Some structural features of potent angiotensin converting enzyme (ACE) inhibitory peptides.
Hypertension or elevated blood pressure (BP) is a global health concern, thought to affect up to 30 % of the adult population in developed and developing countries. It is defined by a BP measurement of 140/90 mmHg or above. Hypertension is a major risk factor concomitant with cardiovascular disease (CVD) states such as coronary heart disease, peripheral artery disease and stroke, and kidney disease. Essential hypertension, the most common type of hypertension and to which 90-95% of cases belong, is manifested as an increase in an individual’s BP due to an unknown cause. This class of hypertension can be improved with lifestyle choices such as regular exercise, heart-healthy eating, non smoking, reducing sodium intake and reducing the level of stress . For these reasons it is defined as a controllable risk factor of CVD. At present there is a range of synthetic drugs on the market for treatment of hypertension including diuretics, adrenergic inhibitors such as α- and β-blockers, direct vasodilators, calcium channel blockers, angiotensin II (Ang II) receptor blockers and angiotensin converting enzyme (ACE) inhibitors. However, although hypertension can be controlled by pharmacological agents, it represents a major burden on annual global healthcare costs. According to the Centre for Disease Control and Prevention (CDC) , it was estimated that hypertension-related costs reached $76.6 billion in the USA in 2010. It is thought that prevention through lifestyle choices and early treatment for individuals with mild hypertension can significantly reduce global health-care costs.
Food proteins contain numerous biologically-active peptides (BAPs). These BAPs can exert positive physiological responses in the body beyond their basic nutritional roles in the provision of nitrogen and essential amino acids. Many bioactivities have been found including peptides with antihypertensive capabilities. This has led to significant research on the discovery and generation of peptides with antihypertensive properties
2. Hypotensive mechanisms of action
The regulation of BP is complex, involving a variety of intertwining metabolic pathways. By far, the most studied BP control pathways with regard to food-derived peptides involve those shown to inhibit ACE
2.1. ACE inhibition
ACE inhibition is an excellent physiological target for clinical hypertensive treatment due to its involvement in two BP related systems, the RAS and the KNOS. The RAS is thought to be one of the predominant pressor systems in BP control. In the RAS the N-terminus of the prohormone angiotensinogen, which is derived from the liver, is cleaved by renal renin to produce the decapeptide angiotensin I (Ang I). ACE then removes the C-terminal dipeptide HL to form Ang II, a potent vasoconstrictory peptide which acts directly on vascular smooth muscle cells. Thus, inhibition of ACE consequentially leads to BP reduction. Ang II binds to AT1 and AT2 receptors which are located in peripheral tissues around the body and in the brain. The vasocontriction produced by Ang II is mediated by the AT1 receptor. [5-7]. In the KNOS, ACE inactivates the vasodilatory peptides bradykinin and kallidin. Kallidin is synthesised from kininogen by kallikrein, and its further action on kallidin leads to the formation of bradykinin among other vasoactive peptides. Bradykinin binds to β-receptors which lead to an eventual increase in intracellular Ca2+ level. The binding of bradykinin to β-receptors and the increase in Ca2+ stimulates nitric oxide synthase (NOS) to convert L-arginine to nitric oxide (NO), a potent vasodilator. ACE can therefore, indirectly inhibit the production of NO as it hydrolyses bradykinin into inactive fragments .
There are a number of widely-used synthetic ACE inhibitors currently on the market that serve as the first line of approach for the treatment of hypertension. Such inhibitors include Captopril, Enalapril and Lisinopril. However, their use is associated with a range of side-effects including cough, skin rashes, hypotension, loss of taste, angiodema reduced renal function and fetal abnormalities . Natural ACE inhibitory peptides from food are not associated with the side-effects brought about by the synthetic drugs. They are not as potent inhibitors of ACE as the synthetic inhibitors which can have IC50 values in the nM region. As they inhibit ACE to a lesser extent, this potentially allows for safer levels of bradykinin in the body. Thus, for this reason, ACE-inhibitory peptides have gained interest as potential preventative agents for hypertension control.
ACE-inhibitory peptides have been identified in a range of food proteins including casein, whey, ovalbumin, red algae, wakame, soy, gelatin, chicken muscle, dried bonito, corn, sardines, rapeseed, potato, chick pea, tuna muscle, pea albumin, garlic, wheat germ, sake, porcine haemoglobin and squid. The ACE inhibitory peptides found in different food proteins has been extensively reviewed (for review see [9-12; 3; 131]. Examples of recently reported food protein ACE inhibitory peptide sources include loach (
ACE inhibitory peptides can work in three ways and are classed as inhibitor-type, substrate-type or prodrug-type based on changes in ACE inhibitory activity after hydrolysis of peptides by ACE . Inhibitor-type peptides are ACE inhibitory peptides whose activity is not significantly altered as the peptides are resistant to cleavage by ACE. Substrate-type ACE inhibitors show a decrease in ACE activity due to cleavage by ACE. Prodrug type refers to the conversion to potent ACE inhibitors following hydrolysis of larger peptide fragments by ACE itself. The resulting peptides tend to produce long-lasting hypotensive effects
Inhibition of ACE is by far the most studied mechanism of BP control with regard to food-derived biologically-active peptides. Most peptides have been found to inhibit ACE to some degree. However, in most cases, it has yet to be answered whether this is the BP mechanism being employed
2.2. Renin Inhibition
Renin inhibition is another potential target for BP control. It is thought that inhibition of renin could provide a more effective treatment for hypertension it prevents the formation of Ang-I, which can be converted to Ang-II in some cells independent of ACE, by the enzyme chymase . In addition, unlike ACE which acts on a number of substrates in various biochemical pathways, angiotensinogen is the only known substrate of renin. Therefore, renin inhibitors could ensure a higher specificity in antihypertensive treatment compared to ACE inhibitors [31-32]. Food peptides have recently been found to be inhibitors of renin. Peptides from enzymatic flaxseed fractions were found to inhibit both human recombinant renin and ACE. The study concluded that such peptides with the ability to inhibit both ACE and renin may potentially provide better antihypertensive effects
2.3. Calcium channel blocking effects
Calcium channel blockers interact with voltage-gated calcium channels (VGCCs) in cardiac muscle and blood vessel walls, reducing intracellular calcium and consequently lowering vasoconstriction. It has been shown in various studies that peptides can have the ability to act as calcium channel blockers. Fifteen synthetic peptides based on Trp-His skeleton analogues were tested for their vasodilatory effects in 1.0 μM phenylephrine-contracted thoracic aortic rings from Sprague-Dawley rats. It was previously reported that Trp-His induced the most potent vasodilation among 67 synthetic di-and tripeptides. The study demonstrated that His-Arg-Trp had an endothelium-independent vasorelaxative effect in the phenylephrine-contracted thoracic aorta. It was also shown that His-Arg-Trp, at a concentration of 100 μM, caused a significant reduction in intracellular Ca2+ concentration. The increase intracellular [Ca2+], brought about by the action of Bay K8644 or Ang II, was significantly inhibited by His-Arg-Trp (>30%). It was proposed that His-Arg-Trp may have supressed extracellular Ca2+ influx through voltage-gated L-type Ca2+ channels . Another recent study reported a similar result with Trp-His which was also found to block L-type Ca2+ channels. Trp-His at 300 μM elicited an intracellular Ca2+ reduction of 23 % in 8 week-old male Wistar rat thoracic aortae smooth muscle cells. In addition, the reduction in [Ca2+] brought about by Trp-His was eliminated by verapamil indicating that Trp-His specifically works on L-type Ca2+ channels .
2.4. Opioid peptide vasorelaxive effects
Food-derived peptides have also been found to be sources of opioid like-activities. These peptides bind to opioid receptors to produce morphine-like effects. Natural opioid peptides include endorphins, enkephalins and dynorphins. In humans opioid receptors are found in the nervous, endocrine and immune systems, and in the intestinal tract. These receptors may be involved in various regulatory processes in the body including the regulation of circulation which can affect BP [37; 38]. Nurminen
2.5. Endothelin-1 and endothelin converting enzyme (ECE) inhibition
The vasoconstrictory peptide endothelin-1 (ET-1) is released from big endothelin-1 (big ET-1) by the action of endothelin-converting enzyme (ECE). ET-1 mediates vasoconstriction via 2 receptors, ETa and. ETb. Both receptors mediate contractions on smooth muscle, but ETb also induces relaxation of endothelial cells by the production of nitric oxide. ET-1 is known to have a greater vasocontrictive effect than Ang II [44; 7]. Endothelial-dependent release of NOS was found to be the mechanism of action for the antihypertensive egg protein derived ovokinin (f2-7) peptide (Arg-Ala-Asp-His-Pro-Phe). Dilation of isolated SHR mesenteric arteries was found to be inhibited by L-NAME but not by indomethacin, demonstrating NO release from the endothelial cells . A later study showed that ovokinin (2–7) modulates a hypotensive effect through interaction via B2 bradykinin receptors .
It has been found that food proteins have the ability to act as inhibitors of ECE. Okitsu
3. Structure activity relationships
An understanding of the relationship between a peptide and its bioactivity allows for the targeted release of potentially potent peptide sequences. This would eliminate the need for the time-consuming conventional peptide discovery strategy. There is limited knowledge on the structure-activity relationship of hypotensive peptides. To date, the main focus with regard to bioactive peptide research has been on the generation and characterisation of these peptides. ACE inhibition is by far the most widely studied biomarker with regard to antihypertensive effects of bioactive food peptides. ACE can work on a wide range of peptide substrates, and appears to have a broad specificity. Some structural features that influence the binding of a peptide to the ACE active site have been recognised (Table 1). However, potent inhibitory peptides of ACE are generally short sequences, i.e., 2-12 amino acids in length. However, some larger inhibitory sequences have been identified. Studies have indicated that binding to ACE is strongly influenced by the substrate’s C-terminal tripeptide sequence. Hydrophobic amino acid residues with aromatic or branched side chains at each of the C-terminal tripeptide positions are common features among potent inhibitors. The presence of hydrophobic Pro residues at one or more positions in the C-terminal tripeptide region seems to positively influence a peptide’s ACE inhibitory activity. Tyr, Phe and Trp residues are also present at the C-terminus of many potent ACE inhibitors, especially with di- and tripeptide inhibitors . It has been suggested that Leu residues may also contribute to ACE inhibition . Furthermore, the positive charge on the side chains of Arg and Lys residues at the C-terminus have been noted to contribute to the ACE inhibitory potential of a peptide [50-51; 9]. An L-configured amino acid at position three at the C-terminus of the inhibitory peptide may be a requirement for potent inhibition. A study showed that the IC50 for the tripeptide D-Val-Ala-Pro (2 µM) increased to 550 µM with L-Val-Ala-Pro, yet only a slight increase in IC50 was seen for the peptide L-Phe-Val-Ala-Pro (17 µM; Maruyama
|Hydrophobic residues||2-12 amino acids in length|
Peptide conformation important for longer peptides
Bulky hydrophobic residues
Aromatic or branched side chains
Proline at one or more positions
Positively charged residues in position two, Arg, Lys
Tyr, Phe, Trp, Leu
L-configured residue in position three
Both domains of ACE (C- and N-domains) contain an active site containing the sequence His-Glu-XX-His. These active sites are located within the cleft of the two domains, and are protected by an N-terminal ‘lid’. This ‘lid’ blocks access of large polypeptides to the active site. This is thought to explain why small peptides are more effective in inhibiting ACE. In addition, ACE inhibition may include inhibitor interaction with subsites on the enzyme that are not generally occupied by substrates or with an anionic inhibitor binding site that is different for the catalytic site of the enzyme. With the catalytic sites of ACE having different conformational requirements, this could indicate that for a more complete inhibition of ACE, there may be a need to use a variety of peptide inhibitors each with slightly different conformational features [52-53].
Quantitative computational tools are increasingly been applied in medicinal and pharmaceutical drug discovery. Recently it has been acknowledged that such models could be adapted to food-derived bioactive peptide sequences. Quantitative structure-activity relationship modelling (QSAR) and substrate docking can be used as an effective tool to assess
Substrate docking involves the docking of molecules (ligands) to a receptor or into a protein target such as an enzyme. All possible docking or binding conformations are assessed for their binding affinity to a molecule, and their potential as high affinity binding ligands is estimated by use of a scoring function. An integrated QSAR and Artificial Neural Network (ANN) approach was used to assess the ACE-inhibitory potential of 58 dipeptides present in the sequence of defatted wheat germ protein. The model was used to investigate preferred structural characteristics of ACE-inhibitory dipeptides and following this, appropriate proteases were successfully selected to produce the dipeptides predicted to be potent inhibitors by the QSAR-ANN model. The QSAR model predicted that the C-terminal of the peptide had principal importance on ACE inhibitory activity, with hydrophobic C-terminal residues being essential for high potency. Furthermore, proteins with a high abundance of hydrophobic residues were considered to be good substrates for the production of potent ACE inhibitory peptides . Recently, the ability of docking to predict ACE inhibitory dipeptide sequences was assessed using the molecular docking program AutoDock Vina. All potential dipeptides and phospho-dipeptides were docked and scored. Phospho-dipeptides were predicted by the program to be good inhibitors of ACE. However, the experimentally determined IC50 results for selected phospho-dipeptides did not correlate and the study concluded that phospho-dipeptides may not be potent inhibitors of ACE
4. Peptide bioavailability
The potential antihypertensive effect of a peptide depends on the peptides ability to reach their target organ intact and in an active form. However, there are several barriers which lie in the way of this outcome. Antihypertensive peptides must be resistant to digestive proteinases and peptidases; they must be able to be transported through the bush border membrane intact and must be resistant to serum peptidases. With regard to ACE inhibition, while there have been many studies focusing on the production, isolation and characterisation of ACE-inhibitory peptides, to date little attention has been placed on their bioavailability. It is therefore difficult to determine the relationship between
Bioactive peptides when taken orally may be inactived by several digestive proteinases and peptidases including pepsin in the stomach, and the pancreatic enzymes trypsin, elastase, α-chymotrypsin and carboxypeptidase A and B in the small intestine. A number of studies have been carried out investigating ACE-inhibitory peptides and their ability to resist gastrointestinal digestion by these enzymes. These studies involve simulating the gastrointestinal process by sequential hydrolysis of ACE inhibitory peptides with pepsin and Pancreatin™, each concluding the importance of gastrointestinal digestion analysis in the ACE inhibitory activity of the peptide [61-66; 49; 21; 20] . It has been noted that certain protein/peptide structures are resistant to gastrointestinal digestion due to the composition and position of amino acids in their primary chains. The rate of hydrolysis of a peptide is also dependent on the peptide’s amino acid composition. Peptides containing Pro and hydroxy Pro residues have been found to be resistant to hydrolysis. Furthermore, glycosylated peptides and peptides which have undergone changes during food processing such as during the formation of Maillard reaction products have been shown to be resistant to GI tract enzyme cleavage . Once the peptides reach the brush border membrane of the large intestine, they may also be subjected to further cleavage by a variety of membrane anchored epithelial cell intestinal peptidases. These include a number of aminotripeptidases and several dipeptidases, each with varying specificities . However, it has been found that certain free amino acids released during gastrointestinal breakdown may in turn serve as inhibitors of the brush border membrane dipeptidases, Moreover, it has been reported that during gastrointestinal proteolysis at the brush border membrane, the large variety and high concentration of peptides present would exceed the apparent
Absorption of peptides across the brush border membrane can be studied by Caco-2 cell monolayers, the representative model for human intestinal epithelial cell barrier. The intestinal transport of pea and whey ACE inhibitory peptides was also studied using a Caco-2 monolayer. It was found that only minor ACE inhibitory activity crossed the Caco-2 cell monolayer in 1 h. However, it was concluded that the extent of ACE inhibitory peptides that may be transported
Following absorption of a peptide into the blood stream, it may undergo hydrolysis by serum peptidases. The ACE inhibitory peptide may need to be able to with-stand hydrolysis in order to reach their target organs intact and yield their antihypertensive effect. It has been suggested that potent ACE inhibitors may be produced in circulation by the action of serum peptidases on less potent inhibitors of ACE and by the action of ACE itself. These peptides have been referred to pro-drug type inhibitors of ACE [78; 60].
Thus, the bioavailability of ACE-inhibitory peptides is essential for their activity. Several approaches to aid in peptide delivery are been considered. Peptides may be chemically modified in order to reduce the rate of enzymatic degradation and to increase bioavailability while also in some cases enhancing bioactivity. The half-life of unmodified peptides in the blood is in most cases very short. They also generally have poor bioavailability in tissues and organs, limiting their ability as preventative therapeutic agents [79; 80]. Modifications such as end changes, glycosylation, alkylation, and conformational changes to amino acids within the peptide may therefore have potential for ACE inhibitory peptides [80; 81]. These approaches have already been adapted to opioid peptides . There is significant scope for these modifications to also be applied to ACE inhibitory and antihypertensive peptides. Encapsulation via nanoparticles and liposomes is also a strategy previously employed for opioid peptides that has possibility for adaption to for ACE inhibitory peptides. These approaches may aid in the passage of a peptide through the GI tract and may enhance the plasma half-life of the peptides. Furthermore, there is potential for bioactive peptides to be produced by microorganisms through genetic engineering to be delivered to target organs
Ex-vivoand in vivoanimal studies
The first step employed to determine if a peptide is hypotensive is to conduct trials with small animals such as SHRs, the accepted model for human essential hypertension. A bioactive peptide can only be referred to as ‘antihypertensive’ after a significant decrease in BP is observed in trials with SHR. There have been many studies carried out in animals to elucidate whether food-derived ACE-inhibitory peptides can lead to an antihypertensive effect
Ile-Pro-Pro (β-casein f74-76; κ-casein f108-110) and Val-Pro-Pro (β-casein f84-86) were among the first dietary peptides found to have a hypotensive effect in SHR. The peptides were first isolated from milk fermented with
Other rat models, such as the normotensive WKY rat, have been used to evaluate the effect of food peptides on arterial BP. However, a significant hypotensive effect is not always observed in WKY. Single oral administration of Ameal S containing the LTPs decreased BP from 6 to 8 h after administration in SHR. However, no change in SBP was observed in normotensive WKYs . Similarly, the ACE inhibitory peptide Leu-Arg-Pro-Val-Ala-Ala from bovine lactoferrin was found to have a significant antihypertensive effect in SHR but no change in BP was found when the peptide was administered via intravenous injection to WKY rat . Thus, the hypotensive effect of some food-derived peptides may be specific to the hypertensive state of the animal. The effect of fermented milk with LTPs on BP and vascular function in salt-loaded type II diabetic Goto–Kakizaki rats has also been assessed. GK rats are characterized by impaired glucose-induced insulin secretion, abnormal glucose regulation, insulin resistance and polyuria. They are normotensive but when on a high-salt diet can develop hypertension. The study showed a significant decrease in BP and enhanced endothelium-dependent relaxation of mesenteric arteries .
There are wide variations in BP responses from different food proteins. These variations may be due to the different food sources themselves but also may be due to differences in experimental models such as the type of animal used, the dosage of peptide required for a significant decrease in BP, duration of administration and administration route, i.e., oral versus intravenous administration. In general, it has been found that peptides administered intravenously have a higher decrease in BP than peptides administered orally. This may be due to lower bioavailability of these peptides in the blood stream as transport of the peptides across the brush border membrane in an intact state may not be possible. Hydrolysis or partial hydrolysis of the peptides by GI and serum enzymes may lead to inactive or less active hypotensive peptide forms. Thus, bioavailability studies are essential to assess the antihypertensive potential of a peptide .
Furthermore, it must be noted that although dietary peptides have lower ACE IC50
6. Human studies
The majority of the clinical trials regarding the antihypertensive effects of milk-derived peptides to date have been carried out on the LTPs, Ile-Pro-Pro and Val-Pro-Pro. Although some conflicting results exist, the majority of these trials have reported a significant decrease in BP. Their effect on office BP has been well documented (for reviews see 101; 3; 25). It is essential that the BP of test subjects is evaluated in comparison to placebo values and not to baseline values of the test product. As with test products, placebo groups have been found to often decrease in BP over the test period . Furthermore, the 24-h ambulatory BP monitoring (ABPM), of patients BP is thought to be a more reliable method for evaluation of BP as it reduces the ‘white coat effect’. Recent clinical trials with LTPs have employed ABPM [102-104; 91]. ABPM was used to assess the effects of LTP administration on dipper (where BP decreases at night time) and non-dipper (where BP does not decrease at night time) hypertensive subjects. Non-dippers are thought to have a higher cardiac vascular risk and BP monitoring in the morning and night can help predict cardiac events such as stroke and myocardial infarction. Twelve patients received a fermented milk product containing Ile-Pro-Pro (1.52 mg) and Val-Pro-Pro (2.53 mg) daily for 4 weeks. The study reported a significant reduction in night-time and early-morning SBP for nondipper subjects but not for dipper subjects . A range of hemodynamic parameters was recently evaluated for 52 human subjects with high-normal BP or first-degree hypertension. These included office BP and ABPM, stress-induced BP increase and cardiac output-related parameters. Subjects were treated with LTPs (3 mg/day) for 6 weeks. The study reported a reduction in office SBP as well as an improvement in pulse wave velocity (an instrumental biomarker for vascular rigidity), stroke volume and stroke volume index (markers of cardiac flow) and acceleration and velocity index (markers of cardiac contractility). No effect on ABPM and BP reaction to stress was observed . LTPs have also been reported to reduce arterial stiffness in humans. In a double-blind parallel group intervention study, 89 hypertensive subjects received daily milk containing a low dose of 5 mg/day of Ile-Pro-Pro and Val-Pro-Pro for 12 weeks and a dose of 50 mg/day for the following 12 weeks. Arterial stiffness, measured by the augmentation index (AI), decreased in the peptide group by -1.53% compared to 1.20% in the placebo group at the end of the second intervention period . A similar result was seen in a study by Nakamura
Other hypotensive peptides and food preparations used in human trials include peptides from casein , whey , dried bonito , fermented milk containing gamma-aminobutyric acid (GABA; 114) sardine muscle  and wakame (
7. Hypotensive peptides as functional food ingredients
The main considerations which need to be taken into account in the utilisation of antihypertensive peptides as functional ingredients in food products include characterisation of their organoleptic and physicochemical properties. In the first instance, establishment of the optimal method for peptide release via protein hydrolysis is required. Industrial scale processing of BAPs requires that peptides be able to withstand and retain their bioactivity during multi-step processing including pasteurisation, homogenisation, pressure-driven membrane-based processing such as ultrafiltration and nanofiltration, dehydration by spray-drying or freeze-drying, and peptides must also be stable during long-term storage. Little data exists on the effects of different processing techniques on BAPs in food products. Dehydration via spray drying has been found to produce changes in peptide confirmation, a reduction in amino acid content and may also lead to non-enzymatic browning reactions . The industrial-scale production of a casein hydrolysate containing the antihypertensive peptides Arg-Tyr-Leu-Gly-Tyr (αs1-CN f90-94) and Ala-Tyr-Phe-Tyr-Pro-Glu-Leu (αs1-CN f143-149) and the stability of the hydrolysate incorporated in a yoghurt to processing conditions, i.e. drying, homogenisation and pasteurisation, and to storage at 4°C was recently investigated. The study showed the hydrolysate to be stable after processing as both
It has been shown that heat treatments and mechanical damage can reduce peptides bioactivity. As a result of changes in protein structure, the profile of peptides released may differ as digestive enzymes may be capable of digesting these regions of the protein that were previously inaccessible to the enzyme. This has been previously shown to be the case for whey protein [121-122]. Furthermore, ACE inhibitory peptides from whey protein isolates (WPI) pretreated at 65 °C were shown to have greater inhibitory activity than peptides from WPI pretreated at 95 °C. This result can be explained by the formation of whey aggregates .
Optimisation of the hydrolytic process should also be considered when planning to up-scale the production of BAPs. Bioactive peptides may be produced by enzymatic or microbial hydrolysis. However, it is thought that enzymatic hydrolysis is more suited for food-grade BAP production over microbial fermentation . Enzyme immobilisation offers several advantages over the addition of soluble enzymes directly to the product. They can be recycled and the use of immobilized enzymes potentially avoids the generation of interfering metabolite products due to autolysis of the enzymes. Furthermore, protein hydrolysis using immobilized enzymes can also be carried out in milder more controlled conditions and does not need to be inactivated by heat or acidification, which may be damaging for the product [124; 123]. The use of membrane bioreactors may be a substitute for the development of functional materials from food proteins. This system integrates a reaction vessel with a membrane separation system allowing for the recycling of the enzyme, separation, fractionation and/or concentration of the bioactive compound. The use of membrane bioreactors for the development of functional materials from sea-food processing wastes has been recently reviewed .
BAP fractionation and enrichment steps include membrane processing incorporating ultrafiltration and liquid chromatography, ion exchange, gel filtration and reverse phase matrices. Electro-membrane filtration (EMF), a combination of conventional membrane filtration and electrophoresis, may be a consideration for industrial scale isolation of BAPs. EMF is more selective than conventional membrane filtration (ultrafiltration) and is less costly than chromatography .
As mentioned earlier, a large portion of antihypertensive peptides are of low molecular weight and many contain hydrophobic residues, attributes which have been classically associated with bitterness in foods. Hence, this is notably an obstacle that must be resolved during the processing of antihypertensive food products. A number of strategies have been applied with the aim of debittering protein hydrolysates including absorption of bitter peptides on activated carbon, selective extraction with alcohols and chromatographic removal using different matrices. Peptidase-mediated debittering has also been applied. This involves the concomitant or sequential incubation of the protein hydrolysates with exopeptidases, with priority cleavage at hydrophobic residues . However, these debittering strategies may lead to the loss of some amino acid residues from hydrolysates. As bioactivity relies greatly on peptide sequence, these debittering methods may not therefore be suitable for debittering of BAPs including antihypertensive peptides, as hydrolysis may result in loss of activity. Changes in peptide structure may also have implications for absorption at the brush border membrane. Therefore, enzymatic debittering strategies need to be approached on a case-by-case basis.
The widespread commercialisation of antihypertensive food products is dependent on the availability of scientific data from
Antihypertensive peptides have major potential as functional ingredients aiding in the prevention and management of hypertension. Although these peptides have been found to be less potent than antihypertensive synthetic drugs, as part of the daily diet they could play an important part as natural and safe BP control agents. Further detailed mechanistic studies on food protein-derived antihypertensive peptides must be carried out to elucidate the BP mechanism(s) involved. With regard to ACE-inhibitory peptides, a better understanding of the interactions involved in the binding of peptides to the active site of ACE is required such that more effective food peptide-based inhibitors of ACE can be discovered. The use of bioinformatics and