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.