Proteolysis in UHT Milk: Causes and Remedies
Authors: Ranvir Suvartan Gautam, Navan Sampath Kumar

What is UHT Milk

Ultra-high temperature (UHT) is one of the potential method involving short time heat treatment (~140–145°C for 2 sec) which renders milk effectively sterile, so that when packaged aseptically, makes products stable at ambient temperature for several months

Methods for UHT treatment of milk

There are two principal methods of UHT treatment:

1. Direct Heating

2. Indirect Heating

Direct heating systems: The product is heated by direct contact with steam of potable or culinary quality. The main advantage of direct heating is that the product is held at the elevated temperature for a shorter period of time. For a heat-sensitive product such as milk, this means less damage.

Direct heating system is further two type

  • Injection
  • Infusion
Injection Type: High pressure steam is injected into pre-heated liquid by a steam injector leading to a rapid rise in temperature. After holding, the product is flash-cooled in a vacuum to remove water equivalent to amount of condensed steam used. This method allows fast heating and cooling, and volatile removal, but is only suitable for some products. It is energy intensive and because the product comes in contact with hot equipment, there is potential for flavour damage.

Infusion Type: The liquid product stream is pumped through a distributing nozzle into a chamber of high pressure steam. This system is characterized by a large steam volume and a small product volume, distributed in a large surface area of product. Product temperature is accurately controlled via pressure. Additional holding time may be accomplished through the use of plate or tubular heat exchangers, followed by flash cooling in vacuum chamber.

Proteolysis: The breakdown of proteins or peptides into amino acids by the action of native milk alkaline proteinase (plasmin) and heat-stable extracellular bacterial proteinases produced by psychotropic bacterial.

Causes of proteolysis in UHT milk

Proteolysis in UHT milk can cause the development of bitter flavour 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. The gel that forms in UHT milk is a 3-dimensional (3D) protein matrix formed by the whey proteins, particularly β-lactoglobulin, interacting with casein, chiefly κ-casein, of the casein micelle. The major proteinaceous linkages that develop during the heat treatment result in formation of β-lactoglobulin-κ-casein complexes (βκ-complexes).

Enzymatic mechanism of gelation, according to (McMahon 1996),

The proteinases do not act directly on the βκ-complex but cleave the peptide bonds that anchor the κ-casein to the casein micelle, facilitating release of the βκ-complex. This dissociation of βκ-complexes from the casein micelles by proteinases is considered to be the 1st stage in a 2-stage mechanism of age gelation. The 2nd stage involves the subsequent aggregation of the βκ-complexes and formation of a 3D network of cross-linked proteins.

Non- Enzymatic mechanism of gelation, according to Andrews and Cheeseman 1972

Gelation caused by polymerization of casein and whey proteins by Maillard reactions that are promoted by higher storage temperatures

Lack of gel formation during storage of UHT milk at temperatures above 35 ᴼC does not corroborate their suggestion

Samel et al., 1971

    • Blockage of ε-NH groups of lysine residues in casein micelles of UHT milk prevents micelles from interacting with each other
    • May retard age gelation due to modification of the charge on the casein micelles According to another hypothesis
    1. Gelation of UHT milk results from changes in the free energy of casein micelles
    2. Differences in potential energy promote aggregation of the casein micelles,
    3. The extent of this depends upon the probability of contact and the number of low potential micelles both of which increase with storage time
    4. Micelle aggregation leads to increased viscosity of the UHT milk. Factors affecting the gelation behaviour of UHT milk
      1. Age of cow: Plasmin activity in milk increases with the age of the cow, plasmin activity in milk remains constant throughout lactation of first lactation cows, but increases during lactation in milk collected from older cows. As a consequence, the milk from older cows gels faster than that of young cows.
      2. Stage of lactation: UHT-treated early-lactation milk is more susceptible to age gelation than the corresponding late-lactation milk.
      3. Mastic: Mastitic milk (i.e. with high somatic cell count and plasmin activators more present in Somatic cell) subjected to UHT treatment is more susceptible to gelation than normal milk
      4. Season: Seasonal variation in the composition of milk may indirectly affect the gelation behaviour of UHT sterilized milk. Summer milk has been reported to give a more stable UHT product than winter milk
      5. Storage temperature : In general, gelation occurs more readily at room temperatures than at low
        6. Ways to Improve Shelf Life of UHT Milk

        1. Quality of raw milk
          • Use high quality raw milk, milk with a high pre-processing microbial count is more susceptible to gel formation than milk with a low count
          • Storage of raw milk at low temperature (<4 ◦C) for a minimum period of time (≤48 h) minimizes growth of psychrotrophic bacteria and, consequently, the amount of extracellular bacterial proteinases produced in the milk before heat treatment
          • Use of milk with a low somatic cell count ensures a minimum level of plasmin and plasminogen activators in the milk
          • Raw milk bacterial count is <25000 CFU/mL, then the raw milk SCC will be the most important determinant of shelf life in pasteurized extended shelf life milk with respect to development of off-flavors
          1. Low-temperature inactivation of proteinases
            • The low temperature inactivation treatment of 55 C for 1 h has been proposed as a feasible means to inactivate these heat-resistant proteases in sterile skim milk.
            • Effectiveness of such “low-temperature-inactivation” is independent of proteinase concentration and does not significantly alter flavor of milk
              • The method can be applied before or after sterilization
              • Effective when used in milk at least 1 d after UHT treatment
              • LTI at 55 ᴼC is only effective up to 60 min; thereafter inactivation rate of LTI when combined with UHT treatment is similar to UHT treatment alone
              • Combination of LTI and UHT sterilization can prolong the shelf life (up to 3 times) of sterile skim milk containing psychrotrophic bacterial proteinase
              • LTI did not alter the flavour or protein content of the milk and k-casein was affected by the protease
              • Such a process, if feasible on a commercial scale, could offer the best solution to the problem presented by heat-stable proteases
              • LTI treatment of 55 C for 1 h offers the best solution to the problems presented by heat-resistant proteases in UHT sterilized skim milk and is feasible commercially under close control
            1. Heat treatment during preheating and sterilization
              • Adequate heating required for denaturation of β -lactoglobulin and complexation with casein. Such high heat treatment also inactivates plasmin. For the same bactericidal effect, indirect heating produces milk that is more stable to gelation than produced by direct heating
              1. Addition of sodium hexametaphosphate (SHMP)
                  • Addition of 0.05% calcium chloride or 0.1% SHMP to milk before UHT milk considerable increase in stability with no gelation evident after 500 d at 25⁰C.
                  • Addition of low level SHMP facilitate bridging between ionized ionic bond that would not otherwise form an ionic bond. It hold the κ-casein more tightly to the casein micelle that delay release of the βκ-complex, thus retarding gelation
                1. ISI Heater
                    • 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 heater, 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
                    1. Sulfhydryl (SH) group-blocking agent
                      • N-ethylmaleimide Added to milk before heating inhibits denaturation of whey proteins and interaction of these proteins with caseins. Hong (1984) showed that UHT milk, containing 0.5 g/L NEM, and processed by direct heating, gelled later (at 52 wk) than indirectly heated milk with the same additive (at 18 wk); this was in contrast to the corresponding directand indirect-processed control milks that gelled at 18 and 40 wk, respectively. The reason for the opposite effects of NEM in the 2 milks is unexplained.
                      1. High-pressure treatments
                        • The application of high pressure, rather than heat, to food enables destruction of microorganisms without causing significant changes to the colour, flavour and nutritional attributes of the food
                        • Plasmin system is very pressure stable at room temperature
                        • Synergistic effect of pressure and temp treatment observed in the range 300 to 600 MPa and 35 to 65ᴼC and stabilization above 600 MPa Advantages of HPP
                        • Thermal degradation of heat sensitive foods can be avoided.
                        • High retention of colour, aroma and nutritional value.
                        • No re-contamination as treatment is given after final packaging of product.
                        • Environmental friendly as no chemicals added.
                        • Positive consumer acceptance
                        • Operates at room temperature
                        1. High pressure homogenization (HPH) or ultra-HPH
                          • Similar Principle of conventional homogenizers except it works at higher pressures (up to 400 MPa)
                          • If milk treated at 200 MPa at 30⁰C had the longest microbial shelf life (21 d)
                          • Changes colour, viscosity, pH, acidity, texture, and mouthfeel of milk Conclusion
                            • Advantages of UHT milk include reduced energy consumption, extended shelf life, and ambient storage and distribution conditions
                            • Age gelation is a major factor limiting the shelf life of UHT milk
                            • Proteolysis, by native milk plasmin or bacterial proteinases, accelerates gelation by facilitating release of the complex from the micelle
                            • Shelf life of UHT milk by using techniques such as ISI-heating, LTI, membrane processing, UHPH


                            References:

                            1. www.ndsu.edu/pubweb/~mcclean/plsc431/students98/fleck.htm
                            2. Human Gene Therapy: Current Opportunities and Future Trends - Page 176 - By G. M. Rubanyi



                            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.