Biochemical, microbiological, and structural evaluations to early detect age gelation of milk caused by proteolytic activity of Pseudomonas fluorescens

Abstract

Heat–stable peptidase AprX, released by Pseudomonas species in raw milk during cold storage, can cause gelation of UHT milk since it is able to split caseinomacropeptides (CMPtot) from κ-casein, so inducing aggregation of casein micelles. Identifying raw milk susceptibility to gelation would allow UHT milk manufacturers to select appropriate processing conditions or give the milk a different destination. Two approaches, i.e., detection of free CMPtot and evidence of casein aggregates, were evaluated as possible indicators for early detecting milk destabilization. With this aim, microfiltered milk was inoculated with a P. fluorescence strain and incubated at either 4 or 25 °C. The presence of CMPtot was detected using capillary electrophoresis after 96 and 24 h at the two temperatures, respectively, when milk also became heat unstable and small flocks of protein appeared. Confocal laser scanning microscopy evidenced initial aggregates of casein micelles after 48 and 24 h at 4 and 25 °C, respectively. Keeping the milk at 25 °C/24 h could be a useful condition to accelerate milk destabilization. Despite the similar timing of instability detection, presence of CMPtot was the only trait specific for AprX activity.