Proteolytic enzyme activity in lactococci grown in different pretreated milk media

Lactococcus lactis ssp. lactis MG1363, harbouring plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, was used to study the effect of two different treatments of the growth medium milk on the activity levels of PrtP and the intracellular localized aminopeptidase PepN and X-prolyl-dipeptidyl aminopeptidase PepXP. All three proteolytic enzymes showed lower activity levels in cells grown in high heat-treated milk as compared to cells grown in non-heat-treated milk. Highest activity levels of the three studied enzymes were found in cells grown in milk heat treated for 30 min at 63 degrees C. Using cells of strain L. lactis ssp. lactis MG1363, harbouring plasmid pNZ544, which encodes reporter gene gusA under control of the prtP promoter, it was demonstrated that the regulation of PrtP takes place at the transcription initiation level. After separation of the pH 4.6 soluble fraction of high heat-treated milk with reverse phase HPLC, it was found that the hydrophilic small peptide fraction of the milk was responsible for this regulation. Amino acid analysis of this fraction confirmed that this fraction consisted of peptides only. Ultrafiltration of milk, which increases the dry matter of the milk specifically through increase of its protein content, only significantly affected the levels of PrtP and PepN in cells of strain MG1363. Highest activity was found during growth in unconcentrated milk, and the lowest level was found during growth in four times concentrated retentate. Using cells of strain MG1363(pNZ544), it was demonstrated that also in this case regulation of PrtP takes place at the level of transcription initiation. Approximately 40% of the decrease in activity of PrtP and PepN could be explained by the presence of higher amounts of purified whey proteins and higher amounts of dry mass. This suggests the presence of another factor, concentrated by ultrafiltration, which controls the production different proteolytic enzymes in concentrated retentate.