Changes in Protein Fluorescence in a Lipid-Protein Co-oxidizing Oleogel.

High and low levels of lipid-induced protein oxidation (tuned by the addition of 0%-8.4% water) were investigated in oleogels, using excitation-emission matrix (EEM) fluorescence spectroscopy, coupled with a partial least-squares (PLS) regression and lipid hydroperoxide data. In high-level oxidation models, the intrinsic tryptophan fluorescence decreased and the emission maxima increased from 352.5 to 356.0 nm indicating the presence of protein modifications, which was further supported by size-exclusion chromatography. PLS recognized 3 latent components, with several excitation-emission points of interest. These apparent compounds include a region associated with radical mediated protein modifications (approximately 325 and 410 nm), lipid oxidation product adducts (approximately 350 nm and 420-425 nm), and malondialdehyde adducts (approximately 375 and 425 nm). The separate evaluation of these apparent compounds, at a 420 nm emission, indicated that lipid oxidation promotes protein lipid adduct fluorescence at high water levels, rather than radical mediated protein fluorescence.

Cooxidation of proteins and lipids in whey protein oleogels with different water amounts.

Protein- and lipid oxidation were investigated in whey protein based oleogels with varying water addition. Lipid oxidation was low (~30 mmol O2/kg lipid hydroperoxides after 6 weeks) in gels with < 0.23% water and a high (>1,000 mmol O2/kg lipid hydroperoxides after 4 weeks) in gels with > 2.4% water addition. In systems with > 2.4% water addition fluorescence (excitation 325 nm / emission 410 nm) as indicator of tyrosine oxidation and carbonyl content significantly increased and remained at low levels in oleogels with < 0.23% water addition. Primary amines as indicator for protein backbone breakage increased in early stages of oxidation in high water oleogels and decreased after 28 days. Degradation has been suggested to occur through interactions with reactive secondary lipid oxidation products and was confirmed by spiking experiments using respective compounds. The results suggest that secondary lipid oxidation markers are masked dependent on water addition in the presence of proteins.

The partitioning of emulsifiers in o/w emulsions: a comparative study of SANS, ultrafiltration and dialysis.

The partitioning of SDS and CTAB in o/w emulsions was investigated by ultrafiltration (UF), dialysis and small-angle neutron scattering (SANS). It was possible to measure the monomeric and the micellar concentrations of the emulsifiers in the filtrate and permeate in the UF and dialysis experiments, respectively. In addition, the interfacial concentration was calculated as the difference to the initial concentration. SANS experiments provided data, from which the micellar concentrations were obtained, followed by the calculation of the interfacial concentrations. The three methods were compared on the basis of the area, which is occupied by each emulsifier molecule at the interface. Good agreement was shown for both emulsifiers studied. Micellation started at total emulsifier concentrations of approx. 10 mM in emulsions containing either CTAB or SDS. At saturation (>10 mM SDS in a 10% o/w emulsion), the area per SDS headgroup at the interface was between 48 and 64 A2, depending on the method. In emulsions with CTAB, saturation of the interface was not achieved. The minimum headgroup area was determined by UF to be 33 A2 at a concentration of 30 mM CTAB in a 10% o/w emulsion.

The location of phenolic antioxidants and radicals at interfaces determines their activity.

To characterize parameters influencing the antioxidant activity at interfaces a novel ESR approach was developed, which facilitates the investigation of the reaction stoichiometry of antioxidants towards stable radicals. To relate the activity of antioxidants towards the location of radicals at interfaces NMR experiments were conducted. Micellar solutions of SDS, Brij and CTAB were used to model interfaces of different chemical nature. The hydrophilic Fremy's radical was found to be solubilized exclusively in the aqueous phase of SDS micellar solution but partitioned partly into the hydrophilic headgroup area of Brij micelles. In contrast the hydrophobic galvinoxyl was exclusively located in the micellar phase with the increasing depth of intercalation in the order SDS < Brij < CTAB. Gallates revealed a higher stoichiometric factor towards galvinoxyl in CTAB systems, which is accounted to a concentration effect of antioxidant and radical being both solubilized in the palisade layer. In contrast, in SDS solutions hardly any reaction between galvinoxyl and gallates was found. SDS acted as a physical barrier between radical (palisade layer) and antioxidant (stern layer). The influence of the hydrophobic properties of the antioxidant was clearly seen in Brij micelles. Elongation of the alkyl chain in gallate molecule resulted in increasing stoichiometric factors in the presence of galvinoxyl being located in the deeper region of the bulky headgroup area. The reverse trend was found in the presence of Fremy's radical being located in the hydrated area of the micelles.

Investigating the location of propyl gallate at surfaces and its chemical microenvironment by (1)H NMR.

The location and the resulting chemical microenvironment of the antioxidant propyl gallate (PG) was studied in micellar solutions using the cationic emulsifier cetyl trimethyl ammonium bromide (CTAB), the anionic emulsifier sodium dodecyl sulphate (SDS) and the non-ionic emulsifier Brij 58 (polyoxyethylene-20-cetyl ester). T (1) relaxation time of the aromatic protons of PG was investigated in micellar solutions and compared with that in aqueous solution in the absence of emulsifier. The relaxation time of the PG portion that is solubilized in the micelle (T (1,eff)) was calculated from the partition behavior of PG in micellar solution. From the 1D-(1)H spectrum, the alteration in the electron density of the aromatic protons and the alteration in the peak shape of the emulsifier headgroup and alkyl chain proton signals were indicative of the location of propyl gallate in the different micelles. Nuclear Overhauser effects (NOE) made it possible to deduce the exact location of PG by calculation of the relative NOEs. Marked differences were found for the location of PG in CTAB, SDS and Brij 58 micelles. PG was found to be located in the palisade layer of CTAB micelles, in the region of the polyoxyethylene chain of Brij micelles and in the Stern layer of SDS micelles. For careful study of the location of antioxidants and therefore to be able to characterize the chemical microenvironment of the antioxidants is crucial for understanding differences in antioxidant activities as a function of lipid surfaces. The application of spectroscopic methods may help to optimize the antioxidant activity to inhibit lipid oxidation at surfaces that are formed in a wide range of foods (emulsions), cosmetics, pharmaceuticals (emulsions and carrier systems) and of biological membranes (LDL-particles).

The more--the better? Estimating the inhibitory activity of alpha-tocopherol towards lipid oxidation.

Tocopherols are considered to be powerful antioxidants, but prooxidative effects are discussed for higher concentrations. The aim of this in vitro study was to investigate the dose-dependent inhibition of oxidation product formation caused by alpha-tocopherol, and to estimate the range of maximum antioxidant activity of alpha-tocopherol at different stages of lipid oxidation. Alpha-tocopherol was added to rapeseed oil triglycerides (ROTG, purified rapeseed oil) in concentrations ranging from 25 to 1500 micromol/kg ROTG. The inhibitory activity of alpha-tocopherol increased up to a concentration of 100 micromol/kg ROTG. A concentration of 125 micromol alpha-tocopherol/ kg ROTG did not result in an improved antioxidant effect. The formation of volatile secondary oxidation products followed the same trend, and the maximum inhibitory effect was also found for 100 micromol alpha-tocopherol/kg. Further, concentrations between 250 and 1500 micromol alpha-tocopherol/kg ROTG clearly caused increased formation of hydroperoxides during the induction period. However, compared to the control, all tested alpha-tocopherol concentrations resulted in a reduction of hydroperoxide formation and no prooxidative effects were observed.