Discipline: quality; Key words: milk flocculation, protease activity, Bacillus, plasminogen, heat treatment.

The investigation into the causes of milk flocculation is an important R & D Programme of Milk SA. In the contribution of 2017-12-08 attention was given to the influence of fat content of milk on protease activity and the progress on tests to study the success of various chemicals and gases as methods to reduce the activity. In this contribution the protease activity of more bacteria species was investigated together with activation of plasminogen by heat treatment. The protease activity results in protein breakdown and depending on severity and time, may result in flocculation. These studies are done at the UFS with Dr Koos Myburgh as project leader.

Objective 1: To determine RP-HPLC profiles of proteases from a wider range of psychrotrophic bacteria. The goal was to determine, in addition to profiles already obtained from Bacillus and Pseudomonas strains, HPLC enzyme profiles from a wider range of proteolytic psychrotrophic bacteria. Presumably this should enable better comparisons between microbial and plasmin activity profiles.

Various bacterial species which produce heat-resistant proteases were used in order to cultivate proteolytic enzymes. Protease extracts were produced from Bacillus licheniformis, Pseudomonas fluorescens, Pseudomonas fragi, Bacillus cereus and Bacillus subtilis. The results showed that all the cultivated proteases can be distinguished from one another since each protease exhibited unique HPLC peaks.  The main difference between the peptide profiles for Pseudomonas and Bacillus proteases was that characteristic peaks for Pseudomonas proteases were mainly at elution times between 5-45 minutes whereas for Bacillus proteases the unique peaks were at elution times between 45-75 minutes.  It was, therefore, concluded that the cultivated proteases consist of their own unique peptide profile with distinguishable differences, indicating that Pseudomonas and Bacillus proteases can be successfully distinguished from one another.

Objective 2: To determine the possible activation of plasminogen by psychrotrophic microbial proteases and peptides. This should provide more knowledge of whether and to what extent milk plasminogen may be activated by microbial proteases and bioactive peptides released during microbial protease action.

Low-Fat UHT milk was treated with commercial Bacillus protease.  The supernatant was then freeze-dried and the resulting peptides were used for further experimental use. The results showed that protease activity was present in all the samples which means that plasminogen was activated to plasmin.  These results are a major breakthrough and clearly shows that peptides liberated by Bacillus protease are able to activate plasminogen to plasmin. This implies that it is possible that bacterial contamination may contribute to flocculation and age gelation in UHT milk. An adjunct to this study was that KIO3 solution was added to the plasminogen solution in one UHT milk sample which did not contain freeze-dried Bacillus peptides. This peptide profile also indicates the plasmin peak at an elution time of 60 minutes and it can thus be concluded that KIO3 also has the ability to activate plasminogen which is in accordance with the literature.

Objective 3: To study the possible deactivation of plasmin by heat pre-treatment. Heat treatment in the range of 50 to 60oC should result in the release of highly reactive S-H (sulfhydryl) groups from ß-lactoglobulin (a whey protein).  This, in turn, may cause an irreversible denaturation of plasminogen and plasmin

Milk samples were pre-heat treated at 60°C for 1 hour.  The purpose of this was to investigate the impact of pre-heat treatment on the plasmin activity within milk. The protease assay was performed on the various samples in order to evaluate whether plasminogen activation was indeed enhanced by the pre-heat treatment step and to establish the protease activity level for each sample. The pre-heat treatment enhanced proteolytic activity since the samples that received pre-heat treatment consist of higher proteolytic values than the samples without the application of pre-heat treatment. All the results obtained from this experiment (protease assay and RP-HPLC) supported the literature which states that pre-heat treatment of milk between 50-60°C enhances plasminogen activation.