Discipline: quality; Keywords: Protein instability, Alizarol test, Pseudomonas, potassium, APC method, plasmien.

Milk flocculation and gelation describe the phenomena relating to protein instability that could lead to the rejection of milk for processing or to defects in long-life milks such as UHT milk. The term flocculation which is the topic here, applies to the protein precipitate that is formed in the test tube when ethanol unstable milk is subjected to the Alizarol test.  The ethanol concentration in the test varies from 68% to 76% ethanol and milk that flocculates is then declared unfit for further heat processing and rejected. Milk flocculation required studies at the cow feeding level, the bacterial contamination level and the enzymatic proteolytic level in milk. The cow feeding studies were done at Outeniqua and the microbiological/biochemical studies at UFS. 

In the study at Outeniqua, high potassium (K) levels due to pasture fertilization were hypothesized as being contributory. Thus, K levels were progressively increased in the concentrate mix. The pasture grazed was of high quality and contained a K content of 5.2%. With the supplementation, this resulted in a K content in the total diet of the cows on the high K treatment of more than 5%. The higher K levels in the diet significantly reduced the alcohol stability of the milk and also reduced the protein, lactose and MUN content in the milk. The reduced milk protein of 0.2% on the high K treatment compared to the low K treatment may result in a R0.20 to R0.30/ℓ decrease in milk price depending on the milk buyer. The high K level also resulted in reduced milk Ca, K, P and Mg. The lower milk P content may partly explain the reduced protein stability of the milk as P linkages are present in the structure of casein micelles and are crucial to maintain stability of milk protein. It was concluded that by reducing the intake of K, the problem of alcohol instability of milk should be alleviated. Farmers, therefore, should determine the mineral status of all paddocks on the farm. Additionally, they should limit K fertilization to the minimum required for plant growth, from paddocks with high K levels remove and ensile the material and avoid these paddocks, especially during warm weather. They should also not use chicken manure as fertilizer.

At the processing plant, the most important reason for flocculation (as determined by the Alizarol test) of milk entering the plant is contamination of equipment and pipe lines on the farm. Ineffective or inadequate cleaning of pipe lines will favour development of biofilms on milk contact surfaces. Psychrotrophs are mostly implicated as it was shown in the UFS work that they are the most important organisms affecting protein instability in fresh milk if contamination results. Pseudomonas spp., particularly Ps. fluorescens, was the prevalent genus at the time of flocculation. Psychrotrophic bacteria have the ability to proliferate at cold storage temperature of less than 7oC. While growing, these bacteria produce proteolytic enzymes which destabilize casein. The commonly used psychrotrophic count method employs incubation at 7oC for 10 days which means that by the time the results are known, the raw milk will already have been spoiled. The research group, therefore, focused on evaluating methods to rapidly and reliably detect psychrotrophic bacteria in raw milk.

The results from the project showed that the rapid qualitative Psychro-Fast test, which gives results within 48 hours, can be used to not only indicate the presence of psychrotrophic bacteria in raw milk, but the pink colour intensity can be used to indicate the degree of psychrotrophic bacterial contamination. For the detection of proteolytic psychrotrophic counts, the standard method casein agar (SMCA) or skim milk agar (SMA) can be used, but the SMCA will give more accurate results. The study further indicated that lower counts of the total bacteria, total coliforms and the Pseudomonas count of raw milk resulted in milk with a better quality which in turn resulted in delayed flocculation. Both the APC method and the Pseudomonas count may be used as reliable and rapid methods to indicate how fast the milk will flocculate.

As indicated above, proteolytic enzymes from external proteases from mainly psychrotrophic bacteria which may not have been killed before heat exposure have been shown as causative factors in the condition. Intrinsically, plasmin in the milk is another factor. It is important to firstly develop or apply suitable detection methods to establish the cause, and secondly to effectively distinguish between proteolysis induced by plasmin and proteolysis induced by psychrotrophic bacterial contamination. This was another focus at UFS.

The researchers developed computer assisted software to enable the use of the proteolytic peptide profiles obtained from the RP-HPLC methodology to establish protein peptide profile fingerprints for the different enzymes. The proteolytic enzymes investigated were the intrinsic plasmin and the proteases of Bacillus licheniformis and Pseudomonas fluorescens. The software created chromatograms representative of peptide peaks liberated by each proteolytic enzyme and distinguished satisfactorily between the bacterial protease profiles and the plasmin profiles present in the milk. Furthermore, it could also distinguish between the proteases of the two bacterial species tested. It was established that the sensitivity of the method should make it possible to detect whether the origin of the flocculation is from psychrotrophic bacteria (contamination) which can survive low temperatures, or whether it results because of heat induced proteolysis in the milk manufactured for extended shelflife.


Meissner, H.H., 2021. MSA: Summary of Research Progress with Milk Flocculation and Age Gelation 2017 to 2021. Report to DRDC Meeting on 25 August 2021, Milk SA, Pretoria.