activity, with IC50 values of 0.076 mg/ml for pea and 0.048 mg/ml for whey protein. The degree of proteolysis did not correlate with the ACE inhibitory activity and pea protein was more susceptible to hydrolysis by gastrointestinal proteases than whey protein. In a semi-continuous reactor model of gastrointestinal digestion (pre-SHIME), response surface methodology was used to investigate the influence of temperature, incubation time in the stomach phase and incubation time in the small intestine phase on the ACE inhibitory activity and the degree of proteolysis. For the pea protein, a central composite design could constitute a linear model for the degree of proteolysis and a quadratic model for the ACE inhibitory activity, expressed as log IC50. Within the model, maximal degree of proteolysis was observed at the highest temperature and the longest incubation time in the small intestine phase, while maximal ACE inhibitory activity was obtained at the longest incubation times in the stomach and the small intestine phase. For the whey protein, no significant model for both responses could be designed. Yet, the overall results demonstrate that the ACE inhibitory activity of pea and whey protein hydrolysates can be controlled by the conditions of the in vitro gastrointestinal digestion. This has implications in food processing, where optimal digestion conditions can be set, as well as in physiological digestion, where pathological defects can hamper release of bioactivity.

Next, the evolution of ACE inhibitory activity, degree of proteolysis and protein degradation was investigated during in vitro physiological digestion. A 2.73 mg/ml pea digest showed maximal ACE inhibitory activity (%) already in the early stomach phase and its IC50 further decreased in the small intestine phase. For whey digest, however, the level of 100% ACE inhibitory activity was only attained in the small intestine phase. Subsequent supplementation of a rat intestinal acetone powder, which simulated the digestion by brush border enzymes, resulted in an increase in IC50 value for both proteins. This decrease in ACE inhibitory activity was less pronounced for pea compared to whey. Yet, a substantial amount of peptides were still ACE inhibitory active after digestion of pea and whey protein by gastrointestinal and brush border enzymes and the final IC50 values were 0.093 mg/ml for pea and 0.128 mg/ml for whey. The degree of proteolysis presented a different evolution and augmented more for pea than for whey in both the stomach and small intestine phase, while the brush border peptidases caused a smaller increase in the degree of proteolysis of pea compared to whey. SDS-PAGE showed that the major part of the proteins in pea was already broken down in the stomach phase. The major whey protein b-lactoglobulin was only degraded from the small intestine phase onwards. The presence of known ACE inhibitory peptides in the pea and whey protein sequences was studied using a dedicated computer program. This program makes use of a database of about 500 reported ACE inhibitory sequences and their IC50 values, and produces a theoretical score, weighted on the total number of amino acids, for the potential of ACE inhibitory peptides in a given source protein. For pea, vicilin and albumin PA2 obtained scores as high as 10 on ACE inhibitory activity, compared to 16 for b-casein taken as a reference protein. b-lactoglobulin exceeded all other proteins with a score of 26. To our knowledge, this is the first described large ACE inhibitory peptide database that attributes scores for the potential of ACE inhibitory activity to protein sequences. In silico digestion of these proteins by pepsin, trypsin and chymotrypsin directly released two ACE inhibitory peptides from PA2, one from vicilin and one from b-lactoglobulin with an IC50 lower than 100 µM. The high ACE inhibitory peptide score of b-lactoglobulin and the fact that this protein resists digestion by pepsin are consistent with the substantial decrease in IC50 for the whey digest during the small intestine phase digestion. The database and in silico digestion facilitate the efficient release of ACE inhibitory peptides and the study of proteins on a large scale.

The ACE inhibitory activity of pea and whey hydrolysate, obtained by in vitro gastrointestinal digestion, with IC50 values of 0.070 mg P/ml and 0.041 mg P/ml, was further increased upon purification by ultrafiltration-centrifugation and subsequent fractionation of the permeate by RP-HPLC. The most active RP-HPLC fractions eluted at 24-28% acetonitrile and therefore contained short, hydrophobic peptides, which is in accordance with the structure-activity relationship of ACE inhibitory peptides. The IC50 values of these fractions amounted to 0.016 mg P/ml for pea and 0.003 mg P/ml for whey. In this way, the ACE inhibitory activity of the pea digest was more than 4 times enriched, while that of the whey digest was more than 13 times augmented. This may suggest that in whey hydrolysate very potent ACE inhibitory peptides were present next to low active peptides, while in pea digest all peptides had similar ACE inhibitory activity.

The intestinal transport of active compounds in the digests and permeates was investigated in Caco-2 cell monolayers. All samples retained relatively high ACE inhibitory activity after 2 h incubation in the presence of Caco-2 homogenates. Addition of 9 mg P/ml digests and 10 mg P/ml permeates in the apical compartments of the Caco-2 cell monolayers resulted in the detection of no or only little ACE inhibitory activity in the basolateral compartments after 1 h transport experiment, even after concentration of the sample. Some ACE inhibitory activity was observed in the transport experiments after addition of 45 mg P/ml digests and 50 mg P/ml permeates in the apical compartments, but these were associated with compromised cell monolayer integrity as indicated by decreased transepithelial electrical resistance and increased sodium fluorescein fluxes. Hence, not the degradation by intestinal peptidases, but the uptake by the cell monolayer seemed to be the decisive factor with respect to the transport of active compounds in the ACE inhibitory digests and permeates. As the Caco-2 model is tighter than intestinal mammalian tissue, transport of these peptides in substantial quantities might still take place in vivo. Overall, these observations suggest that the protein digests can reach the blood stream in an active form

After intravenous administration of a dose of 50 mg P/kg BW in spontaneously hypertensive rats, pea permeate exerted a transient, but strong antihypertensive effect of 44.4 mmHg. Whey permeate exerted no effect at a dose of 50 mg P/kg BW. Further research into potential positive effects in the prevention and treatment of hypertension, for example after daily administration over a longer period of time or after addition of a single higher dose, is necessary.

In conclusion, this work presented a profound investigation on the release of ACE inhibitory activity from pea and whey protein and the maintenance of this activity in the oral delivery route. For the first time, the formation of high ACE inhibitory activity from pea protein is described. This knowledge can be applied in functional foods for the prevention and treatment of hypertension.

References

* V. Vermeirssen, J. Van Camp, L. Devos & W. Verstraete (2003). Release of Angiotensin I Converting Enzyme (ACE) inhibitory activity during in vitro gastrointestinal digestion: from batch experiment to semi-continuous model. Journal of Agricultural and Food Chemistry, 51(19), 5680-5687.
* V. Vermeirssen, J. Van Camp, K. Decroos, L. Van Wijmelbeke & W. Verstraete (2003). The impact of fermentation and in vitro digestion on the formation of ACE inhibitory activity from pea and whey protein. Journal of Dairy Science, 86(2), 429-438.
* V. Vermeirssen, J. Van Camp & W. Verstraete (2002). Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides. Journal of Biochemical and Biophysical Methods, 51(1), 75-87.
* V. Vermeirssen, B. Deplancke, K.A. Tappenden, J. Van Camp, H.R. Gaskins & W. Verstraete (2002). Intestinal transport of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg through a Caco-2 Bbe monolayer. Journal of Peptide Science, 8(3), 95-100.