Diagnosing the cause of proteolysis in UHT milk
Introduction
Age gelation of UHT milk is a major concern of the dairy industry since it limits the shelf-life and market potential of the milk. One of the causes of age gelation is proteolysis of caseins by either native milk proteinase or bacterial proteinase, or both (Harwalkar, 1992; Datta & Deeth, 2001).
Proteolysis of UHT milk causes the development of bitter flavours and leads to increases in viscosity, with eventual formation of a gel. These changes are caused, or at least accelerated, by hydrolysis of caseins, releasing the β-lactoglobulin–κ-casein complex (βκ-complex), formed during heat treatment, from the micelle. The released complex subsequently aggregates and forms a three-dimensional network of cross-linked proteins, which causes the milk to gel (McMahon, 1996). Any processing or storage conditions of UHT milk that accelerate (or delay) release of the βκ-complex from the casein micelle will ultimately accelerate (or delay) age gelation of UHT milk.
Proteolysis of UHT milk has been attributed to the natural milk alkaline serine proteinase, known as plasmin, and to proteinases produced by psychrotropic bacterial contaminants of raw milk. Milk plasmin is associated with the casein micelle and the milk fat globule membrane in milk, and is highly heat-resistant (Visser, 1981). It exists in milk in both its active form as well as its enzymatically inactive precursor form, plasminogen. Its activity in milk is controlled by a system of enzyme activators and inhibitors (Richardson, 1983). Plasminogen is converted to plasmin by plasminogen activators (PAs) during storage of UHT milk. The activity of the plasminogen activators increases after heat treatment due to heat inactivation of their inhibitors (Richardson, 1983). Plasmin, plasminogen and the PAs are more heat-stable than the inhibitors. The plasminogen activators are only slightly inactivated by UHT processing conditions. Plasminogen is more heat-stable than plasmin, which some researchers have found to be almost completely inactivated by (indirect) UHT processing (Cauvin, Sacchi, Rasero, & Turi, 1999).
The milk proteins most susceptible to attack by milk plasmin are β-casein (3 times more susceptible than αs1-casein) and αs2-casein and, to a lesser extent, αs1-casein, while extracellular proteinases of microbial origin attack predominantly κ-casein with the formation of para-κ-casein (Snoeren & Riel, 1979), followed by extensive nonspecific hydrolysis (Law, Andrews, & Sharpe, 1977).
Both plasmin and bacterial proteinase can accelerate age gelation. They do not hydrolyse the βκ-complex, but they hydrolyse the proteins that attach the βκ-complex to the micelles, thus facilitating release of the βκ-complex from the micelles. When the βκ-complex is primarily attached to αs1-casein, any enzymatic reaction of αs1-casein promotes the release of the complex and subsequent gelation (McMahon, 1996).
It is evident that only trace levels of proteinase are required to cause gelation of UHT milk during storage. For example, Richardson and Newstead (1979) reported that UHT milk containing as little as 1 ng bacterial proteinase/mL may have a shelf-life of only 3 months.
In order to address a problem of proteolysis in UHT milk, it is necessary to determine the nature, and hence origin, of the enzyme(s) responsible. This paper discusses the use of reversed-phase (RP)-HPLC and the fluorescamine method for analysing the peptides soluble in trichloroacetic acid (TCA) and at pH 4.6, and how these results enable differentiation between the products of plasmin and bacterial proteinase action on milk proteins.
Section snippets
Proteinases
Bacterial proteinase was obtained by culturing Pseudomonas aureofaciens (API 20NE V5.1 Code 1057547; Deeth, Khusniati, Datta, & Wallace, 2002) in UHT skim milk at 30°C for 3 d and centrifuging at 1000×g for 30 min. The supernatant containing the extracellular proteinase was used as the Pseudomonas proteinase. Plasmin was from Sigma Chemical Company (Cat. No. P-7911).
Fresh UHT milk
Raw milk, with total bacterial count less than 5×103 cfu/mL, was collected from the University of Queensland dairy herd within 24 h of
Appearance
During incubation at 40°C for 3 h, Pseudomonas proteinase caused the formation of a hard gel, while plasmin caused partial digestion of the casein as evidenced by turbidity in the digested sample. The clarified samples indicated extensive breakdown of casein while the gelled samples indicated limited proteolysis. The hard gel is similar to that caused by rennet, which, like Pseudomonas proteinase, preferentially hydrolyses the hydrophilic glycomacropeptide from κ-casein on the outside of the
Conclusion
The peptides produced by bacterial proteinase are less hydrophobic and elute early in the RP-HPLC, while the peptides produced by the native milk plasmin are more hydrophobic and elute later. Acid precipitation to pH 4.6 and with 12% TCA allows the peptides to be fractionated in a similar manner. Determination of the peptides in the fraction soluble at pH 4.6, using the fluorescamine method, provides a measure of the total amount of proteolysis from both plasmin and bacterial proteinases, while
Acknowledgements
The authors wish to thank Miss Katherine Raymont for her excellent technical assistance, and the Australia Dairy Research and Development Corporation for financial support.
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