Introduction
The present study is focused on characterization of linseed oil body
emulsions for prevention of polyunsaturated fatty acid (PUFA) oxidation
in food products and supplements. Its importance is due to healthy
nutrition requires consumption of the omega-3 polyunsaturated fatty
acids (PUFA) α-linolenic acid (ALA),
eicosapentaenoic
acid (EPA) and
docosahexaenoic
acid (DHA) that are key nutrients involved in normal growth and
development of various human tissues (Zarate et al., 2017; Yesiltas et
al., 2019). They, however are not synthesized within the body but can be
obtained from plant and marine sources, such as linseeds that have been
widely used since ancient times for different practical and valuable
applications including healthy foods. Linseeds health feature is their
high oil content of PUFAs, including omega-3 (ALA) and omega-6 fatty
acids. Since the consumption of omega-3 fatty acids has diverse
beneficial human health effects (Abozid and Ayimba, 2014) linseed oil is
used in pharmaceutical products, cosmetics and food supplements. Other
important linseed components: the fiber fractions of the seeds contain
the antioxidant lignan that may contribute to the prevention of certain
cancers (Thompson and Cunnane, 2003). They also contain mucilages with
anti-inflammatory effects on the intestine and seem to decrease
cholesterol and glucose levels in the blood (Thakur et al., 2009).
Linseed proteins, rich in arginine, are thought to have antioxidant and
antiatherogenic properties.
Linseed oils due to its high PUFA levels are well known to undergo
oxidation polymerization, conferring drying properties, which are used
in diverse technical applications, including paints, mastic formulations
and wood treatment (Jhala and Hall, 2010; Humar and Lesar, 2013).
Paradoxically, PUFAs though needed for health, also undergo facile
oxidation during storage and digestion into toxic oxidative products
(Yehuda et al., 1999). Therefore, methods to overcome oxidation such as
protective encapsulation and the use of antioxidants is extensively
studied (Jackowski et al., 2015 and Jacobsen, 2015). One delivery
approach is based on PUFA-rich oils encapsulated in gelatin capsules,
but for salmon fish oil, because of its high EPA and DHA concentration
it is still sensitive to shelf life oxidation. An alternative relatively
novel approach to PUFA encapsulation is based on natural seed oil bodies
(OB), that can spontaneously, under external force, organize in a water
matrix to form emulsions (Goyal et al., 2015). Linseed is an excellent
source of such oil body emulsion products (Fabre et al 2015).
Dispersion of aqueous extracts of intact seed oil bodies (OBs) in water
is a novel way of producing natural and oxidatively stable food
emulsions with minimal use of synthetic antioxidants and emulsifiers, as
there is growing interest in natural food emulsions containing
unsaturated oil (Shen et al., 2012). Within linseeds, the oil is stored
in OB, which protects the PUFA triacylglycerides (TAG) (97.6% of the
mass of the oil body) by their encapsulation with a membrane composed of
phospholipids (0.9%) and proteins (1.34%) (Tzen and Huang, 1992). The
oil bodies are stabilized and protected against coalescence by the
proteins present in the membrane; they can be directly incorporated into
formulations and this process is facilitated in some cases by the
emulsifying compounds present in the seed. Oil bodies and tensioactive
proteins are generally extracted in water from the seed. The exceptional
physical stability of Linseed OBs has been attributed to unique
amphipathic proteins called oleosins that occur within the oil body
surface (Huang, 1994). They are thought to stabilize OBs by steric
hindrance and electronegative repulsion (Jolivet et al., 2009). It has
been suggested that the entire surface of the OB is covered by oleosin
such that the compressed OBs rarely coalesce or aggregate in the cells
of a mature seed (Tzen and Huang, 1992). The storage of TAGs within OBs
not only prevents the TAG droplets from aggregation and coalescence but
also appears to protect them from lipid oxidation. OBs protect lipid
reserves against oxidation and hydrolysis until seed germination and
seedling establishment (Purkrtova et al., 2008). Oils of linseed,
sunflower and echium contain highly levels of PUFA that very susceptible
to oxidative deterioration resulting in a rapid decrease in
palatability, nutritional quality and shelf-life of foods into which
they are incorporated. Intact OBs offer a novel and effective route to
the preparation of oxidatively stable food emulsions from such unstable
PUFA oils without necessarily using synthetic antioxidants for
stabilization. Current international dietary guidelines advocate
replacement of saturated fats in food with unsaturated fats which
heightens the need to develop more efficient and improved methods for
stabilization of unsaturated oils incorporated in to emulsion-based
foods.
In nuts and seeds, lipids are stored in oil bodies called oleosomes that
store energy in the form of TAGs for use during germination and to
protect the lipids against physical and chemical stresses in the seeds
(Nikiforidis, 2019). Nature has evolved oleosomes as efficient mechanism
against lipid oxidation via an encapsulation by relatively complex
phospholipid/protein membranes, that are the only membranes that consist
only of a monolayer of phospholipids anchored with proteins
(Karefyllakis et al., 2019). The phospholipid layer has a thickness of
about 0.9 nm and composes about 2 wt% of the total mass of the seed
oleosomes (Millichip et al., 1996). Within and on this layer are
proteins (e.g., caleosins, and steroleosins (Nikiforidis, 2019;
Karefyllakis et al., 2019; Wahlroos et al., 2015). The proteins
N-terminal (N) and C-terminal (C) domains associate with the
phospholipid polar heads.
The present study characterizes linseed ALA fatty acid OBs, as an
encapsulating carrier of relatively high levels of EPA and DHA from fish
oil that are highly susceptible to oxidation. The OB’s phospholipids and
amphiphilic proteins and peptides of oleosin from linseed are well known
to spontaneously form an encapsulating shell around the oil emulsion
particles, with antioxidant characteristics (Fisk et al., 2008). These
linseed’s emulsion of linseed oil and the amphiphilic components of the
linseed (phospholipids and proteins) that stabilize the emulsion formed
by sonication of the seeds within water will be characterized by the
methodologies described below and will be correlated to its antioxidant
properties for the encapsulated PUFA’s. In addition, the different
components of linseeds can contribute to the emulsion’s lipophilic
antioxidants/vitamins, and arrange according to interfacial forces such
as tocopherol and lipophilic phenols and polyphenols within the oil
phase, and within the water the phase are water soluble
antioxidants/vitamins such as hydrophilic polyphenols and ascorbic acid
(Shen et al., 2012).
Numerous chemical and physical analytical methods have been developed to
asses lipid oxidation such as conjugated diene values, peroxide values
(PV), alcohols, epoxides, p-anisidine assay, HBR titration, iodometric
titration, xynol orange, total polar components (TPC) by high
performance liquid chromatography (HPLC), fatty acid composition
determined by gas chromatography-mass spectrometry (GC-MS), fourier
transformation infrared spectroscopy (FTIR), volatile product
determination by gas chromatography, dimer/polymers by size exclusion
chromatography (SEC), and electron spin resonance (ESR) (Jacobsen, 2015;
Hwang et al., 2017; Velasco et al., 2005). There is however a lack of
consistency in many of the results, because most of these analytical
methods are designed to detect one type of oxidation product while lipid
oxidation is a very complicated process producing numerous products at
different times of oxidation. Hence, as suggested by Hwang et al.
(2017), the development of methods that combine the concomitant
detection of different types of oxidation products is necessary for the
consistent assessment of lipid oxidation. In this respect, as described
below, LF-1H-NMR spectroscopy technology has a significant potential in
elucidating molecular structures of oxidation products from lipids and
in revealing the mechanisms of lipid oxidation.
Methods using high field 1H NMR relaxation were found
by Sun et al. (2011) and Bakota et al. (2012) to correlate well the
various parameters associated with lipid oxidation (e.g., free fatty
acid; polar materials in heated oils; solid fat content). Hwang et al.
(2017) proposed that ”there are molecular structure and composition
changes in oil during oil oxidation and degradation process affecting
the chemical environment surrounding the protons. Thus, the proton
mobility affecting the NMR energy relaxation time values changes as oil
degrades”. High field 1H NMR was also used to analyze
aldehydes produced in various heated oils (Guillen and Uriarte, 2009).
These researchers reported on the ability to analyze a list of aldehyde
products in linseed oil heated at 190 °C for 20 h, and also determined
acyl groups’ iodine value and polar compounds. Merkx et al. 2018
reported a broad band selective 1H NMR method for
determination of both hydroperoxides and aldehydes in oxidized oils.
Furthermore, based on electron spin resonance (ESR) system, combined
with a free radical standard and trapping agents (TEMPO and PBN) was
released for determination of peroxides in the early fast initiation
phase of oil oxidation (Velasco et al., 2005). Blumich (2016) developed
compact low field 1H LFNMR systems and Gouilleux et
al. (2016) developed an automate LF-NMR system. However, one of the
remaining problems of 1H LF -NMR and especially 2D
T1-T2 systems is the relatively long experimental and data processing
time required to finalize the results. Therefore, these systems were not
suitable for high throughput applications such as real-time reaction
monitoring or rapid screening of oil oxidation (Hwang et al., 2017).
In the present work, 1H LF-NMR recently developed data
processing was used to characterize both the chemical and
physical/morphological structures directly on a single graph (Weisman et
al., 2018, Resende et al 2019a,b). Our preliminary studies have shown
how the 1H LF-NMR energy relaxation time technology
with L1/L2 norm regularization parameters can characterize and
monitor PUFA rich oil oxidation, by generating 2D chemical and
morphological spectra (Campisi et al. 2018, 2019; Resende et al.,
2019a,b, 2020). In effect by using our recently developed primal‐dual
interior method for the convex objectives (PDCO) optimization solver for
computational processing of the energy relaxation time
signals T1 (spin–lattice)
and T2 (spin–spin), 2D spectra of the morphological and
chemical arrangements could be reconstructed. The LF‐NMR signal
reconstruction into 2D T1 vs. T2 graphs
effectively characterizes the chemical and morphological domains of
complex materials (Resende et., al 2019a) such as the 2D
spectra generated for butter, rapeseed oil, soybean oil, and linseed
oil. In order to correlate the chemical and morphology arrangements of
these emulsified aggregate structures we used our previous work on TD
NMR energy relaxation time analysis that can monitor changes in highly
complex systems. These studies also showed how the different degrees of
unsaturation of fatty‐acid oils affects their chemical and morphological
domains and influences their oxidative susceptibility (Resende et al.,
2019a).
The aim of the present study is to demonstrate the capability of1H LF-NMR energy relaxation time sensor to monitor the
TD fingerprints of chemical and structural changes of linseed oil bodies
emulsion (LSE) and linseed emulsion enriched with fish oil (LSFE) in
thermal autoxidation conditions and to test the efficacy of LSE as an
autoxidation protected fish oil PUFA-rich delivery system. Specific
objectives include characterization of structural and chemical changes
and stability using TD peak assignment, under thermal oxidizing
conditions (55oC for 94 hrs) using1H LF-NMR relaxation application and supportive
methodologies of the following PUFA aggregate structures. A.
Non-emulsion formulations, B. Linseed emulsion formulations, and C.
Linseed emulsions with fish oil.