Biological Activities of Peptides Derived from Milk Proteins
by Mueen Aslam and Walter L. Hurley
Milk is a source of nutrition which provides amino acids, carbohydrate,
fat, vitamins and minerals. Milk also contains a number of enzyme activities
which may act upon these components and alter the biological nature of milk.
One group of enzymes found in milk is the proteinases which breakdown proteins
into smaller fragments. Generally, the small fragments of proteins produced
by the action of proteinases are referred to as peptides. Some peptides derived
by proteolytic breakdown of milk proteins have been found to possess biological
In the early stages of lactation, milk has low activity of proteinases.
However, as lactation progresses the concentration of proteinase increases and
is highest near the end of lactation. Various types of proteinases are present
in milk, including plasmin, acid milk proteinases, and proteinases derived from
leukocytes (somatic cells) entering the milk during mastitis. Plasmin contributes
90% of proteolytic activity in cow milk. It is derived from plasminogen which
is the inactive form of plasmin. Plasminogen is converted into plasmin by the
action of another enzyme called plasminogen activator. Milk plasmin and plasminogen
are mostly associated with the casein (CN) micelle, and can be dissociated from
CN micelles under different ionic, temperature, and pH conditions. For example,
plasmin is released from CN micelles in the presence of one molar sodium chloride.
Storing milk at 4° C does not effect the release of plasmin from CN, however,
storage of milk at 30 or 37° C dissociates plasmin from the CN micelles. Plasmin
and plasminogen are also dissociated from CN micelles at pH 4.6. These are important
factors during the handling and storage of milk. Under normal circumstances,
excessive proteolysis of milk protein is prevented by the presence of proteinase
inhibitors in the milk including plasmin inhibitor, plasminogen inhibitor, and
plasminogen activator inhibitor which can inhibit the activity of plasmin, plasminogen,
and plasminogen activators, respectively.
The major milk proteins are the caseins, which comprise about
80% of all protein in cow milk, and a-lactalbumin and ß-lactoglobulin which
are the major whey proteins. Casein in cow milk consists of the four isoforms
as1-CN, as2-CN, ß-CN, and k-CN. ß-Casein is the most susceptible
to proteolysis, although the other CN isoforms are also broken down by plasmin.
Plasmin breakdown the milk proteins, which occurs both in the mammary gland
prior to milk harvest, as well as post-harvest, results in production of a number
of distinct peptides. For example, plasmin breakdown of ß-CN produces several
peptides referred to as the ?-caseins and the proteose peptone fraction. Proteose
peptone consists of several peptides derived from ß-CN which are acid soluble
and heat stable, unlike most intact casein protein. The reason for higher susceptibility
of ß-CN to proteolysis by plasmin may be the presence of numerous basic amino
acid residues in the protein which are important for the proteolytic specificity
of plasmin. Although susceptibility of the other isoforms of CN to proteolysis
is lower than for ß-CN, plasmin can breakdown as1-CN, as2-CN
and K-CN to form several distinct peptides.
Increased somatic cell counts (SCC) resulting from mastitis result
in increased proteolysis of CN due to increased plasmin activity in milk. Breakdown
of CN resulting from mastitis is of particular concern in terms of the cheese
yield of milk. Proteinases other than plasmin are also involved in the proteolysis
of milk proteins. For example, milk acid proteinase has maximal activity at
acidic pH 4.0 and can breakdown as1-CN, ß-CN and k-CN to produce
Ingestion of milk results in exposure of the milk proteins to
another set of proteolytic enzymes. Enzymes found in the gastrointestinal tract
generally breakdown milk proteins into very small peptides (3-12 amino acids).
These enzymes include trypsin, chymotrypsin, carboxypeptidase, and pepsin. Some
of the small peptides generated in the intestine by breakdown of milk proteins
may have biological activity, as well as acting as a source of nutritionally
valuable amino acids. Casomorphins are peptides produced from the breakdown
of CN and possess opioid activity. The term opioid refers to morphine-like effects
which include signs of sedation, tolerance, sleep induction, and depression.
Peptides with opioid activity can be produced by proteolysis of ß-CN, as1-CN,
and k-CN, as well as the whey proteins a-lactalbumin and ß-lactoglobulin. Some
of these peptides also have biological activities other than opioid activity
such as immunomodulatory effects, antihypertensive activities or antibacterial
ß-Casomorphins with opioid activity are generated in the intestine
by breakdown of CN, but they are highly resistant to further proteolysis by
intestinal enzymes. Once absorbed into the blood, these peptides can pass to
the brain and various other organs to elicit an opioid effect. Some actions
of the casomorphins may also occur in the intestine. Casomorphin peptides absorbed
through the intestinal wall often appear in the circulation as larger molecules
than the final peptide with opioid activity, suggesting that the absorbed forms
of the casomorphins are further cleaved to the active peptide at the target
cell. In contrast to the casomorphins, peptides produced by breakdown of k-CN
function as opioid antagonists; that is, they can inhibit the effect of morphine-like
In addition to the opioid effects of ß-casomorphins, some of those
peptides inhibit the activity of an enzyme involved in vasoconstriction of blood
vessels, a normal part of blood flow regulation. Peptides produced from cow
ß-CN, as1-CN, and human k-CN are potent inhibitors of this enzyme
and can function as antihypertensive agents.
Peptides derived from casein may also have antidiarrheal effects.
Infusion of casein peptides into the abomasum of steers reduces the frequency
and duration of intestinal motility. Similar effects of casomorphins have been
reported in humans and dogs where casomorphins decrease intestinal motility
and prolong gastrointestinal transit. The antidiarrheal effect of these peptides
could result from the enhancement of water and electrolyte absorption stimulated
by the casomorphins. Milk-derived casomorphins may contribute to the regulation
of nutrient assimilation by decreasing intestinal motility and improving digestion
Bioactive peptides produced from milk proteins are also implicated
in immunomodulatory and bactericidal activities. Some of these peptides stimulate
the phagocytic activity of macrophages by causing biochemical changes in the
cell. Several small peptides derived from bovine ß-CN enhance macrophage phagocytic
activity. Another peptide derived from human a-lactalbumin stimulates polymorphonuclear
oxidative metabolism and increases the phagocytic activity of macrophages. Increased
phagocytic activity of immune cells confers protection against bacterial infections.
Bovine lactoferrin is an antibacterial protein normally found
in low concentrations in cow milk. Digestion of lactoferrin by gastric pepsin
yields an antibacterial peptide called lactoferrin, which inhibits and inactivates
various types of bacteria. The bactericidal effect of this peptide is similar
to the antibiotic polymyxin B and seems to act by disruption of the bacterial
membrane. Peptides produced from proteolytic digestion of milk lactoferrin after
ingestion by the newborn may provide defense against infections.
In conclusion, milk proteins are degraded by various proteinases
present either in the milk or in the intestine. Proteinases present in the milk
are important because they alter the physical and chemical characteristics of
the milk and dairy products. Intestinal digestion of milk proteins mostly yields
amino acids used by the animal in normal growth and metabolism. However, some
peptides are also produced during breakdown of milk proteins, and some of these
peptides may possess biological activities such as opioid, immunostimulatory,
antihypertensive, or antibacterial effects. Available knowledge about these
peptides suggests that some may have value as therapeutic agents or food additives.
However, further studies are needed to elucidate the role of these peptides
in the young animal ingesting milk or in dairy foods.