《现代食品工程高新技术》课程教学资源（文献资料）食品粉碎、微胶囊包埋新技术 Encapsulation, protection, and delivery of bioactive proteins and peptides using nanoparticle and microparticle systems：A review David Julian McClements.pdf_P6-P8

the permeability of the cell membranes (transcellular) or that open up the tight junctions separating the cells (paracellular), thereby promoting greater protein absorption [54]. Second, efflux inhibitors can be included in a protein delivery system that blocks the active transport mechanisms within the cell membrane that normally expel proteins or particles out of the epithelium cells [36]. Third, the proteins can be encapsulated within colloidal particles that are absorbed by the cells, and then released into the systemic circulation [37,55]. The design of colloidal delivery systems to achieve this goal depends on a good understanding of the various cellular absorption mechanisms, and the factors that impact them (such as particle size, shape, charge, and polarity). 4. Product requirements Once the molecular and physicochemical properties of the bioactive proteins have been clearly defined, and the challenges to their delivery have been identified, it is then necessary to specify the requirements of the end product, which will depend on the particular application. In the case of medical foods, functional foods, or supplements, one should define the required appearance, texture, mouthfeel, and stability characteristics of the end product. For example, the bioactive protein may be delivered in the form of a cloudy low viscosity beverage, a transparent gummy type product, or an opaque solid tablet. In addition, it is important to define the functional attributes of the end product. For instance, it may have to protect the bioactive protein from degradation within the end product, mouth and stomach, but then release it within the small intestine. Some of the most important factors that should be considered when developing a delivery system for bioactive proteins intended for oral ingestion are highlighted in this section [56]. 4.1. Matrix compatibility If the delivery system is going to be incorporated into a medical food, functional food, or supplement intended for oral ingestion, then it should not adversely impact the desirable quality attributes of the end product, such as its appearance, texture, mouthfeel, taste, or shelf-life (see later). Particle characteristics, such as their concentration, size, shape, and charge, will determine their impact on end product properties. 4.2. Product stability The delivery system should prevent any undesired changes in the activity of the bioactive protein during the manufacture, storage, and utilization of the end product. The approach used to ensure protein stability will depend on the nature of the product, e.g., whether it is a fluid, gel, or solid. Moreover, the delivery system itself should be resistant to any undesired changes in its properties throughout the lifetime of the end product. Consequently, the delivery system may have to be designed to be resistant to changes in pH, ionic strength, temperature, light, oxygen, and mechanical stresses. This can be achieved by careful selection of the composition and structure of the colloidal delivery system, as well as by controlling the composition and structure of the food matrix and packaging material. 4.3. Dose The delivery system should be capable of encapsulating the level of bioactive proteins required to have the intended biological effect, and then consistently delivering the proteins to the intended site of action at this level. The level of bioactive proteins delivered will depend on the loading capacity, retention, and release properties of the colloidal particles. Factors that may affect the reliability of the dose received should also be carefully considered, and the delivery system should be designed to overcome any problems, e.g., variable processing or storage conditions, or food matrix effects. 4.4. Gastrointestinal stability After ingestion, the delivery system should be designed to protect the bioactive proteins from degradation within certain regions of the GIT (such as the mouth and stomach), but then release them in other regions (such as the small intestine). In addition, the delivery system may have to be designed to have a prolonged residence time in the region of the GIT where the bioactive proteins are supposed to be absorbed, which may require that the colloidal particles have mucoadhesive properties. 4.5. Ingredient selection Colloidal delivery systems intended for oral ingestion may be fabricated from a variety of synthetic and/or natural constituents, including surfactants, phospholipids, proteins, polysaccharides, and lipids. For certain applications, it may be important to select particular types of ingredients, e.g., for individuals who have vegan, vegetarian, Kosher, or non-allergenic dietary requirements. In addition, the cost, shelf-life, ease of use, and reliability of the ingredients used to assemble the colloidal delivery system should be considered. 4.6. Production economics and feasibility The colloidal delivery system should be capable of being consistently produced at an appropriate scale and cost. Many of the methods of producing colloidal delivery systems described in the literature involve ingredients or processing operations that are too costly or inappropriate for commercial applications. 5. Particle characteristics Once the properties of the bioactive proteins to be encapsulated have been clearly defined, and the requirements of the end product have been specified, then it is necessary to establish the particle characteristics required to create an appropriate oral delivery system [56]. In this section, some of the most important particle properties that may impact the efficacy of colloidal delivery systems for bioactive proteins are highlighted. 5.1. Composition Colloidal particles can be assembled from a variety of food-grade ingredients, including proteins, polysaccharides, lipids, surfactants, and minerals [57–59]. The ingredients used to fabricate the colloidal particles impact their functional attributes, and so ingredient selection is an important consideration when developing colloidal delivery systems for bioactive proteins. For instance, the composition of colloidal particles impacts the region they are digested in the GIT, as well as their ability to inhibit protein degradation. Some of the most important ways that particle composition impacts the encapsulation, protection, and release of bioactive proteins are discussed in later sections. 5.2. Size and shape The size and shape of the colloidal particles used to encapsulate bioactive proteins should also be carefully selected for the particular application. The size of colloidal particles may vary from around 10 nm for small nanoparticles (such as microemulsions) to around 1 mm for large microparticles (such as hydrogel beads), and depends on the ingredients and processing operations used to fabricate them. The colloidal particles in delivery systems are often spherical, but they can have other shapes, such as ellipsoid, cylindrical, or irregular, which can impact the optical, rheological, stability, and release characteristics of colloidal dispersions. The impact of the size and shape of colloidal 6 D.J. McClements / Advances in Colloid and Interface Science 253 (2018) 1–22

particles on their ability to encapsulate and deliver bioactive proteins is discussed in later sections. 5.3. Interfacial properties The interfacial properties of colloidal particles can be manipulated by fabricating them from different ingredients, or by coating them with other ingredients after they have been formed. Consequently, the thickness, composition, charge, and permeability of the interfacial layer can be controlled, which allows one to manipulate the retention, protection, and release of encapsulated bioactive proteins. The interfacial properties of colloidal particles with some lipophilic character (such as lipid droplets or hydrophobic protein nanoparticles) can be modified after fabrication by adsorbing surface-active emulsifiers to their surfaces, such as surfactants, phospholipids, proteins, or polysaccharides [60,61]. The interfacial properties of colloidal particles with an electrical charge can be altered by depositing oppositely charged substances, such as biopolymers or solid particles, onto their surfaces [62]. This electrostatic deposition method can be used to coat colloidal particles with multiple layers of charged substances, which allows the thickness, permeability, and electrical properties of the interfacial layers to be manipulated. 5.4. Aggregation state Colloidal particles may be present within a colloidal dispersion as individual entities or as clusters (“flocs”). The aggregation state of the particles in a system may have a major impact on their functional attributes. For example, it influences the stability of the particles to gravitational separation (flocs usually move faster than individual particles because of their larger size) and rheology (flocs usually give a higher viscosity than individual particles because they trap more solvent). Moreover, aggregated particles may be digested more slowly that individual particles in the GIT, which can impact the stability and release of bioactive proteins [63,64]. Consequently, it is often important to control the aggregation state of the colloidal particles in the product, as well as in the GIT. 6. Particle functionality In this section, the most important functional attributes of the particles in colloidal delivery systems are highlighted, with special reference to their relevance to the encapsulation, protection, and delivery of bioactive proteins. 6.1. Loading An important property of any protein delivery system is the maximum amount of bioactive protein that can be successfully loaded into the colloidal particles, i.e., the loading capacity (LC) [60]. The LC will determine the level of colloidal particles required to achieve the intended dose of the bioactive protein. Moreover, the fraction of bioactive protein that is actually incorporated into the colloidal particles (rather than remaining outside of them) during the encapsulation process is also important, i.e., the encapsulation efficiency (EE). The EE will determine the fraction of bioactive protein that is lost during the manufacturing process, which obviously has important economic consequences. The following expressions can be used to calculate these values for a particular bioactive protein-colloidal particle combination: LC ¼ mB;E=mP ð1aÞ EE ¼ mB;E=mB;T ð1bÞ Here, mB,E and mB,T are the masses of the encapsulated and total bioactive protein used to produce the colloidal delivery system, and mP is the total mass of the colloidal particles (carrier material + bioactive proteins). The loading capacity and encapsulation efficiency depend on the molecular and physicochemical properties of the bioactive proteins, as well as of the carrier materials used to assemble the colloidal delivery system. Most bioactive proteins are predominantly hydrophilic and so colloidal particles should have some hydrophilic domains within them, which means they must be assembled from ingredients that have appreciable numbers of polar groups (such as hydroxyl, carboxyl, sulfate, phosphate, or amino groups). Some examples of colloidal delivery systems with hydrophilic domains (usually water) are reverse micelles, W/O microemulsions, W/O emulsions, W/O/W emulsions, liposomes, and microgels (Fig. 1). Bioactive proteins may also be held inside colloidal particles by electrostatic interactions between oppositely charged groups or by hydrophobic interactions between non-polar groups (see next section). In this case, the ingredients used to form the interior of the colloidal particles should have electrically charged or non-polar groups that are strongly attracted to the bioactive proteins. In some cases, the sign or magnitude of the interactions between bioactive proteins and carrier materials can be altered by changing environmental conditions (such as pH, ionic strength or temperature), which can be used to develop triggered release mechanisms. As a specific example, the encapsulation of bioactive proteins within W1/O/W2 emulsions is considered, where W1 and W2 are the internal and external aqueous phases, and O is the oil phase (Fig. 1). Assuming that the bioactive proteins cannot diffuse through the oil phase and that the internal water droplets are not disrupted during the second homogenization stage, the encapsulation efficiency would be relatively high (EE ≈ 100%) because all of the proteins would remain trapped within the internal water phase. The loading capacity depends on the highest level of protein that can be dissolved in the internal water phase and the highest level of water droplets that can be incorporated in the W/O emulsion. If it is assumed that the maximum amount of protein that can be dissolved into an aqueous solution is around 20% and the maximum level of water droplets that can be packed into an oil phase is around 50%, then the loading capacity (LC) of the capsules in a W/O/W emulsion would be around 10%. Similar kinds of calculations could be carried out for other kinds of colloidal delivery systems. However, in practice it is always important to measure the masses of encapsulated and non-encapsulated bioactive protein to determine the EE and LC values more accurately. 6.2. Retention/release Colloidal delivery systems are often designed to retain bioactive proteins under one set of environmental conditions, but then release them when exposed to another set of conditions, such as a change in pH, ionic strength, temperature, or enzyme activity [60]. A number of physicochemical mechanisms can be utilized to control the retention and release of bioactive proteins (Fig. 4). 6.2.1. Simple diffusion In the simplest case, bioactive proteins may be released from polymeric colloidal particles by simple diffusion. To a first approximation, the release of a bioactive protein from a spherical particle due to simple diffusion can be described by the following expression [65]: M tð Þ Mð Þ ∞ ¼ 1− exp −1:2π2DP r2 t ð2Þ Here, M(t) and M(∞) are the concentrations of the bioactive protein trapped within the colloidal particles at time t and at equilibrium (infinite time), r is the radius of the colloidal particles, and DP is the diffusion coefficient of the bioactive proteins through the colloidal particles. Bioactive proteins are often encapsulated within biopolymer microgels whose interior consists of a network of crosslinked polymer molecules with a certain pore size. In this case, the diffusion coefficient can be obtained using the following expression, D.J. McClements / Advances in Colloid and Interface Science 253 (2018) 1–22 7

which describes the diffusion of small molecules through a network of polymer chains [66,67]: DP ¼ DW exp −π rH þ rf ξ þ 2rf ! ! ð3Þ Here, DP and DW are the diffusion coefficients of the bioactive protein through the polymer network and through pure water, rH is the hydrodynamic radius of the bioactive protein,rf is the cross-sectional radius of the polymer chains, and ζ is the pore diameter. The translational diffusion coefficient of proteins through water can be estimated using the following equation: Dw ¼ kBT=6πηrH ð4Þ Here, kB is Boltzmann's constant, T is absolute temperature, and η is the viscosity of water. These equations can provide valuable insights into the impact of the pore size and external dimensions of polymeric colloidal particles on protein retention and release. A prediction of the influence of the protein dimensions relative to the pore size (rH/ζ) on the normalized diffusion coefficient (DP/DW) is shown in Fig. 5. This prediction indicates that the bioactive proteins must have dimensions appreciably larger than those of the pores in the polymer network before their diffusion is appreciably retarded. Consequently, polymeric colloidal particles would only retain proteins when they have pore sizes appreciably smaller than that of the bioactive proteins. This may be difficult to achieve for small peptides (d b 1 nm), but may be possible for larger proteins or protein aggregates. This equation also predicts that the release of bioactive proteins from colloidal particles decreases as the size of the particles increases (Fig. 6), since the proteins have a greater distance to travel before they reach the external environment. Conversely, the retention of proteins will increase as the particle size Fig. 4. Encapsulated bioactive proteins can be released from a colloidal particle due to a variety of mechanisms, including changes in molecular interactions, an increase in pore size, particle dissociation (network disintegration), or simple diffusion. Fig. 5. The restricted diffusion of bioactive proteins through a hydrogel depends on the hydrodynamic radius of the proteins (rH) relative to the pore size (ζ) of the polymer network. Here the diffusion coefficient of the proteins through the polymer network (DP) is normalized relative to their diffusion coefficient in water (DW). 8 D.J. McClements / Advances in Colloid and Interface Science 253 (2018) 1–22