Editor's note: We are pleased to present in this issue 3 of 6 papers presented in symposia at the IFT09 annual meeting in Anaheim, Calif. that were organized by Jochen Weiss, Univ. of Hohenheim, and Kumar Mallikarjunan, Virginia Polytechnic & State Univ., with coordination by Professor M.A. Rao, Scientific Editor of the Nanoscale Food Science, Engineering, and Technology section of the Journal of Food Science. The Concise Reviews and Hypotheses section of JFS continues to serve as a mechanism to publish several peer-reviewed papers as a group. We look forward to receiving other opportunities to bundle papers together to serve the scientific community.
–Daryl Lund, Editor in Chief
ABSTRACT: There is currently a lack of effective delivery systems to encapsulate, protect, and release bioactive lipophilic components, such as ω-3 fatty acids, conjugated linoleic acid, tributyrin, vitamins, antioxidants, carotenoids, and phytosterols, which is holding back the development of functional foods designed to combat diseases such as coronary heart disease, diabetes, hypertension, and cancer. Delivery systems consisting of lipid droplets encapsulated by nano-laminated biopolymer coatings have great potential for use in the food industry for the encapsulation, protection, and release of bioactive lipids.
This article reviews the potential impact of the physicochemical characteristics of nano-laminated biopolymer coatings on the bioavailability of encapsulated lipids. The effects of layer thickness, composition, electrical charge, permeability, and environmental responsiveness on digestion, release, and absorption of lipophilic components are highlighted. The possibility of designing nano-laminated biopolymer coatings to increase, decrease, or control the bioavailability of encapsulated lipids is shown. Data generated from in vitro digestion models and animal feeding studies are presented. This knowledge could be used by the food industry to produce functional foods designed to improve human health and wellness.
There is increasing interest in the development of nutraceuticals and functional foods designed to combat specific diseases, such as cancer, heart disease, hypertension, obesity, and diabetes. A number of lipophilic components have been identified as providing potential health benefits when consumed regularly at appropriate concentrations, including ω-3 fatty acids, conjugated linoleic acid (CLA), butyrate, phytosterols, carotenoids, antioxidants, coenzyme Q, and vitamins A and D (Ubbink 2002; Shefer and Shefer 2003; Chen and others 2006; Ubbink and Kruger 2006; McClements and others 2009a, 2009b). It is often beneficial to deliver these bioactive lipophilic components within an aqueous medium because this increases their palatability, desirability, and bioactivity. Thus, a bioactive lipophilic component might be incorporated within a beverage or food that could easily be consumed by drinking or eating. Nevertheless, there are often a number of technical challenges that must be overcome before a bioactive lipid can be successfully incorporated into an aqueous-based food. Lipophilic bioactive components come in a wide variety of different molecular forms, which lead to differences in their physicochemical and physiological properties, such as chemical stability, physical state, solvent solubility, rheology, optical properties, and bioavailability. Consequently, different delivery systems are usually needed to address specific molecular, physicochemical, and physiological concerns associated with each type of bioactive lipid component.
Generally, an edible delivery system for lipophilic bioactive components must have a number of specific attributes:
- (i) The delivery system should be capable of encapsulating a sufficiently high amount of bioactive lipid and efficiently retaining it during storage. In other words, it should have a high loading capacity, encapsulation efficiency, and retention efficiency.
- (ii) The delivery system may have to protect a chemically labile bioactive lipid from chemical degradation so that it remains in its active state. For example, it is often necessary to prevent oxidative degradation of encapsulated lipophilic compounds, such as ω-3 fatty acids, lycopene or β-carotene.
- (iii) The delivery system should be compatible with the food or beverage matrix that it will be incorporated into, without causing any adverse affects on product appearance, texture, mouth feel, flavor, or shelf life.
- (iv) The delivery system should be resistant to the environmental stresses that the food or beverage it is incorporated into experiences during its production, storage, transport and utilization, for example, thermal processing, chilling, freezing, dehydration, light exposure, or mechanical agitation.
- (v) The delivery system should be prepared entirely from generally recognized as safe (GRAS) ingredients using processing operations that are legally acceptable within the country of manufacture and sale, for example, a production facility that conforms to good manufacturing practices (GMP).
- (vi) The additional value contributed to the final product by encapsulating the bioactive lipophilic components should be sufficient to outweigh the additional expenses associated with encapsulation. Thus, the ingredients and processes used to produce the delivery system should be inexpensive, reliable and robust.
- (vii) The delivery system may have to be designed to control the release and/or absorption of the bioactive lipophilic component at a particular site within the gastrointestinal tact, such as the mouth, stomach, small intestine, or large intestine.
A wide variety of different types of delivery system have been developed to encapsulate lipophilic bioactive agents, including simple solutions, association colloids, emulsions, biopolymer matrices, powders, and so on (McClements and others 2009b). Each type of delivery system has its own advantages and disadvantages for encapsulation, protection, and delivery of functional agents, as well as in its cost, regulatory status, ease of use, biodegradability, biocompatibility, and so on.
This article begins by providing a brief overview of the physicochemical and physiological processes that may occur when a structured delivery system passes through the human gastrointestinal tract, as well as defining the concept of lipid bioavailability with regards to lipophilic bioactive components. An overview of various structured delivery systems based on emulsion technology that can be used to encapsulate, protect and release lipophilic bioactive agents is then given. The article then focuses on the design, fabrication and potential applications of nano-laminated coatings produced using the layer-by-layer (LbL) electrostatic deposition method for producing novel encapsulation and delivery systems for bioactive lipids.
Physicochemical Basis of Lipid Digestion
Some knowledge of the basic physicochemical and physiological processes that occur when a food passes through the human gastrointestinal (GI) tract is required to design effective structured delivery systems to control the bioavailability of lipophilic components (McClements and others 2007a, 2007b; McClements and others 2009a, 2009b; Singh and others 2009). After ingestion a delivery system experiences a complex series of physical and chemical changes as it passes through the mouth, stomach, small intestine, and large intestine (Figure 1). Some of the key changes are summarized subsequently:
pH. There are considerable variations in the pH of the aqueous medium surrounding the lipid droplets as they pass through the digestive tract: mouth (pH ≈ 7); stomach (pH ≈ 1 to 3); small intestine (pH ≈ 6 to 7); large intestine (pH ≈ 6 to 7). These variations may cause changes in the electrical charge on the lipid droplet surfaces, as well as causing alterations in the properties and interactions of other charged species (such as enzymes, proteins, amino acids, polysaccharides, and lipids).
Ionic composition. There may be considerable variations in the type and concentration of ions surrounding the lipids droplets, which may impact the electrostatic interactions in the system through electrostatic screening or binding effects. For example, long chain fatty acids may precipitate in the presence of calcium ions, thereby removing them from the lipid droplet surface (which facilitates further digestion), but which may also reduce their absorption (Zangenberg and others 2001).
Surface active components. There are a variety of endogeneous (for example, proteins, phospholipids and bile salts), exogeneous (for example, surfactants, proteins), and internally generated (for example, lipid digestion products) surface active substances present within the aqueous phase surrounding the lipid droplets. These substances compete with the surface-active substances already present at the oil-water interface, potentially leading to changes in interfacial composition and properties (Reis and others 2008, 2009).
Enzyme activity. There are various kinds of enzymes in the mouth, stomach, and small intestine that can digest food components, such as lipids (lipases), proteins (proteases), and starch (amylases) (Tso 2000). The ability of these enzymes to interact with their specific substrates within a structured particle may impact lipid digestibility, and therefore the absorption of encapsulated lipophilic bioactive components. Enzyme accessibility to a substrate may be controlled by creating physical barriers between the encapsulated substrate and the surrounding aqueous phase where the digestive enzymes are normally located, for example, coating a lipid droplet with a dietary fiber layer (Mun and others 2006a).
Temperature. There may be a large change in temperature from the initial food to the human body, which may cause changes in the physical state, molecular conformation, or interactions of specific components that impact digestibility, for example, fat melting or biopolymer conformational changes.
Flow/force profile. The encapsulated lipids may be exposed to various kinds of forces and flow profiles during their passage through the human body. These processes will mix the various components together, as well as potentially breaking down various structures (lipid droplets, protein particles, hydrogel matrices, and so on).
Knowledge of how structured delivery systems behave under these different conditions may be used to design foods that either increase, decrease, or control lipid bioavailability.
It is useful to define the term “lipid bioavailability” since this will help understand how lipid digestion and absorption processes can be controlled using physicochemical and structural approaches. The term bioavailability has been defined as the fraction of an ingested component (or its products) that eventually ends up in the systemic circulation (Versantvoort and others 2004; Holst and Williamson 2008). For lipophilic components, the bioavailability (F) can be defined as (Versantvoort and others 2004):
Here, FB is defined as the bioaccessibility coefficient or fraction of the lipophilic components that is released from the food matrix into the juices of the gastrointestinal tract, FT is defined as the transport coefficient or the fraction of the released lipophilic components that are transported across the intestinal epithelium; and FM is the fraction of the lipophilic components that reaches the systemic circulation without being metabolized. The value of FM depends on the pathway that the lipophilic components follow to reach the systemic circulation, for example, short and medium chain fatty acids go through the portal vein and liver (where they may be metabolized) while long chain fatty acids go through the lymph system (thereby bypassing the liver) (Tso 2000; Porter and others 2007). After the lipophilic components reach the systemic circulation they may be distributed between different tissues, where they may be stored, utilized, or excreted (Verhagen and others 2004; Holst and Williamson 2008). The relative rates of these various processes determine the time-dependence of the concentrations of the lipophilic component and its metabolites at specific locations within the body. The concentration-time profile of a specific lipid component at a particular site-of-action will determine its beneficial or adverse affects on human health and wellness. Consequently, it is usually important to measure the concentration of a lipid component at a particular location to establish its efficacy (Verhagen and others 2004). When determining the bioavailability of bioactive lipophilic components, it is important to account for the fact that they may be chemically modified during passage through the gastrointestinal tract prior to or after absorption, for example, triglycerides are converted to monoglycerides and free fatty acids, whereas some lipophilic bioactives (such as polyphenolics) may be chemically derivitized (Scholz and Williamson 2007; Holst and Williamson 2008).
Emulsion-Based Structured Delivery Systems
A variety of structured delivery systems can be produced based on the application of emulsion science and technology (Figure 2 and 3). Many of these systems can be utilized to encapsulate, protect and deliver lipophilic bioactive components. In this section, a brief overview of some of the potential structured delivery systems is given, with an emphasis on systems that are dispersible in an aqueous medium, for example, oil-in-water (O/W), water-in-oil-in-water (W/O/W), or oil-in-water-in-water (O/W/W) systems. All of these systems are thermodynamically unstable systems (unlike microemulsions), and will eventually destabilize over time (McClements and others 2007b). Consequently, they must be designed to remain kinetically stable over the lifetime of the product.
Basic particle types
A number of different particles can be used as the basic building blocks of structured delivery systems (Figure 2).
Lipid droplets Lipid droplets are the basic particle type in conventional emulsions and nanoemulsions. An O/W emulsion consists of emulsifier-coated lipid droplets dispersed within an aqueous medium, with the mean droplet diameter typically being within the range 100 nm to 100 μm for conventional emulsions and 10 to 100 nm for nanoemulsions. At a sufficiently small diameter (typically < 50 nm), this type of emulsion becomes optically transparent, which is useful for applications in clear foods and beverages (Wooster and others 2008). Emulsions may be formed using either high energy (homogenization) or low energy (phase inversion and spontaneous phase separation) techniques (Kesisoglou and others 2007; Gutierrez and others 2008), with homogenization techniques being the most widely used in the food industry (McClements and others 2007b). Special care must be taken to optimize the sample composition and homogenization conditions to efficiently produce very small droplets in nanoemulsions (Wooster and others 2008).
Lipid particles Solid lipid particles are the basic particle type in solid lipid particle (SLP) suspensions. Like O/W emulsions, SLP consist of emulsifier-coated lipid particles dispersed within an aqueous medium (Figure 2), but in this case the lipid phase is either fully or partially solidified (Weiss and others 2008). In addition, the morphology and packing of the crystals within the lipid phase is usually controlled. SLP are formed by homogenizing an oil and water phase together in the presence of a hydrophilic emulsifier at a temperature above the melting point of the lipid phase. The emulsion is then cooled (usually in a controlled manner) so that some or all of the lipids within the droplets crystallize. The structure, polymorphic form and spatial arrangement of the fat crystals with the lipid droplets can be controlled by manipulating the lipid phase type and composition, the emulsifier type and composition, and the cooling conditions (Muller and others 2000; Müller and others 2002; Bunjes and others 2007).
Hydrogel particles The basic particle in this type of system consists of a network of associated (gelled) biopolymer molecules that traps solvent molecules (usually water) (Burey and others 2008). This system can therefore be considered to be a type of water-in-water (W1/W2) emulsion, where W1 and W2 represent the hydrogel particle and surrounding aqueous phase, respectively (Figure 2). There are a number of different ways to form this kind of system based on aggregative or segregative phase separation of single or mixed biopolymers in solution (Augustin and others 2001; Tolstoguzov 2002; McClements 2006). Hydrogel particles may be made from proteins (such as milk, egg, plant, fish, or meat proteins) and/or polysaccharides (such as starch, cellulose, pectin, alginate, carrageenan, or agar).
Liposomes This type of particle consists of phospholipid molecules organized into a structure similar to that found in the lipid bilayer membrane of cells, that is, a hydrophobic ring that traps water inside, but that is also surrounded by water outside (Taylor and others 2005; Flanagan and Singh 2006). The phospholipid molecules are arranged so that their hydrophilic heads point into either the inner or the outer water phase, and their hydrophobic tails associate closely with each other to reduce the unfavorable hydrophobic effect (Figure 2).
Other particle types A variety of other particles can also be used to fabricate delivery systems designed to encapsulate lipophilic materials, including various types of organic matter (starch granules, oil bodies, pollen, viruses, bacteria) and inorganic matter (titanium dioxide, calcium carbonate) (Iwanaga and others 2007; Paunov and others 2007; Iwanaga and others 2008; Campbell and others 2009).
The functionality of the basic particle types can often be either improved or extended by filling them with other functional materials, for example, lipid droplets can be filled with water droplets to form multiple emulsions, hydrogel particles can be filled with lipid droplets to form filled hydrogel particles, or liposomes can be filled with lipid droplets to form filled liposomes (Figure 3). In this section, we highlight 2 of the most common forms of filled particles: multiple emulsions and filled hydrogel particles. It should be stressed, that many other kinds of substances could be used as either the filler material or the carrier particles.
Multiple emulsions Multiple emulsions of the W/O/W type consist of small water droplets trapped within larger oil droplets that are dispersed in an aqueous medium (Figure 3). They are normally produced using a 2-step procedure (Garti 1997; Khan and others 2006; Muschiolik 2007). First, a W/O emulsion is produced by homogenizing water, oil and an oil-soluble emulsifier together. Second, a W/O/W emulsion is produced by homogenizing the W/O emulsion with an aqueous solution containing a water-soluble emulsifier. These emulsions are particularly useful for encapsulating hydrophilic components within the internal aqueous phase or for reducing the fat content (Muschiolik 2007).
Filled hydrogel particles Filled hydrogel particles consist of oil droplets contained within hydrogel particles that are dispersed within an aqueous continuous phase (Malone and Appelqvist 2003; Burey and others 2008). They can therefore be considered to be a type of oil-in-water-in-water (O/W1/W2) emulsion. This type of system is usually formed by mixing an O/W emulsion with a biopolymer system that phase separates into 2 different aqueous phase regions, for example, through either aggregative or segregative mechanism (Norton and Frith 2001; McClements 2006; Norton and others 2006; McClements and others 2009b). The system is designed so that the lipid droplets partition into the aqueous phase that forms the inner water droplets (W1). The conditions are then changed to promote gelation of the inner water phase to form hydrogel particles, for example, by changing temperature or adding a cross-linking agent.
The functionality of the basic particle types can also be improved or extended by coating them with other functional materials, for example, lipid droplets can be coated with biopolymers or colloidal particles (Guzey and McClements 2006a; McClements and others 2007a). In this section, we highlight 2 of the most common form of coated particles: multilayer emulsions and colloidosomes. Again, we note that various other kinds of substances could be used as either the carrier particles or the coatings to form a wide range of other coated particles.
Multilayer emulsions Multilayer O/W emulsions consist of emulsifier-coated oil droplets dispersed in an aqueous medium, with each droplet being surrounded by one or more layer of biopolymer molecules (Guzey and McClements 2006a; McClements and others 2007a). Multilayer emulsions are usually formed using a multiple-step procedure. First, an O/W emulsion is prepared by homogenizing an oil and aqueous phase together in the presence of an ionized water-soluble emulsifier. Second, an oppositely charged biopolymer is added to the system so that it adsorbs to the droplet surfaces and forms a 2-layer coating around the droplets. This procedure can be repeated to form oil droplets coated by nano-laminated interfaces containing 3 or more biopolymer layers by successively adding charged materials with opposite charges. The same procedure can also be used to coat other types of particles, such as solid particles, oil bodies, liposomes, or hydrogel particles (Iwanaga and others 2008; Laye and others 2008).
Colloidosomes Colloidosomes usually consist of large particles surrounded by a coating of smaller particles (Figure 3). These systems are typically formed by mixing together a suspension containing large droplets with one containing small droplets under conditions where there is a sufficiently strong attractive force between the different types of particles (Gu and others 2007; Shilpi and others 2007). This attractive force may simply be a free energy decrease upon adsorption, or it may be primarily hydrophobic or electrostatic in origin. The preparation conditions have to be carefully controlled to form colloidosomes rather than obtaining a highly aggregated system. The large and small particles can be made from either the same or different materials, and may consist of liquid droplets, solid particles, liposomes, hydrogel particles, or other types of colloidal particles.
Designing structured particles for functional performance
The functional performance of a particular emulsion-based structured delivery system can be controlled by varying the properties of the particles (Figure 1):
Composition—structured particles can be prepared using different types and amounts of water, lipids, emulsifiers, biopolymers and minerals, depending on the method used to fabricate them;
Structure—structured particles can be prepared that have various types of internal structure, including homogeneous, dispersion and core-shell structures (Figure 2);
Dimensions—the dimensions of the various components used to construct a structured particle can often be controlled, for example, particle diameters, particle shapes, and coating thicknesses;
Physical state—the ratio of solid to liquid fractions within a particular structured component can sometimes be varied, for example, using a lipid phase that crystallizes at a particular temperature;
Partitioning—the relative polarity of the various phases within a structured particle can be varied to control the equilibrium partition coefficients (K) of any encapsulated components, and thereby their distribution within the structured particle and the surrounding medium.
Physical integrity—the physical integrity of a particle within a particular environment can often be controlled by manipulating the type of components within it and the forces holding it together (for example, electrostatic, hydrogen bonding or hydrophobic forces). Thus a particle can be made to swell, shrink, or disintegrate when the pH, ionic strength, of temperature is changed.
Permeability—the ability of molecules (for example, encapsulated materials, enzymes or digestion products) to be transported in and out of a structured particle depends on the permeability of the various components from which it is constructed. For example, the diffusion of an enzyme into a biopolymer particle can be controlled by manipulating the dimensions of the biopolymer network mesh size relative to those of the enzyme.
Digestibility—the ability of a structured particle to resist changes within the gastrointestinal tract will depend on its susceptibility to enzyme digestion, for example, lipases, proteases, and amylases. This susceptibility can be controlled by selecting components with different enzyme digestibility to construct it, for example, proteins are primarily digested in the stomach or small intestine, dietary fibers, and resistant starches are digested mainly in the colon, and regular starches are mainly digested in the mouth, stomach, and small intestine. The relative location of these components within the structured particle will therefore be important for controlling digestibility.
Multilayer Emulsion-Based Delivery Systems: Lipid Droplets Coated by Nano-laminated Coatings
In the remainder of this article, we focus on the utilization of multilayer emulsions for the encapsulation and delivery of lipophilic bioactive components. Conventional O/W emulsions are usually manufactured by homogenizing a lipid phase with an aqueous phase in the presence of a water-soluble emulsifier (Friberg and others 2004; McClements 2005a). The emulsifier rapidly adsorbs to the surfaces of the lipid droplets formed during homogenization, thereby reducing interfacial tension, facilitating further droplet disruption, and retarding droplet coalescence. Many different emulsifier types are available for utilization in foods, including various small molecule surfactants, phospholipids, proteins, and polysaccharides. Each emulsifier type varies in its effectiveness at producing small droplets during homogenization, the amount needed to form a stable emulsion, and its ability to prevent droplet aggregation under different environmental stresses, such as pH, ionic strength, heating, freezing, drying, and mechanical agitation. Food emulsifiers also differ in their cost, reliability, ease of utilization, ingredient compatibility, label friendliness, and legal status. For these reasons, there is no single emulsifier that is ideal for use in every kind of food product. Instead, the selection of a particular emulsifier (or combination of emulsifiers) for a specific food product depends on the type and concentration of other ingredients that it contains, the homogenization conditions used to produce it, and the environmental stresses that it experiences during its manufacture, storage and utilization.
Using conventional food emulsifiers and homogenization techniques there are only a limited range of functional attributes that can be engineered into emulsion-based delivery systems. This has motivated a number of researchers to examine alternative means of improving emulsion stability and performance. One strategy has been to create O/W emulsions containing lipid droplets surrounded by multi-component nano-laminated interfacial coatings consisting of emulsifiers and/or biopolymers (Moreau and others 2003; Ogawa and others 2003a, 2003b, 2004; Gu and others 2004a, 2005, 2006; Guzey and others 2004; Aoki and others 2005; Klinkesorn and others 2005a, 2005b, 2005c; McClements 2005c; Mun and others 2005; Guzey and McClements 2006b; Harnsilawat and others 2006a, 2006b). In this LbL electrostatic deposition approach, an ionic emulsifier (E) that rapidly adsorbs to the surface of lipid droplets during homogenization is used to produce a primary emulsion containing small droplets, then an oppositely charged biopolymer (B) is added to the system that adsorbs to the droplet surfaces and produces secondary emulsions containing droplets coated with an emulsifier-biopolymer interfacial layer (Figure 4). This latter procedure can be repeated to form lipid droplets covered by coatings consisting of 3 or more layers, for example, E–B1/B2, where B1 and B2 are oppositely charged biopolymers Emulsions containing lipid droplets surrounded by multi-layered interfacial coatings have been found to have better stability to environmental stresses than conventional O/W emulsions under certain circumstances. For example, they have been found to provide protection against pH changes, high ionic strengths, thermal processing, dehydration, and freezing (Ogawa and others 2003a; Gu and others 2004b; Aoki and others 2005; Klinkesorn and others 2005a, 2005b, 2005c; Surh and others 2005; Guzey and McClements 2006b, 2007). They can also be used to protect lipophilic functional components within lipid droplets from chemical degradation (Klinkesorn and others 2005b, 2005c) or to develop controlled, or triggered release systems (Ogawa and others 2003a; Gu and others 2006).
The LbL-electrostatic deposition method therefore offers a promising way to improve the stability and performance of emulsion-based delivery systems. Nevertheless, the choice of an appropriate combination of emulsifier and biopolymers is essential to the success of this approach, as well as determination of the optimum preparation conditions (for example, droplet concentration, biopolymer concentration, pH, ionic strength, order of addition, stirring speed, washing, floc disruption, and temperature) (Ogawa and others 2003b; McClements 2005c; Mun and others 2005). In this section, an overview of recent research that has been carried out in our laboratory on the development, characterization and application of O/W emulsions containing lipid droplets surrounded by nano-laminated coatings of emulsifier and biopolymer is given. In particular, the use of the LbL electrostatic deposition technique to create emulsion-based delivery systems to control the digestion and release of lipophilic functional components will be highlighted.
Preparation of multilayered emulsions
Multilayer emulsions containing lipid droplets surrounded by nano-laminated interfacial coatings are usually prepared by a multiple-step process (McClements 2005c; Guzey and McClements 2006b). For example, the following procedure could be used to create emulsion droplets coated by 3 layers, for example, E–B1/B2 (Figure 4). First, a primary emulsion containing electrically charged lipid droplets surrounded by a layer of emulsifier is prepared by homogenizing oil, aqueous phase and a water-soluble ionic emulsifier together. Second, a secondary emulsion containing charged droplets stabilized by E-B1 layers is formed by incorporating biopolymer 1 into the primary emulsion. Biopolymer 1 normally has to have an opposite electrical charge than the droplets in the primary emulsion (although this is not always necessary if there are significantly large patches of opposite charge on the lipid droplet surface). If necessary mechanical agitation is applied to the secondary emulsion to disrupt any flocs formed because of bridging of droplets by biopolymer molecules. In addition, the secondary emulsion may be washed (for example, by filtration or centrifugation) to remove any free biopolymer remaining in the continuous phase. Third, tertiary emulsions containing droplets stabilized by E–B1–B2 interfacial layers are formed by incorporating biopolymer 2 into the secondary emulsion. Biopolymer 2 normally has to have an opposite electrical charge than the droplets in the secondary emulsion (but see above). If necessary mechanical agitation is applied to the tertiary emulsion to disrupt any flocs formed, and the emulsion may be washed to remove any nonadsorbed biopolymer. This procedure can be continued to add more layers to the interfacial coating, with the outer layer normally controlling the overall charge of the nano-laminated droplets formed, for example, E–(B1/B2)n/B1 will have a net charge determined by B1, while E–(B1/B2)n/B1/B2 will have a net charge determined by B2.
The adsorption of the biopolymers to the droplet surfaces can be conveniently monitored using ζ-potential measurements, whereas the stability of the emulsions to flocculation can be monitored by light scattering, microscopy, or creaming stability measurements (Figure 5) (Mun and others 2005; Guzey and McClements 2006b; Harnsilawat and others 2006a, 2006b). The principle of forming nano-laminated coatings with different properties is relatively straightforward. Nevertheless, the creation of stable delivery systems requires a good understanding of the physicochemical mechanisms underlying their assembly, as well as careful control over the experimental preparation procedures. In particular, biopolymer and droplet concentrations used to prepare the coated lipids must be carefully controlled to avoid particle aggregation (due to charge neutralization, bridging or depletion effects), and mechanical agitation may be needed to disrupt any flocs formed (McClements 2005c).
Designing the functional properties of nano-laminated coatings
One of the most powerful attributes of multilayer emulsions as structured delivery systems is that the properties of the nano-laminated coatings surrounding the lipid droplets can be controlled to produce different kinds of functionality. This can be achieved in a variety of ways:
- (i) Changing the nature of the emulsifier used to prepare the initial lipid droplets;
- (ii) Changing the nature of the biopolymers used to form the individual layers within the nano-laminated coatings;
- (iii) Changing the total number of electrostatic deposition steps used to prepare the nano-laminated coatings, that is, the number of layers;
- (iv) Changing the order that the various biopolymers are deposited onto the lipid droplet surfaces (for example, E–B1/B2/B3 compared with E – B3/B2/B1).
- (v) Controlling the properties of the solutions used during the preparation and storage of the coated lipids (for example, pH, ionic strength, temperature, shearing).
- (vi) Cross-linking one or more of the adsorbed biopolymer layers, for example, physically, chemically, or enzymatically.
A range of different food-grade emulsifiers and biopolymers can be used to assemble nano-laminated coatings around lipid droplets, including various surfactants, phospholipids, proteins, and polysaccharides. Food-grade emulsifiers and biopolymers with different molecular characteristics (for example, electrical charge, molecular weight, conformation, flexibility, hydrophobicity, thermal stability) and functional properties (for example, solubility, viscosity, gelation, surface activity, digestibility) can be used, which gives one a great deal of scope in designing the functional performances of nano-laminated coatings (for example, thickness, charge, packing, environmental responsiveness). The design of the functional performance of the nano-laminated coating interface will depend on the specific application:
Ensuring bioavailability. If a nano-laminated coating is used to encapsulate and protect a bioactive food component (for example, ω-3 fatty acids, carotenoids, phytosterols) during storage, then it is essential that it is broken down within the GI tract so that the bioactive component is released and absorbed, otherwise the potential benefits of the bioactive ingredient will not be realized.
Reducing bioavailability. If a nano-laminated coating can be designed to be completely indigestible within the GI tract then it could be used to reduce the calorie content of foods by preventing digestive enzymes (that is, lipases) from accessing encapsulated lipids. This approach could be used to design reduced-calorie foods for tackling obesity and related diseases, while maintaining good sensory acceptability.
Targeted release. If a nano-laminated coating can be designed so that its integrity or permeability changes in response to specific biological triggers (for example, pH, ionic strength, polar lipids, enzymes) then it is possible to design delivery systems that release a functional food component to a specific site-of-action where it is most effective, such as the mouth, stomach, small intestine, large intestine or colon (Angelatos and others 2007).
An improved understanding of how nano-laminated coatings behave within the human GI tract could be used for the rational creation of delivery systems that control the bioavailability of lipophilic components. For example, if encapsulated triacylglycerols (TAG) are going to be bioaccessible it is necessary for lipases to adsorb to the lipid droplet surfaces and catalyze their conversion to diacylglycerols (DG), monoacylglycerols (MG), and free fatty acids (FFA) (Tso 2000). If water-insoluble encapsulated bioactive lipids (such as carotenoids or phytosterols) are to become bioaccessible it is usually necessary for the TAG carrier oil surrounding them to be digested first, so that they can be released into the surrounding aqueous phase and taken up by mixed micelles (Tso 2000). In addition, the digestion products from TAG digestion may themselves become part of the mixed micelles, thereby increasing the overall solubilizing capacity of the small intestine (Porter and others 2007). Hence, the access of lipases to encapsulated lipids is a critical step in determining their bioactivity. The most important properties of nano-laminated coatings that could potentially influence these processes are (Figure 6):
Coating integrity. If a lipid droplet is surrounded by a coating of material that prevents the enzymes from reaching the encapsulated lipids, then the lipid phase may not be digested and bioactive components released. The integrity of a nano-laminated coating may be greatly altered during the passage of an encapsulated lipid through the GI tract due to changes in pH, ionic strength, levels of surface-active components, and enzyme activities (McClements 2005b). Surface active components may displace biopolymer layers through a competitive adsorption process. Changes in pH and ionic strength may cause biopolymer layers to become detached by weakening electrostatic interactions. Enzymes such as proteases or amylases may degrade certain biopolymer layers. The bioavailability of encapsulated lipids may therefore be controlled by controlling the responsiveness of a coating's integrity to different biological fluids (for example, saliva, gastric, or pancreatic juices).
Coating permeability. Even if the nano-laminated coating remains attached around the lipid droplets within the GI tract, the digestibility of the encapsulated lipids will still depend on the ability of the digestive enzymes to penetrate through the coating and access the encapsulated lipid phase (Angelatos and others 2007). The permeability of a nano-laminated coating will depend on its pore size, as well as specific interactions between the enzyme molecules and biopolymers in successive layers. Previous studies have shown that the permeability of nano-laminated coatings formed by the LbL method depends strongly on ionic strength and pH because they are held together by electrostatic interactions. For example, when the solution pH or ionic strength is changed so that the electrostatic interactions between the polymer layers are weakened, a coating may swell considerably, thereby increasing its permeability to small molecules (Rubner 2003; Schlenoff 2003). It is therefore possible to “tune” the permeability of coatings by careful control of biopolymer type and assembly conditions (Angelatos and others 2007). Consequently, it may be possible to control the bioavailability of an encapsulated lipid by controlling the responsiveness of a coating's permeability to different biological fluids (for example, saliva, gastric, or pancreatic juices).
This areas is still in its infancy in the food industry and research is needed to establish: (i) how can nano-laminated coatings with different integrities and permeabilities be produced?; (ii) how do these coatings respond to the conditions found within the human GI tract?; (iii) how can this information be used to design delivery systems that can be used to control the bioavailability of lipophilic food components? Our laboratory is currently carrying out research to better understand how nano-laminated biopolymer layers can be rationally designed to control the bioavailability of encapsulated lipids.
Characterizing the functional performance of nano-laminated coatings
A combination of in vitro and in vivo experiments is required to study the efficacy of nano-laminated coatings for controlling the bioavailability of encapsulated lipids. In vitro studies enable one to obtain mechanistic insights into the basic physicochemical properties impacting the functional performance of coatings. On the other hand, it is extremely difficult to accurately mimic the complex physiochemical and physiological processes that occur when foods pass through animal or human digestive tracts using in vitro models. Consequently, it is always necessary to utilize animal feeding studies or human trials to test the efficacy of delivery systems that have been demonstrated to have good potential for controlling the bioavailability of encapsulated lipids.
A summary of the in vitro models currently used in our laboratory for testing lipid digestion and the bioavailability of lipophilic components is shown in Figure 7 and 8. Typically, an emulsion-based delivery system is passed through a series of steps that are designed to mimic passage of a food through the human GI tract: (i) mouth (pH 6.8, 1 min, simulated saliva juice); (ii) stomach (pH 2, 2 h, simulated gastric juice); small intestine (pH 6.8, 2 h, simulated duodenal, and bile juices) (Figure 7). The change in the properties of the lipid droplets is typically measured as they pass through the digestion model, for example, their size, structure, charge, aggregation, digestion, and chemical composition. We have used this model to examine a variety of factors that impact the digestion of emulsified lipids, including emulsifier type (Mun and others 2007), interfacial coating (Mun and others 2006a), interfacial cross-linking (Sandra and others 2008), lipid phase crystallization (Bonnaire and others 2008), and the presence of soluble dietary fibers (Beysseriat and others 2006).
A more sophisticated in vitro model is required to measure the bioavailability of lipophilic bioactive components (Figure 8). This method enables one to determine: (i) the rate and extent of digestion of the carrier lipid in the small intestine using pH-Stat measurements (Dahan and Hoffman 2006); (ii) the fraction of lipophilic components solubilized within mixed micelles after digestion, which is related to the fraction that is bioaccessible FB (Failla and others 2007); and, (iii) the fraction of solubilized lipophilic components absorbed into cell culture model cells, which is related to the fraction transported FT into the human intestinal epithelial cells (Failla and others 2007). A variety of sophisticated commercial in vitro testing instruments have also been developed to study lipid digestion and bioavailability, including the TIM model (TNO, The Netherlands) and Dynamic Gastric Method (IFR, Norwich, U.K.).
In vitro testing models are particularly useful for screening different formulations, and for establishing the physicochemical mechanisms that impact the bioaccessibility and absorption of specific bioactive components. Nevertheless, they cannot mimic the complex physiological and physicochemical events that occur during the passage of foods through the human GI tract. For this reason, it is necessary to use in vivo animal models to test the absorption and bioavailability of lipophilic components. These animal models more realistically mimic the complex events occurring within the human GI tract, but they also have limitations because there are important differences between animal and human GI tracts. In these models, one can monitor changes in the composition, structure, and properties of particles as they travel along the digestive tract of animals by removing samples from different regions of the GI tract at different times after food ingestion, for example, stomach, small intestine, large intestine, or faeces. In addition, it is possible to measure kinetic changes in the concentration of specific bioactive molecules or biomarkers at different locations within the animal or within its waste products, for example, blood, liver, kidneys, brain, faeces, urine, breath. Finally, it is possible to directly measure the outcomes of the bioactivity of lipophilic components after long-term feeding studies, such as changes in body weight, heart disease, diabetes, blood pressure, or tumor growth.
Potential Efficacy of Nano-laminated Coatings
We have carried out a number of preliminary studies on the impact of nano-laminated coatings on the digestibility of encapsulated lipids (Mun and others 2006b; Park and others 2007). Initially, we examined the influence of coating properties on the in vitro digestion of encapsulated lipids by pancreatic lipase. The electrostatic LbL deposition technique was used to prepare corn O/W emulsions (3 wt% oil) that contained droplets coated by (i) lecithin, (ii) lecithin-chitosan, or (iii) lecithin-chitosan-pectin. Pancreatic lipase and/or bile extract were added to each emulsion and the particle charge, droplet aggregation and free fatty acids released due to digestion were measured. In the presence of bile extract, the amount of fatty acids released was much lower in the emulsions containing droplets coated by lecithin-chitosan (38 ± 16 μmoL mL−1) than those containing droplets coated by lecithin (250 ± 70 μmoL mL−1) or lecithin-chitosan-pectin (274 ± 80 μmoL mL−1) (Figure 9). In addition, there was much more extensive droplet aggregation in the lecithin-chitosan emulsion than in the other 2 emulsions (Mun and others 2006a). We postulated that lipase activity was reduced in the lecithin-chitosan emulsion due to the formation of a relatively thick cationic layer around each droplet, as well as the formation of large flocs, which restricted the access of the pancreatic lipase to the lipids within the droplets. The observation that a lecithin-chitosan-pectin coating did not inhibit lipase activity was attributed to desorption of a chitosan/pectin complex from the lipid droplet surfaces thereby enabling the digestive enzymes to access the lipid surfaces (Figure 9). This study highlighted the potential for nano-laminated coatings to be used to modulate the bioavailability of encapsulated lipids. Nevertheless, in more recent in vitro studies we have found that a single layer of chitosan around lipid droplets only has limited ability to protect the encapsulated lipids against digestion (Klinkesorn and McClements 2009). This effect can be attributed to the fact that chitosan loses most of its electrical charge at neutral pH so that the electrostatic attraction with the droplet surface is weakened, which may cause it to become detached from the droplet. In addition, chitosan itself may be partially digested by lipase (as seen by an increase in glucosamine content during digestion), which may also cause it to become detached from the droplet surfaces (Klinkesorn and McClements 2009).
The limited ability of a single layered chitosan coating to protect encapsulated lipids against digestion was also demonstrated using an animal feeding study (Park and others 2007). The electrostatic LbL deposition technique was used to prepare soybean O/W emulsions that contained lipid droplets coated by either lecithin or by lecithin-chitosan. Mice were divided into 4 groups and fed treatment diets for 4 wk; atherogenic diets supplemented with (A) nonemulsified fat, without chitosan (control); (B) nonemulsified fat, with chitosan; (C) emulsified fat, without chitosan; or, (D) emulsified fat encapsulated by chitosan. We found no significant differences in fecal fat contents between all treatment groups, with total fat absorption being greater than 90% in all treatments. These results therefore suggested that encapsulation of lipids by a single layer of chitosan did not inhibit their in vivo digestibility. This has important consequences for using chitosan as an encapsulating material. A chitosan coating may be used to physically or chemically protect an encapsulated bioactive component within a food product, but still release it in the digestive tract after ingestion, thereby ensuring its bioaccessibility.
To delay or prevent the digestion of a lipid within the stomach or small intestine it is necessary to use additional interfacial engineering approaches to prevent the lipases from reaching the encapsulated lipid. We are currently examining 2 different approaches:
Multiple layers—we hypothesized that by increasing the number of layers of dietary fiber within a nano-laminated coating would decrease the rate of lipid digestion. Preliminary experiments in our laboratory support this hypothesis. We prepared lipid droplets coated with different numbers of alginate (anionic)/chitosan (cationic) layers, and then measured the rate of lipid digestion in a pH-Stat (Figure 10). These experiments indicated that the rate of lipid digestion decreases as the number of biopolymer layers increases, suggesting that it is possible to control the release of encapsulated components at different locations within the GI tract.
Cross-linking layers—we hypothesized that cross-linking of dietary fiber layers within a nano-laminated coating would decrease the rate of lipid digestion by restricting the access of the enzyme lipase to the encapsulated lipid phase. Again, recent experiments in our laboratory support this hypothesis (Figure 11). We prepared three different emulsions containing lecithin-stabilized lipid droplets: 1°– uncoated; 2°– coated with chitosan; 2°-X coated with chitosan cross-linked by tripolyphosphate (TPP). The rate of lipid digestion of these systems decreased in the following order: 1° > 2° > 2°-X, indicating that cross-linking of the dietary fiber layer may help to prevent lipase from accessing the encapsulated lipid.
Our research so far has shown that stable emulsions containing nano-laminated lipid droplets can be prepared using a simple cost-effective method and food grade ingredients. These multilayered emulsions may be particularly suitable for controlling the bioavailability of lipids within the gastrointestinal tract. The properties of the nano-laminated coatings can be designed to have particular functional attributes by selecting appropriate emulsifiers, biopolymers and preparation conditions: sign and magnitude of emulsifier; molecular weight, charge density, flexibility, and digestibility of biopolymers; order of biopolymer addition, number of adsorption steps stirring speed, ionic strength, and pH. Preliminary experiments suggest that it is possible to design nano-laminated coatings that can control the digestibility of encapsulated lipids. These coatings could be used to maintain, decrease or control the release of bioactive lipophilic components within the human GI tract. Our laboratory is currently working to better understand the functional performance of these systems.
The author would like to thank the following people for important discussions and insights in this study, including Eric Decker, Hang Xiao, Yeonhwa Park, Jochen Weiss, Yan Li, Min Hu, Jean Alamed, Demet Guzey, and Young Hee Cho. This material is partly based upon research supported by United States Dept. of Agriculture, CREES, NRI Grants, and a Univ. of Massachusetts CVIP award.