Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolytic activity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified as Streptococcus uberis (strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified as Weissella confusa FT424, W. hellenica FT476, Leuconostoc citreum FT671 and Lactobacillus plantarum FT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain was L. plantarum FT723 that inhibited Penicillium expansum in modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed against Yarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified as Enterococcus faecalis (strains FT132 and FT522) and Lactobacillus paracasei FT700 were confirmed by SDS–PAGE, as visualized by the digestion of caseins and whey proteins (β-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolytic activity or production of antimicrobials) in dairy foods production.

The presence of yeasts in milk may cause physical and chemical changes limiting the durability and compromising the quality of the product. Moreover, milk and dairy products contaminated by yeasts may be a potential means of transmission of these microorganisms to man and animals causing several kinds of infections. This study aimed to determine whether different species of yeasts isolated from bovine raw milk had the ability to develop at 37°C and/or under refrigeration temperature. Proteinase and phospholipase activities resulting from these yeasts were also monitored at different temperatures. Five genera of yeasts (Aureobasidium sp., Candida spp., Geotrichum spp., Trichosporon spp. and Rhodotorula spp.) isolated from bovine raw milk samples were evaluated. All strains showed one or a combination of characteristics: growth at 37°C (99·09% of the strains), psychrotrophic behaviour (50·9%), proteinase production (16·81% of the strains at 37°C and 4·09% under refrigeration) and phospholipase production (36·36% of the isolates at 37°C and 10·9% under refrigeration), and all these factors may compromise the quality of the product. Proteinase production was similar for strains incubated at 37°C (16·81% of the isolates) and room temperature (17·27%) but there was less amount of phospholipase-producing strains at room temperature (15·45% of the isolates were positive) when compared with incubation at 37°C (36·36%). Enzymes production at 37°C by yeasts isolated from milk confirmed their pathogenic potential. The refrigeration temperature was found to be most efficient to inhibit enzymes production and consequently ensure better quality of milk. The viability of yeasts and the activity of their enzymes at different temperatures are worrying because this can compromise the quality of dairy products at all stages of production and/or storage, and represent a risk to the consumer.

A complete developmental sequence of Leishmania mexicana has been produced in axenic culture for the first time. This was achieved by manipulation of media, pH and temperature conditions over a period of 16 days. All experiments were initiated with lesion amastigotes that were transformed to multiplicative promastigotes by culture in HOMEM, 10% foetal calf serum, pH 7·5, at 25 °C. Metacyclogenesis was induced by subpassage in Schneider's Drosophila medium, 20% foetal calf serum, pH 5·5, and the resulting forms transformed to axenically growing amastigotes by subpassage in the same medium and raising the temperature to 32 °C. Parasites from each day were characterized with respect to their general morphology using light microscopy of Giemsa-stained smears, and biochemically by analysis of total protein content, proteinases, nucleases and secretory acid phosphatase. The results demonstrated that the three main stages identified - amastigotes, multiplicative promastigotes and metacyclic promastigotes - each exhibited the expected suite of biochemical properties. Further, the changes in morphology observed as the developmental sequence proceeded from stage to stage were accompanied by appropriate changes in biochemical properties. These results provide both useful biochemical markers and a culture system in which to examine the regulation of differentiation and transformation during the Leishmania life-cycle.

Blastocrithidia culicis is an insect trypanosomatid that presents bacterial endosymbionts. The cell-associated and secreted proteinases of the endosymbiont-bearing and aposymbiotic strains were compared through the incorporation of proteinaceous substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Few qualitative changes could be detected in the proteolytic zymograms in the 2 strains studied when gelatin, casein, haemoglobin or bovine serum albumin (BSA) were tested. However, the level of proteolytic activities was significantly higher in the aposymbiotic strain. Some of the B. culicis proteins reacted in Western blots with antibodies raised against gp63, a zinc-metalloproteinase, and cruzipain, a cysteinyl-proteinase, which are virulence factors of the human pathogenic trypanosomatids, Leishmania spp. and Trypanosoma cruzi, respectively. The anti-cross-reacting determinant (CRD) antibody recognized 2 polypeptides (50 and 58 kDa) in the spent culture media and in the supernatant from glycosylphosphatidylinositol-phospholipase C (GPI-PLC)-treated cells, suggesting that these proteins are GPI-anchored to the plasma membrane. In addition, the anti-gp63 reacted with the 50 kDa protein. The identification of protein homologues in trypanosomatids with distinct life-cycles may help to determine the importance of proteinases in trypanosomatids.