Discipline: quality; Keywords: Phosphate, Alizarol test, Psychrotropic bacteria, NIR, biofilms, gelation, UHT milk.

Priority 1: The literature shows that almost any factor which has an influence on phosphate metabolism will affect the stability of milk. In the cow heat stress can reduce the availability of phosphate in the udder by up to 50%. It is also well known that the addition of phosphate salts can improve the stability of milk. One of the questions which should be addressed is whether milk not passing the 72% alizarol test has less phosphate than milk passing the 80% alizarol test. If so, it can be expected that low availability of phosphate plays a role during milk synthesis. This should then be established by testing different levels of P and Ca during periods of heat stress or in the feed. It will also be worthwhile to add phosphate to fresh milk to determine whether flocculation is really associated with instability of the Ca/ PO4/casein complex. It is interesting to note that addition of 0.05% Na-phosphate + 0.05% Na-citrate to milk destined for UHT (to increase the heat stability during UHT processing) prevents milk from being precipitated in the tubes. The success though depends on the initial quality of the fresh milk. 

Priority 2: The most important reason for flocculation (as determined by the Alizarol test) of milk entering the processing plant is contamination of equipment and pipe lines on the farm. Mostly, this results because of poor or irregular cleaning, or not adhering to the specifications of the cleansing agent. In the minority of cases it could also result because the cleansing agent is not really efficient (an example: Psychrotrophic bacteria such as Pseudomonas are resistant to quaternary ammonium compounds). Ineffective or inadequate cleaning of pipe lines will favour development of biofilms on milk contact surfaces. Psychrotrophs are mostly implicated. From our previous work, it was also shown that they are the most important organisms affecting protein instability in fresh milk if contamination results. It can prove useful to incorporate accelerated psychrotrophic bacterial plate counts, tests for Pseudomonas fluorescens and outomated bacterial counts based on Near Infrared (NIR) principles as well as rapid tests for proteolytic activity in milk in quality testing regimes of the raw milk. Such tests will enable the accurate prediction of raw milk shelf-life and greatly supplement the alizarol platform test.

It should be taken into account that psychrotrophs are resistant to low temperatures, that they can form biofilms on milk contact surfaces and that this can make them even more resistant to cleansing agents. Therefore, if they are allowed to increase in biofilms and thereby in fresh milk, they will produce proteolytic enzymes which are associated with the destabilization of the casein complex. Normally, such destabilization will be detected by the alizarol test, but in these circumstances if the test is not strict enough contaminated milk can be accepted and result in fouling of heat treatment equipment in the processing plant. Although the bacteria themselves will be killed during pasteurization, their enzymes are resistant, even to the high temperature in UHT processing resulting in possible gelation in the UHT milk. The proposal is then that, in addition to possibilities mentioned above, the sensitivity of psychrotrophs to cleansing agents be tested with variables that include agent type, concentration, speed of washing/cleansing, temperature and exposure time. In this context, it does appear that the presence of specific types of psychrotrophs and biofilms at farm level may be one of the most important factors which have not received sufficient attention. This could be preceded by a comprehensive literature review.                                                                                   

 Priority 3: Although it is accepted that high numbers of psychrotrophic bacteria are the main cause of flocculation, the intrinsic proteolitic enzyme plasmin in milk does play a significant role in the destabilization of the casein complex. Plasmin is normally in the milk in the form of the inactive pro-enzyme plasminogen, but can be converted to plasmin by particular activators. One of these is free fatty acids such as oleic acid which in our work thus far has proved to be a major activator. These fatty acids are released by milk lipase and also lipase-type enzymes excreted by psychrotrophs. All activators thus far have not been identified by our research and further studies in this regard should be preceded by a thorough literature investigation. Plasmin is also resistant to pasteurization and UHT treatment and therefore may cause gelation in UHT milk. Gelation is not the same as flocculation. Gelation can occur over time and affects the shelf life of long-life milk. It results because of proteolytic enzymes excreted by psychrotrophs or by plasmin through activators as explained above. This should be investigated. A further instigator of gelation is spore-forming Bacillus spp which survive pasteurization and can establish themselves as biofilms in tanks, fillers and pipe lines in the processing plant. The cells which then gather in the heat treated milk can grow unrestrained at room temperature within the packed UHT milk on the shelf and their proteolytic enzymes may then cause gelation.