Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives

Abstract

Phytosterols are plant sterols recommended as adjuvant therapy for hypercholesterolemia and tocopherols are well-established anti-oxidants. However, thermo-sensitivity, lipophilicity and formulation-dependent efficacy bring challenges in the development of functional foods, enriched with phytosterols and tocopherols. To address this, we developed liposomes containing brassicasterol, campesterol and β-sitosterol obtained from canola oil deodorizer distillate, along with alpha, gamma and delta tocopherol. Three approaches; thin film hydration-homogenization, thin film hydration-ultrasonication and Mozafari method were used for formulation. Validated liquid chromatographic tandem mass spectrometry (LC-MS/MS) was utilized to determine the entrapment efficiency of bioactives. Stability studies of liposomal formulations were conducted before and after pasteurization using high temperature short time (HTST) technique for a month. Vesicle size after homogenization and ultrasonication (<200 nm) was significantly lower than by Mozafari method (>200 nm). However, zeta potential (-9 to -14 mV) was comparable which was adequate for colloidal stability. Entrapment efficiencies were greater than 89% for all the phytosterols and tocopherols formulated by all three methods. Liposomes with optimum particle size and zeta potential were incorporated in model orange juice, showing adequate stability after pasteurization (72 °C for 15 s) for a month. Liposomes containing phytosterols obtained from canola waste along with tocopherols were developed and successfully applied as a food additive using model orange juice.

Keywords: canola oil deodorizer distillate; liposomes; model orange juice; phytosterols; tocopherols.

Figure 1

Particle size of liposomes prepared at different B/L ratio by homogenization method expressed as mean ± standard deviation.

Figure 2

Transmission electron microscopy (TEM) analysis of liposomes prepared by; (A) homogenization method, (B) Ultrasonication and (C) Mozafari method. Sample of unilamellar vesicles are shown with a dotted arrow while aggregates are indicated by solid arrows. Scale bar in the figure A, B and C indicates 200nm, which represents the size of vesicle.

Figure 3

LC-MS/MS chromatogram of tocopherols: 1-δ tocopherol, 2-γ tocopherol, 3-α tocopherol; and phytosterols: 4-brassicasterol, 5-campesterol, 6-stigmasterol and 7-β-sitosterol. A-Rac tocol and B-cholestanol are internal standard.

Figure 4

Effect of addition of sucrose as a cryo-protectant on particle size of liposomes before and after lyophilization expressed as mean ± standard deviation where * represents statistical significant (*p < 0.05) in particle size after lyophilization in comparison to before lyophilization and after lyophilization in presence of sucrose.

Figure 5

Particle size of pasteurized and non-pasteurized liposomes incorporated in model orange juice during storage period of 1 month at 4 °C expressed as mean ± standard deviation.