3.3 Comparison of different methods
The selection of OBs extraction
method mainly depends on the extraction yield, complexity, production
cost, environmental friendliness, and safety. Aqueous extraction is a
traditional method widely applied for extraction of OBs has the
advantages of no chemical pollution and low energy consumption, however,
there are certain limitations that it requires large amount of solvents,
and thus makes up-scaling difficult. Although the enzymatic extraction
of OBs requires a long reaction
time, and the enzyme has strict requirements on temperature and pH, the
extraction efficiency of OBs is higher. There are major problems that
must be solved to develop special enzyme, reduce the dosage of enzyme
and ultimately reduce the cost of enzyme in the industrialization of
enzymatic extraction of OBs technology. Furthermore, the extraction
procedures require simple equipment that can be operated safely to
facilitate industrial production and commercial application of OBs.
3.4 The main influencing
factors ofextraction
process
In order to further improve the OBs
yield, reduce the cost and obtain high quality and purity OBs, the key
parameters in the extraction process can be controlled. Generally, there
are many factors affecting the extraction procedures including
pretreatment, pH, purify, medium ratio and centrifugal force.
3.4.1 Pretreatment
The pretreatment of plant
materials usually involves
grinding and
soaking. Grinding provides better
exposure of OBs to the water as a
result of cells structure rupture, and soaking allows water molecules to
penetrate into the network of cells for more efficient extraction. For
seed raw materials, mechanical grinding is usually carried out in the
extraction medium after soaking for 8~72 h. For the
processing by-products such as wheat bran and rice bran, after sieving
and removing impurities can be soaked or not for a short time, followed
by mixed with the extraction medium for homogenous extraction of OBs
(Lan et al., 2020; Nantiyakul, Furse, Fisk, Tucker & Gray, 2013; Tzen
et al., 1992; White et al., 2006).
At present, pretreatment methods such as
mechanical
crushing,
extrusion,
high pressure, and ultrasound have been successfully used for aqueous
oil extraction, but it is worth noting that these pretreatment methods
have the potential to improve the OBs extraction yield (Mat Yusoff,
Gordon & Niranjan, 2015; Koubaa, Mhemdi, Barba, Roohinejad, Greiner &
Vorobiev, 2016). The crushing
degree of raw materials directly affects the extraction yield of OBs.
The size of raw material cells should be considered during mechanical
crushing. When the particle size
of the material is smaller than
the cell size, the cell wall can be destroyed and the contact area
between the OBs cells and the extraction medium will be enlarged. While,
excessive crushing may destroy the complete structure of the
OBs. Nikiforidis et al. (2009)
showed that the extraction yield of OBs was the highest at any pH and
number of extraction steps applied when the corn particle size was less
than 0.8 mm. However, there was a possibility that a single OB merge
into a larger droplet.
The
extrusion
process increases the pressure generated by compression to exceed the
maximum shear stress that the plant cell wall can withstand, causing the
cell wall to rupture (Peng et al., 2021; Li, Zhou, Zhang, Wang & Cong,
2020). It is not conducive to the solvent percolation for the fine
powder raw materials. Extrusion can also reshape the material into
porous expanded particles, which increases percolation rate of the
solvent and hence better efficiency of OBs extraction (Liu et al.,
2020). Romero-Guzmán, Jung, Kyriakopoulou, Boom & Nikiforidis (2020)
used a twin-screw press to extract
OBs at pH 7.0, and showed that the
yield can reach 90% and twin-screw press was a promising alternative to
scale-up the OBs aqueous extraction and the water usage was
significantly reduced.
It has been reported that the high pressure-processing transmitted
isostatic pressure (100~400 MPa) to plant materials
could be useful for destroying cells and increasing the solubility of
bioactive compounds (Bueno, Gallego, Chourio, Ibáñez, Herrero &
Saldaña, 2020; Ninčević Grassino et al., 2020). However, the structure
of the product changed at a pressure of around 600 MPa, resulting in a
decrease in extractability (Butz, Edenharder,
Garcı́a, Fister, Merkel &
Tauscher, 2002; Dobrinčić, Repajić, Garofulić, Tuđen, Dragović-Uzelac &
Levaj, 2020). Kapchie et al. (2008) demonstrated that the highest OBs
yield of 71.39% was obtained with the ultrahigh pressurizations of
soybeans flours at 200 MPa for 5
min at 25 °C, while, the OBs yield was 21.82% with material pressurized
at 500 MPa.
Ultrasound
induced impacts which can be attributed to the cavitation phenomenon
referring to bubble formation, growth and implosion during the
propagation of the ultrasonic wave into medium. The bubble implosion
will create a hot spot with a temperature of up to 5000 K and a pressure
of 5000 atm (Khadhraoui, Ummat, Tiwari, Fabiano-Tixier & Chemat, 2021).
This may result in slight or high effects on cell walls, enhancing the
penetration of the solvent into the internal structure and facilitating
the release of the target compound. In addition, the strong shear forces
and turbulence generated by the propagation of the ultrasonic waves
appear to further accelerate the exchange between the raw material and
the surrounding extraction solvent. These mechanisms lead to ”an
increase in the depth and speed of solvent penetration into plant
internal structures” (Soria & Villamiel, 2010; Chemat et al., 2017).
Loman, Callow, Islam & Ju (2018) found that 1.5 W/mL pulsed ultrasound
treatment for 5 min every 3 h could significantly improve the
performance and separation of OBs from protein during the enzyme
processing. It was studied that ultrasonic pretreatment would increase
the OBs yield, but the yield showed a downward trend after a certain
time, because the ultrasonic energy was not uniformly distributed in the
pretreatment process (Albu, Joyce, Paniwnyk, Lorimer & Mason, 2004;
Kapchie et al., 2008; Toma, Vinatoru, Paniwnyk & Mason, 2001).
3.4.2 pH
pH is an important parameter in the
OBs
extraction process, which affects the separation of exogenous proteins
and the integrity of OBs. The isoelectric point of the OBs is between
4.0~6.0 due to the existence of proteins on the
surface
(Wang et al., 2019; Tzen et al., 1993). Therefore, it is not considered
that the extraction pH is lower than 6.0. At this point near the
isoelectric point of the proteins, the natural and independent OBs in
the cells will aggregate after separation. The pH value of the medium
when extracting OBs is usually between 6.5~11.0. In this
pH range, the surface of the OBs
maintains a negative charge, and electrostatic repulsion and steric
hindrance effect exist between the OBs. When the pH value is higher than
9.0, both proteins and OBs are soluble and the extraction yield is
improved (De Chirico et al., 2018). Study have indicated that an
extraction yield as high as 95% could be reached when a finely
comminuted germ material was extracted 3 times at pH 9.0
(Nikiforidis
et al., 2009).
It is known that the pH value of the extraction buffer not only has a
direct influence on the extraction yield of OBs, but also has a great
influence on the protein composition of the extracted OBs, which affects
the OBs properties, and then
affects the OBs
utilization. Zhao, Chen, Chen, Kong
& Hua (2016) studied jicama, sunflower, peanut, castor bean, rapeseed,
and sesame to explore the effects of pH (6.5~11.0) on
protein compositions of OBs. The results showed that there were many
extrinsic proteins (globulins, 2S
albumins and enzymes) presented in
pH 6.5-extracted OBs. Globulins was mostly removed at pH 8.0 and 2S
albumins were removed at pH 11.0. At pH 11.0, highly purified OBs were
obtained from jicama, sunflower, peanut, and sesame, whereas there were
still enzymes remained in the castor bean and rapeseed OBs. Therefore,
the extraction pH could be selected according to the properties of OBs
product: for high protein-containing OBs products, neutral or even
acidic pH should be selected; for highly purified OBs products, high
alkaline pH should be
selected.
It was clear that as the extraction pH increases, the exogenous protein
was gradually reduced, however, it is not clear whether oleosins could
also be removed by alkaline pH (Chen & Ono, 2010). Cao, Zhao, Ying,
Kong, Hua & Chen (2015) confirmed that alkaline pH not only removed
contaminated proteins but also oleosins, and more and more oleosins were
removed with increasing alkaline pH. The research showed that pH also
affected the rheological properties of OBs, gels were formed from OB
emulsions with solid content of 40% except pH 11.0-OBs, for liquid-type
OBs products, pH 11.0 should be selected; for solid-type OBs products,
pH 9.5 should be selected (Zhao, Chen, Yan, Kong & Hua, 2016). Zaaboul
et al. (2018) first investigated the main compounds of peanut OBs and
found that oleic acid and linoleic acid were the major fatty acids in
OBs regardless of pH. Tocopherol content went from 270.76 to 278.2 mg/g
when pH was increased. On the contrary, phytosterols content decreased
when pH was increased, with 631.49 μg/g at pH 6.8 and 614.96 μg/g at pH
11.0.
3.4.3Purified OBs
It is necessary to determine whether pure
OBs or a mixture of OBs and
storage proteins are needed when extracting
OBs. For some applications,
especially foods, such as salad dressings, where proteins have been used
to modulate the macro properties of the system, so mixtures containing
OBs and storage proteins are beneficial (Nikiforidis, Biliaderis &
Kiosseoglou, 2012; Karefyllakis, Octaviana, van der Goot & Nikiforidis,
2019). However, in other applications, pure OBs may be required. To
obtain pure OBs, several cleaning steps are required including
urea washing, sucrose
washing, deionized water washing,
salt washing, alkali washing and buffer solution washing including
Tris-HCl washing and PBS washing. Generally, the washing medium is
similar to the extraction medium, while the medium used in extracting
crude OBs and obtaining pure OBs
can be different. It was reported that
OBs washed in (9 M) urea were
significantly enriched in lipids and low in proteins compared with
unwashed, water-washed, and salt-washed OBs and washing significantly
reduced the total phenolic content
of the oat OBs but significantly
increased concentrations of vitamin E (White et al., 2006). For soybean
OBs from different varieties, the contents of
vitamin E and total phenolics were
decreased by urea washing (Fisk et al., 2011). Murphy & Cummins (1989)
explored the influence of washing times on
OBs found that the composition of
OBs
was not affected by washing times, but multiple washing times would
greatly reduce the final production of
OBs. The OBs apolipoprotein was
tightly bound to the surface of OBs and could not be removed by washing
and pure OBs fraction could be obtained by two floatation steps at most.
3.4.4
Other influencing factors
The key factors affecting OBs
extraction also include medium
ratio, centrifugal force,
incubation time, temperature, stirring speed and so on.
The
viscosity of the system is determined by the ratio of the solid to the
disperse medium. When the viscosity is too low, it may result in less
force borne by OBs during the recovery process and reduce the collision
and damage of OBs particles. However, when the water content of the
system is low, the viscosity is high and OB is not easy to free,
resulting in low yield. It had been confirmed that the OB particle size
distribution presented a wide distribution at a high solid-phase loading
ratio, and droplet aggregation was observed in the optical image, while,
in a more diluted seed grinding system, the OB was smaller and integrity
(De Chirico et al., 2018). The centrifugal force can affect the size
distribution of the OBs, If the centrifugal force is too low, small OBs
will be emulsified or too high,
OBs trend to coalesce. Current
studies select centrifugal forces ranging from 5000 RCF to 20000 RCF
(Zhang et al., 2017). Some
researchers modified the traditional extraction
method by giving the raw materials
a certain incubation time and
temperature under a stirring speed (Niu et al., 2021; Nantiyakul et al.,
2012). These operations affected the denaturation of proteins, the
dispersion and aggregation of OBs,
and also affect the extraction yield of
OBs. For enzymatic extraction, the
amount of added enzyme is also an important factor. Generally, there are
many factors affecting the yield of OBs during the extraction process,
while systematic and statistical optimization of the significant factors
has not been attempted to obtain the optimal extraction conditions and
the ideal yields.
4.Stabilityof OBs emulsions
OB is mainly dispersed in an aqueous media to form a natural
emulsion system, which can deliver
stable preemulsified oil into appropriate food systems to obtain
products. This will reduce the need to extract and purify the oil using
organic solvents and then emulsify it using a homogenizer, thereby
achieving more sustainable and environmentally friendly processing
operations. The utilization of OBs in food products requires a thorough
understanding of their functional properties under complex environmental
conditions. During food processing, storage, transport and utilization,
the pH value of the system may change. For taste, preservation and
modification of physicochemical properties, salt is added to the food.
Many emulsion-based food products may undergo various kinds
of thermal treatments such as
pasteurization, sterilization, temperature fluctuations, baking and
cooking and freezing and thawing process.
Studies have shown that the rheology
and stability of OBs suspensions
are susceptible to the effects of pH and salt concentration, resulting
in reduced electrostatic repulsion between OBs and instability such as
flocculation (White, Fisk, Mitchell, Wolf, Hill & Gray, 2008).
Therefore, to utilize
OBs
commercially in food systems, the stability of OBs must be enhanced in
order to adjust to environmental changes (in terms of pH, ionic strength
and thermal treatment).
OBs and
polysaccharides
have similar types of charges which can cause the effect of
electrostatic deposition and steric repulsion of polysaccharides between
emulsion droplets (Iwanaga, Gray, Decker, Weiss & McClements, 2008).
Many researchers have attempted to use a method that changed interfacial
composition by coating OBs with a
layer of polysaccharides to
improve the stability of OBs to environmental stresses. The most
commonly reported polysaccharides are pectin, carrageenan, xanthan gum
and gum
arabic.
The formation of polysaccharide-OB complex on the surface layer depended
on the pH values, polysaccharide concentration, and temperature. Laccase
cross-linked beet pectin coated soybean OBs had better stability at pH
change (3.0~7.0), NaCl addition (0~500
mM), and freeze-thaw cycle (-20 ℃ for 22 h; 40 ℃ for 2 h) (Chen,
McClements, Gray & Decker,
2010).
It had been shown that soybean OBs emulsions stabilized with
ι-carrageenan were more stable to
creaming due to depletion flocculation than the emulsions stabilized
with κ or λ-carrageenan after 7 d storage and the
soybean OBs emulsions by coating a
ι-carrageenan layer at pH 3.0 and 7.0 had improved stability to
environmental stresses (Wu et al., 2012; Wu, Yang, Teng, Yin, Zhu & Qi,
2011). Lan et al. (2020) screened
xanthan
gum from ten stabilizers to stabilize safflower OBs, and determined that
when the addition amount was 0.3%, the OBs coated with xanthan gum had
thermal
stability, but was affected by ultrasonic strength (Sukhotu et al.,
2016). Ding et al. (2019)
successfully and effectively encapsulated soybean OBs by using
maltodextrin (MD)–chitosan (CS)- Epigallocatechin- 3-gallate (EGCG)
covalent conjugates (CSEG) as coating material and applying spray drying
technology, and the emulsifying activity and thermal stability of OBs
microparticles were improved and significantly reduced the amount of oil
released during the whole digestion process.
5.Delivery
system of OBs
5.1Emulsions-based
delivery system of OBs
Emulsions-based delivery system in food industry is widely recognized to
be a promising method of encapsulating and delivering bioactive
compounds in product manufacturing and storage processes for preventing
chemical degradation and increasing bioavailability (McClements, 2015).
Liu, Wang, He, Cheng & Ma (2020) reported that soybean OBs could be
used as the novel carriers in delivering the
curcumin to improve the stability
of curcumin and its release rate during
digestion. Chiang, Chen, Liou &
Chao (2019) found that the
self-assembly nanoscale
OBs enabled targeted delivery
curcumin, and curcumin-loaded nanoscale OBs displayed a strong
anti-proliferative effect on tumor cells. Studies also have showed that
OBs also were effective carriers for volatile
flavor compounds. Fisk, Linforth,
Taylor & Gray (2011) indicated that OBs offered the enhanced flavor
delivery through elevated headspace flavor persistence. Water-washed OBs
were spray dried further stabilized with capsules to embedment volatile
lipophilic actives (D-limonene), with a retention rate of 55.59% (Fisk,
Linforth, Trophardy & Gray, 2013).
5.2
Emulsion-gels
Emulsion-gel
is a typical semi-solid food system consisting of gel matrix filled with
oil/fat droplets (Dickinson, 2012). Proteins such as gelatin, soy
protein, casein and polysaccharides such as starch, carrageenan,
pectins, alginate and flax gum are the most recently used as the matrix
(Bi, Chi, Wang, Alkhatib, Huang & Liu, 2021; Dickinson, 2012;
Fontes-Candia, Ström, Lopez-Sanchez, López-Rubio & Martínez-Sanz, 2020;
Hu, Karthik & Chen, 2021; Li,
Gong, Hou, Yang & Guo, 2020; Saavedra Isusi, Madlindl, Karbstein & van
der Schaaf, 2020). Emulsion-gels have broad application prospects in
food industry such as fat reduction, probiotics release and flavor
control due to their diversity in structure and composition (Lin, Kelly
& Miao, 2020). OBs can replace the traditional
oil/fat droplets in the gel due to
their nutritive value and natural emulsification properties resulting in
great application prospects in food industry (Nikiforidis et al, 2014).
However, to the best of our knowledge, there are very few studies on the
emulsion-gels filled with OBs.
Kirimlidou,
Matsakidou, Scholten, Nikiforidis & Kiosseoglou (2017) revealed that
filling the gelatin matrix with
OBs instead of
oil/fat droplets, the OBs could be
well dispersed in the gel network, and had no negative effect on the
rheological properties of the composite gels. Mert & Vilgis (2021)
adopted xanthan gum and pectin to stabilize natural
OBs suspension based on
electrostatic deposition, and converted it into soft solid oleogel
structure. This discovery not only improved the stability of OBs
suspension, but also provided a new idea for the design of gel structure
composed of OBs. Yang et al. (2020)
constructed an emulsion-gel with soybean OBs as filling oil/fat droplets
and κ-carrageenan as gel matrix, and found that OBs emulsion-gels
exhibited better lubrication properties and an ultralow boundary
friction coefficient (μ) was achieved, which was significant to study
the oral processing of OB
emulsion-gels when it was used in semi-solid food. Nikiforidis &
Scholten (2015) prepared a high internal phase emulsion gel with volume
fractions of 0.91 and elastic properties by using natural OBs, and its
shear elastic modulus was between 102 and
105 Pa.
5.3 Edible films
Edible films based on biodegradable
materials, such as proteins, polysaccharides, lipids have potential uses
in food packaging and as carriers of active compounds like antioxidants
and antimicrobials (Jeya Jeevahan et al., 2020). However, the water
barrier property of water-soluble hydrocolloid edible films is poor. It
has been reported that vegetable
oil was added into the biopolymer film matrix to reduce the water vapor
permeability (Jeevahan & Chandrasekaran, 2019). The biopolymer-oil
mixture needs to be homogenized to obtain a uniform distribution with
small-sized droplets, thus increasing the tortuosity factor and
improving the water barrier performance of the film (Vargas, Perdones,
Chiralt, Cháfer & González-Martínez, 2011). To avoid the
homogenization, small sized natural OBs can be added to the initial
biopolymer solution to replace vegetable oil. OB is a kind of natural
composite film based on the special structure, but it has reported that
the initial OB film was less elastic and easy to tear, and then used
Tris-HCl plasticizer, Ca2+/Mg2+crosslinking agent and carboxymethyl cellulose could effectively modify
the mechanical properties of OBs membranes, making them have a wide
range of tensile strength and elongation properties (Wang, 2004). The
interaction between the surface of maize
germ OBs and caseinate molecules
resulted in the effective binding of the OBs to the protein matrix, and
then the composite film with milky
white appearance, strong hydrophobicity, relative viscosity and
flexibility was prepared. The fact that mechanical and optical
characteristics of the composite films marked alterations upon storage
due to water uptake or OBs movement (Matsakidou, Tsimidou &
Kiosseoglou, 2018; Matsakidou, Biliaderis & Kiosseoglou, 2013).
5.4 Reconstituted
OBs
According
to relative proportions of TAGs, PLs and proteins, these three basic
components can be used to
reconstruct the stable OBs by
ultrasonic technology (Tzen et al., 1992).
Recently,
the
reconstituted OBs
expression/purification system has been developed for the purification
of recombinant proteins or enzymes immobilization in one step by linking
a desired protein or enzyme to oleosin on the surface of OBs (Bai, Yan,
Zhang, Yu & Bai, 2014; Bettini, Santino, Giancane & Valli, 2014;
Chiang, Chen, Chao & Tzen, 2005). This novel technique provides a
promising alternative for purifying recombinant proteins regarding to
the equivalent purification efficiency at a lower cost (Tseng, Huang,
Huang, Tzen, Chou & Peng, 2011). The reconstituted OBs can be used like
natural OBs to carry nutrients (such as curcumin), probiotics and
medicines (Bettini et al., 2013; Santiago & Devanadera, 2016).
By
changing the amount of three components, it is possible to obtain
reconstituted OBs of different sizes and the physicochemical stability
and structure of the reconstituted OBs with different sizes are also
different (Peng, Lin, Lin & Tzen, 2003). Typically, nanoscale
reconstituted OBs are generated by self-assembly by changing the ratio
of matrix oil to oleosins to target the delivery of hydrophobic drugs
(Chiang, Lin, Lu & Wang, 2011; Chiang, Lin, Yang & Chao, 2016).
6.Digestion of OBs
OBs emulsions can be used as carrier to transport natural, minimally
processed and pre-emulsified oil to appropriate food system, and also
contain lipophilic bioactive components such as tocopherol, oryzanol and
sterol. The
digestive behavior of
OBs
emulsions is similar to that of
protein-stabilized emulsions, with flocculation of the OBs occurring
under gastric conditions. Under intestinal conditions, bile salt
replaces interfacial peptides and phospholipids and destroys the
flocculate and the hydrolysis of triglycerides leading to the
spontaneous formation of a novel multiple emulsion (Gallier, Tate &
Singh, 2013). In general, lipids digestion in OBs is slow and the
digestive efficiency may affect by the following three aspects (Wang, Ye
& Singh, 2020). Firstly, the OBs membrane has a negative impact on the
digestion efficiency. Secondly, the appearance of new prunin isoforms in
oleosins and the rearrangement of
protein profile may limit the lipids bioaccessibility. Thirdly, long
chain fatty acids, the main lipolytic products, accumulated at the
surface of the OBs limited the activity of pancreatic lipase (Gallier &
Singh, 2012; Trombetta et al., 2020). While, in the complex food matrix,
a protective layer is formed around
OBs under the influence of
macromolecules such as protein and polysaccharide, which promote the
flocculation of OBs droplets and inhibit the ability of pepsin and
lipase, so as to reduce the absorption rate of
lipids
and related lipophilic compounds
(Wu et al., 2012). The digestion rate of
lipids in OBs-emulsion food
products is slower than that in free
OBs
emulsion, which will affect
gastrointestinal tract physiology and may result in increasing satiety,
effectively helping to reduce calorie intake (White, Fisk, Makkhun &
Gray, 2009). As carriers for the delivery of bioactive compounds and
pharmaceutical drugs, in the process of digestion,
OBs form mixed micelles to
dissolve functional factors and improve their
bioavailability (Zheng, Zhang, Peng
& Julian McClements, 2019).