Milk is an excellent source of well balanced nutrients and also exhibits a range of biological activities that influence digestion, metabolic responses to absorbed nutrients, growth and development of specific organs, and resistance to disease. These biological activities are mainly due to the peptides and proteins in milk. However, some of the biological activity of milk protein components is latent, and is released only upon proteolytic action. Bioactive peptides are produced during digestion of milk in the gastrointestinal tract, and also during fermentation and food processing.
Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions and may ultimately influence health. Upon oral administration, bioactive peptides, may affect the major body systems—namely, the cardiovascular, digestive, immune and nervous systems. The beneficial health effects may be classified as antimicrobial, antioxidative, antithrombotic, antihypertensive, antimicrobial or immunomodulatory (FitzGerald and Meisel, 2003; Korhonen and Pihlanto, 2003a).
The activity of these biofunctional peptides is based on their inherent amino acid composition and sequence. The size of active sequences may vary from two to twenty amino acid residues, and many peptides are known to have multifunctional properties (Meisel and FitzGerald, 2003) e.g., peptides from the sequence 60-70 of ß-casein show immunostimulatory, opioid and angiotensin I converting enzyme (ACE)-inhibitory activities. This sequence has been defined as a strategic zone (Migliore-Samour and Jolles, 1988; Meisel, 1998). The sequence is protected from proteolysis because of its high hydrophobicity and the presence of proline residues. Other examples of the multifunctionality of milk-derived peptides include the αs1-casein fraction 194-199 showing immunomodulatory and ACE-inhibitory activity, the opioid peptides α- and ß-lactorphin also exhibiting ACE-inhibitory activity and the calcium-binding phosphopeptides (CPPs), which possess immunomodulatory properties (Korhonen and Pihlanto, 2003a).
Source of bioactive peptides
Milk is a rich source of protein. Casein and whey proteins are the two main protein groups in milk, caseins comprises about 80 percent of the total protein content in bovine milk and are divided into α-, ß- and κ-caseins. Whey protein is composed of ß-lactoglobulin, α-lactalbumin, immunoglobulins (IgGs), glycomacropeptides, bovine serum albumin, and minor proteins such as lactoperoxidase, lysozyme and lactoferrin. Each of the subfractions found in casein or whey has its own unique biological properties. Milk proteins can be degraded into numerous peptide fragments by enzymatic proteolysis and serve as source of bioactive peptides.
Production of bioactive peptides
Bioactive peptides are inactive within the sequence of the parent protein and can be released in three ways: (a) enzymatic hydrolysis by digestive enzymes, (b) food processing and (c) proteolysis by enzymes derived from microorganisms or plants.
a) Enzymatic hydrolysis
The cleavage of latent bioactive peptides from milk proteins normally occurs during digestion by pepsin and pancreatic enzymes (trypsin, chymotrypsin, carboxy and aminopeptidases), producing active peptide fragments in the gastrointestinal tract of the milk-consuming individual (Schlimme and Meisel, 1995). The physiological effects of bioactive peptides depend on their ability to reach their target sites intact, which may involve absorption through the intestinal epithelium prior to travel to the peripheral organs (Vermeirssen et al., 2004).
Many of the known bioactive peptides have been produced in vitro using gastrointestinal enzymes, usually pepsin and trypsin. ACE-inhibitory peptides and CPPs, for example, are most commonly produced by trypsin. Other digestive enzymes and different enzyme combinations of proteinases—including alcalase, chymotrypsin, pancreatin, pepsin and thermolysin as well as enzymes from bacterial and fungal sources—have also been utilized to generate bioactive peptides from various proteins.
b) Food Processing
The structural and chemical changes that occur during the processing of food proteins may result in the release of bioactive peptides. In particular, heat and/or alkali treatment can generate additional inter-and intramolecular covalent bonds that are resistant to hydrolysis. Such processing conditions also promote the racemic conversion of L-amino acids to D-isomers and consequently, lead to indigestible peptide bonds. The potential formation of indigestible peptide sequences during food processing is noteworthy, because this may promote both formation and absorption of bioactive peptides that do not occur naturally in the precursor protein. Such bioactive peptides can be generated during manufacture of several milk products and may thus be ingested as food components. For example, partially hydrolyzed milk proteins for hypoallergenic infant formulae and for clinical applications in nutrition consist exclusively of peptides and contain bioactive peptides. Cheese contains phosphopeptides as natural constituents and secondary proteolysis during cheese ripening leads to formation of various ACE inhibitory peptides.
c) Microbial fermentation
Many industrially utilised dairy starter cultures are proteolytic to some extent. Bioactive peptides can, thus, be generated by the proteolytic activities of the strains of starter and non-starter bacteria e.g. Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactococcus lactis, Streptococcus thermophilus used in the manufacture of fermented dairy products. The proteolytic system of lactic acid bacteria (LAB) is well characterised. This system consists of a cell wall-bound proteinase and a number of distinct intracellular peptidases, including endopeptidases, aminopeptidases, tripeptidases and dipeptidases. Extracellular proteinases cause degradation of casein into oligopeptides. The longer chain oligopeptides may be a source of bioactive peptides when further degraded by intracellular peptidases of lysed-lactic acid bacteria.
The single most effective way to increase the concentration of bioactive peptides in fermented dairy products is to ferment or co-ferment with highly proteolytic strains of LAB. The choice of strains influences the release of effective bioactive peptides. The strain should not be too proteolytic otherwise the product will be destroyed and must have the right specificity to give high concentrations of active peptides. The concentration of ACE-inhibitory peptides seems to rely on a balance between their formation and further breakdown into inactive peptides and amino acids that in turn depends on storage time and conditions. Various bioactive peptides including ACE-inhibitory or antihypertensive peptides, immunomodulatory, antioxidative, antimutagenic peptides have been released from milk proteins through microbial proteolysis (Gobbetti et al., 2004; Korhonen and Pihlanto, 2001; Korhonen and Pihlanto, 2004, Matar et al., 2003). The best known ACE-inhibitory peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), have been identified in milk fermented with strains of Lb. helveticus and Saccharomyces cerevisiae. In addition to live microorganisms, proteolytic enzymes isolated from LAB have been successfully employed to release bioactive peptides from milk proteins.
Occurrence of bioactive peptides in dairy products
It is now well documented that bioactive peptides can be generated during milk fermentation by the proteolytic activity of starter cultures. As a result, peptides with various bioactivities can be found in the end-products, such as various cheeses and fermented milks. These traditional dairy products may under certain conditions have specific health effects when ingested as part of the daily diet. A list of bioactive peptides found in dairy products is given in table 1.
Table 1. Bioactive peptides identified from milk products
|
Product |
Origin |
Biofunctional role |
Reference |
|
Cheddar cheese |
αs1- and ß -casein fragments |
Several phosphopeptides with a range of properties including the ability to bind and solubilise minerals |
Singh et al. (1997) |
|
Italian cheeses: Mozzarella, Crescenza, Italico, Gorgonzola |
ß-CN f (58–72) |
ACE inhibitory |
Smacchi and Gobbetti (1998) |
|
Yoghurt type products |
αs1- , ß- and κ-CN fragments |
ACE inhibitory |
Yamamoto et al. (1999) |
|
Gouda cheese |
αs1-CN f (1–9), ß -CN f (60–68) |
ACE inhibitory |
Saito et al. (2000) |
|
Festivo cheese |
αs1-CN f (1–9), f (1–7), f (1–6) |
ACE inhibitory |
Ryhänen et al. (2001) |
|
Emmental cheese |
αs1- and ß -casein fragments |
Immunostimulatory, mineral binding and solubilising , antimicrobial |
Gagnaire et al. (2001) |
|
Manchego cheese |
Ovine αs1-, αs2 - and ß-casein fragments |
ACE inhibitory |
Gomez-Ruiz et al. (2002 |
|
Sour milk |
ß-CN f (74–76, f (84–86), κ-CN f (108–111) |
ACE inhibitory/ Antihypertensive |
Nakamura et al. (1995a) |
|
Dahi |
Ser-Lys-Val-Tyr-Pro |
ACE inhibitory |
Ashar and Chand (2004) |
Physiological effects of bioactive peptides
Effects on cardiovascular system
Hypertension is one of the major risk factors for cardiovascular disease and stroke. Because diet has a role in the prevention and treatment of this disease, there is interest in developing foods with antihypertensive activity. ACE is a multifunctional ectoenzyme that is located in many tissues and plays an important role in blood pressure regulation and in turn hypertension. Therefore, ACE inhibition mainly results in a hypotensive effect. Recent research has shown that enzymatic digestion of casein and whey proteins generate peptides that have the ability to inhibit ACE. The best known ACE-inhibitory peptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) with IC 50 values (concentration of peptides mediating 50% inhibition of ACE activity) of 9 and 5 µMoles respectively have been identified from a Japanese sour milk drink (Calpis ®) fermented with Lb. helveticus and Saccharomyces cerevisiae strains (Nakamura et al., 1995). In a placebo-controlled study, the blood pressure of hypertensive patients decreased significantly after 4 weeks of daily ingestion of 95 ml of sour milk that contained these two tripeptides. This was equivalent to an ingested dose of ACE inhibitory peptides of about 2.6 mg per day (Hata et al., 1996). Similarly, a milk product Evolus ® fermented with Lb. helveticus LBK-16H (Valio Ltd, Finland or Kaiku Vitabrand ®, Spain) exerted significant antihypertensive effects in humans at daily doses of 150 ml. Two other commercial products, a casein hydrolysate containing the peptide FFVAPFPEVFGK (αs1-casein f23-34; Casein DP, Kanebo, Ltd, Japan, and C12 peptide, DMV, The Netherlands) and a whey protein hydrolysate (BioZate, Davisco, US) were also claimed to lower blood pressure in humans (FitzGerald et al., 2004).
Effects on immune system
Diet is known to play an important role in the body's defense mechanism. Research concerning the role of functional peptides on the immune system is quite recent but seems to be promising. The two main activities being studied are the immunomodulatory (stimulation of immune system) and antimicrobial (inhibition of microorganisms) effects of bioactive peptides. Several casein and whey protein derived peptides display an immunomodulatory role. Immunomodulating peptides have been found to stimulate the proliferation of human lymphocytes, the phagocytic activities of macrophages and antibody synthesis. Also, it has been suggested that immunomodulatory milk peptides may alleviate allergic reactions in humans and enhance mucosal immunity in the gastrointestinal tract (Korhonen & Pihlanto, 2003a). In this way immunomodulatory peptides may regulate the development of the immune system in newborn infants. Furthermore, it has been suggested that immunopeptides formed during milk fermentation may contribute to the antitumor effects of fermented milk (Matar et al., 2003).
The antimicrobial properties of milk have been widely acknowledged for many years. The antimicrobial activity of milk is mainly attributed to immunoglobulins, and to non-immune proteins, such as lactoferrin, lactoperoxidase and lysozyme. One of the most potent antimicrobial peptides described so far corresponds to a fragment of the whey protein lactoferrin, named lactoferricin (Bellamy et al., 1992). More recently, other whey proteins such as α-lactalbumin and ß-lactoglobulin have also been considered as potential precursors of bactericidal fragments. Similarly, antibacterial fragments have also been derived from αs1-, αs2- and κ-casein (Lahov and Regelson, 1996; Recio and Visser, 1999b). These peptides have been found to be active against a broad range of pathogenic organisms e.g. Escherichia, Helicobacter, Listeria, Salmonella and Staphylococcus, yeasts and filamentous fungi. Depending on the target microorganism, inhibitory concentrations of peptides vary, eg: antimicrobial peptides αs2 -CN f183-207 and f164-179 exhibited inhibition against Gram-positive and -negative bacteria with minimal inhibitory concentrations (MICs) ranging from 8 to 95 µmol/l (Recio and Visser, 1999).
Effects on nervous system
Recent studies have provided evidence that peptides exist in dairy products which play an active role in the nervous system; these are known as opioid peptides. The first major opioid peptides discovered were ß-casomorphins, fragments of ß-casein (Smacchi and Gobbbetii, 1998). Once absorbed into blood, these peptides can travel to the brain and various other organs and elicit pharmacological properties similar to opium or morphine. This may be the reason why human neonates generally become calm and sleepy after drinking milk. In contrast to the casomorphins, some peptides produced by the break down of κ-casein function as opioid antagonists, that is, they can inhibit the effect of morphine like substances.
Effects on nutritional status and dental health
Casein-derived phosphorylated peptides, CPPs, can form soluble organophosphate salts and lead to enhanced mineral uptake. Calcium absorption is enhanced by limiting the precipitation of calcium in the distal ileum. The charged side chains, in particular the phosphate groups of amino acids can bind minerals e.g. Ca, Mg, Fe and Zn (Meisel, 1998). Since CPPs can bind and solubilise minerals, they may have value in the prevention of osteoporosis, dental caries, hypertension and anemia. CPPs can have an anticariogenic effect by promoting recalcification of tooth enamel, whereas glycomacropeptide (GMP) derived from κ-casein seems to contribute to the anticaries effect by inhibiting the adhesion and growth of plaque-forming bacteria on oral mucosa. Various dental care products containing CPPs and/or GMP are now commercially available.
Other functional roles
Recent studies have shown that peptides with antioxidative properties can be released from caseins by hydrolysis with digestive enzymes and by proteolytic LAB in fermented milks (Korhonen and Pihlanto, 2003a). Most these were derived from αs-casein and have been shown to possess free radical-scavenging activities and to inhibit enzymatic and non-enzymatic lipid peroxidation . In the future, antioxidative peptides may find applications as ingredients in different fields, e.g. in the prevention of oxidation in fat-containing foodstuffs, cosmetics and pharmaceuticals. More research is needed to demonstrate if peptides produced during fermentation can prevent oxidative damage in vivo .
Nagaoka et al.(2001) identified a hypocholesterolemic peptide (Ile-Ile-Ala-Glu-Lys) from the tryptic hydrolysate of ß-lactoglobulin. This peptide suppressed cholesterol absorption by Caco-2 cells in vitro and elicited hypocholesterolemic activity in vivo in rats upon oral administration of the peptide solution. The mechanism of the hypocholesterolemic effect remains to be clarified.
Future perspectives for bioactive peptides
Fermented dairy products and other foods containing bioactive peptides would appear to have the potential to offer specific health benefits to consumers. While there is a need for further basic research to clarify why these peptides have physiological effects, commercial products containing bioactive peptides are now commercially available. Food and pharmaceutical companies are actively considering how to exploit bioactive peptides in both human nutrition and in health promotion.
Bioactive peptide preparations have the potential to be used in the formulation of functional foods, cosmetics and as potent drugs having well defined pharmacological effects. With the rise of consumer concerns about the deleterious effects of chemical preservatives and the increasing preference for natural components, milk derived bioactive substances may have value in food preservation and nutraceuticals.
Application of enrichment protocols such as membrane processing and chromatographic isolation may also be an area of future interest in the extraction of potent biofunctional peptides from fermented dairy products and their subsequent utilization as functional food ingredients.
Molecular studies are required to study the mechanisms by which the bioactive peptides exert their activities. Ultimately this research may be helpful in understanding, preventing and treating life-style related diseases such as cardiovascular disease, cancers, osteoporosis, stress and obesity.
Summary
Recent research has shown that milk proteins can yield bioactive peptides with opioid, mineral binding, cytomodulatory, antihypertensive, immunostimulating, antimicrobial and antioxidative activity in the human body. Bioactive peptides are encrypted in milk proteins and are only released by enzymatic hydrolysis in vivo during gastrointestinal digestion, food processing or by microbial enzymes in fermented products. At present significant research is being undertaken on the health effects of bioactive peptides. A variety of naturally formed bioactive peptides have been found in fermented dairy products, such as yoghurt, sour milk and cheese. In particular, antihypertensive peptides have been identified in fermented milks, whey and ripened cheese. Some of these peptides have been commercialized in the form of fermented milks. Bioactive peptides have the potential to be used in the formulation of health-enhancing nutraceuticals, and as potent drugs with well defined pharmacological effects.
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