Fig. 4 Microscopic images of LSE and LSFE. (a & c) Confocal oil bodies image; (b1 & b2) Cryo-SEM intact LSE oil bodies; (d1 & d2) Cryo-SEM LSFE oil body.
In the linseed emulsion (LSE) and linseed fish oil enriched emulsion (LSFE) of this study, the supramolecular assembly of the lipid molecules is characterized by the oil body’s (OB) physical and chemical aggregated organization . This structural organization contains polar head groups forming the outside as the surface of the oil bodies in contact with water, while the hydrophobic tails are inside and away from the aqueous matrix. The OB’s interfacial surface coating layer contains the LS’s phospholipids and amphiphilic proteins such as oleosins and residual linseed mucilage components, that together stabilize the OB (Goyal et al., 2015; Fabre et al., 2015). Thus LSE chemical and morphological structures are oil body vesicles with a multicomponent amphiphilic surface and lipophilic core of encapsulated PUFA-rich oil
The 1H LF-NMR energy relaxation TD fingerprinting for LSE before and after thermal oxidation at 55 oC for 96 hours are shown in Fig. 5. This figure shows that before LSE oxidation the oil’s T1 and T2 energy relaxation time consists of two different parts. One is related to the PUFA-rich oil molecules within the OB’s core and the other on the OB’s interfacial surface. The T1 and T2 energy relaxation time of the oil body’s vesicle surface is not significantly affected by the water because the oil’s energy relaxation time rate is much faster than water’s energy relaxation time (Kleinberg et al., 1994). The water T1 and T2 energy relaxation time rates are primarily within the continuous bulk water phase. The high T1 and T2, of bulk water are out of the range of the 1H LF-NMR energy relaxation graphics of Fig. 5.
It is interesting that in both samples of LSE before and after thermal oxidation at 55 oC for 96 hours very minimal phase separation is observed (see supplemental 1). Similarly in Fig. 5 a similar peak pattern/arrangement is seen on the 1H LF-NMR energy relaxation TD graphics of LSE before and after thermal oxidation at 55 oC for 96 hours. Both samples before and after oxidation, have a 1D T1 energy relaxation time spectrum characterized by three peaks corresponding to the oil molecules in the core of the vesicles and one peak of higher intensity related to oil’s PUFA energy relaxation in the vesicle’s surface volume. Their 1D T2 energy relaxation time spectrum has four small peaks assigned to the four segmental motions of the oil molecules in the core of the oil body’s vesicles and one peak of higher intensity related to oil’s PUFA energy relaxation in the vesicle surface. The 2D energy relaxation time spectrum of LSE (T 0 h) and LSE (T 96 h) presents a peak of high intensity assigned to the energy relaxation of the oil bodies vesicle’s surface and small peaks assigned to the PUFA-rich oil molecules in the core of the oil bodies (Fig. 5, Table 3). Although the pattern of the 1D T1 and T2 and 2D relaxation time graphics of LSE (T0 h) and LSE (T 96 h) are very similar, the analysis of the T1 and T2peaks values presented in Table 3 showed that the sample of LSE after T 96 h oxidation has somewhat lower relaxation times than the sample of fresh LSE at T 0 h, most probable due to the thermal oxidation process and resulting increasing internal viscosity.
Average droplet particles size distribution as determined by dynamic light scattering (DLS) system shows an increase of oil bodies size from 803.2 nm at T 0 h to 1363 nm after T 96 h of thermal heating (Table 4). Microscopic images of fresh LSE sample versus LSE sample after 96 h of thermal heating further demonstrate this observation (see in supplemental material section). Indeed this pattern of vesicles size increase is well explained by a process of vesicles fusion after addition of heat energy (Millichip et al., 1996; Nikiforidis, 2019). However, this relatively small increase in the average oil bodies size may be correlated with the results of a small reduction in T1 and T2 proton energy relaxation times shown in Fig. 5 due to oxidation, which may indicate that the oil bodies emulsion preparation may be further improved to enhance oxidative stability. Furthermore the results of zeta potential assay show values of -29.1 and -28.7 for T 0 h and T 96 h, respectively. Usually it is accepted that emulsion preparations with values of zeta potential above -40 are considered as very stable and values of about -30 are considered to represent moderate stability (Nikovska, 2012; Chanamai and McClements 2002). Testing of self-diffusion of LSE samples before and after thermal heating for 96 h show some minimal decrease from 2.750 to 2.383 10^-9m*m/s, in respectively. These supportive results are also well correlate with previous data described above for linseed oil. The ratio of T1/T2 that is taken directly from the T1-T2 TD fingerprinting map show the similar pattern of a relative small change from 2.10 to 2.82 for fresh and 96 h heated LSE samples. This change in T1/T2 ratio values under oxidative conditions represent the chemical and structural changes effects on the signals obtained from the 1H LF-NMR that may be used as a fast tool to monitor the oxidation of the multicomponent complex emulsion samples.