Identification of the most proteolytic Pseudomonas strains that can produce heat-resistant proteases and contribute to the Ultra High Temperature (UHT) milk destabilization is of great interest. In the present study, among the 146 Pseudomonas isolates that encoded the aprX gene, five isolates with the highest proteolytic activity were selected and identified based on 16S rRNA, rpoD and gyrB gene sequences data. The identification results were confirmed by phylogenetic analysis based on multilocus sequence analysis and identified the representative isolates as P. jessenii (two isolates) and P. gessardii (three isolates). Casein zymography demonstrated the ability of these species to produce heat-resistant enzymes, AprX, with molecular mass of about 48 kDa during storage at 7° C for 72 h. In sterilized milk samples, the residual activity of AprX caused a considerable enhancement in the degree of protein hydrolysis, non-protein nitrogen and non-casein nitrogen contents of the samples during a two-month storage. This enhancement was slightly higher in samples containing enzyme produced by P. jessenii compared to P. gessardii ones, resulting in earlier onset of sterilized milk destabilization. Hence, this study revealed that P. jessenii and P. gessardii can play a considerable role in deterioration of Iranian commercial long-life milk.

Proteinase activity of Lactobacillus fermentum IFO3956 cells was higher when they were grown on milk-based media than on 10% reconstituted skim milk. The lowest protease activity was observed when cells were grown on milk-free media. The extraction of milk-induced cell-bound proteases from Lb. fermentum IFO3956 was most efficient using 1% Tween 80 while the use of 1% SDS inhibited all proteolytic activity. Two bands of ∼35 and >100 kDa were observed by zymogram, indicating that proteolytic activity corresponded to the presence of at least two types of enzymes or two molecular forms of one enzyme. Mass spectrometry analyses of αS1-casein hydrolysates detected 24 peptides with sizes ranging from 5 to 36 amino acids, including 9 phosphorylated peptides, resulting from the fermentation of Lb. fermentum IFO3956 of αS1-casein. Most of the identified peptides originated from the N-terminal portion of αS1-casein. The studied bacterial strain could hydrolyze αS1-casein in many sites including the epitopes triggering the allergic reactions against αS1-casein e.g. at the positions 23, 30, 41, 71, 91, 98, 126, 179. After hydrolysis of αS1-casein with Lb. fermentum IFO3956 the recognition and the binding of this casein to IgE from the pooled sera of 18 patients with cow's milk allergy was significantly reduced.

The goal of this work is to report a novel assay that preserves antigenicity of extracts with high protease content without using enzymatic inhibitors. A great reduction of proteolytic activity in the insoluble chloroform/methanol and heated amoebic fraction (IC[ratio ]MC) was obtained by this method, even in the presence of sodium dodecyl sulphate and 2-mercaptoethanol. The substrates azo-casein and the hide powder azure were used to determine the reduction of proteolytic activity of IC[ratio ]MC compared with the activity of crude extract and crude extract with iodoacetamide. The IC[ratio ]MC SDS-PAGE pattern shows a higher quantity of bands than extract with the inhibitor iodoacetamide or than crude extract. In addition, anti-Entamoeba histolytica antibodies from amoebic liver abscess patients recognized a richer antigenic Western blot pattern in the IC[ratio ]MC fraction than in crude extract alone or with inhibitor. The described method has proved to be suitable to preserve amoebic antigens for its use in diagnostic tests and it can be used for immunological response studies against E. histolytica antigens. Furthermore we propose that this method to obtain the IC[ratio ]MC fraction can be applied for the study of other microorganisms or cells with high enzymatic content.