《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

Historical perspective Encapsulation, protection, and delivery of bioactive proteins and peptides using nanoparticle and microparticle systems: A review David Julian McClements Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA article info abstract Available online 16 February 2018 There are many examples of bioactive proteins and peptides that would benefit from oral delivery through functional foods, supplements, or medical foods, including hormones, enzymes, antimicrobials, vaccines, and ACE inhibitors. However, many of these bioactive proteins are highly susceptible to denaturation, aggregation or hydrolysis within commercial products or inside the human gastrointestinal tract (GIT). Moreover, many bioactive proteins have poor absorption characteristics within the GIT. Colloidal systems, which contain nanoparticles or microparticles, can be designed to encapsulate, retain, protect, and deliver bioactive proteins. For instance, a bioactive protein may have to remain encapsulated and stable during storage and passage through the mouth and stomach, but then be released within the small intestine where it can be absorbed. This article reviews the application of food-grade colloidal systems for oral delivery of bioactive proteins, including microemulsions, emulsions, nanoemulsions, solid lipid nanoparticles, multiple emulsions, liposomes, and microgels. It also provides a critical assessment of the characteristics of colloidal particles that impact the effectiveness of protein delivery systems, such as particle composition, size, permeability, interfacial properties, and stability. This information should be useful for the rational design of medical foods, functional foods, and supplements for effective oral delivery of bioactive proteins. © 2018 Elsevier B.V. All rights reserved. Keywords: Microencapsulation Insulin Lipase Lactase Nanoparticles Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2. Protein characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2.1. Molecular dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.2. Electrical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.3. Polarity, solubility, and surface activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.4. Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3. Challenges to oral protein delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.1. Product stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.1.1. Challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.1.2. Potential solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.2. Gastrointestinal stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.2.1. Challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.2.2. Potential solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.3. Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.3.1. Challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.3.2. Potential solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4. Product requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.1. Matrix compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.2. Product stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.3. Dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.4. Gastrointestinal stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Advances in Colloid and Interface Science 253 (2018) 1–22 E-mail address: mcclements@foodsci.umass.edu. https://doi.org/10.1016/j.cis.2018.02.002 0001-8686/© 2018 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Advances in Colloid and Interface Science journal homepage: www.elsevier.com/locate/cis

4.5. Ingredient selection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4.6. Production economics and feasibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5. Particle characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5.1. Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5.2. Size and shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5.3. Interfacial properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5.4. Aggregation state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 6. Particle functionality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 6.1. Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 6.2. Retention/release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 6.2.1. Simple diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 6.2.2. Swelling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 6.2.3. Specific molecular interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 6.2.4. Particle dissociation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 6.3. Bioactive protection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 6.4. Particle stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 6.4.1. Gravitational stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 6.4.2. Aggregation stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 6.5. Particle permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 7. Particle impact on end product quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 7.1. Appearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 7.2. Texture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 7.3. Mouthfeel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 8. Delivery system selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 8.1. Microemulsions and emulsified microemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 8.2. Emulsions, nanoemulsions, and multiple emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 8.3. Solid lipid nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 8.4. Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 8.5. Biopolymer microgels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 9. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 9.1. Hormones: insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 9.2. Digestive enzymes: lipase and lactase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 9.3. Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 9.4. Antimicrobials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 9.5. ACE inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 1. Introduction There is great interest in the oral delivery of various types of bioactive proteins and peptides because of their potential health benefits, such as hormones, enzymes, vaccines, antimicrobials, and nutraceuticals [1–7]. For the sake of concision, these types of biologically active proteins and peptides will be referred to collectively as “bioactive proteins” for the remainder of this article. Bioactive proteins exhibit a broad spectrum of biological activities, which make them of interest for application in foods, supplements, and medicines. For instance, oral delivery of lactase aids in the breakdown of lactose into galactose and glucose within the small intestine, which is important for individuals with lactose intolerance [8,9]. Similarly, oral delivery of lipase can help patients with pancreatitis, i.e., the inability to breakdown lipids within the small intestine [10]. Bioactive proteins may also include various kinds of hormones, such as insulin and glucagon-like peptide-1 (GLP-1) which are used to treat diabetes [11] or angiotensin converting enzyme (ACE) inhibitors which are used to treat hypertension [12]. Certain peptides have strong antimicrobial activity, and can therefore be utilized as therapeutic agents [5]. However, there are a number of important technical challenges that have to be overcome before these bioactive proteins can be successfully delivered through the oral route. Typically, bioactive proteins must have a specific three-dimensional structure to exhibit their beneficial biological activities [13]. Proteins may undergo appreciable changes in their molecular structure within commercial products (such as functional foods, supplements, and drugs) during manufacturing, transport or storage, as well as inside the gastrointestinal tract (GIT) after ingestion. These structural changes may be brought on by alterations in environmental conditions, such as pH, ionic strength, denaturants, temperature, and enzyme activity [14]. In particular, many proteins are susceptible to degradation within the highly acidic and protease-rich environment of the human stomach [15]. Consequently, bioactive proteins often have to be encapsulated so as to protect them during storage and after ingestion, but then release them at the appropriate site of action within the human body [3,16,17]. Numerous types of colloidal delivery systems with different structural designs have been developed to encapsulate bioactive proteins (Fig. 1), with each system having its own advantages and disadvantages. The selection of the most efficacious oral delivery system for a specific application depends on a thorough understanding of the factors that impact the loading, retention, stability, and release of the proteins in that specific system. The aim of this review article is therefore to provide a critical evaluation of some of the most important factors that impact the development of oral delivery systems for bioactive proteins based on food-grade nanoparticles and microparticles. This type of colloidal particle is assembled from food-grade ingredients (such as proteins, polysaccharides, lipids, surfactants, and mineral oils) using food-grade processing operations. These colloid systems could therefore be widely utilized in medical foods, functional foods, or supplements specifically designed to deliver bioactive proteins via the oral route. 2. Protein characteristics The first factor to consider when identifying an appropriate colloidal delivery system for a specific application is the molecular and physicochemical properties of the bioactive proteins to be encapsulated. 2 D.J. McClements / Advances in Colloid and Interface Science 253 (2018) 1–22

Bioactive proteins vary considerably in their molecular weights, conformations, electrical characteristics, polarities, and stabilities [13], which will impact their loading, retention, stability, and release in colloidal delivery systems. In this section, a brief overview of the impact of molecular and physicochemical properties of bioactive proteins that may impact the design of colloidal delivery systems is given. 2.1. Molecular dimensions The dimensions of proteins in aqueous solutions depend on their molecular weight, conformation, and aggregation state, and may have a major impact on their retention and release within colloidal delivery systems. The molecular weight of individual bioactive proteins may vary from around 1 kDa for relatively small peptides to around 100 kDa for relatively large proteins. The conformations of bioactive proteins are mainly determined by their specific biological functions, and can be conveniently classified as globular, random coil or helical [13]. At the same molecular weight, the dimensions of proteins in solution depend strongly on the configuration they tend to adopt, with globular proteins being considerably smaller than random oil or helical proteins. Proteins may exist as individual molecules, small clusters, or large aggregates depending on solution conditions, such as pH, ionic strength, and temperature [18,19]. Consequently, proteins may vary considerably in their molecular dimensions, from a few nm (for small isolated globular proteins) to a few 100 nm (for aggregated proteins). Knowledge of the molecular dimensions of proteins under different solution conditions is therefore important for developing appropriate delivery systems. For W/O microemulsions or emulsions (Fig. 1), a bioactive protein should be smaller than the hydrophilic domains (water droplets) inside this kind of colloidal delivery system if it is going to be successfully encapsulated [20]. Conversely, for polymeric colloidal particles such as microgels (Fig. 1), a bioactive protein should be considerably larger than the pore size if it is going to be trapped inside the particles through steric hindrance effects. The impact of the molecular dimensions of proteins on their retention and release from polymeric colloidal particles is discussed in a later section. 2.2. Electrical characteristics The electrical characteristics of proteins are also important in determining their encapsulation properties, as changes in electrostatic interactions between proteins and colloidal particles are often used to tune their retention and release properties [21–23]. The electrical properties of proteins depend on the number of exposed anionic (e.g., \\COOH ↔ \\COO− + H+) and cationic (e.g.,\\NH2 + H+ ↔\\NH3 +) groups on their surfaces, and the pH of the surrounding solution. Typically, the electrical charge goes from positive to neutral to negative as the pH is increased from below to above the isoelectric point (pI) of the protein (Fig. 2). Some of the isoelectric points of common bioactive proteins are included in Table 1. Experimentally, the electrical characteristics of proteins can be conveniently characterized by measuring the change in ζ-potential with pH using micro-electrophoresis methods. Knowledge of the electrical characteristics of bioactive proteins can be extremely important in designing effective colloidal delivery systems. For instance, the retention and release of a bioactive protein from a polymeric colloidal particle depends on the charge characteristics of the biopolymers from which it is constructed. Thus, proteins will be attracted to networks consisting of anionic biopolymers (such Fig. 1. Schematic diagrams of some common types of colloidal delivery systems for encapsulation of hydrophilic bioactive proteins so that they can be incorporated into aqueous-based products. D.J. McClements / Advances in Colloid and Interface Science 253 (2018) 1–22 3

as alginate, carrageenan, or pectin) at pH values below their pI, but released at higher pH values [24,25]. Conversely, it would be expected that proteins would be attracted to networks consisting of cationic biopolymers (such as chitosan or polylysine) above their pI, but released at lower values. It should be highlighted that the strength of electrostatic interactions is weakened in the presence of salts due to electrostatic screening effects [26]. Consequently, for practical applications, it may be difficult to retain bioactive proteins inside colloidal particles using this approach because of the relatively high salt contents found in many commercial products. 2.3. Polarity, solubility, and surface activity The polarity of proteins is important because it determines their solubility characteristics, as well as their interactions with each other and with other substances. Proteins vary from being very hydrophilic to very hydrophobic depending on the relative proportion of polar and non-polar groups exposed at their surfaces [27]. Consequently, bioactive proteins may be either soluble or insoluble in aqueous solutions depending on their surface polarities (Table 1). Some bioactive proteins have good surface activity because they have an appropriate balance of polar and non-polar groups on their surfaces, i.e., they can adsorb to air-water, oil-water, or solid-water interfaces [28]. Knowledge of the polarity of proteins can be particularly important for designing effective colloidal delivery systems. 2.4. Stability The native structure of proteins may be altered appreciably when environmental conditions are changed, such as pH, ionic composition, or temperature [29,30]. Changes in protein conformation often lead to a loss in biological activity, and therefore it is important to clearly establish the major physical and chemical factors that impact the stability of the protein to be encapsulated. Globular proteins undergo appreciable conformational changes when they are heated above their thermal denaturation temperature (Tm), whose value depends on protein type and local environmental conditions (such as pH, ionic strength, and dielectric constant) [27]. In addition, they may undergo conformational changes when they adsorb to certain interfaces, which is known as surface denaturation, and can also lead to a loss of activity [31,32]. They may also become denatured under highly acidic or alkaline conditions [33], when exposed to certain types of salts [34], or in the presence of certain types of surfactant [35]. These changes in structure and activity may be reversible or irreversible depending on the nature of the protein and the environmental conditions. Consequently, it is important to identify the range of temperatures, pH values, and ingredient interactions where a bioactive protein retains its activity. Colloidal delivery systems can sometimes be designed to extend this range of conditions, and therefore enhance protein stability and functionality. 3. Challenges to oral protein delivery There are a number of major hurdles that must be overcome before bioactive proteins and peptides can be successfully delivered via the oral route [36,37]. In this section, some of the most important hurdles are highlighted, as well as some possible strategies to overcome them. 3.1. Product stability 3.1.1. Challenge Bioactive proteins may be incorporated into functional foods, supplements, or medical foods that have different physicochemical properties and storage requirements. For instance, proteins may be delivered in the form of fluids, gels, pastes, powders, or bulk solids, which may be exposed to different temperature, light, oxygen, and humidity levels. As a result, the proteins may become denatured, aggregated, or hydrolyzed during the manufacture, storage, transport or utilization of commercial products, thereby reducing their biological activity and efficacy [38–41]. Consequently, knowledge of the composition and structure of the matrix surrounding proteins in commercial products is important, as well as information about the environmental stresses that they might encounter during the lifetime of the product. In addition, the range of conditions where the bioactive proteins maintain their structure and activity should also be clearly defined. 3.1.2. Potential solutions Knowledge of the environmental factors and ingredient interactions that adversely alter the structure and activity of a specific bioactive protein can be utilized to design a product matrix and processing operations that will minimize any damage to it. For instance, if the temperature, pH, and water-activity ranges that promote protein denaturation are known, then the product can be designed to avoid these Table 1 Molecular and physicochemical properties of some important proteins and peptides that may need to be delivered orally. Note, thermal denaturation data may depend on protein type, pH and ionic strength, and should just be used as a guide. Protein MW (Da) Conformation pI Tm (°C) Polarity Biological activity Lipase (pancreatic) 51,000 Globular 4.9 70 Amphiphilic Digestive enzyme: hydrolyzes lipids Lactase 465,400 Globular (tetramer) 4.61 86 Amphiphilic Digestive enzyme: hydrolyzes lactose Amylase 55,000 Globular 6.5–7.0 61 Amphiphilic Digestive enzyme: hydrolyzes starch Insulin 5808 Dimer 5.3 76 Amphiphilic Hormone: modulates glucose levels in the blood GLP-1 3298 Flexible coils 4.6 – Amphiphilic Hormone: enhances insulin secretion Ghrelin 3371 Flexible coils 11.5 – Amphiphilic Hormone: regulates appetite Nisin 3354 Flexible coils 8.5 – Hydrophobic Antimicrobial: inhibits microorganisms Lysozyme 14,000 Globular 11 72 Amphiphilic Antimicrobial: inhibits microorganisms Lactoferrin 77,000 Globular 8.7 61 and 93 Amphiphilic Antioxidant: inhibits oxidation of lipids Fig. 2. The electrical charge on proteins typically goes from positive at low pH to negative at high pH, with a point of zero charge at intermediate pH, which is known as the isoelectric point. Key: β-Lg – β-lactoglobulin; LF = lactoferrin. (Data from, Mao and McClements, 2011, Food Hydrocolloids 25 (2011) 1201–1209). 4 D.J. McClements / Advances in Colloid and Interface Science 253 (2018) 1–22

environmental stress factors. In some cases, encapsulation of bioactive proteins in delivery systems can be used to improve the stability of bioactive proteins by altering their local environment [42,43]. 3.2. Gastrointestinal stability 3.2.1. Challenge Many bioactive proteins are highly susceptible to denaturation, aggregation, and hydrolysis when exposed to gastrointestinal fluids [39,41]. In particular, the highly acidic environment of the gastric fluids within the stomach may promote protein denaturation and aggregation [44,45]. The extent of these effects depends on the structure and properties of the bioactive proteins, as well as the nature of any foods consumed with them. In addition, digestive enzymes (such as proteases) in the mouth, stomach, and small intestine can promote hydrolysis of proteins [46,47]. The extent, rate, and nature of hydrolysis depend on the molecular structure of the bioactive proteins involved, as well as their environment. Consequently, many bioactive proteins may lose their biological activity when they are exposed to the fluids within the gastrointestinal tract. 3.2.2. Potential solutions Knowledge of the impact of specific GIT conditions on the structure and activity of a bioactive protein can be utilized to design an effective delivery system that inhibits these changes. For instance, if a bioactive protein is normally denatured and hydrolyzed in the stomach due to the high acidity and enzyme activity of the gastric fluids, then a delivery system can be developed to isolate it from these stressors. For instance, it has been shown that digestive enzymes (such as lactase and lipase) can be trapped inside biopolymer microgels that maintain a neutral internal pH under gastric conditions, which greatly enhances their stability and activity [42,43]. 3.3. Absorption 3.3.1. Challenge Another major factor that limits the efficacy of bioactive proteins is their relatively low absorption within the gastrointestinal tract [39,41]. The proteins must diffuse through the gastrointestinal fluids and across the mucus layer before they reach the surfaces of the epithelium cells (Fig. 3). The rheological properties of the gastrointestinal fluids impact the mixing and transport of the bioactive proteins, thereby impacting their residence time within certain regions of the GIT, as well as their absorption rate. The gastrointestinal fluids may vary from relatively low viscosity fluids to highly viscous gels depending on the type and amount of foods consumed [48–50]. The mucus layer consists of a highly viscous network of cross-linked mucin molecules and other substances (e.g., enzymes, lipids, and mineral ions) that coats the GIT and protects it from damage [51–53]. Bioactive proteins (or the colloidal particles containing them) may not be able to enter the mucus layer, or they may be trapped within the mucus layer and hydrolyzed by digestive enzymes before reaching the epithelium cells [37]. Moreover, once the bioactive proteins do encounter the epithelium cells they must be absorbed, which is often challenging because of their relatively large size and polarity. In principle, absorption may occur through a variety of physiological mechanisms, including transcellular (passive or active), paracellular (T-junctions), and endocytosis mechanisms [37]. Proteins (or colloidal particles containing them) may be absorbed by enterocytes or M-cells depending on their dimensions and surface characteristics. Typically, the overall extent of intact protein absorption is relatively low because of these barriers, and so special approaches must be developed to increase it. 3.3.2. Potential solutions A number of different methods can be used to increase the absorption of bioactive proteins by the epithelium cells. First, permeation enhancers can be included in a protein delivery system that increase Fig. 3. Proteins or protein-loaded particles must move through the lumen and mucus layer and be absorbed by the epithelium cells before they can reach the systemic circulation. D.J. McClements / Advances in Colloid and Interface Science 253 (2018) 1–22 5