The bactericidal potencies of saturated and unsaturated fatty acids (FAs) and monoglycerides (MGs) against Helicobacter pylori were determined following short incubations with freshly harvested cells over a range of pHs. FAs and their derivatives with an equivalent-carbon number of 12 were the most potent: lauric acid had a minimum bactericidal concentration (MBC) at pH 7.4 of 1 mM, myristoleic and linolenic acid were the most potent unsaturated FAs (MBCs of 0.5 mM, pH 7.4), and monolaurin was the most potent MG (MBC 0.5 mM). Potencies of saturated FAs were increased sharply by lowering pH, and a decrease of only 0.5 pH units can cause a change from non-lethal to lethal conditions. Conversely, the bactericidal action of monolaurin was not pH-dependent. The bactericidal potencies of unsaturated FAs increased with degree of unsaturation. When more than one FA or FA plus MGs were present, their combined action was additive. Urea and endogenous urease did not protect H. pylori from the bactericidal action of FAs. These results suggest that H. pylori present in the stomach contents (but not necessarily within the mucus barrier) should be rapidly killed by the millimolar concentrations of FAs and MGs that are produced by pre-intestinal lipase(s) acting on suitable triglycerides such as milk fat.

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Enzymatic conjugation with fatty acids including omega-3 polyunsaturated fatty acids (ω-3 PUFAs) derived from fish oil to three citrus fruit-derived flavonoids: grapefruit extract, naringin, and neohesperidin dihydrochalcone were investigated. The conversions were achieved over 85% under the catalysis of lipase Novozyme 435 in acetone at 45°C at semi-preparative scale. The conjugates were purified via solvent partition and silica gel chromatography and achieved 90–98% in purity. The NMR analysis of the conjugates confirmed that the fatty acid carbon chain was linked onto the primary –OH group on the glucose moiety of the flavonoids. The purified flavonoid conjugates alongside their original flavonoids were analyzed for antioxidant activities via 2,2-diphenyl-1-picrylhydrazyl scavenging assay, and anti-peroxidation test via peroxide values measured during a 1-week fish oil storage trial. Vascular endothelial growth factor (VEGF) assay was conducted with 1, 10, and 100 μM of naringin and grapefruits and their conjugates, respectively, and total VEGF levels were measured at 24 and 48 h, respectively, using ELISA and dot blot analysis. The results from these functionality experiments demonstrated that flavonoid FA conjugates have at least comparable (if not higher) antioxidant activity, anti-peroxidation activity, and anti-angiogenic activity.

Free fatty acids and monoglycerides released from milkfat by partial pregastric lipase-catalysed hydrolysis are bactericidal towards Helicobacter pylori. Two milkfat preparations were investigated: a normal bovine milkfat, and a fractionated milkfat preparation, termed ModFat, enriched in triglycerides containing short-and medium-chain fatty acids. The released products were tested for bactericidal potency against H. pylori. The potencies of the respective preparations were consistent with expected potencies calculated from individual free fatty acid and monoglyceride concentrations and their lauric acid equivalence factors (K i ). ModFat products were more bactericidal, in accordance with release of free fatty acid types of high potency, and addition of the surfactant Tween 80 to the hydrolysed lipid increased potency eight times more than did addition of lecithin. Tween 80 micelles have smaller aggregation numbers, and the mixed micelles of Tween 80/free fatty acids would be more likely to expose the bacteria to higher apparent free fatty acid concentrations.

This novel, recombinant keratanase-II meets all performance requirements and can be produced in a rapid and reproducible manner. We speculate that other related bacterial enzymes of biomedical or industrial interest may be amenable to similar engineered enhancements.

A new assay for the determination of lactosylceramide-2,3-sialyltransferase (SAT I, EC 2.4.99.9) and monosialoganglioside sialyltransferase (SAT IV, EC 2.4.99.2) is described. The assay utilised the commercially available fluorophore labelled sphingolipids, boron dipyrromethene difluoride (BODIPY) lactosylceramide (LacCer), and BODIPY-monosialotetrahexosylganglioside (GM1) as the acceptor substrates, for SAT I and SAT IV, respectively. HPLC coupled with fluorescence detection was used to analyse product formation. The analysis was performed in a quick and automated fashion. The assay showed good linearity for both BODIPY sphingolipids with a quantitative detection limit of 0.05 pmol. The high sensitivity enabled the detection of SAT I and SAT IV activities as low as 0.001 μU, at least 200 fold lower than that of most radiometric assays. This new assay was applied to the screening of SAT I and SAT IV activities in ovine and bovine organs (liver, heart, kidney, and spleen). The results provided evidence that young animals, such as calves, start to produce ganglioside sialyltransferases as early as 7 days after parturition and that levels change during maturation. Among the organs tested from a bovine source, spleen had the highest specific ganglioside sialyltransferase activity. Due to the organ size, the greatest total ganglioside sialyltransferase activities (SAT I and SAT IV) were detected in the liver of both bovine and ovine origin.

A commercial extract from the tongue and epiglottal region of suckling calf was partially purified to yield a calf pregastric enzyme with esterase activity against 4-nitrophenylalkanoate esters (C2-C12) at 370C, pH 7.2. The Km against 4-nitrophenylacetate was 0.023 mM, however, when 4-nitro-phenyldodecanoate was used as the substrate, the Km was 1.06 AM. The maximum activity was achieved at ca. 1.6 AM, which is similar to its critical micelle concentration. The reactivity is dependent upon pH. A pK of 7.23 which was obtained in the pH range 5.5-9.0 is indicative of a single ionizable residue. The activity dependence upon temperature of the partially purified enzyme was determined within the range of 25°C to 48°C and Eyring parameters were calculated.