One-step separation system of bio-functional lipid compounds from natural sources.

Lipids are a very heterogeneous class of biomolecules with distinct structures and functions. Total lipids (TLs) obtained from natural sources are regularly further separated into lipid subclasses, with the two major ones being the polar lipids (PLs) and neutral lipids (NLs). Traditional analytical methods for fractionating TLs into NLs, PLs, and their subclasses, usually comprise difficult, costly and time-consuming steps. Instead, several benefits and applications are derived by implementing a novel one-step semi-preparative and reversed-phase HPLC-analysis for separating TLs into all kinds of lipid subclasses. This method allows a one-step separation/fractionation of several subclasses of bio-functional PLs (i.e. phospholipids, glycolipids, phenolic compounds, N-acyl-homoserine-lactones, etc.) and NLs (i.e. triacylglycerols, fatty acids, esters, etc.) from TL-extracts of a natural source, prior to further testing them for their bio-functionality (i.e. in bioassays/cell models) and structure-activity relationships (i.e. LC-MS/GC-MS).•This method can be applied in several natural sources, such as animal and marine sources, plants, microorganisms of biotechnological and agricultural interest, foods, beverages and related products, and by-products.•This method can also be applied for separating specific bio-functional lipids from complex medical and pharmaceutical samples (i.e. cells, tissues, blood, plasma, liposomes, etc.), either for evaluating their role in diseases (i.e. PAF/PAF-like molecules) or by elucidating their protective roles (i.e. PLs rich in ω3 PUFA) for supplements and nutraceuticals' applications.

Nutraceutical phycocyanobilin binding to catalase protects the pigment from oxidation without affecting catalytic activity.

Phycocyanobilin is a dark blue linear tetrapyrrole chromophore covalently attached to protein subunits of phycobiliproteins present in the light-harvesting complexes of the cyanobacteria Arthrospira platensis (Spirulina "superfood"). It shows exceptional health-promoting properties and emerging use in various fields of bioscience and industry. This study aims to examine the mutual impact of phycocyanobilin interactions with catalase, a life-essential antioxidant enzyme. Fluorescence quenching experiments demonstrated moderate binding (Ka of 3.9 × 104 M-1 at 25 °C; n = 0.89) (static type), while van't Hoff plot points to an enthalpically driven ligand binding (ΔG = -28.2 kJ mol-1; ΔH = -41.9 kJ mol-1). No significant changes in protein secondary structures (α-helix content ~22%) and thermal protein stability in terms of enzyme tetramer subunits (Tm ~ 64 °C) were detected upon ligand binding. Alterations in the tertiary catalase structure were found without adverse effects on enzyme activity (~2 × 106 IU/mL). The docking study results indicated that the ligand most likely binds to amino acid residues (Asn141, Arg 362, Tyr369 and Asn384) near the cavity between the enzyme homotetramer subunits not related to the active site. Finally, complex formation protects the pigment from free-radical induced oxidation (bleaching), suggesting possible prolongation of its half-life and bioactivity in vivo if bound to catalase.

Melanin and humic acid-like polymer complex from olive mill waste waters. Part I. Isolation and characterization.

A water soluble humic acid and melanin-like polymer complex (OMWW-ASP) was isolated from olive mill waste waters (OMWW) by ammonium sulfate fractionation to be used as natural additive in food preparations. The dark polymer complex was further characterized by a variety of biochemical, physicochemical and spectroscopic techniques. OMWW-ASP is composed mainly of proteins associated with polyphenols and carbohydrates and the distribution of its relative molecular size was determined between about 5 and 190 kDa. SDS-PAGE shows the presence of a well separated protein band of 21.3 kDa and a low molecular weight peptide. The OMWW-ASP complex exhibits a monotonically increasing UV-Vis absorption spectrum and it contains stable radicals. Antioxidant activity measurements reveal the ability of the OMWW protein fraction to scavenge both the cationic 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(+)) radical, as well as the stable nitroxide free radical 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL).

Influence of nanoreactor environment and substrate location on the activity of horseradish peroxidase in olive oil based water-in-oil microemulsions.

Oxidative enzymatic reactions using horseradish peroxidase (HRP) were carried out in water-in-oil (w/o) microemulsions composed of olive oil/lecithin/1-propanol/water, a model biomimetic system. The substrates used (gallic acid, octyl gallate and 2,2'-azino-bis[3-ethylbenzo-thiazoline-6-sulfonic acid] (ABTS)) have different hydrophobicities and possible locations in the microemulsion system. HRP reactivity with reference to substrate hydrophobicity and structural characteristics of the microemulsions is discussed. The nature of the enzyme microenvironments was examined using dynamic light scattering (DLS), differential scanning calorimetry (DSC) and diffusion NMR (DOSY) methodologies while the location of various enzymatic substrates in the microemulsion phase was assessed by solubility measurements and by taking pressure-area isotherms of mixed monolayers of the substrates with dipalmitoyl-phosphatidylcholine (DPPC), which is a major constituent of lecithin. In contrast to the bulk aqueous phase, in the severely restricted environment of the polar domains of the microemulsion HRP reacted faster with octyl gallate, a substrate that is solubilized at the lipid interfaces. HRP was deactivated in the olive oil microemulsions within a few hours, a phenomenon that has also been observed in other microemulsion systems.

Anticarcinogenic compounds of olive oil and related biomarkers.

Olive oil, one of the oldest vegetable oils consumed without any refining, is associated with a reduced risk of a number of common cancers. Minor constituents of virgin olive oil have been suggested to be among the major chemopreventive components. A brief overview is presented of recent findings concerning the bioavailability of certain important olive oil minor components including efficient antioxidant polyphenols, the triterpene hydrocarbon squalene and beta-sitosterol, considered as putative nutritional biomarkers, in relation to the incidence of cancer.

Biocompatible microemulsions based on limonene: formulation, structure, and applications.

The preparation of biocompatible (w/o) microemulsions based on R-(+)-limonene, water, and a mixture of lecithin and either 1-propanol or 1,2-propanediol as emulsifiers was considered. The choice of the compositions of the microemulsions used was based on the pseudo-ternary phase diagrams of the four-component system determined at 30 degrees C for different weight ratios of the components. When 1-propanol was considered as co-surfactant, the area of the microemulsion zone was remarkably increased. Interfacial properties and the dynamic structure of the emulsifier's monolayer were studied by electron paramagnetic resonance (EPR) spectroscopy using the spin-labeling technique. The rigidity and polarity of the interface were affected by the nature of the alcohol used as co-surfactant. When 1-propanol was used, the emulsifier's interface was much more flexible, indicating a less tight packing of lecithin molecules than in the case of 1,2-propanediol. In addition, the membrane's polarity was decreased when the diol was added as co-surfactant in the microemulsion system. To evaluate the size of the dispersed aqueous domains as a function of water content and other additives concentration, dynamic light scattering (DLS) measurements were carried out. Radii in the range from 60 to 180 nm were observed when 1-propanol was used as co-surfactant, and the water content varied from 0 to 12% w/w. Electrical conductivity measurements of R-(+)-limonene/lecithin/1-propanol/water microemulsions with increasing weight fractions of water indicated the appearance of a percolation threshold at water content above 4% w/w. Lipase from Rhizomucor miehei was solubilized in the aqueous domains of the biocompatible microemulsions, and the esterification of octanoic, dodecanoic, and hexadecanoic acids with the short-chained alcohols used as co-surfactants for the formulation of microemulsions was studied. The enzyme efficiency was affected by the chain length of the carboxylic acids and the nature of the alcohol. In the case of 1-propanol, a preference for the long-chain carboxylic acids was observed. On the contrary, when 1,2-propanediol was used formulation of the corresponding esters was not observed. This behavior could be possibly attributed to either the specificity of the lipase toward the alcohol employed for the esterification of the acids or the structural changes induced in the system when 1-propanol was replaced by 1,2-propanediol.

Olive oil microemulsions: enzymatic activities and structural characteristics.

Microemulsions composed of olive oil, either extravirgin (EVOO) or refined (ROO), as the continuous oil phase, water as the dispersed phase, and a mixture of lecithin-propanol as the emulsifier were prepared and investigated as potential biocompatible media for biotransformations. The area of the microemulsion zone increased considerably by increasing the lecithin to propanol weight ratio in both EVOO- and ROO-based systems. However, the nature of the oil used does not seem to affect the ability of the system to incorporate water. The catalytic activities of two oxidizing enzymes that have been detected in virgin olive oil, namely, tyrosinase and peroxidase, and the activity of a proteolytic enzyme such as trypsin were studied in olive oil microemulsions. In all cases a reduced catalytic activity was observed when ROO was considered as the continuous oil phase. The interfacial properties of lecithin layers were studied by electron paramagnetic resonance spectroscopy employing the nitroxide spin probe 5-doxylstearic acid. By varying the weight ratio of lecithin to propanol and the water content of the microemulsions, the mobility of the probe and the rigidity of the interface were altered. Droplet sizes were measured by dynamic light scattering. At higher water content of the system the size of the droplets was increased. When EVOO was considered as the oil phase, smaller aqueous droplets were formed. Lecithin-based olive oil microemulsions were also characterized with regard to the phenomenon of electrical percolation. At a water content above 3% (w/w) and a lecithin/propanol weight ratio of 2, a sharp increase in conductivity was observed, indicating a structural transition in the bicontinuous form.