Preparation of Structure Lipid Containing Nervonic Acid via Enzymatic Acidolysis and Its Entrapment in Nanostructure Lipid Carriers

Abstract

This research aimed to incorporate nervonic acid (NA) into soybean oil via lipase-catalyzedacidolysis. The Lipozyme TL IM immobead 150 was selected as the lipase to catalyze the reaction in the sn-1,3 positions, and the critical reaction parameters were optimized to producestructured lipids with maximum nervonic acid incorporation. The optimal conditions were as follows: temperature of 60 ℃, substrate molar ratio (soybean oil:NA) of 1:4, the reactiontimeof4 h, and enzyme load of 10 w/w%. Under these conditions, the incorporation of NAwas 65.57±0.99 w/w%. The modified NA-TAG oil was produced, and the fatty acid composition was analyzed by gas chromatography-flame ionization detector (GC-FID). The total saturated fatty acid was 9.58 w/w%, and the monounsaturated fatty acid and polyunsaturated fatty acidwere72.69 w/w% and 17.73 w/w%, respectively. Subsequently, purification of the NA-TAGoil mixture was carried out by silica gel column chromatography to remove the free fatty acids. The encapsulation of lipophilic compounds in a nanocarrier system greatly enhances their biological efficiency and controlled delivery. The study further focused on producing the optimized nanostructured lipid carriers (NLCs) that entrapped the purified NA-TAGoil. The optimized NLCs was successfully prepared by hot high-shear homogenization, followed by sonication and recrystallization. The NLCs contained 3% Tween 80, soybean oil as liquidlipid, glyceryl monostearate (GMS) as solid lipid, NA-TAG concentration of 1%, and lipid phase to aqueous phase ratio of 10:90. Results showed that the nanoparticles have a mean size 82.11±0.14 nm, polydispersity index of 0.26, the zeta potential of -50.47 ± 0.40 mV and achieve the highest entrapment efficiency of 99.82 ± 0.01%. The optimized NLCs with NA-TAG oil entrapped were lyophilized by the freeze-drying method, followed by analyzing via Differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy. The DSC results showed a less-ordered crystalline structure in NLCs. FT-IR indicated no chemical interaction between the NA-TAG oil and the other NLC components. Short-term storage trials revealed good physical and chemical stabilities of the lyophilized NLC at 4 and 25 °Cf or 42 days. The storage stability test showed that significant aggregation of lyophilized NLCs was observed at 45 °C after the storage time. Lipid oxidation enhanced significantly with storage time and temperature. The in vitro digestion was performed to investigate the behaviours of lyophilized NLCs during transient in the gastrointestinal tract (GIT), i.e., mouth, stomach, and small intestine. Extensive droplet flocculation was observed in the gastric phase, which would restrict lipase access to lipid droplet surfaces. The electrical charge of the droplets changed in different phases. The potential gastrointestinal fate of oil-in-water emulsions containing NA-TAG oil was characterized by the free fatty acid released profile versus time, generated by the pH-stat method. 46%of free fatty acid was released from NA-TAG-loaded NLC. The results enhance the understanding of the physicochemical changes of emulsified droplets containing NA within the gastrointestinal tract and provide information concerning the bio-accessibility of NA-TAG-loaded NLCs. This research has provided scientific data for a comprehensive understanding of the synthesis of NA-TAG structured lipid using the immobilized lipase Lipozyme TL IM immobead 150as the biocatalyst and the entrapment of the NA-TAG oil into nanostructured lipid carriers (NLCs)