Proteases in Milk
by Mueen Aslam and Walter L. Hurley
TAKE HOME MESSAGE
- In addition to the highly structured milk proteins, milk also contains enzymes (proteases) that degrade the milk protein structure.
- The plasmin system is the major milk protease system.
- Plasmin activity is increased during mastitis and mammary involution, and is affected by numerous other factors.
- Breakdown of milk casein by plasmin and other proteases can result in off-flavor and bitterness in milk, as well as decreased cheese yeild.
Milk contains a number of proteins. The major proteins are in cow milk are the caseins which have three subclasses including as-casein, ß-casein, and k-casein. Cow milk also contains whey proteins such as a-lactalbumin and ß-lactoglobulin. While these major milk proteins are synthesized in the mammary gland, several minor proteins are transferred from blood to milk. These include immunoglobulins and serum albumin. Milk also contains a group of small peptides which are referred to as the proteose-peptone fraction, and which are mainly derived from breakdown of casein by the action of proteases. Proteases are a type of enzyme which act on proteins (in this case milk proteins) and cause their breakdown to produce smaller fragments. When ingested, stomach and intestinal proteases breakdown the milk proteins to small peptides (several amino acids long) or individual amino acids. However, milk proteins may be attacked by proteases found in the mammary tissue even as the milk is being produced.
There are several different types of proteases present in the bovine milk. Some originate from contaminating microorganisms and some are transferred from blood to milk. These proteases have been studied from the perspective of their physiological significance in milk or the mammary gland, their effect on the processing of dairy products, and their nutritional and antimicrobial roles. Numerous factors affect the concentration of these enzymes in the milk, including breed of cow, nutritional and lactational effects, and disease conditions such as mastitis. Bovine milk contains several proteases including plasmin, plasminogen, plasminogen activators, thrombin, cathepsin D, acid milk proteases, and aminopeptidase. Milk also contains several proteases which are derived from leukocytes (somatic cells) in the milk. Some proteases are secreted in an inactive precursor form. Under physiological conditions these precursors are converted into the active form by autolysis (self activation) or by limited proteolysis by another protease. For example, plasmin is the active enzyme form which is produced from the inactive zymogen called plasminogen. Conversion of plasminogen to plasmin occurs by specific action of plasminogen activators, which are also proteases. The result of plasmin activation is breakdown of some of the milk proteins, especially casein.
THE PLASMIN SYSTEM IN MILK
Plasmin is generally the major protease activity in milk. Plasmin, plasminogen, and plasminogen activators are associated with the casein micelles and milk fat globule membranes in milk. Activity of this enzyme is controlled by certain inhibitors such as plasmin inhibitors, or inhibitors of plasminogen activators which inhibit the activity of plasminogen activators so that plasminogen cannot be converted into plasmin. Most plasmin inhibitors are present in the serum phase of milk.
The concentrations of plasmin, plasminogen, and plasminogen activators are low during early lactation. However, concentrations of these enzymes in milk increase with advancing lactation, and are highest during the dry period of the cow. The increased concentration of these enzymes during the dry period is attributed to increased permeability of blood-milk barrier where these enzymes are passed from blood to milk. Plasmin activity in milk is higher in older cows and increases with increasing lactation number. Holsteins generally have higher activity than Jerseys and Aryshires. Plasmin activity is also affected by mastitis when the increased number of somatic cells contributes to increased plasminogen activators which in turn activate plasminogen into plasmin.
Plasmin activity in milk can also be altered by post-harvest handling procedures. Plasminogen converts into plasmin faster at body temperature than under refrigeration. Pasteurization at 72°C for 15 seconds reduces milk plasmin activity only by 10-17 percent. However, storage of pasteurized milk leads to increased plasmin activity due to inactivation of inhibitors of plasminogen activators present in milk. To completely inhibit plasmin during storage it is necessary to heat milk at 142°C for 18 seconds or 120°C for 15 minutes.
Proteolysis of milk proteins is the major activity attributed to plasmin in the milk. Casein is most susceptible to breakdown by this enzyme. Among the various classes of casein, ß-casein is more susceptible than as-casein, and k-casein is somewhat resistant to breakdown by the plasmin. Breakdown fragments of casein can produce off-flavor and bitterness in milk. In contrast, milk whey proteins such as a-lactalbumin and ß-lactoglobulin are fairly resistant to the action of plasmin.
Increased plasmin activity decreases the viscosity of a caseinate solution. This is especially noticed in milk from older cows which has higher plasmin activity. Plasmin affects cheese making characteristics of milk such as the rennet coagulation time, curd strength, and curd syneresis. Mozzarella cheese made from late lactation milk, which contains higher amounts of plasmin, has inferior stretchability and melting ability. Proteolysis of casein by plasmin also increases the moisture content in cheddar cheese. Swiss cheese contains higher plasmin activity than cheddar cheese because the higher cooking temperature required for Swiss cheese destroys the inhibitors of plasmin and plasminogen activators. As a result, more breakdown of casein occurs during ripening of Swiss cheese as compared to cheddar cheese.
Thickening, gelation, and coagulation of milk occurs during storage. This is also attributed to the proteolytic activity either from milk proteases such as plasmin or proteases of bacterial origin. Proteolytic activity caused by plasmin has a particular effect on the taste of milk (appearance of bitter peptides). Bitter peptides produced by the action of proteases influence the flavor of UHT milk, UHT cream, and cheese.
Raw milk is refrigerated during its transport and during storage before processing. Refrigeration mainly suppresses the growth of acid producing bacteria, while psychotropic bacteria (which survive at cold temperature) such as Pseudomonas fluorescens can produce highly active proteases. Proteases from bacterial origin can produce undesirable changes in the milk and dairy products. Bacterial proteases predominantly affect k-casein, while ß-casein and as-casein are less susceptible to bacterial proteases.
Milk contains various proteolytic enzymes which degrade milk proteins. These proteases are either secreted into the milk during milk synthesis or originate from other sources such as bacteria or leukocytes. Plasmin is the major protease in milk. The level of plasmin activity is controlled by availability of the precursor plasminogen and by the levels of several activators and inhibitors. Plasmin activity in milk is affected by factors such as stage of lactation, breed of cow, disease condition, and nutritional and lactational effects. Breakdown of casein by plasmin results in off-flavor, bitterness and altered characteristics of dairy products. Milk also contains proteases derived from bacteria and leukocytes in the milk. Although these proteases are present in small amounts, they can degrade casein and affect the processing of dairy products. Breakdown of milk proteins by proteases affects milk clotting, cheese ripening, and flavor and texture of dairy products.