No abstract

The analysis of sterols in biological fluids allows the clinical study of cholesterol related diseases. This research is focused on reducing the sample processing time of the determination of free and bonded sterols in human serum. Ten sterols were studied: cholesterol precursors (desmosterol, lanosterol, and cholestanol); phytosterols (stigmasterol, campesterol, sitosterol, and sitostanol) and oxysterols (7-α-hydroxy-4-cholesten-3-one, 24-hydroxycholesterol, and 27-hydroxycholesterol). Ultrasound assistance was used to diminish the reaction time during the alkaline hydrolysis for determining total sterols. Different retention mechanisms of solid-phase extraction were compared, two reversed-phase sorbents DSC-18 and polymeric Oasis-HLB and a novel zirconia-coated silica phase. DSC-18 and zirconia-coated silica were the most suitable sorbents to analyze these metabolites. The resulting extracts were analyzed by liquid chromatography coupled to mass spectrometry. The analytical parameters were determined and better values were observed with DSC-18 cartridges for most sterols. LOQ were in the low ng/mL level. Recoveries were in the range 85-99%. Average intermediate precision was 15%. Accuracy for both cartridges was more than 92%. Zirconia-coated silica showed better performance for the oxysterols, with recoveries around 90%. The procedure allows the determination of free and bonded sterol precursors, phytosterols, and oxysterols in human serum.

The aim of this work was to elucidate the role of the secondary metabolites produced by B. amyloliquefaciens BUZ-14 against B. cinerea, M. fructicola, M. laxa, P. digitatum, P. italicum and P. expansum both in vitro and in planta. The entire cell free supernatant (CFS) and the lipopeptide fraction (LPF) showed similar antifungal activities, completely inhibiting all the fungi at dilutions of 1:24 or even lower, whereas the nonbutanolic fraction (NBF) barely inhibited the fungi. However, when the LPF and CFS were applied on fruit, only brown rot in peaches and blue rot in apples was totally inhibited. The main families of metabolites in the LPF were iturin A, fengycin and surfactin with maximum concentrations of 407, 853 and 658 µg mL-1 , respectively. Subsequently, a TLC-bioautography revealed iturin A as the key metabolite in the inhibitions and allowed us to establish in vivo MICs of 16.9 and 33.9 µg mL-1 for Monilinia species and P. expansum, respectively. The application of 24 hold BUZ-14 cultures supressed brown rot in peaches and also blue rot in apples but failed to inhibit the other diseases. However, BUZ-14 was only able to grow and produce iturin A in peaches so we can deduce that the amount of iturin A brought with the cultures (36 ± 14 µg mL-1) could be enough to control both diseases. The strong antifungal activity of the iturin A present in the BUZ-14 CFS suggests that it could be successfully used for postharvest disease control. However, future research is necessary to maximize the iturin A production by B. amyloliquefaciens BUZ-14 in order to optimize a commercial application.

For many years, the adulteration of milk from sheep, goats or water buffalos with cows' milk has been a widespread practice due to the higher cost of milk from those other species. Because of this, great concern has been shown by many Protected Designation of Origin councils that have to assure the quality and genuineness of the cheese produced by their associates. Therefore, the whole production chain needs analytical tools that allow the control of potential adulteration. Rapid methods to be used in the field are scarce and have not been validated according to international guidelines. The aim of this work has been to validate a rapid test based on lateral flow immunochromatography to detect cows' milk in milk from other species, including buffalo's milk, according to AOAC guidelines. No false-positive result was found after analysing 146 known negative samples from individual animals. The lowest level of adulteration with a Probability of Detection (POD) of 1.00 (confidence interval between 0.94 and 1.00) was found at 0.5% of cows' milk. This level is below the current EU allowed level of cows' milk, set at 1%. Variations in the time of assay, volume of the analysis buffer and different batches of the test were evaluated to detect any effect on the false-positive rate or on the limit of detection of the test. The effects of compositional factors (such as high level of fat, protein and somatic cell counts) were also evaluated. The new rapid test to detect cows' milk in milk from other species is shown to be an adequate tool to control milk quality in routine analysis. This kind of test is very easy to use and it can be performed by untrained staff during milk collection at the farm or upon arrival at dairies.