Discipline: Milk quality Keywords: age gelation, UHT milk, Alizarol test, psychrotolerant bacteria, Pseudomonas, rapid tests. 

Age gelation is a process in which there is formation of a three-dimensional protein network within UHT milk which occurs during storage, and is marked by increasing viscosity before observable gelation. In South Africa, the dairy industry loses millions of Rands annually as a result of the condition in UHT milk. As such, there is a need to study and evaluate the possible correlation of variables that may contribute to the occurrence of age gelation in UHT milk. Therefore, the aim of this project by the authors cited below were to determine whether the alizarol test, mineral concentration, PsychroFast test, accelerated psychrotolerant count, Pseudomonas count and the proteolytic activity of milk samples can be used to rapidly determine the instability of milk which may result in the age gelation of UHT milk.

Milk samples from six farms in three geographical different regions, which were sampled each month from August until November, were used to determine the following correlations: a) alizarol results with mineral (ionic calcium, calcium, magnesium, sodium, potassium, phosphate) concentrations, b) alizarol results with the microbial methods (PsychroFast, accelerated psychrotolerant count, Pseudomonas count), and c) alizarol results with the proteolytic activity in the milk samples. All the milk samples were evaluated with all the mentioned methods, on the day of arrival at the laboratory (fresh) and on the day that the milk samples flocculated with the alizarol test (flocculated).

The alizarol test and its correlation with other methods used in this study, were regarded the most rapid (within minutes) and inexpensive method to predict the microbial quality of the raw milk. Although the fresh alizarol test results were not correlated with the fresh proteolytic activity of milk samples, it did correlate with the flocculated proteolytic activity, indicating that the alizarol method can be used to predict high proteolytic activity. 

When the minerals, ionic calcium, calcium, magnesium, sodium, potassium and phosphate were evaluated as possible indicators of protein instability using the alizarol test, it was clear that increased concentrations of ionic calcium played the most important role in milk being susceptible to age gelation because of its relation with the casein micelle. According to Anema (2019), when the pH is lower than 6.65 and/or the ionic calcium level is higher than 2.7 mg/100 mL (1.5 mM) at the time of UHT processing, calcium-induced aggregation of ĸ-casein-depleted casein micelles may occur, causing sedimentation in UHT milks very quickly during storage. 

Psychrotolerant bacteria, especially Pseudomonas spp., grow at refrigeration temperatures and produce heat-stable proteolytic enzymes that survive the UHT process and then have the ability to cause age gelation of UHT milk. This study evaluated the PsychroFast test, the accelerated proteolytic psychrotolerant count and the Pseudomonas count as methods that can rapidly determine the amount of psychrotolerant bacteria present in the raw milk. Of the three microbial methods evaluated, the PsychroFast was a rapid (within 30 hours), inexpensive, qualitative method to determine the psychrotolerant bacterial quality of raw milk. It also correlated relatively well with the alizarol test results and the flocculated proteolytic activity results. Both the accelerated proteolytic psychrotolerant count and Pseudomonas count were regarded excellent methods to determine the psychrotolerant quality of the milk and will give results within 48 hours. These two methods also correlated with the fresh proteolytic activity.

When the azo-casein and PierceTM proteolytic activity methods were validated, the commercial PierceTM method were the best in terms of linearity, sensitivity and precision and the azo-casein method was not sensitive enough for the determination of proteolytic activity in the milk samples. Although the PierceTM method gave excellent, rapid (on the day of arrival at the laboratory) results in terms of proteolytic activity, it might be considered a costly method (± R4000.00/50 samples of milk).

There was no correlation between the fresh alizarol test results and the fresh proteolytic activity, which indicated that the alizarol test will not be able to predict whether the fresh milk has high concentrations of proteolytic enzymes. However, there was a significant (p < 0.001) and good correlation (0.6132) of the fresh proteolytic activity with the flocculated proteolytic activity. This indicated that, if proteolytic enzymes were present in the raw milk, the concentration of the proteolytic activity will increase during storage of the milk at 4 – 7o C and may lead to age gelation of UHT processed milk.

In conclusion, the alizarol test, PsychroFast test, accelerated proteolytic psychrotolerant count and Pseudomonas count methods evaluated in this study, may all be used by UHT processors to rapidly determine the psychrotolerant bacterial quality of the raw milk before UHT processing. If the results of these methods indicate normal pH with the alizarol test, a white to light pink colour in the PsychroFast test and accelerated proteolytic psychrotolerant counts and Pseudomonas counts of less than 2 log cfu/mL (< 100 cfu/mL), the milk may be UHT processed. Otherwise, it is recommended that the milk should only be pasteurized. The study also stressed the importance of the quality of the raw milk in terms of psychrotolerant bacteria and their proteolytic enzymes. It is important that the milk, from production on the farm until it reaches the UHT processing facility, have as low as possible psychrotolerant counts, that the cold chain stay intact until the milk reaches the UHT processing facility and that the time from production of the milk until processing of the milk, be as short as possible.


Celia J Hugo, Prof A Hugo, Lesego Xaba, Lianca Uys, Lican Ndhlovu & Mbali Jiyane, 2022.  Evaluation and validation of methods for the detection of psychrotolerant bacteria and proteolytic enzymes in milk. FINAL PROJECT REPORT. Milk SA Project number: PRJ-0298-2021