Date of Award


Degree Name

MS in Agriculture - Dairy Products Technology


Animal Science


Philip S. Tong



Influence of chelating agents on proteolysis

of micellar casein slurries

Azucena Becerril Díez

The focus of this research was to analyze the effect of Ca chelators on the proteolytic activity of rennet during ripening of a micellar casein slurry. This research was divided in two phases. During the first phase, preliminary studies were performed to understand the behavior of rennet and the milk system in the presence of chelating agents. A solution of reconstituted skim milk at 10% (w/v) was prepared. Individual samples of this solution were divided in two study groups, samples with chelators and rennet and samples with only chelators. Two different chelators were used, sodium hexametaphosphate (SHMP or NaHMP) at concentration levels of 2.5 and 10 mM; and sodium citrate (SC) at concentration levels of 5 and 25 mM. These samples were incubated in a water bath at 32°C to measure coagulation time (CT) and Urea-PAGE electrophoresis was performed to identify presence of caseins and smaller peptides. For the second experiment, micellar casein slurries were prepared with a 25% protein content to simulate protein content in cheese. Samples were classified in two study groups, samples with chelators and rennet and samples with only chelators. Percentage of water soluble nitrogen analysis (%WSN) was performed following Kjeldahl procedures to measure the break down of caseins into free soluble nitrogen and Urea-PAGE electrophoresis was used to measure the appearance of smaller peptides due to protein break down. Bands with a higher density on electrophoretic gels were an indication of a higher concentration of peptides due to possible increased exposure of caseins to proteolysis. For the second phase, samples with the 2 most significant chelator levels were selected to add a protease and measure with the same tests if there was a higher rate of proteolysis. The data showed no particular trend or significant difference between treatments (p>0.05) for the %WSN test while some treatments showed a significant effect in samples treated for Urea-PAGE gels. In the Urea-PAGE analysis a different ANOVA was conducted on the percentage of alpha, beta, and gamma caseins for treatments before and after the incubation period separately and for the difference between both periods. These ANOVAs showed that the percentage difference in each casein before and after incubation was affected by the choice and level of chelator, and that these effects were modified by the presence or absence of rennet (p<0.001).