Discipline: quality; Key words: age gelation, UHT milk, plasmin. Proteases, psychro-tolerant bacteria, free fatty acids, chemicals, gases.
The Dairy Industry loses millions of Rand per annum due to age gelation of milk that reduces its shelf life. Age gelation can be induced by anything that has an impact on the suspension of the negative charge on the casein micelle during storage of long life (UHT) milk. This includes the enzymatic or chemical processes that occur during storage. The enzymatic processes involve the action of plasmin (native enzyme) or heat stable microbial proteases. With regard to the latter, psychro-tolerant bacteria, predominantly the Pseudomonas and Bacillus species, start to grow in the milk and produce protease enzymes during storage at the processing plant or on the farm. In addition, at the processing plant, inactive plasminogen in milk can be activated to plasmin protease during storage at 5°C. The heat stable proteases are not destroyed during UHT treatment and remain active. This can result in gelation being observed within a few weeks after storage of UHT milk. In order to implement means to combat the age gelation problems, knowledge of plasminogen activators that mediate the conversion of plasminogen to plasmin after heat treatment is required. This was the objective of the study cited below.
Throughout the study, it was clear that milk-fat plays an important role in the susceptibility of casein towards proteolytic attack. It was evident that UHT fat-free milk is more susceptible to bacterial protease than UHT low-fat and full-cream milk. In contrast, plasmin protease hydrolysed UHT fat-free, low-fat and full-cream milk equally. During the treatment of milk fat with Pseudomonas lipase enzyme, it was evident that the free-fatty acids (FFA’S) released during fat hydrolysis activate plasminogen to plasmin protease. From these findings, further investigations were performed to study the individual and combined roles of FFA’s on the activation of plasminoged to plasmin protease. Surprisingly, it was found that most of the FFA’s between C4-C18:1 had the ability to activate plasminogen to plasmin. This activation by FFA’s is astonishing, and it will bring in a totally new approach towards the understanding of the age gelation phenomenon.
Often milk is stored in the cold chain at temperatures of the order of 5°C. According to enzyme kinetics, low levels of casein hydrolysis should occur under these conditions in comparison to room temperature. However, controversy exists in the literature. In this study, it was found that proteolysis by plasmin protease (activated by KIO3) occurs to a greater extent at refrigeration temperature than at room temperature. Thus, it is clear that not only psychro-tolerant bacterial protease results in proteolysis at lower temperatures, but native plasminogen can be activated during storage under refrigeration temperatures and as a result, plasmin can effectively contribute to age gelation. To inhibit the proteolytic attack, chemicals and gases were evaluated. EDTA has an inhibitory effect on Bacillus protease due to its ability to bind metals (Bacillus protease is a metallo-enzyme) and therefore inhibit enzyme activity. In the case of plasmin, which is a serine enzyme, EDTA had no impact. However, SHMP and CaCl2inhibited both Bacillus protease and plasmin protease. Sodium citrate, on the other hand, enhanced the activity of both plasmin and Bacillus protease.
However, the addition of additives/chemicals is only of academic interest as the addition thereof is prohibited. An alternative option is to bubble gases through the milk. Both Bacillus protease and a plasmin protease were retarded mainly by CO2and He in raw full-cream milk. Nitrogen gas, on the other hand, was mainly effective against proteolytic attack induced by bacterial protease (Bacillus) and plasmin protease more in UHT fat-free milk than in UHT full-cream milk. The effectiveness of N2 is however short-lived (only effective up to one week) and thus not ideal. It is also doubtful whether it can be practically applied in the usual milk storage and transport lines.
S. Dube, 2019. Age gelation within UHT milk. Master of Science (Food Science) thesis, Department of Microbial, Biochemical and Food Biotechnology, UFS, Bloemfontein.