Rotimi E. Aluko
The primary purpose of protein consumption is to provide essential amino acids, which are used by the body to synthesize various structural (muscles, bones, hair) and functional (enzymes, hormones) proteins required for homeostasis maintenance. However, the increasing popularity of functional foods and nutraceuticals has led scientists to seek protein-derived fragments (peptides) that could prevent or even treat chronic metabolic disorders. The ability of these peptides to influence biological reactions and physiological conditions is what makes them ‘bioactive’. A bioactive peptide consists of a certain number of amino acids (2-20) that are usually encrypted (hence the term ‘cryptides’) within the linear protein chain (Fig. 1) and remain inactive until released by digestion. A protein chain can contain several cryptides that may be similar (same length and amino acid sequence) or dissimilar (same or different length with different amino acid sequence). Thus, it follows that under appropriate gastrointestinal tract (GIT) digestion conditions, food proteins could yield bioactive peptides. Peptide absorption from the GIT is then facilitated by specific or non-specific transporters, cellular endocytosis and by simple translocation (passive diffusion) into the blood circulatory system1. However, the level of such bioactive peptide release during regular food digestion is considered very low and of little consequence to human health. Therefore, the most useful approach involves customized in vitro protein digestion with human or microbial proteases in order to enhance bioactive peptides production. Upon completion of the enzymatic hydrolysis, the digest is centrifuged and the soluble portion (supernatant) is isolated as the protein hydrolysate (contains the released cryptides) while the undigested portion (precipitate) can be discarded. High peptide solubility increases absorption potential during oral consumption and hence ensures a more effective bioavailability. This method produces a ‘peptide soup’ called a protein hydrolysate that contains cryptides and non-cryptides with peptides of different sizes, amino acid composition or sequence and activity. The protein hydrolysate can be tested for desirable activity and if positive may be used directly to formulate functional foods and nutraceuticals. High levels of cryptides will enhance bioactive properties of the protein hydrolysate. Therefore, subsequent separation techniques (membrane ultrafiltration, column chromatography) can be used to enrich the product with highly active peptides (fractions B, C, D) through removal of the inactive or less active components (fraction A) as shown in Fig. 1. Peptide separation is based mainly on size, net charge and hydrophobicity to produce distinct and homogenous fractions with better bioactive properties than the original protein hydrolysate. However, in some cases, peptide separation actually produces fractions with reduced bioactive properties than the original protein hydrolysate. The strong bioactive properties of such protein hydrolysates has been attributed to synergistic effects whereby the peptide interactions produce stronger effects than the sum of individual peptides. Therefore, loss of synergy as a result of peptide separation causes reductions in bioactive effects of the peptide fractions; in such cases, use of the protein hydrolysate without further peptide separation is preferred.
Safety of bioactive peptides: Apart from the demonstrated health benefits, one of the main attractions of food protein-derived peptides is the low risk of negative side effects that are normally associated with drug therapy. For example, several human intervention trials with bioactive milk products have shown little or no negative side effects2,3. Also important is the low to nil risk of adverse health effects or permanent health damage from peptide overdose unlike drug overdose. This was illustrated in a randomized human intervention trial where consumption of 5 times the normal dose of casein antihypertensive peptides was shown to have no negative effect4. Therefore, even though peptides are less active on a weight basis, they can be used as preventive or therapeutic agents at relatively higher doses than possible with drugs. The relative safety of food protein-derived peptides may be attributed to faster clearance from the blood circulatory system since they are susceptible to peptidase-dependent degradation. Moreover, unlike drugs, peptides are not stored ‘as is’ in tissues but rather are used for the biosynthesis of new proteins within the body. Unlike drugs, peptides do not require liver detoxification and are not excreted in the urine; therefore, potential damages to these organs are minimized during peptide therapy, even at high doses. The following examples illustrate some of the potential health benefits of bioactive peptides.
Antihypertensive: The mammalian blood pressure is regulated in part by the renin-angiotensin system (RAS), which consists of two main catalytic agents, renin and angiotensin-converting enzyme (ACE). Renin converts angiotensinogen to an inactive angiotensin I, which is then acted upon by ACE to release angiotensin II, a highly potent vasoconstrictor. Under homeostatic conditions, the catalytic activities of the two enzymes ensure moderate blood levels of angiotensin II and hence blood vessel contraction (systolic pressure <140 mmHg) is followed by adequate relaxation (diastolic pressure <90 mmHg) to maintain normal blood pressure. However, under certain metabolic or disease conditions, there are excessive activities of renin and ACE, which lead to abnormally high blood angiotensin II level. At high angiotensin II levels, blood vessel contraction is stronger and longer while relaxation period is reduced, hence high pressure develops and ultimately manifests as a hypertensive state along with associated negative health effects.
How do peptides work to reduce blood pressure? Traditional treatment of hypertension involves the use of chemical agents (drugs such as captopril, enalapril, lisinopril) that bind to ACE (ACE inhibitors) and prevent excessive production of angiotensin II. Recently, a renin-inhibitory drug (aliskiren) was also approved as an antihypertensive agent. Other drug intervention methods involve agents that inhibit the interaction between angiotensin II and its cellular receptor (angiotensin receptor blockers) and those that enhance nitric oxide (NO) production within vascular walls. NO is a well-known vasodilator and high levels could improve vascular wall relaxation for blood pressure-lowering effects. Initial food protein-derived peptides were targeted at ACE inhibition and were produced through milk fermentation during which the proteins are degraded by fermenting microbial proteases5. The first set of ACE-inhibitory peptides (VPP and IPP) were isolated from fermented milk and have been demonstrated as effective blood pressure-reducing agents. Fermented milk products have exhibited antihypertensive effects during oral consumption by human subjects, which indicates absorption of these peptides occurred6. Following identification of fermented milk as a source of ACE-inhibitory peptides, several works have adapted the principle by using microbial or mammalian proteases as effective in vitro digestion tools to release antihypertensive peptides from several food proteins7-13. In addition to ACE inhibition, some of these peptides also have renin-inhibitory properties and could act similar to antihypertensive drugs. In fact, plasma activities of ACE and renin were decreased when spontaneously hypertensive rats (SHR) were orally administered an antihypertensive hemp seed protein hydrolysate14. Apart from RAS inhibition, food protein-derived peptides have also produced plasma NO-boosting effects, which were accompanied by blood pressure decreases15-17. A recent report showed that a lactoferrin derived peptide has both ACE-inhibitory and human angiotensin AT1 receptor inhibitory activities, which limited angiotensin II-receptor interactions in an in vitro model18. While the specific modes of action of several food protein-derived peptides remain to be elucidated, it is possible that the antihypertensive effects are due to certain combinations of ACE and/or renin inhibition, angiotensin II receptor blocking and enhanced NO levels in the blood and tissues. Blood pressure reductions of up to 50 mmHg have been reported after oral administration of food protein-derived peptides to SHR1. However, the antihypertensive effects are less in humans with a previous report of ~5 mmHg reduction after oral ingestion of 3 g pea protein hydrolysate (PPH) for 3 weeks19.
Antioxidant: Under normal physiological conditions, the body produces several reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) through various metabolic reactions occurring as a part of cellular reactions. Some of these ROS and RNS are used as cellular communication messengers while others provide defensive tools against pathogenic microorganisms. Thus, ROS and RNS species constitute important tools for maintaining homeostatic conditions. However, these species are highly reactive and can cause irreversible oxidative damages to critical cell biopolymers that include proteins, lipids and especially DNA. For example, DNA damage if left unchecked may lead to cancer cell development and associated
pathological damages. Normally, the cell maintains an effective mechanism that includes enzymes (superoxide dismutase, catalase), a peptide (glutathione) and others (ascorbic acid, tocopherols), which are used to scavenge and neutralize these toxic reactive species before cellular damage can occur. However, under certain conditions (diet, age, diseases) the balance is tilted towards higher levels of ROS and RNS, such that the cell loses effective scavenging ability, which then leads to an oxidative stress state. During oxidative stress, destruction of vital cellular components can accelerate disease (hypertension, cancer, diabetes, kidney disease) development and lead to increased morbidity. To combat oxidative stress, the endogenous antioxidant deficit can be compensated for by using exogenous antioxidant supply, especially in the form of peptides. Therefore, various food protein-derived peptides have been investigated and shown to possess antioxidant properties, both in vitro and in vivo.
Structure and function of antioxidative peptides: Usually after protein hydrolysis, the peptides are screened for antioxidative activity using reactions that involve scavenging of free radicals (2,2 diphenyl-1 picrylhydrazyl or DPPH, superoxide, hydroxyl, hydrogen peroxide), metal (iron or copper) chelation, iron (III) reduction, inhibition of lipid (linoleic acid) oxidation20,21 and DNA damage prevention22. Peptide fractions with strong in vitro antioxidant properties are then tested for in vivo effectiveness using appropriate animal models. For example, oral administration of hemp seed peptides to SHR led to reduced plasma oxidative stress factors23 while a fish protein hydrolysate effectively reduced alcohol-induced oxidative stress in rats24. Peptides act as antioxidants through two main mechanisms, either by donating electrons or hydrogen atom transfer, which neutralizes free radicals and abolishes oxidative capacity. Protein hydrolysis enhances production of peptides that contain excess electrons, especially through the presence of carboxyl groups from hydrolyzed peptide bonds and others present in acidic amino acid groups. The acidic group confers excellent electron-donating capacity on the peptides and is responsible for the excellent antioxidant properties of peptides that contain glutamic and aspartic acids25. Hydrogen atom transfer is believed to be facilitated when peptides contain aromatic or ring-like structure, hence the excellent antioxidant properties of peptides that contain histidine, phenylalanine, tryptophan and tyrosine26,27. Other structural properties such as presence of hydrophobic (leucine, isoleucine, valine, alanine) or sulfur-containing amino acids (methionine and cysteine) have been suggested to positively influence peptide antioxidant properties28,29. In addition to providing physiological benefits, bioactive peptides may also serve as effective replacements of synthetic antioxidants in foods. Through metal chelation, peptides could reduce lipid peroxidation and help to maintain food freshness during storage21,28. Moreover, as ROS and RNS scavengers, addition of antioxidant peptides to foods could also prevent radical species-catalyzed quality deterioration. Thus, peptides that exhibit both metal chelation and radical scavenging activities provide ideal antioxidative agents for preventing quality deterioration during food storage28,30.
Antidiabetic: The inability to maintain regular influx of glucose into muscles leads to accumulation of glucose in blood that could cause degenerative diseases if not rectified. Effective control of blood glucose requires insulin, which enhances cellular absorption. In type 1 diabetes, the pancreatic insulin production is compromised, hence reduced capacity to sensitize muscle cells for adequate glucose uptake. In type 2 diabetes, causative factors are more complex but essentially there is also reduced glucose uptake by muscles. Peptides have been proposed as potential agents that can enhance glucose uptake in muscles and reduce the pathological intensity of diabetes. For example, insulin-dependent glucose uptake by L6 myocytes (muscle cells) was significantly enhanced in the presence of a flaxseed peptide fraction31. More importantly, addition of the flaxseed peptides to myocytes only (no insulin) also improved glucose uptake when compared to the cells without peptides. Thus, the flaxseed peptides could be a useful therapeutic agent to reduce muscle cell dependence on insulin for glucose uptake and be of benefit in disease situations like type 1 diabetes where inadequate insulin production is an important causative factor.
Inhibition of a critical pro-diabetes factor: Peptides can also work as antidiabetic agents through inhibition of dipeptidyl peptidase IV (DPP-IV) activity. DPP-IV is an enzyme that causes rapid degradation of incretins such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), which reduces insulin production and release efficiency by the pancreatic β cells. Therefore, DPP-IV inhibition will boost physiological levels of GLP-1 and GIP, which leads to increased insulin production for better blood sugar control. DPP-IV inhibitory peptides have been generated from enzymatic hydrolysis of cowpea seed32, canary seed33 and bovine whey34 proteins. The active cowpea peptides consisted mainly of threonine, alanine and branched-chain amino acids. In contrast, the presence of proline at the 3rd position from the N-terminal seem to potentiate the DPP-IV inhibitory properties of whey peptides. While these works have been restricted to in vitro inhibitory assays, future experiments could determine the in vivo DPP-IV activity changes in response to oral administration of peptides to animals. Since active DPP-IV inhibitory peptides have been shown to consist of mostly short chain peptides (3-14 amino acids),32,34 there is a high probability for bioactivity in living animal tissues.
Other potential health benefits: Peptides that reduce cell proliferation may be important tools in the fight against cancer. Using various cancer cell lines (liver, breast and cervical), rapeseed peptides obtained through fermentation significantly reduced cell proliferation but without cell toxicity35. Similarly, a peptide (Trp-Pro-Pro) isolated from an enzymatic hydrolysate of blood clam muscle showed toxicity and antiproliferative effects when incubated with various cancer cell lines36. In the presence of Trp-Pro-Pro (5 and 15 mg/ml), apoptosis of human prostate cancer cells (PC-3) increased 2-fold, which suggests anticancer potentials for the peptide. Cowpea protein hydrolysates have demonstrated potential cholesterol-reducing abilities through in vitro inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the enzyme that catalyzes the rate determining step during hepatic cholesterol synthesis37. If absorbed from the GIT, the cowpea protein hydrolysate could interfere with and decrease in situ cholesterol synthesis, which would lead to lowering of blood cholesterol and associated health benefits. The cowpea protein hydrolysate also interfered with micellar solubilization of cholesterol during in vitro tests. Since cholesterol solubilization is an important prerequisite for absorption from the GIT into blood circulation, consumption of the micellar-disrupting cowpea protein hydrolysate may enhance fecal removal of cholesterol and promote low blood cholesterol levels. Protein hydrolysates have also been tested for immune-modulating activities, especially to promote cellular expression of anti-inflammatory hormones (cytokines). Using a lipopolysaccharide/interferon γ-stimulated RAW 264.7 NO(-) macrophages, PPH treatment was shown to inhibit NO production by up to 20% when compared to non-treated cells38. The PPH also significantly inhibited secretion of the pro-inflammatory cytokines, TNF-α and IL-6, by 35 and 80%, respectively. Mice that received an oral administration of PPH displayed enhanced peritoneal macrophages phagocytic activity in addition to stimulated gut mucosa immune response. The enhanced immune response was typified by an increased number of IgA+ cells in the small intestine lamina propria as well as higher numbers of IL-4+, IL-10+ and IFN-γ+ cells38. The authors concluded that the PPH may be used as an alternative therapy to prevent inflammatory-related diseases.
Summary: Protein hydrolysates are potential sources of ingredients that can be used to formulate functional foods and nutraceuticals against several human chronic diseases. The low cost and high proteolytic efficiency of microbial enzymes serve as useful aids in developing novel protein hydrolysates. One area of critical need is multifunctional peptides that can elicit simultaneous effects against various metabolic disorders or diseases. For example, it will be more efficient to have a single protein hydrolysate that can reduce oxidative stress and at the same time act against RAS enzymes to decrease blood pressure. Or a single protein hydrolysate that can reduce blood sugar level while also reducing body weight as a means of effective treatment for type 2 diabetes. Therefore, additional research works are needed that will extend current highly promising in vitro results into animal or human intervention trials to confirm bioactive properties and enhance commercialization.
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Rotimi E. Aluko, Professor,
Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2