DISTINGUISHING BETWEEN BACTERIAL PROTEASE AND PLASMIN INDUCED MILK FLOCCULATION.

Milk flocculation is a major problem in the dairy industry and consequently it is the subject of an intensive research programme funded by Milk SA. It is observed as a physical change in milk when exposed to extreme destabilizing conditions such as low storage temperatures but more often observed after heat exposure as introduced in extended shelf life processing. The condition is initiated with changes at the surface of casein micelles, resulting in the micelles losing their colloidal stability and forming a three-dimensional protein network. The end result is decreased fluidity, increased viscosity and the formation of flakes, which results in the milk being rejected in the market.

Proteolytic enzymes such as plasmin in the milk and external proteases from psychrotrophic bacteria which may not have been killed before heat exposure are postulated as causative factors in the condition. 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 the goal of the study by the authors referenced below.

The authors developed computer assisted software to enable the use of the proteolytic peptide profiles obtained from the relevant RP-HPLC methodology to establish protein peptide profile fingerprints for the different enzymes. The computer assisted software technique was used since the differences in peaks and form within the chromatograms of the RP-HPLC are not always visible with the naked eye. Chromatograms of 20 repetitions of a specific experiment were drawn into the programme to construct an average chromatogram for the specific experiment. This was repeated using the proteolytic enzymes investigated (plasmin, and the proteases of Bacillus licheniformis and Pseudomonas fluorescens). The computer assisted 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.

Bottom line: The sensitivity of the method makes 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.

Reference:

Hattingh, A & Myburgh, J., 2017. Computer assisted identification of proteolytic peptide profiles using MILQC software. In: Proc. of the 50^th SASDT Symposium “Dare to Dairy”, Kievitskroon, Pretoria, 8-11 May 2017.