Inactivation of Proteinase during Milk Processing
Authors: Ranvir Suvartan Gautam , Navan Sampath Kumar

About 60 indigenous enzymes have been reported in normal bovine milk. The indigenous enzymes are constituents of the milk as excreted and arise from three principal sources: (a) the blood via defective mammary cell membranes; (b) secretory cell cytoplasm, some of which is occasionally entrapped within fat globules by the encircling fat globule membrane (MFGM); and (c) the MFGM itself, the outer layers of which are derived from the apical membrane of the secretory cell, which in turn originates from the Golgi membranes; this is probably the principal source of the enzymes. Thus, most enzymes enter milk owing to peculiarities of the mechanism by which milk constituents, especially the fat globules, are excreted from the secretory cells. Milk does not contain substrates for many of the enzymes present, while others are inactive in milk owing to unsuitable environmental conditions such as pH.

Enzymes used in Dairy Industries The application of enzymes (proteases, lipases, esterases, lactase, and catalase) in dairy technology is well established. Proteases of various kinds are used for acceleration of cheese ripening, for modification of functional properties and for modification of milk proteins to reduce the allergenic properties of cow milk products for infants. Lipases are used mainly in cheese ripening for development of lipolytic flavors.


“Proteinases are enzymes which carried out hydrolysis of peptide bonds of proteins in to peptides & amino acids”. Milk contains at least two proteinases, plasmin (alkaline milk proteinase) and cathepsin D (acid milk proteinase) and possibly several other proteolytic enzymes, e.g., two thiol proteinases, thrombin, and an aminopeptidase. In terms of activity and technological significance, plasmin is the most important of the indigenous proteolytic enzymes and has been the subject of most attention


Milk contains the complete plasmin system: plasmin, plasminogen, plasminogen activators (PAs) and inhibitors of PAs and of plasmin. Plasmin itself is a heat-stable enzyme that survives pasteurization and many UHT processes. The inhibitors present in fresh milk are heat labile, whereas the activators are known to be heat stable. Consequently, heat treatment of milk alters the natural balance between the activators and inhibitors in favor of the activators. This can lead to enhanced proteolysis in heated milk. This system enters milk from blood and plasmin activity increases during a mastitic infection and in late lactation, when there is an increased influx of blood constituents into milk. All components in the PL system work together to regulate the proteolytic activity of PL. In milk, there is about four times as much plasminogen as plasmin and both, as well as plasminogen activators, are associated with the casein micelles, from which they dissociate when the pH is reduced to ~4.6. The inhibitors of plasmin and of plasminogen activators are in the milk serum (Fox 2015). Plasmin in milk occurs mainly as inactive precursor plasminogen, activated through proteolysis by plasminogen activators which are known to be also very heat stable. Bulk raw milk contains 0.07–0.15 µg/ml -1 plasmin and 0.7–2.4 µg/ml -1 plasminogen. Milk plasmin is associated with casein micelles and milk fat globule membrane The presence in the fat globule membrane is actually due to presence of casein in the membrane. Plasminogen is also associated with casein micelles which also play a roll as an immobilization matrix for activation of plasminogen.

Action of plasmin on caseins

  • Plasmin is highly specific for peptide bond
  • Plasmin specifically break peptide bond at carboxylic group of lysine or arginine
  • Plasmin more susceptible for ß-Casein among the other milk proteins
  • Plasmin also act on α-s2 or α-s1-caseins
  • Plasmin very less susceptible towards the k–casein
Bacterial proteinases

Three main sources of bacterial contamination have been found for raw milk: the interior of the udder, cow’s teats, and milking and storage equipment. The contaminants are almost entirely psychrotrophs, mainly species of Pseudomonas. Proteinase production by psychrotrophs is normally at a maximum in the late exponential or stationary phase of growth. The majority of Pseudomonas species produce only one type of proteinase, typically a neutral zinc metallo-proteinase with pH optimum of 6.5-8.

Advantages and Disadvantages of plasmin in milk and milk products


  • Primary proteolysis in high cooked cheese as well as some extent in low-cooked cheeses
  • Activity high in high cooked cheese as compare to low-cooked cheeses
  • Activity less in cheeses made from UF-concentrated milk
  • In cheese it gives characteristic flavour

  • Development of off flavour in pasteurized milk as well as UHT milk
  • In cheese excess proteolysis by plasmin cause bitterness
  • proteinases responsible to increase rennet coagulation time of milk in cheese
  • Formation of a gel during storage UHT milk What is the need for inactivation of plasmin?
1. The enzymes responsible for the proteolysis in UHT milk are the native milk alkaline proteinase, plasmin and heat-stable extracellular bacterial proteinases produced by psychotropic bacterial. The majority of the psychrotrophic bacteria (excluding Bacillus spp.) are destroyed by pasteurization, but they produce extracellular enzymes that are extremely thermostable. Proteolysis in UHT milk can cause the development of bitter flavor and lead to an increase in viscosity, with eventual formation of a gel during storage, which is a major factor limiting its shelf life and market potential.

Proteinase inactivation during milk processing

  • Heat Treatment: Pasteurized at various temperatures (65–75°C) and times (15–30 s) Commercially sterilized UHT processing (135–150°C for few seconds)
  • Thermal treatment associated with pasteurization of milk has been shown to increase PL levels and PG activation. Further heat treatment has been shown to reduce PL levels
  • This initial increase in PL activity has long been attributed to inactivation of PI and PAI
  • Possible explanations for the decrease in PL levels with further heat treatment have included interactions of PL, PG, or PA with denatured whey proteins
  • Plasmin and PG can fully survive pasteurization conditions and are somewhat resistant (20–40% remaining activity) to certain UHT heat treatments
  • The temperature range for denaturation of PG is between 50.1°C and 61.6°C
  • In this temperature range, PG loses its naturally occurring tertiary structure but is not yet inhibited
  • Above 100oC, the time needed to obtain 90% of enzyme inactivation is ~100 s
  • It partly survives UHT sterilization and is inactivated by heating at 80°C x 10 min at pH 6.8
  • Inactivation proteases by innovative steam injector (ISI)
    • The ISI heater is a new type of steam injection that enables fast heating (shorter than 0.2 s holding time) and high temperatures (150 to 180 ◦C)
    • In the ISI, the product is pumped through a pipe with a narrow end (nozzle, 1 to 2 mm)
    • The wall of this pipe contains several small openings through which high-pressure steam is injected enabling very fast heating of the product
    • The milk can be heated at 80◦C before (preheated) or after (postheated) the heat treatment with the ISI
    • After heating, the product can be instantaneously cooled using flash cooling
  • Protease inactivation by Pulse Electric Field
PEF treatment will generate Minimal amount heat, depending on the selected dosage and media used. The induced heat will in turn cause increase in temperature that, when high enough, may cause thermal inactivation of enzymes.



Treatment conditions



Plasmin Simulated milk ultrafiltrate 15–45 kV/cm, 10–50 pulses Up to 90% inactivation Vega-Mercado et al., 1995
Protease from P. fluorescens Casein solution (CS) Skim milk (SM) 14–15 kV/cm, up to 98 pulses SM: 60% inactivation CS: no significant effects Vega-Mercado et al., 1995
Protease from B. subtilis SMUF Skim milk 16.4–27.4 kV/cm, up to 100 pulses SMUF: 10% inactivation SM: no significant effects Bendicho et al., 2001
  • Protease inactivation by Ultrasound
  • Ultrasound is a form of energy generated by cyclic sound pressure waves of frequencies that are greater than upper limit of human hearing range typically above 20 kHz
  • The application of ultrasound waves is called sonication Principle
  • When ultrasound waves are passed through a liquid substance alternating regions of high and low pressure i.e. compression and expansion which induce cavitation and form gas bubbles
  • These gas bubbles expand because of increased gas diffusion during the expansion cycle
  • Rapidly condense at one point when the energy of the ultrasound waves is insufficient to retain the vapour phase in the gas bubbles
  • These gas bubbles expand because of increased gas diffusion during the expansion cycle and rapidly condense (implosion) at one point when the energy of the ultrasound waves is insufficient to retain the vapour phase in the gas bubbles
  • The condensed molecules collide violently, resulting in shock waves (mechanical or shear forces), regions of high temperature and pressure, and generate free radicals through water sonolysis
  • In the fluid that is subject to sonication, cavitation exerts rotational forces and stresses on the cells in the vicinity of the gas bubbles
  • These result in a microscopic fluid movement called micro streaming
  • Cavitation is thus able to increase heat and mass transfer


for 2.5 min

Reduced Protease activity in Skim Milk Medium

Reduced Protease activity in Reduced-Fat Milk

Reduced Protease activity in Whole Milk

107 μm




119 μm




133 μm




  • High Pressure Processing (HPP)
  • High-pressure–induced enzyme inactivation is a very complex phenomenon
  • It involves a series of events like
  • Formation or disruption of numerous interactions
  • Change in the native structure of enzymes by folding and/or unfolding
  • Under the high-pressure environment, the mechanism of enzyme inactivation can be hypothesized similar to protein denaturation
  • The application of pressure may induce reversible (Possibility B in Figure 1) or irreversible (Possibility A in Figure 1) and partial or complete unfolding of the native structure of the enzyme
  • This eventually leads to a change in enzyme activity as its specificity is related to the structure of its active site
  • In general, enzymes are much less affected by high-pressure treatment than by heat
  • Plasmin very pressure stable at room temperature
  • Synergistic effect of pressure and temp treatment at 300 to 600 MPa and 35 to 65ᴼC
  • Stability of the plasmin system at pressures above 600 MPa
  • High-pressure thermal inactivation of plasmin in the 300 to 600 MPa and 36 to 65 ᴼC range
  • Inactivation proteases low-temperature treatment Inactivated heat-resistant enzymes:
    • Low temperature 55 ᴼC for 30 to 60 min potential treatment to inactivate the protease in milk
    • Proteinase undergoes a unique conformational change
    • Followed by aggregation of altered proteinase with casein
    • Form an enzyme-casein complex that causes inactivation of the enzyme
  • LTI can be applied before or after sterilization
  • Effective when used in milk at least 1 d after UHT treatment
  • Heat-resistant proteases undergo a unique inactivation at sub-UHT and suggested that heating skim milk at 55 ᴼC for 1 h might be an effective method for inactivating these proteases
  • The average inactivation by LTI was 87 to 90%.

  • Milk & milk product quality mostly affected by endogenous & exogenous milk enzymes specially proteinases
  • It plays an important role in characteristic flavour development in high cooked cheeses and also some extent in low cooked cheeses
  • It also plays an important role in detoriation of the quality & shelf of UHT milk.
It can be controlled by proper handling, appropriate storage temperature, proper processing conditions and appropriate inactivation methods

About Author / Additional Info:
I am currently pursuing PhD in Dairy Chemistry from National Dairy Research Institute Karnal. I have also worked with Mother Dairy as a Senior Executive for 2 years.