Understanding the progression of lipid oxidation in oil-in-water emulsions from the aspect of the food quality and safety, consumer satisfaction and cleaner food label is of importance, because most of the food emulsions are oil-in-water emulsions. There is an increasing tendency in the food industry to incorporate unsaturated oils into food products, but that results in shortened shelf-life. Therefore, studying the factors and consequences of the oxidative instability provides beneficial insight into prolonging the antioxidative stage and inhibiting undergoing oxidation processes to improve the food quality and increase the shelflife of the food products.
In the present work, lipid oxidation in oil-in-water emulsions was studied via conventional analytical and via novel state-of-the-art techniques. For the first time, the effect of mixing emulsions made of saturated medium-chain triglyceride (MCT) oil and unsaturated linseed oil (LSO) was studied and it was observed that mixing had a substantial effect on the oxidative stability – mixed oil emulsions and mixed emulsions showed decreased rates of lipid oxidation. Saturated MCT oil in the presence of unsaturated LSO, either as in isolated lipid droplets or as intra-mixed into the same oil droplet, had lipid oxidation inhibiting effect. Moreover, investigation of the chemical composition of the mixed emulsion by Raman microspectroscopy showed the oil droplets remained intact, and the contents of the oil droplets did not mix – droplets contained either MCT oil or LSO.
One novel state-of-the-art technique involved incorporation of the radical-sensitive
fluorescent probe BODIPY665/676 into the oil droplets to investigate the lipid oxidation in emulsions by confocal laser scanning microscopy (CLSM). Radicals were generated either from 2,2’-azobis(2,4-dimethyl)valeronitrile (AMVN) or from di-tert-butyl peroxide (DTBP). BODIPY665/676 evoked a change in the fluorescence caused by the reaction with radicals, and subsequently, probe oxidation. Upon oxidation, the main peak at excitation wavelength 670 nm/675 nm diminished, but the secondary peak at excitation wavelength 580 nm appeared, and increased with increasing collisions between the probe and the radicals. The results obtained from AMVN and DTBP using fluorometric technique, showed the response of BODIPY665/676 was the highest in the oxidatively stable saturated oil, and moderate in the oxidatively unstable unsaturated oil. This was an unexpected outcome. It was suggested that it might have been due to the high steady state concentration of the radicals in the saturated oil,
while in the unsaturated oil a competition between radical attacks towards lipids and the probe decreased the concentration of the radicals. Moreover, the bulky unsaturated lipids might be sterically hindered from interacting with the probe. When the lipid oxidation was initiated with the two-photon laser irradiation, then the results were opposite to fluorometrically obtained data. It was observed that the higher the degree of unsaturation, the higher the response of BODIPY665/676. Assembling these results, it was suggested the concentration of radicals had the highest effect on the probe. In unsaturated oil, the concentration of radicals was very high, which enhanced the radical-radical interactions, but also accelerated the formation of the secondary oxidation products. BODIPY665/676 is not sensitive to secondary oxidation products, thus the response was modest. In saturated oil, the concentration of radicals was lower, decomposition of hydroperoxides did not occur, thus the response of
BODIPY665/676 was high.
Moreover, the experiments with the AMVN indicated that the AMVN-derived radicals were able to diffuse from the droplets, where they were generated, move through the interface, and aqueous phase, and initiate lipid oxidation in the neighboring droplets, while the experiments with DTBP did not show any mobility of radicals.
The fluorescent probe BODIPY665/676 possesses potential to be used in non-polar neutral lipid domains as a reporter probe for lipid oxidation. The highly lipophilic and radical-sensitive probe is providing with new understanding of the radical mobility and radical reactions, thus contributing to the fundamental knowledge of lipid oxidation processes.