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.