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.