Biotransformation of Flavonoid Conjugates with Fatty Acids and Evaluations of Their Functionalities.

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.

A new assay for determining ganglioside sialyltransferase activities lactosylceramide-2,3-sialyltransferase (SAT I) and monosialylganglioside-2,3-sialyltransferase (SAT IV).

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.

The products from lipase-catalysed hydrolysis of bovine milkfat kill Helicobacter pylori in vitro.

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 (Ki). 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.

Antibacterial actions of fatty acids and monoglycerides against Helicobacter pylori.

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.

The antimicrobial properties of milkfat after partial hydrolysis by calf pregastric lipase.

Studies on the kinetic characteristics of calf pregastric lipase (EC 3.1.1.3) have shown that it preferentially releases short chain fatty acids (SCFAs) from bovine milkfat. The released fatty acids form mixed micelle structures. The aim of this investigation has been to test whether hydrolysed milkfat is antimicrobial, and how the state of the emulsion alters the bactericidal or bacteriostatic effects. Partial hydrolysis of milkfat by pregastric lipase was carried out in two types of emulsion systems, containing either Triton X-100 or casein/lecithin, plus milkfat in citrate/phosphate buffer (pH 5.0-6.0). The concentrations and compositions of fatty acids were determined by gas chromatography. The minimum percentages of hydrolysed milkfat which affected growth and survival of selected Gram-positive and Gram-negative bacteria were measured. The bacterial experiments were repeated using pure fatty acids at similar concentrations. Lauric acid (C12:0) was found to be the most potent bactericidal fatty acid against Enterococcae (Gram-positive), and caprylic acid (C8:0) was the most potent against coliforms (Gram-negative). Use of Triton X-100 for milkfat emulsification provided a more compatible medium for studying bacterial growth in the hydrolysed milkfat than did use of casein/lecithin. The results also show that the antimicrobial effects of individual fatty acids released from hydrolysed milkfat were at least additive and suggest that hydrolysis of milkfat may be a significant factor in controlling growth of organisms imbibed with food in pre-weaned animals. The amount of pregastric catalyzed triglyceride hydrolysis in the digestive tract is sufficient to produce an antibacterial concentration of fatty acids and monoglycerides.