Abstract:
Oil bodies (OBs) are micron- or submicron-sized sub-organelles widely found in plants seeds and nuts. The structure OBs is composed of a core of triglycerides covered by a phospholipid-protein layer, which ensures the stability of the OBs under extreme environmental conditions and further protects core lipids as energy reserves. As naturally pre-emulsified oil-in-water emulsions, OBs have been gradually applied to replace synthetically engineered oil droplets. In this paper, the recent research on the composition, extraction, stability, delivery system, digestion, food applications and future perspectives of plant OBs are reviewed. Recent studies have focused on the OBs surface protein identification and function, large-scale extraction techniques such as enzyme assisted, high pressure, ultrasound, and extrusion and the reconstituted OBs. Electrostatic deposition of polysaccharides significantly improves the stability of OBs emulsions. OBs emulsions have promising applications to encapsulate bioactive compounds, deliver targeted drugs, and prepare gels and edible functional films. The digestive behavior of OBs emulsions is similar to that of protein-stabilized emulsions, which can increase the satiety, effectively help reduce calorie intake and improve the bioavailability of functional factors. It has also promoted the development of simulated dairy, spices and meat products.
Keywords : Oil bodies; Composition; Extraction; Stability; Delivery system; Digestion; Food applications
Introduction
Organisms store lipids in sub-organelles as energy reserves. These micron- or submicron-sized sub-organelles can be found in plants seeds and nuts, as well as few in some animal cells, fungi and insects (Huang, 1996) called oil bodies (OBs). Since 1970, scientists have reported on the structure, extraction and application of OBs from various natural sources. OBs either intracellularly or in isolated preparations have exhibited unique physical and chemical stability due to the presence of a surface membrane composed of phospholipids (PLs) and hydrophobic proteins such as oleosins preventing their aggregation or coalescence and protecting core lipids from extreme environmental conditions (Nikiforidis, Matsakidou & Kiosseoglou, 2014). It has isolated more than twenty kinds of seed OBs in the range of 0.5~2.0 μm, including peanut, sunflower and walnut with high oil content and soybean, corn, germ and flax with low oil content (Huang, 2018). The small size of OBs provides a large surface area per unit of triacylglycerols (TAGs) and facilitate binding to lipase or other subcellular structures for rapid TAGs mobilization during germination (Tzen, Cao, Laurent, Ratnayake & Hung, 1993). OBs is extracted from plant materials by using aqueous media and the difference of specific gravity and solubility between OBs and other components. The isolated OBs droplets can be dispersed in aqueous environments to form natural emulsions. As a naturally pre-emulsified oil-in-water emulsions, OBs can be widely applied in food production, which is beneficial to the low energy consumption and it has excellent stability and emulsifying performance (Nikiforidis, 2019).
This paper reviews the research advances of OBs derived from plants. It provides a general description of OBs composition and structure and the methods to maintain the stability of OBs emulsions. The technologies for large-scale extraction and delivery systems of OBs are highlighted. In addition, the digestive behavior and current applications of plant-based OBs mainly in food research are discussed. The review reports the future research direction and development potential of natural OBs.
2.Composition and structure of OBs
The composition of the OBs conforms to a formula describing a spherical particle surrounded by a shell of a monolayer of phospholipids embedded with proteins. The spherical particle is a neutral lipid core consisting of TAGs as a rich source of oil in storage form, a small amount of diglycerides, free fatty acids, and Vitamin E, etc (Abdullah, Weiss & Zhang, 2020). The neutral lipids of the OBs account for about 92%~98%, the contents of PLs and proteins are about 0.6~2.0% and 0.6~3.0%, respectively (Ding et al., 2019; Tzen et al., 1993).
2.1 Neutral lipids
The lipid composition of OBs was examined by chromatography. It was found that the content of TAGs was the highest, and palmitic acid, stearic acid, oleic acid, linoleic acid were the main fatty acids (Zaaboul, Raza, Chen & Liu, 2018; Xu et al., 2021). TAGs can not only be as energy reserves for germination and post germinative growth of the seedlings, but also are important for cell division and expansion, membrane lipid remodeling and organ formation (Yang & Benning, 2017). If the seeds have been stored for a long time, the hydrolysis of TAGs and PLs by internal or external lipase or nonspecific acyl hydrolases may lead to the increase of minor lipids, especially free fatty acids, causing rancidity and quality degradation (Huang, 1992).
2.2 PLs
The electron microscope results showed that the core of OBs was an electronically opaque TAGs matrix surrounded by one electron-dense layer. This single electron-dense layer contrasted with the two parallel electron dense layers presented in the unit membrane of the two PLs layers that composed of the cell membrane. It was determined that the surface of the OBs was surrounded by a ”half-unit” membrane of one PLs layer, in which the hydrophobic tail faced inward to interact with the TAGs, and the hydrophilic head was exposed to the cytoplasm (Tzen & Huang., 1992). Typically, PLs accounted for more than 80% of the surface area of the OBs, and the thickness was 0.9 nm~2.5 nm (Purkrtova, Jolivet, Miquel & Chardot, 2008; Yang, Su, Zhang, Jia & Phillips, 2020). It was determined by TLC that the major PLs in Echium plantagineum OBs was phosphatidylcholine (PC; 52.4%), followed by phosphatidylserine (PS; 32.6%), phosphatidylethanolamine (PE; 12.3%) and phosphatidylinositol (PI; 4.2%) (Payne, Lad, Foster, Khosla & Gray, 2014). 31P NMR measured the highest content of PC (50.6%) in peanut OBs, followed by PI (13.6%), PE (12.7%), PS (10.8%) and phosphatidic acid (PA; 10.0%) (Niu, Chen, Liu & Duan, 2021). PC had a stronger hydrophobic interaction with the OBs membrane proteins, therefore, the OBs were much more stable. Tzen et al. (1993) analyzed the PLs in the OBs from seven kinds of oilseeds found that they contained substantial amounts of the uncommon negatively charged PS and PI. It has been proposed that these negatively charged PLs can interact with the basic amino acid residues of the oleosins on the surface of the PLs layer. The fatty acid composition of the PLs in the OBs was determined to be high in saturated fatty acids, which contributed to the anchorage of the oleosins and thus to the stability of the OBs (Furse et al., 2013; Katavic, Agrawal, Hajduch, Harris & Thelen, 2006; Payne et al., 2014).
2.3 Proteins
It was studied that the PLs on the surface of the natural OBs could not be approached by external phospholipase A2 or phospholipase C. The integrity of the OBs was due to the shielding effect of proteins, while the OBs termed to polymerized after trypsin treatment. Experiments also showed that the artificially prepared particles of TAGs surrounded by a layer of PLs would polymerize rapidly. However, when the particles contained proteins on their surfaces, like natural OBs, they became stable and did not aggregate or coalesce, even they were brought to squeeze against one another in vivo and in vitro . This confirmed the importance of proteins in maintaining OBs stability, since proteins provided not only amphiphilic surfaces but also steric hindrances (Tzen et al., 1992). At present, the research mainly focuses on the identification and function of OB interface proteins.
The dominant OBs-associated proteins are called oleosins. Oleosin has a low molecular mass of 15~26 kD with three structural regions. Two amphiphilic N- and C-terminal regions moieties located at the surface of the OBs, with the positively charged residues interacts with the negative charge of the phosphate molecule towards the lumen of the organelle and the negatively charged residues facing the cytosol, which makes the OBs surface negatively charged and generates electrostatic repulsion to maintain the stability of the OBs, also can prevent external phospholipase from acting on the PLs membrane (Napier, Stobart & Shewry, 1996). A central hydrophobic region of about seventy residues formed by two approximately 11 nm long antiparallel strands connected by a “proline knot” is inserted in the acyl moieties of PLs and the TAGs matrix and forms a hairpin-like structure (Tzen, Lie & Hung, 1992).
Caleosin is another group of protein on the OBs surface, with a molecular mass of 25~35 kDa. It has the same molecular structure as oleosin, including a hydrophilic N-terminal group, a hydrophilic C-terminal region, and a central hydrophobic region inserted into the core of TAGs (Næsted, Frandsen, Jauh, Hernandez-Pinzon & Mundy, 2000). Caleosin is characterized by a more hydrophilic N-terminal segment as it contains a Ca2+ binding region (Chen, Tsai & Tzen, 1999). It has been reported that caleosin may be involved in OBs fusion, membrane-fission and/or fusion and lipid intracellular trafficking and metabolism (Frandsen, Mundy & Tzen, 2001; Næsted et al., 2000).
Steroleosin is a comparatively bigger protein with molecular mass more than 35 kDa. It firstly identified as a minor protein in sesame OBs, comprised a hydrophobic anchoring segment at the same length as caleosin, followed by a sterol-binding dehydrogenase domain, which could anchor the soluble sterol binding dehydrogenase domained to the surface of the OBs through the N-terminal hydrophobic fragment (Lin, Tai, Peng & Tzen, 2002).
Tnani, López, Jouenne & Vicient (2011) identified new proteins in maize embryos OBs besides oleosin, caleosin and steroleosin, such as karyopherin-beta and a stress-induced membrane pore protein were involved in membrane transport. Jolivet et al. (2004) found that oleosins accounted for 79% of OBs proteins in Arabidopsis thaliana ecotype WS, and the 18.5 kDa oleosin was the most abundant. Meanwhile, they found a probable aquaporin and a glycosylphosphatidylinositol-anchored protein, which had never been found in plant OBs, without known functions. Plant nsLTP, a soluble protein with a molecular mass of less than 20 kDa, presented in rice bran OBs, which contributed to the transfer of fatty acids, PLs, glycolipids and steroids between membranes, and played a key role in the process of plant resistance to pathogens. Embryo-specific protein belonging to plant antimicrobial peptides family and storage proteins such as gi|31432342, gi|24899397 and gi|12039336 were also identified in rice bran OBs (Xu et al., 2021).
2.4Bioactivecomponents
OBs may contain a small amount of bioactive components that are contribute to their chemical stability, such as tocopherols, phytosterols, γ-oryzanol, essential amino acids and isoflavones depending on the source (Fisk & Gray, 2011; Gallier, Gordon & Singh, 2012; Nantiyakul, Furse, Fisk, Foster, Tucker & Gray, 2012; White, Fisk & Gray, 2006; Zaaboul et al., 2018). Bioactive components make natural OBs with high nutritional value and the development and utilization prospects are very broad.
3. OBsextraction
The microstructure of plant cells was observed, and abundant natural OBs of different sizes were clearly found (Fig. 3). Starch and proteins were distributed around the OBs, and each region was separated by the cell wall to maintain the integrity of the OBs. Therefore, the destruction of cell wall is the key to extracting natural OBs. Due to the hydrophilic nature of the OBs surface, it can be dispersed into aqueous phase to form a uniform emulsion (Nikiforidis, 2019). Based on this characteristic, the isolation process of OBs can be divided into four steps: (1) the structure of plant cell wall is destroyed by mechanical crushing, and the OB is separated from protein body and starch grain to release in aqueous medium; (2) the slurry is filtered to produce a ” milk ”, which is a suspension of OBs in an aqueous medium, with reduced seed particulate material; (3) OB is concentrated into cream by centrifugation of the milk to obtain crude OBs; (4) the crude OB is washed to remove the impurities on the surface and obtain pure OBs (Nikiforidis et al, 2014). Current extraction methods of OBs mainly include aqueous extraction and enzyme assisted extraction based on the different principles of disrupting the cell wall.
3.1Aqueous extraction
The aqueous extraction of OBs requires soaking seeds in an aqueous medium, then blending or pressing to disrupt cell walls and released intracellular materials. Urea, sucrose, deionized water, salt, alkali and buffer solution including Tris-HCl and PBS are often used as the grinding medium for aqueous extraction. In 1992, 0.6 M sucrose solution was first used to extract plant OBs including maize, rice, soybean, rapeseed, jojoba, wheat bran, flax, sesame, tripsacum, teosinte, yucca, mustard, jojoba, sunflower, peanut, palm, castor bean, Brazil nut and oat bran, the grinding medium also contained 1 mM EDTA, 10 mM KCI, 1 mM MgC12, 2 mM DTT, and 0.15 M Tricine buffer (Tzen et al, 1993; Tzen & J., 1992; Tzen et al, 1992). Since 1996, the extraction medium was simplified (Chuang, Chen, Chu & Tzen, 1996; Tzen, Peng, Cheng, Chen & Chiu, 1997). Lacey, Wellner, Beaudoin, Napier & Shewry (1998) found that the OBs extracted by urea (9 M) was not affected by exogenous proteins, and integral oleosin proteins could be completely preserved. Urea can disrupt non-covalent bonds of proteins allowing the removal of passively associated proteins from the OBs surface (Millichip, Tatham, Jackson, Griffiths, Shewry & Stobart, 1996). However, urea is not a food grade material, thus limiting its use. At present, deionized water and buffer solution are often selected as extraction media (Nikiforidis & Kiosseoglou, 2009; Iwanaga, Gray, Fisk, Decker, Weiss & McClements, 2007; Lan et al., 2020), while, the OBs still contain some intracellular substances such as exogenous proteins and phytochemicals, and endogenous proteins are also vulnerable to destruction. Therefore, in order to simplify the subsequent washing steps, De Chirico, di Bari, Foster & Gray (2018) pointed out that the purity of oilseed rape seed OBs extracted with sodium bicarbonate solution (0.1 M) in the grinding and washing steps was the same as that produced by washing a crude preparation with 9 M urea, and the physical stability of the OB was improved. The results provided a new method for the aqueous extraction of intact and pure OBs.
3.2Enzyme assisted extraction
Plant cell walls are composed of cellulose, hemicelluloses, lignin and pectin. Thus, in addition to mechanical means, the use of carbohydrase such as cellulase, hemicellulose, pectinase, xylanase and β-glucanase may also destroy the cell wall. The high specificity of enzymes greatly limits the hydrolysis due to the diversity of cell wall components and raw materials. In this case, the complex enzyme has the activities of multiple enzymes can be considered. During the extraction process, the use of proteases is not considered. Although the recovery of OBs can be improved, the associated proteins may be destroyed into small peptides through hydrolysis. Kapchie, Wei, Hauck & Murphy (2008) compared the efficiency of mixing Multifect Pectinase FE (Pectinase, cellulase, and hemicellulase complex), Cellulase A (Cellulase, β-glucanase, hemicellulase, and xylanase complex) and Multifect CX 13L (β-glucanase complex) in equal proportions with aqueous extraction of soybean OBs found that the total soybean oil recovered from OBs extracted by enzymatic method was 36.42%~63.61% and the yield of soybean oil in OBs could reach 84.65% after three successive extractions of the residue. However, the total soybean oil recovered from OBs was only 28.65%~34.28% by conventional method. Xu et al. (2021) studied the structures, physical properties and chemical compositions of rice bran OBs extracted by plant exacted enzyme, xylanase and their mixture, and showed that the yield of OBs obtained by the mixture of xylanase and plant extracted enzyme was 76.95%. Extracting OBs from plant raw materials is a relatively new research field. It is necessary to invest in developing special equipment and consider the actual production conditions to achieve industrial production. Towa, Kapchie, Hauck, Wang & Murphy (2011) conducted a pilot scale equipment in which the yield of OBs isolated from soybean to 93.40% compared to a laboratory-scale process.