Illini DairyNet Papers
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 activity.
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 various peptides.
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 activities.
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 substances.
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 and absorption.
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.