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).