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Physical approaches for the delivery of active ingredients in foods
Trends in Food Science & Technology 17 (2006) 244–254
Review
Physical approaches for the delivery of active ingredients in foods Job Ubbink* and Jessica Kru¨ger
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Nestle´ Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland (Tel.: C41 21 785 9378; fax: C41 21 785 8554; e-mail: [email protected]) Encapsulation systems in food applications are typically employed to solve formulation problems arising from a limited chemical or physical stability of the active ingredient, an incompatibility between active ingredient and food matrix, or to control the release of a sensorially active compound or the bioavailability of a nutrient. However, the use of encapsulation systems in complex food matrices often fails because their application is mostly based on trial and error. The present article introduces a conceptually new approach in which the target application is analyzed based on physical principles, including materials science, physical chemistry and biophysics in order to develop a range of solution strategies from which the most promising may be selected for final application. The approach is illustrated with two case studies on the stability of active ingredients in glassy carbohydrates.
Introduction The creation of novel functionalities of active ingredients in complex food matrices is of increasing importance for the food industry. Active ingredients which are introduced in a variety of food products include traditional ones such as flavors (Risch & Reineccius, 1988; Ubbink & Schoonman, 2003), vitamins (Lukaski, 2004) and minerals (Lukaski, 2004; Zimmermann, 2004), or relatively novel ones such as probiotic microorganisms (Roy, 2005; * Corresponding author. 0924-2244/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2006.01.007
Richardson, 1996; Ross, Desmond, Fitzgerald, & Stanton, 2005) and various classes of bioactive compounds (Rodriguez-Huezo, Pedroza-Islas, Prado-Barragan, Beristain, & Vernon-Carter, 2004; Shi, Mazza, & Le Maguer, 2002). Application of active ingredients in food products often requires innovative approaches because of their sensitivity to a variety of physical and chemical factors, which causes either the loss of biological functionality, chemical degradation or a premature or incomplete release. The situation is challenging not only because of the high sensitivity of many of the active ingredients, but also because of the complexity of many food products and the conditions prevalent in many food matrices. In addition, product safety, appearance, storage conditions, ease of preparation by the consumer, freshness and sensory properties of the food product are not to be compromised by the incorporation of the active ingredient. Compared to other domains, such as the pharmaceutical, agro-chemical or cosmetic industries, the development of products containing active ingredients often more challenging in the food industry, because of the necessity to simultaneously fulfill these multiple conditions. In most non-food domains, the delivery of the active ingredient is the primary objective of the product, and consequently the composition, processing and storage conditions may be flexibly adapted to maximize the performance of this active ingredient. In addition, in the food industry, regulations with respect to ingredients, processing methods and storage conditions are tight and the price margin is much lower than in, for example, the pharmaceutical industry. An approach increasingly advocated as enabling the introduction of active ingredients in foods is the use of encapsulation systems for protection or controlled release (Bencze´di & Blake, 1999; Gibbs Kermasha, Alli, & Mulligan, 1999; Gouin, 2004; Risch & Reineccius, 1995; Ubbink & Schoonman, 2003). Consequently, extensive technologies have been developed to encapsulate active food ingredients in a variety of food-grade materials including glassy carbohydrates (Ubbink & Schoonman, 2003), lipids (Lamb, 1987; Risch & Reineccius, 1995) and biopolymer complexes (Soper, 1995; Weinbreck, Minor & de Kruif, 2004). The choice of technology and encapsulation materials is however often based on trial and error and not on a fundamental understanding of the physical and chemical phenomena determining stability, release, perception and digestion.
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Whereas for simple food systems under controlled conditions of temperature and water activity (e.g. instant beverages, chewing gum, and dehydrated or frozen foods), trial and error approaches for the incorporation of active ingredients have often proved successful, for more complex food products, such as chilled meals, RTD and shelf-stable beverages, they have generally failed to provide efficient solutions. This is primarily the case because trial and error approaches are not efficient in finding a rational compromise between the large numbers of physical, chemical and biological factors influencing the performance of both the active ingredient and the food matrix. In the present article, a new approach is introduced in which the target application is first analyzed using scientific principles, including materials science, physical chemistry and biophysics. The scientific understanding is used to develop a range of potential solution strategies from which the most promising may be selected for further development. Based on technological considerations, such as cost, ease of manufacturing, adaptability, one of these various possible solutions may finally be implemented in the actual food product. The major advantage of our approach is that it does not focus from the outset on a specific technology to solve an issue, which might be poorly understood, and for which the selected technology may ultimately be ineffective. In contrast, the emphasis is on the final application and on a clear specification of the requirements to be fulfilled by a prospective technology. It will be apparent that such developments are facilitated when based on a scientific foundation. Even though the field is in significant development at present, we attempt to illustrate the principle of our approach and the role of scientific developments in the search for a rational delivery strategy by way of two case studies. Both focus on the stability of active ingredients as an important subtopic of the overall delivery field and we limit the scientific discussion of encapsulation materials to carbohydrates in the glassy state. From the two case studies, several functionalities appear which would be highly desirable in the context of the encapsulation of active ingredients. A number of such functionalities and their potential are listed, but also their limitations. The potential impact of recent developments in carbohydrate physics is discussed. Finally, we discuss the implications of our approach for the development of delivery systems for food applications. ‘Retro’-design of delivery systems In a retro-design approach to the delivery of active ingredients, the food application is placed at the center and from there one systematically works back to find a feasible technology to introduce the active ingredient in the food product. The principle of retro-design, as developed in organic synthesis, allows the systematic evaluation of all steps and routes starting from the final product down to the raw materials (Corey & Cheng, 1989). In organic chemistry,
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such a retro-design allows the evaluation of all possible reaction pathways and intermediates leading to the desired product and facilitates the choice of the favored synthesis route based on a rational compromise between reaction yields, number of reaction steps, and availability of starting materials. In addition, the approach has proved useful as it allows the definition of chemical transformations which do not yet exist but whose development may then be attempted (Corey & Cheng, 1989). In the food field, we have experienced that a similar retro-approach is very useful, in particular in the development of complex food products containing active ingredients. In adopting a retro-design approach towards the delivery of active ingredients in foods, one starts by defining precisely the functionality and performance of the active ingredient desired in the final application (Diagram 1). This target sets the required functionality and performance of the active ingredient. This could for instance be the release of a sufficient amount of properly balanced flavor at a given moment during the preparation or consumption of a food product, the presence of a defined quantity of vitamins or a minimal number of live probiotic microorganisms in a food product at the moment of consumption, or the maintenance of a bioactive ingredient in its proper bioactive form during the shelf life and the digestion of a food product. Subsequently, the physical, chemical and biological properties of the active ingredient and the conditions prevailing in the food matrix are analyzed (Diagram 1). Such properties of active ingredients include the physical properties, including phase behavior and molecular mobility, chemical reactivity and conditions under which the physiological and sensory characteristics are maintained and they effectively determine the application window of the active ingredient. If the target can be satisfied is then determined by the conditions prevailing in the food matrix, during manufacturing, storage and consumption. In general, for a specific food product, we have only a very limited flexibility in adapting the conditions in the food matrix to match the window of application of the active ingredient. Therefore, often a significant discrepancy exists between the window of application of the active ingredient and the limits set by product developers on changes in product composition, processing routes and storage conditions. Consequently, often conditions in the food matrix have to be accepted which are detrimental to the stability and functionality of the active ingredient. In such situations, the active ingredient and the product may be said to be incompatible and a successful product development satisfying the original requirements on the functionality and performance of the active ingredient turns out to be impossible. However, having clearly identified both the conditions required for maintenance of the performance of the active ingredient and the limits to the conditions set by the food matrix, one may define the functionality needed to resolve the causes of the incompatibility of active ingredient and
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Diagram 1. Outline of a retro-design approach towards the optimization of the functionality and performance of active ingredients in complex food matrices.
food matrix. The functionality is defined solely based on the analysis of the interaction of active ingredient and food matrix and it does not yet relate to a specific technology. In fact, a functionality defined in this way may not even have an existing associated technology. The major advantage of introducing the concept functionality is that it postpones the selection of a technology to a later stage in order to allow the optimal matching of the different factors indicated in Diagram 1. It thereby enables a clear definition of the requirements a novel technology should fulfill in satisfying the specific application. Based on the defined functionalities, one is in a position to select or develop an appropriate technology. This may involve the use of delivery or encapsulation systems, but also the reformulation of a food product using different (active) ingredients and raw materials, or the induction of a different structure in the food product (Diagram 2). In addition, having clearly identified the causes for the incompatibility between active ingredient and food product, it sometimes turns out that a small adjustment of the manufacturing process will already enhance the performance of the active ingredient. By using any of these options, one can attempt to eliminate the incompatibility of active ingredient and food product. The approach based on ‘retro-design’ has a number of important advantages over the more established trial and
error evaluation of encapsulation systems in the development of advanced food products containing active ingredients. In the first place, the focus is on the food product and its functionality and not on an, often arbitrary, encapsulation technology. In the second place, following the ‘retro-design approach’, the choice for an optimized encapsulation system or food manufacturing process is made in an advanced
Diagram 2. Technologies for the delivery of active ingredients in foods. Encapsulation is the isolation of the active ingredient within the food product using food-grade materials. Formulation encompasses the structuring of the active ingredient, often on molecular or nanoscale levels using food-grade ingredients interacting with the active ingredient. By adaptation of the processing conditions, the adverse effects on the active ingredient may be minimized, or the performance of active ingredient may be maximized. Packaging extends beyond a narrow definition of delivery approaches as it generally uses non-food grade materials and processes and is not included in the diagram.
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stage, allowing maximum flexibility with respect to the evaluation of performance, side effects and costs of the various options. In the last place, it stimulates the systematic use of scientific knowledge to resolve concrete issues arising in the development of innovative food products and it helps to efficiently identify technology gaps. Case study I: encapsulation and stabilization of complex flavor mixtures in amorphous food powders Let us consider the case of the stability of a flavor composition or extract in a food powder or granular material. For the purpose of the present discussion, this can be any food product in powder form of which the matrix largely consists of amorphous carbohydrates in the glassy state, like instant drinks, dairy-based powders, culinary powders, or soluble coffee. During storage, the quality and strength of the flavor of food products decreases, leading to a perceived loss in quality of the food product (De Roos & Mansencal, 2003). This decrease in flavor quality and strength is related to losses of flavor compounds by physical and chemical processes. Physical processes primarily comprise the diffusion of flavor compounds out of the food matrix, but also the physical binding of flavor compounds by components of the food matrix. Chemical processes comprise the degradation of flavor compounds by oxidation, hydrolysis and chemical reactions with the food matrix, but also the formation of offnotes (Winkel, 2005). To counteract the loss of flavor, encapsulation systems are routinely used (Risch & Reineccius, 1988; Ubbink & Schoonman, 2003). The use of such systems has been highly successful in situations where the cause of the loss of the flavor is known, and where it can be counteracted by the properties of the encapsulation matrix. Encapsulation systems based on amorphous carbohydrates in the glassy state have been very effective in reducing the rate of release of the flavor during storage, and in minimizing the rate of oxidation of oxygen-sensitive flavors by environmental oxygen. Both processes involve the diffusion of guest molecules through the glassy carbohydrate matrix, which is a very slow process. As the functionality of encapsulation systems based on amorphous, glassy carbohydrates is based on the prevention of diffusion of flavor compounds and oxygen, strategies to optimize such systems are immediately clear. The rates of diffusion can be minimized by control of the material properties, such as the lowering the water activity of the carbohydrate matrix or the selection of the appropriate carbohydrate composition. In addition, by changing the granule morphology, one can minimize the rate of release of the flavor compounds or the rate of uptake of oxygen. In general, the larger and the less porous the structure of granule, the lower the effective transport rates (Fig. 1a). In many of the cases of current interest to the food industry, highly sensitive flavor compositions or extracts are used, for instance topnotes in culinary and bakery products. In addition, the matrix for the entrapment of the flavors is often a
Fig. 1. Diffusion and degradation of flavor compounds encapsulated in a complex food matrix largely consisting of amorphous carbohydrates: (a) granule morphology is an important parameter to optimize when the stability of encapsulated flavors is determined by diffusional losses (evaporation, oxidation by environmental oxygen). When chemical reactions with the matrix are of importance, particle morphology ceases to be a relevant parameter: (I) extruded carbohydrate granules; (II) freeze-dried granules; barZ100 mm; (b) flavor retention in coffee-based carbohydrate matrix in the glassy state after storage for 42 days at 37 8C and a water activity of 0.33. By plotting the flavor retention in a product prototype as function of the molecular volume of the various flavor compounds in the flavor composition, a separation of the flavor retention data into two distinct groups is observed. The open symbols denote the flavor compounds of which the retention is related to their molecular volume. These flavor compounds may be considered to be chemically stable in the matrix and their losses are caused by evaporation of the flavor into the surrounding atmosphere. The retention of a number of flavor compounds is much lower than predicted on basis of their molecular volume (filled symbols). These flavor compounds are chemically unstable in this particular prototype matrix. The fraction of the flavor lost owing to chemical degradation may be estimated by subtracting the calculated physical loss for a hypothetical chemically stable flavor molecule from the total loss; (c) loss of three key compounds from coffee aroma after storage (42 days, 37 8C, water activity Z0.33) for two different spray-dried encapsulation matrices: A, coffee-based matrix; B, maltodextrin matrix (DE-10). The height of the bar indicates the total loss of the flavor compound, divided in physical loss (black bar) and chemical loss (open bar). Adapted with permission from Ubbink and Reineccius (2002).
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Diagram 3. Retro-analysis of the delivery of flavors in food matrices.
chemically and structurally heterogeneous food matrix, such as a cereal product or a soluble coffee matrix. In such complex systems, the loss of flavor is caused by multiple effects acting in concert, which complicates the choice of an effective stabilization strategy. Because of the complexity of the situation, the first step is to develop a quantitative understanding of the processes underlying aroma mobility, degradation and release (Diagram 3). As an example, we consider the stability of a coffee aroma in both a glassy matrix consisting of coffee extract and in a glassy carbohydrate matrix (Fig. 1). The flavor compounds in coffee aroma represent a large variety of chemical functionalities, including alcohols, esters, ketones and thiols, present in the ppb–ppm range. If only physical processes contribute to the loss of flavor, the retention should depend solely on physical properties such as the vapor pressure, the oil-to-water partition coefficient and molecular size. As in glassy systems, the diffusional mobility of a permeant is related to its molecular size (Cohen & Turnbull, 1959; Menting, Hoogstad, & Thijssen, 1970; Vrentas & Duda, 1978), we analyze the retention of the various flavor compounds as a function of the molecular
volume (determined by the Van der Waals radii) (Fig. 1b). A number of the flavor compounds in this case does not correlate with the molecular volume (filled symbols). Consequently, the low retention of these compounds is not caused by diffusional losses of the flavor compounds out of the product, but by chemical reactions occurring within the food system. In this way, we may rapidly identify the relative importance of chemical and physical factors in the loss of the individual flavor compounds for various encapsulation matrices (Fig. 1c). We can now proceed by defining the functionalities needed to reduce loss of flavor. They clearly need to be related to the controlling of chemical reactions, so more detailed chemical knowledge will be important at this moment. Based on the defined functionalities, suitable technologies can be selected, if available (Table 1). For instance, amorphous carbohydrates in the glassy state may be employed to enhance the barrier properties for both the flavor compounds and oxygen. The use of chemically inert matrices may be used to reduce the rate of reactions between flavor molecules and the constituents of the encapsulation matrix. Also, reactive flavor compounds may be individually encapsulated.
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Table 1. Functionalities, working principles and technologies in the delivery of active ingredients in foods Functionality
Principle
Technology
Status
Amorphous carbohydrates in glassy state
Spray-drying Extrusion
Successful in dry state, problematic in moist matrices
Water (vapor and liquid)
Lipid coatings
Spray-chilling Fluidized-bed coating
Limited functionality, in particular in complex food systems or on small particles
Organic molecules (e.g. flavor compounds)
Amorphous carbohydrates in glassy state, polysaccharide films
Various
Successful in dry state, satisfactory in low moisture states
Control of migration Oxygen
Phase partitioning
Effective when large differences in hydrophobicity between organic compound and bulk of food matrix; equilibrium distribution attained
Control of release
Reaction control General
Oxidation
Hydrogels (coacervates, alginate complexes, gelatine capsules)
Complex coacervation, nozzle techniques for bead preparation
Many successful applications known. Shelf life is often an issue.
Emulsions and complex fluids
Emulsification, self-assembly
Emulsions are often used, but have limited functionality. Applications of self-assembly are in infancy.
High molecular weight starches and starch derivatives
Extrusion
Numerous applications developed
Separation of reactants
Phase-separated or fractionated systems
Few examples known; further exploration desirable
Change of local conditions (e.g. pH)
Liposomes/vesicles
Quenching in glassy state
Glassy carbohydrates
Complexation of reactant
Proteins, cyclodextrins hydrocolloids
Antioxidants
Various
Widely used
High-speed homogenization
Sensitivity to changes in environmental conditions; specificity of solutions
Self-assembly Micronization
First applications are developed, similarity to pharmaceutical approaches
Freeze-drying, Spray-drying
Successful in dry state, problematic in moist matrices
Bioavailability of poorly soluble active ingredients Increase of solubility by surfactants, proteins, oils Increase of specific surface area Stabilization of fragile biological materials Heat Amorphous carbohydrates in glassy state Light
Case study II: stability of probiotic cultures in food matrices Probiotics have gained considerable attention because of their postulated health benefits in the gut (Fuller, 1992) and the control of undesirable microorganisms in the intestinal and uro-genital tract (Wood, 1992). In general, probiotics are understood to be living microbial food additives that beneficially affect the host organism by improving its intestinal microbial balance. (Salminen, Ouwehand, Benno, & Lee, 1999) Beside lactic acid bacteria and bifidobacteria, propionibacteria are increasingly being considered as potential probiotics (Mantere-Alhonene, 1995). Probiotics are selected for food applications based on a number of potentially conflicting exigencies, which include safety, efficacy, functionality and, finally, stability (Richardson, 1996).
Most of the probiotic bacteria, in particular bifidobacteria, are sensitive microorganisms with low survival to stresses occurring during the production, storage and consumption of food products. The application of probiotics as active ingredients in food products is thus not straightforward, as a large fraction of the microorganisms will lose their viability already before the moment of consumption of the food product. These losses are commonly larger than 1 log during the shelf life of the product, but may be much higher. A number of technologies and strategies are available or are being developed to support the survival of probiotics in food products during processing and storage, but the range of application of such technologies and strategies is usually restricted to a limited variation in conditions. One solution circumventing the problem of probiotic stability is to use probiotic isolates and extracts instead of
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Diagram 4. Retro-analysis of the delivery of probiotics in food matrices.
viable microorganisms, as several studies have shown that non-viable probiotics can also have beneficial effects on the host (Diagram 4). The current opinion is, however, that probiotic extracts and isolates cannot completely replace live probiotics. Therefore, effective technologies are required to stabilize microorganisms in the food matrix to be able to develop probiotic food products. In order to match the conditions during processing and in the food matrix with the window of probiotic survival as indicated in Diagram 4 and to directly improve their survival capacity under the conditions of processing and consumption, an understanding of the mechanisms by which probiotic cultures lose viability and of the stress response mechanisms of probiotic microorganisms is required (De Angelis & Gobbetti, 2004; Desmond, Fitzgerald, Stanton, & Ross, 2004; Prasad, McJarrow, & Gopal, 2003; Shah, 2000). As an example, we consider the application of probiotic microorganisms in dehydrated food products, with a water activity typically between 0.3 and 0.5. Dehydrated food products have a shelf life at room temperature of typically 1–2 years. Under these conditions, probiotic microorganisms will lose their viability in typically a few months time, via mechanisms, which are not yet fully understood (Potts, 1994; Larochea, Finea, &Gervais, 2005; Kosanke, Osburn, Shuppe, & Smith, 1992). Strategies for the stabilization of probiotic cultures in such dehydrated states may be nevertheless developed based on general observations on the response of the cultures to varying environmental conditions. One obvious solution to the issue of probiotic stability in dehydrated products is to keep the cultures in a very dry
state, as in the dehydrated regime, the probiotic viability rapidly increases with decreasing water activity. This can be achieved by drying the product matrix to the required water content. This is of course a rather costly solution as one loses anything from 1% to about 10% in product weight. In addition, rendering a product extremely dry may alter numerous product characteristics like texture, palatability, and solubility. Another strategy to protect probiotic microorganisms against the effects of moisture is by encapsulation of the dry biomass in materials, which form a barrier towards water. Although a solution for a number of cases, the protection against moisture by coatings of films consisting of food materials is only transient because even hydrophobic food materials like lipids have appreciable rates of moisture migration (Table 1). Even though protection against moisture strongly enhances probiotic viability during storage, the reasons for this improvement are not clear and could be related to the moisture dependence of various reactions, including oxidation, or to the effects of water on the conformations of biological macromolecules. If oxidation processes turn out to appreciably influence the microbial stability (Sigler, Chaloupka, Brozmanova, Stadler, & Ho¨fer, 1999) antioxidants may be applied under conditions where water activity control does not lead to the desired protection. Alternatively, the impact of oxidation reactions on probiotic stability may be limited by encapsulation of the microorganisms in a material with high oxygen barrier properties, such amorphous glassy carbohydrates.
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A specific technology may also have multiple associated functionalities. For instance, glassy carbohydrates are also thought to play an important role in the stabilization of fragile biological structures such as lipid membranes, proteins and nucleic acids by forming a physical stable matrix interacting with the biological structure via hydrogen bonding (Crowe, Carpenter, & Crowe, 1998; Leslie, Israeli, Lighthart, Crowe, & Crowe, 1995). Stabilization strategies complementary to the physicochemical strategies discussed above can be found in microbiology. Strain selection is a first option, but stress adaptation (Beales, 2003), has also become an important strategy to adjust probiotic viability to an application of interest. Stress adaptation consists in the application of a sublethal or gradually increasing doses of stress in order to stimulate an adaptive cellular response that enables the microorganism to resist a similar, but more intense stress at a later stage (Crawford & Davies, 1994). For example, Talwalkar and Kailasapathy (2004) were able to stress adapt several probiotic strains to the high dissolved oxygen present in yogurt by passing cells through gradually increasing concentrations of dissolved oxygen in yogurt. Such adaptive processes are usually of a complex biological nature and may for instance be controlled by either stress specific proteins, such as a group of heat shock proteins (HSPS), which confer stability by promoting the assembly of protein complexes, or by general stress proteins (GSP). Genetic tools, including bioinformatics of entire genome sequences, are increasingly being used to increase the understanding of molecular mechanisms of microbial adaptation and protection (De Angelis & Gobbetti, 2004). Although the issue of probiotic stability in food products is not yet settled, we expect that a structured analysis based on the ‘retro-design’ concept is useful as it offers a rational way to disentangle the various physico-chemical and biological factors determining probiotic stability. The final processing strategy will therefore not be optimal regarding a single process step or ingredient but rather the best compromise in terms of the target product and the requested product specifications.
Role of materials science in establishing encapsulation functionality The desired functional properties of delivery systems are generally based on the need to compensate the incompatibility between an active ingredient and the matrix of application. The most important functions for such systems are summarized in Table 1, pointing out the relation between principle of the delivery function and the potential technology by which the function may be realized. From Table 1, it is immediately obvious that many technologies are applicable only under a limited range of conditions. In addition, for numerous functionalities, no suitable technology may be available yet. In particular, barriers to locally reduce the rate of moisture migration in food systems have
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proven difficult to realize using food-grade materials (Krochta, Baldwin, & Nisperos-Carriedo, 1994). In the functionalities listed in Table 1, the material properties of food materials occupy a central role. Key functionalities like barrier properties, formation of structures physically stabilizing complex biological macromolecules require detailed knowledge on the physical properties of food-grade materials like lipids, proteins, carbohydrates and their mixtures or complexes. Over the last few decades, the materials science of foods has witnessed significant advances (Roos, 1995). Currently, major emphasis is on the elucidation of molecular and nanoscale properties of food materials (Mezzenga, Schurtenberger, Burbidge, & Michel, 2005; Kilburn, Claude, Schweizer, Alam, & Ubbink, 2005). It is expected that this will contribute to an improved understanding of molecular mechanisms underlying
Fig. 2. Materials science of amorphous carbohydrate matrices: (a) in food matrices, the molecular mobility and the rate of reactions depend exponentially on both temperature and water content. Consequently, food properties need investigation as a function of two independent parameters; (b) the glass transition of an amorphous carbohydrate matrix decreases strongly with increasing water content or water activity. An increase of the average molecular weight leads to an increase of the Tg at the constant water content or water activity; (c) a molecular interpretation of the interaction of water with amorphous carbohydrates. In dry state, the hydroxyl groups on the sugar residues form hydrogen bonds (left diagram). Water absorbed into the system leads to plasticization both by interference with the hydrogen bonding between the carbohydrate chains and by creation of additional free volume (right diagram).
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physical phenomena such as barrier properties, diffusion, phase transitions and structural rearrangements. Properties of food materials and food systems generally need to be studied as a function of two independent parameters, the temperature and the water content (Fig. 2a), for reasons outlined below. In the following, we will illustrate this development towards the molecular and nanolevels on the hand of one class of food materials, amorphous carbohydrates in the glassy state. As extensively discussed above, these materials are of importance both as a food matrix (like in milk powder, infant formula, instant coffee, confectionary) and as material for the encapsulation and protection of active ingredients such as flavors, vitamins and polyunsaturated fatty acids (Risch & Reineccius, 1995; Ubbink & Schoonman, 2003). An important concept in the material science of amorphous carbohydrates is the glass transition, which is a well-established concept used to predict the processing and stability of carbohydrate-based food products. The glass transition temperature (Tg), which separates the rubbery from the glassy state (Franks, 1985, chapter 3; Roos, 1995;), is indicative of the degree of molecular mobility of the amorphous matrix. In the rubbery state, translational and rotational motion of the matrix molecules is still possible, but in the glassy state large-scale molecular motion is effectively inhibited (Angell, 1988). The Tg of an amorphous matrix is dependent on its composition, with low molecular weight compounds generally having a lower glass transition temperature than high molecular weight compounds (Levine & Slade, 1986; Roos & Karel, 1991a,b; Slade & Levine, 1995) (Fig. 2b). Of particular importance for amorphous carbohydrates is that the glass transition temperature strongly decreases with increasing water content or water activity (Mateev, Grinberg, & Tolstoguzov, 2000; Roos & Karel, 1991a,b), as illustrated in Fig. 2a. Although large-scale motion of the matrix carbohydrates are effectively blocked in the glassy state, small-scale reorganizations within the matrix are still possible, as is witnessed for instance by the mobility of small molecules such as water (Tromp, Parker, & Ring, 1997), gases (Schoonman, Ubbink, Bisperink, Le Meste, & Karel, 2002; Whitcombe, Parker, & Ring, 2005) and small organic molecules (Goubet, Le Quere, & Voilley, 1998; Ubbink & Reineccius, 2002) in the glassy state. In addition, using spectroscopic techniques such as neutron scattering (Cicerone & Soles, 2004), electron paramagnetic resonance (Van den Dries, Van Dusschoten, Hemminga, & Van der Linden, 2000) and positron annihilation lifetime spectroscopy (Kilburn et al., 2004), evidence is accumulating that temperature changes and changes in water content of an amorphous carbohydrate matrix have profound effect on the local structure and dynamics of a carbohydrate matrix even in the glassy state (Fig. 2c). The structure and dynamics at nanometer and sub-nanometer length scales are of prime importance in understanding the barrier properties of glassy
carbohydrates (Kilburn et al., 2005) and the protective effects of carbohydrates for sensitive biological materials such as proteins (Cicerone & Soles, 2004). Consequently, understanding of processes occurring at the molecular and nanoscales are relevant for optimizing barrier properties of food materials. Such knowledge is of importance for encapsulation systems prepared by a variety of processing techniques such as spray-drying, fluidized-bed drying and melt extrusion, as discussed for instance by Bencze´di and Blake (1999), Gibbs et al. (1999), Gouin (2004), Risch and Reineccius (1995) and Ubbink and Schoonman (2003). Concluding remarks Current demands posed on food developers to satisfy often conflicting requirements with respect to the incorporation of sensitive active ingredients in food products of increasing complexity have provoked a growing interest in the development and application of delivery systems for foodstuffs. In many cases, however, it is difficult to apply established encapsulation technologies without modification to an application of interest, principally because the physical and chemical behavior of the active ingredients and the functionality of the delivery system are poorly understood. In the present article, an alternative approach is proposed which focuses on the desired functionality of the active ingredient in the food application and works then back in a systematic fashion to the various technologies, such as encapsulation technology, which may be utilized to achieve the target product. The advantage of such an approach is that the final product application remains at the center of attention, and not a specific technology itself and it postpones the selection of a technology to a later stage in order to allow matching the different factors determining product quality and stability. In addition, because of the systematic evaluation of various aspects of the delivery issue, gaps in the scientific knowledge become immediately apparent and results from emerging research are more easily adopted. We anticipate that future efforts on the delivery of active ingredients in foodstuffs will increasingly incorporate interdisciplinary scientific developments at the interfaces between biomaterials science, physical chemistry, biophysics and encapsulation technology. References Angell, C. A. (1988). Perspectives on the glass transition. Journal of Physics and Chemistry of Solids, 49, 863–870. Beales, N. (2003). Adaptation of microorganisms to cold temperatures, weak acid preservatives, low pH, and osmotic stress: A review. Comprehensive Reviews in Food Science and Food Safety, 3, 1–20. Bencze´di, D., & Blake, A. (1999). Encapsulation and the controlled release of flavours. Leatherhead Food RA Food Industry Journal, 2. Cicerone, M. T., & Soles, C. L. (2004). Fast dynamics and stabilization of proteins: Binary glasses of trehalose and glycerol. Biophysical Journal, 86, 3836–3845. Cohen, M. H., & Turnbull, D. (1959). Molecular transport in liquids and glasses. Journal of Chemical Physics, 31, 1164–1165.
J. Ubbink, J. Kru¨ger / Trends in Food Science & Technology 17 (2006) 244–254 Corey, E. J., & Cheng, X.-M. (1989). The logic of chemical synthesis. New York: Wiley. Crawford, D., & Davies, K. (1994). Adaptive response and oxidative stress. Environmental and Health Perspectives, 10(Suppl. 10), 25–28. Crowe, J. H., Carpenter, J. F., & Crowe, L. M. (1998). The role of vitrification in anhydrobiosis. Annual review of Physiology, 60, 73–103. De Angelis, M., & Gobbetti, M. (2004). Environmental stress responses in Lactobacillus: A review. Proteomics, 4, 106–122. De Roos, K. B., & Mansencal, R. (2003). Poor performance of flavourings in heat processed products: a problem of poor flavour retention or poor flavour release? Flavour research at the dawn of the twenty-first century, Proceedings of the Weurman Flavor Research Symposium, 10th, Beaune, France, June 25–28, 2002. Desmond, C., Fitzgerald, G. F., Stanton, C., & Ross, R. P. (2004). Improved stress tolerance of GroESL-overproducing Lactococcus lactis and probiotic Lactobacillus paracasei NFBC 338. Applied and Environmental Microbiology, 70(10), 5929–5936. Franks, F. (1985). Biophysics and biochemistry at low temperatures. Cambridge: Cambridge University Press. Fuller, R. (1992). Probiotics: The scientific basis. London: Chapman & Hall. Gibbs, B. F., Kermasha, S., Alli, I., & Mulligan, C. N. (1999). Encapsulation in the food industry: A review. International Journal of Food Sciences and Nutrition, 50, 213–224. Goubet, I., Le Quere, J. L., & Voilley, A. J. (1998). Retention of aroma compounds by carbohydrates: Influence of their physicochemical characteristics and of their physical state. A review. Journal of Agricultural and Food Chemistry, 46, 1981–1990. Gouin, S. (2004). Microencapsulation: Industrial appraisal of existing technologies and trends. Trends in Food Science and Technology, 15, 330–347. Kilburn, D., Claude, J., Mezzenga, R., Dlubek, G., Alam, A., & Ubbink, J. (2004). Water in glassy carbohydrates: Opening it up at the nanolevel. Journal of Physical Chemistry B, 108, 12436–12441. Kilburn, D., Claude, J., Schweizer, T., Alam, A., & Ubbink, J. (2005). Carbohydrate polymers in amorphous states: An integrated thermodynamic and nanostructural investigation. Biomacromolecules, 6, 864–879. Kosanke, J. W., Osburn, R. M., Shuppe, G. I., & Smith, R. S. (1992). Slow rehydration improves the recovery of dried bacterial populations. Canadian Journal of Microbiology, 38(6), 520–525. Krochta, J. M., Baldwin, E. A., & Nisperos-Carriedo, M. O. (1994). Edible coatings and films to improve food quality. Basel: Technomic. Lamb, R. (1987). Spray chilling. Food Flavour Ingredients: Processing and Packaging, 9, 39–43. Larochea, C., Finea, F., & Gervais, P. (2005). Water activity affects heat resistance of microorganisms in food powders. International Journal of Food Microbiology, 97, 307–315. Leslie, S. B., Israeli, E., Lighthart, B., Crowe, J. H., & Crowe, L. M. (1995). Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Applied and Environmental Microbiology, 61(10), 3592–3597. Levine, H., & Slade, L. (1986). A polymer physico-chemical approach to the study of commercial starch hydrolysis products (SHPs). Carbohydrate Polymers, 6, 213–244. Lukaski, H. C. (2004). Vitamin and mineral status: Effects on physical performance. Nutrition, 20, 632–644. Mantere-Alhonene, S. (1995). Propionibacteria used as a probiotic—A review. Lait, 75, 447–452. Mateev, Yu. I., Grinberg, V. Ya., & Tolstoguzov, V. B. (2000). The plasticizing effect of water on proteins, polysaccharides and
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Ubbink, J., & Reineccius, G.A. (2002). Mobility, clustering and phase separation of small molecules in amorphous carbohydrate matrices. Abstracts of Papers, 223rd ACS National Meeting, Orlando, FL, AGFD-035. Ubbink, J., & Schoonman, A. (2003). Flavor delivery systems. KirkOthmer encyclopedia of chemical technology, on-line edition. New York: Wiley. Van den Dries, I. J., Van Dusschoten, D., Hemminga, M. A., & Van der Linden, E. (2000). Effects of water content and molecular weight on spin probe and water mobility in malto-oligomer glasses. Journal of Physical Chemistry B, 104, 10126–10132. Vrentas, J. S., & Duda, J. L. (1978). A free volume interpretation of the influence of the glass transition on diffusion in amorphous polymers. Journal of Applied Polymer Science, 22, 2325–2339.
Weinbreck, F., Minor, M., & de Kruif, C. G. (2004). Microencapsulation of oils using whey protein/gum arabic coacervates. Journal of Microencapsulation, 21, 667–679. Whitcombe, M. J., Parker, R., & Ring, S. G. (2005). Oxygen solubility and permeability of carbohydrates. Carbohydrate Research, 340, 1523–1527. Winkel, C. (2005). Stability of aroma chemicals. In D. J. Rowe (Ed.), Chemistry and technology of flavors and fragrances (pp. 244– 260). Boca Raton, FL: CRC press. Wood, B. J. B. (1992). The lactic acid bacteria in health and disease The lactic acid bacteria (Vol. 1). London: Elsevier. Zimmermann, M. B. (2004). The potential of encapsulated iron compounds in food fortification: A review. International Journal for Vitamin and Nutrition Research, 74(6), 453–461.
Review
Physical approaches for the delivery of active ingredients in foods Job Ubbink* and Jessica Kru¨ger
&
Nestle´ Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland (Tel.: C41 21 785 9378; fax: C41 21 785 8554; e-mail: [email protected]) Encapsulation systems in food applications are typically employed to solve formulation problems arising from a limited chemical or physical stability of the active ingredient, an incompatibility between active ingredient and food matrix, or to control the release of a sensorially active compound or the bioavailability of a nutrient. However, the use of encapsulation systems in complex food matrices often fails because their application is mostly based on trial and error. The present article introduces a conceptually new approach in which the target application is analyzed based on physical principles, including materials science, physical chemistry and biophysics in order to develop a range of solution strategies from which the most promising may be selected for final application. The approach is illustrated with two case studies on the stability of active ingredients in glassy carbohydrates.
Introduction The creation of novel functionalities of active ingredients in complex food matrices is of increasing importance for the food industry. Active ingredients which are introduced in a variety of food products include traditional ones such as flavors (Risch & Reineccius, 1988; Ubbink & Schoonman, 2003), vitamins (Lukaski, 2004) and minerals (Lukaski, 2004; Zimmermann, 2004), or relatively novel ones such as probiotic microorganisms (Roy, 2005; * Corresponding author. 0924-2244/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2006.01.007
Richardson, 1996; Ross, Desmond, Fitzgerald, & Stanton, 2005) and various classes of bioactive compounds (Rodriguez-Huezo, Pedroza-Islas, Prado-Barragan, Beristain, & Vernon-Carter, 2004; Shi, Mazza, & Le Maguer, 2002). Application of active ingredients in food products often requires innovative approaches because of their sensitivity to a variety of physical and chemical factors, which causes either the loss of biological functionality, chemical degradation or a premature or incomplete release. The situation is challenging not only because of the high sensitivity of many of the active ingredients, but also because of the complexity of many food products and the conditions prevalent in many food matrices. In addition, product safety, appearance, storage conditions, ease of preparation by the consumer, freshness and sensory properties of the food product are not to be compromised by the incorporation of the active ingredient. Compared to other domains, such as the pharmaceutical, agro-chemical or cosmetic industries, the development of products containing active ingredients often more challenging in the food industry, because of the necessity to simultaneously fulfill these multiple conditions. In most non-food domains, the delivery of the active ingredient is the primary objective of the product, and consequently the composition, processing and storage conditions may be flexibly adapted to maximize the performance of this active ingredient. In addition, in the food industry, regulations with respect to ingredients, processing methods and storage conditions are tight and the price margin is much lower than in, for example, the pharmaceutical industry. An approach increasingly advocated as enabling the introduction of active ingredients in foods is the use of encapsulation systems for protection or controlled release (Bencze´di & Blake, 1999; Gibbs Kermasha, Alli, & Mulligan, 1999; Gouin, 2004; Risch & Reineccius, 1995; Ubbink & Schoonman, 2003). Consequently, extensive technologies have been developed to encapsulate active food ingredients in a variety of food-grade materials including glassy carbohydrates (Ubbink & Schoonman, 2003), lipids (Lamb, 1987; Risch & Reineccius, 1995) and biopolymer complexes (Soper, 1995; Weinbreck, Minor & de Kruif, 2004). The choice of technology and encapsulation materials is however often based on trial and error and not on a fundamental understanding of the physical and chemical phenomena determining stability, release, perception and digestion.
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Whereas for simple food systems under controlled conditions of temperature and water activity (e.g. instant beverages, chewing gum, and dehydrated or frozen foods), trial and error approaches for the incorporation of active ingredients have often proved successful, for more complex food products, such as chilled meals, RTD and shelf-stable beverages, they have generally failed to provide efficient solutions. This is primarily the case because trial and error approaches are not efficient in finding a rational compromise between the large numbers of physical, chemical and biological factors influencing the performance of both the active ingredient and the food matrix. In the present article, a new approach is introduced in which the target application is first analyzed using scientific principles, including materials science, physical chemistry and biophysics. The scientific understanding is used to develop a range of potential solution strategies from which the most promising may be selected for further development. Based on technological considerations, such as cost, ease of manufacturing, adaptability, one of these various possible solutions may finally be implemented in the actual food product. The major advantage of our approach is that it does not focus from the outset on a specific technology to solve an issue, which might be poorly understood, and for which the selected technology may ultimately be ineffective. In contrast, the emphasis is on the final application and on a clear specification of the requirements to be fulfilled by a prospective technology. It will be apparent that such developments are facilitated when based on a scientific foundation. Even though the field is in significant development at present, we attempt to illustrate the principle of our approach and the role of scientific developments in the search for a rational delivery strategy by way of two case studies. Both focus on the stability of active ingredients as an important subtopic of the overall delivery field and we limit the scientific discussion of encapsulation materials to carbohydrates in the glassy state. From the two case studies, several functionalities appear which would be highly desirable in the context of the encapsulation of active ingredients. A number of such functionalities and their potential are listed, but also their limitations. The potential impact of recent developments in carbohydrate physics is discussed. Finally, we discuss the implications of our approach for the development of delivery systems for food applications. ‘Retro’-design of delivery systems In a retro-design approach to the delivery of active ingredients, the food application is placed at the center and from there one systematically works back to find a feasible technology to introduce the active ingredient in the food product. The principle of retro-design, as developed in organic synthesis, allows the systematic evaluation of all steps and routes starting from the final product down to the raw materials (Corey & Cheng, 1989). In organic chemistry,
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such a retro-design allows the evaluation of all possible reaction pathways and intermediates leading to the desired product and facilitates the choice of the favored synthesis route based on a rational compromise between reaction yields, number of reaction steps, and availability of starting materials. In addition, the approach has proved useful as it allows the definition of chemical transformations which do not yet exist but whose development may then be attempted (Corey & Cheng, 1989). In the food field, we have experienced that a similar retro-approach is very useful, in particular in the development of complex food products containing active ingredients. In adopting a retro-design approach towards the delivery of active ingredients in foods, one starts by defining precisely the functionality and performance of the active ingredient desired in the final application (Diagram 1). This target sets the required functionality and performance of the active ingredient. This could for instance be the release of a sufficient amount of properly balanced flavor at a given moment during the preparation or consumption of a food product, the presence of a defined quantity of vitamins or a minimal number of live probiotic microorganisms in a food product at the moment of consumption, or the maintenance of a bioactive ingredient in its proper bioactive form during the shelf life and the digestion of a food product. Subsequently, the physical, chemical and biological properties of the active ingredient and the conditions prevailing in the food matrix are analyzed (Diagram 1). Such properties of active ingredients include the physical properties, including phase behavior and molecular mobility, chemical reactivity and conditions under which the physiological and sensory characteristics are maintained and they effectively determine the application window of the active ingredient. If the target can be satisfied is then determined by the conditions prevailing in the food matrix, during manufacturing, storage and consumption. In general, for a specific food product, we have only a very limited flexibility in adapting the conditions in the food matrix to match the window of application of the active ingredient. Therefore, often a significant discrepancy exists between the window of application of the active ingredient and the limits set by product developers on changes in product composition, processing routes and storage conditions. Consequently, often conditions in the food matrix have to be accepted which are detrimental to the stability and functionality of the active ingredient. In such situations, the active ingredient and the product may be said to be incompatible and a successful product development satisfying the original requirements on the functionality and performance of the active ingredient turns out to be impossible. However, having clearly identified both the conditions required for maintenance of the performance of the active ingredient and the limits to the conditions set by the food matrix, one may define the functionality needed to resolve the causes of the incompatibility of active ingredient and
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Diagram 1. Outline of a retro-design approach towards the optimization of the functionality and performance of active ingredients in complex food matrices.
food matrix. The functionality is defined solely based on the analysis of the interaction of active ingredient and food matrix and it does not yet relate to a specific technology. In fact, a functionality defined in this way may not even have an existing associated technology. The major advantage of introducing the concept functionality is that it postpones the selection of a technology to a later stage in order to allow the optimal matching of the different factors indicated in Diagram 1. It thereby enables a clear definition of the requirements a novel technology should fulfill in satisfying the specific application. Based on the defined functionalities, one is in a position to select or develop an appropriate technology. This may involve the use of delivery or encapsulation systems, but also the reformulation of a food product using different (active) ingredients and raw materials, or the induction of a different structure in the food product (Diagram 2). In addition, having clearly identified the causes for the incompatibility between active ingredient and food product, it sometimes turns out that a small adjustment of the manufacturing process will already enhance the performance of the active ingredient. By using any of these options, one can attempt to eliminate the incompatibility of active ingredient and food product. The approach based on ‘retro-design’ has a number of important advantages over the more established trial and
error evaluation of encapsulation systems in the development of advanced food products containing active ingredients. In the first place, the focus is on the food product and its functionality and not on an, often arbitrary, encapsulation technology. In the second place, following the ‘retro-design approach’, the choice for an optimized encapsulation system or food manufacturing process is made in an advanced
Diagram 2. Technologies for the delivery of active ingredients in foods. Encapsulation is the isolation of the active ingredient within the food product using food-grade materials. Formulation encompasses the structuring of the active ingredient, often on molecular or nanoscale levels using food-grade ingredients interacting with the active ingredient. By adaptation of the processing conditions, the adverse effects on the active ingredient may be minimized, or the performance of active ingredient may be maximized. Packaging extends beyond a narrow definition of delivery approaches as it generally uses non-food grade materials and processes and is not included in the diagram.
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stage, allowing maximum flexibility with respect to the evaluation of performance, side effects and costs of the various options. In the last place, it stimulates the systematic use of scientific knowledge to resolve concrete issues arising in the development of innovative food products and it helps to efficiently identify technology gaps. Case study I: encapsulation and stabilization of complex flavor mixtures in amorphous food powders Let us consider the case of the stability of a flavor composition or extract in a food powder or granular material. For the purpose of the present discussion, this can be any food product in powder form of which the matrix largely consists of amorphous carbohydrates in the glassy state, like instant drinks, dairy-based powders, culinary powders, or soluble coffee. During storage, the quality and strength of the flavor of food products decreases, leading to a perceived loss in quality of the food product (De Roos & Mansencal, 2003). This decrease in flavor quality and strength is related to losses of flavor compounds by physical and chemical processes. Physical processes primarily comprise the diffusion of flavor compounds out of the food matrix, but also the physical binding of flavor compounds by components of the food matrix. Chemical processes comprise the degradation of flavor compounds by oxidation, hydrolysis and chemical reactions with the food matrix, but also the formation of offnotes (Winkel, 2005). To counteract the loss of flavor, encapsulation systems are routinely used (Risch & Reineccius, 1988; Ubbink & Schoonman, 2003). The use of such systems has been highly successful in situations where the cause of the loss of the flavor is known, and where it can be counteracted by the properties of the encapsulation matrix. Encapsulation systems based on amorphous carbohydrates in the glassy state have been very effective in reducing the rate of release of the flavor during storage, and in minimizing the rate of oxidation of oxygen-sensitive flavors by environmental oxygen. Both processes involve the diffusion of guest molecules through the glassy carbohydrate matrix, which is a very slow process. As the functionality of encapsulation systems based on amorphous, glassy carbohydrates is based on the prevention of diffusion of flavor compounds and oxygen, strategies to optimize such systems are immediately clear. The rates of diffusion can be minimized by control of the material properties, such as the lowering the water activity of the carbohydrate matrix or the selection of the appropriate carbohydrate composition. In addition, by changing the granule morphology, one can minimize the rate of release of the flavor compounds or the rate of uptake of oxygen. In general, the larger and the less porous the structure of granule, the lower the effective transport rates (Fig. 1a). In many of the cases of current interest to the food industry, highly sensitive flavor compositions or extracts are used, for instance topnotes in culinary and bakery products. In addition, the matrix for the entrapment of the flavors is often a
Fig. 1. Diffusion and degradation of flavor compounds encapsulated in a complex food matrix largely consisting of amorphous carbohydrates: (a) granule morphology is an important parameter to optimize when the stability of encapsulated flavors is determined by diffusional losses (evaporation, oxidation by environmental oxygen). When chemical reactions with the matrix are of importance, particle morphology ceases to be a relevant parameter: (I) extruded carbohydrate granules; (II) freeze-dried granules; barZ100 mm; (b) flavor retention in coffee-based carbohydrate matrix in the glassy state after storage for 42 days at 37 8C and a water activity of 0.33. By plotting the flavor retention in a product prototype as function of the molecular volume of the various flavor compounds in the flavor composition, a separation of the flavor retention data into two distinct groups is observed. The open symbols denote the flavor compounds of which the retention is related to their molecular volume. These flavor compounds may be considered to be chemically stable in the matrix and their losses are caused by evaporation of the flavor into the surrounding atmosphere. The retention of a number of flavor compounds is much lower than predicted on basis of their molecular volume (filled symbols). These flavor compounds are chemically unstable in this particular prototype matrix. The fraction of the flavor lost owing to chemical degradation may be estimated by subtracting the calculated physical loss for a hypothetical chemically stable flavor molecule from the total loss; (c) loss of three key compounds from coffee aroma after storage (42 days, 37 8C, water activity Z0.33) for two different spray-dried encapsulation matrices: A, coffee-based matrix; B, maltodextrin matrix (DE-10). The height of the bar indicates the total loss of the flavor compound, divided in physical loss (black bar) and chemical loss (open bar). Adapted with permission from Ubbink and Reineccius (2002).
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Diagram 3. Retro-analysis of the delivery of flavors in food matrices.
chemically and structurally heterogeneous food matrix, such as a cereal product or a soluble coffee matrix. In such complex systems, the loss of flavor is caused by multiple effects acting in concert, which complicates the choice of an effective stabilization strategy. Because of the complexity of the situation, the first step is to develop a quantitative understanding of the processes underlying aroma mobility, degradation and release (Diagram 3). As an example, we consider the stability of a coffee aroma in both a glassy matrix consisting of coffee extract and in a glassy carbohydrate matrix (Fig. 1). The flavor compounds in coffee aroma represent a large variety of chemical functionalities, including alcohols, esters, ketones and thiols, present in the ppb–ppm range. If only physical processes contribute to the loss of flavor, the retention should depend solely on physical properties such as the vapor pressure, the oil-to-water partition coefficient and molecular size. As in glassy systems, the diffusional mobility of a permeant is related to its molecular size (Cohen & Turnbull, 1959; Menting, Hoogstad, & Thijssen, 1970; Vrentas & Duda, 1978), we analyze the retention of the various flavor compounds as a function of the molecular
volume (determined by the Van der Waals radii) (Fig. 1b). A number of the flavor compounds in this case does not correlate with the molecular volume (filled symbols). Consequently, the low retention of these compounds is not caused by diffusional losses of the flavor compounds out of the product, but by chemical reactions occurring within the food system. In this way, we may rapidly identify the relative importance of chemical and physical factors in the loss of the individual flavor compounds for various encapsulation matrices (Fig. 1c). We can now proceed by defining the functionalities needed to reduce loss of flavor. They clearly need to be related to the controlling of chemical reactions, so more detailed chemical knowledge will be important at this moment. Based on the defined functionalities, suitable technologies can be selected, if available (Table 1). For instance, amorphous carbohydrates in the glassy state may be employed to enhance the barrier properties for both the flavor compounds and oxygen. The use of chemically inert matrices may be used to reduce the rate of reactions between flavor molecules and the constituents of the encapsulation matrix. Also, reactive flavor compounds may be individually encapsulated.
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Table 1. Functionalities, working principles and technologies in the delivery of active ingredients in foods Functionality
Principle
Technology
Status
Amorphous carbohydrates in glassy state
Spray-drying Extrusion
Successful in dry state, problematic in moist matrices
Water (vapor and liquid)
Lipid coatings
Spray-chilling Fluidized-bed coating
Limited functionality, in particular in complex food systems or on small particles
Organic molecules (e.g. flavor compounds)
Amorphous carbohydrates in glassy state, polysaccharide films
Various
Successful in dry state, satisfactory in low moisture states
Control of migration Oxygen
Phase partitioning
Effective when large differences in hydrophobicity between organic compound and bulk of food matrix; equilibrium distribution attained
Control of release
Reaction control General
Oxidation
Hydrogels (coacervates, alginate complexes, gelatine capsules)
Complex coacervation, nozzle techniques for bead preparation
Many successful applications known. Shelf life is often an issue.
Emulsions and complex fluids
Emulsification, self-assembly
Emulsions are often used, but have limited functionality. Applications of self-assembly are in infancy.
High molecular weight starches and starch derivatives
Extrusion
Numerous applications developed
Separation of reactants
Phase-separated or fractionated systems
Few examples known; further exploration desirable
Change of local conditions (e.g. pH)
Liposomes/vesicles
Quenching in glassy state
Glassy carbohydrates
Complexation of reactant
Proteins, cyclodextrins hydrocolloids
Antioxidants
Various
Widely used
High-speed homogenization
Sensitivity to changes in environmental conditions; specificity of solutions
Self-assembly Micronization
First applications are developed, similarity to pharmaceutical approaches
Freeze-drying, Spray-drying
Successful in dry state, problematic in moist matrices
Bioavailability of poorly soluble active ingredients Increase of solubility by surfactants, proteins, oils Increase of specific surface area Stabilization of fragile biological materials Heat Amorphous carbohydrates in glassy state Light
Case study II: stability of probiotic cultures in food matrices Probiotics have gained considerable attention because of their postulated health benefits in the gut (Fuller, 1992) and the control of undesirable microorganisms in the intestinal and uro-genital tract (Wood, 1992). In general, probiotics are understood to be living microbial food additives that beneficially affect the host organism by improving its intestinal microbial balance. (Salminen, Ouwehand, Benno, & Lee, 1999) Beside lactic acid bacteria and bifidobacteria, propionibacteria are increasingly being considered as potential probiotics (Mantere-Alhonene, 1995). Probiotics are selected for food applications based on a number of potentially conflicting exigencies, which include safety, efficacy, functionality and, finally, stability (Richardson, 1996).
Most of the probiotic bacteria, in particular bifidobacteria, are sensitive microorganisms with low survival to stresses occurring during the production, storage and consumption of food products. The application of probiotics as active ingredients in food products is thus not straightforward, as a large fraction of the microorganisms will lose their viability already before the moment of consumption of the food product. These losses are commonly larger than 1 log during the shelf life of the product, but may be much higher. A number of technologies and strategies are available or are being developed to support the survival of probiotics in food products during processing and storage, but the range of application of such technologies and strategies is usually restricted to a limited variation in conditions. One solution circumventing the problem of probiotic stability is to use probiotic isolates and extracts instead of
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Diagram 4. Retro-analysis of the delivery of probiotics in food matrices.
viable microorganisms, as several studies have shown that non-viable probiotics can also have beneficial effects on the host (Diagram 4). The current opinion is, however, that probiotic extracts and isolates cannot completely replace live probiotics. Therefore, effective technologies are required to stabilize microorganisms in the food matrix to be able to develop probiotic food products. In order to match the conditions during processing and in the food matrix with the window of probiotic survival as indicated in Diagram 4 and to directly improve their survival capacity under the conditions of processing and consumption, an understanding of the mechanisms by which probiotic cultures lose viability and of the stress response mechanisms of probiotic microorganisms is required (De Angelis & Gobbetti, 2004; Desmond, Fitzgerald, Stanton, & Ross, 2004; Prasad, McJarrow, & Gopal, 2003; Shah, 2000). As an example, we consider the application of probiotic microorganisms in dehydrated food products, with a water activity typically between 0.3 and 0.5. Dehydrated food products have a shelf life at room temperature of typically 1–2 years. Under these conditions, probiotic microorganisms will lose their viability in typically a few months time, via mechanisms, which are not yet fully understood (Potts, 1994; Larochea, Finea, &Gervais, 2005; Kosanke, Osburn, Shuppe, & Smith, 1992). Strategies for the stabilization of probiotic cultures in such dehydrated states may be nevertheless developed based on general observations on the response of the cultures to varying environmental conditions. One obvious solution to the issue of probiotic stability in dehydrated products is to keep the cultures in a very dry
state, as in the dehydrated regime, the probiotic viability rapidly increases with decreasing water activity. This can be achieved by drying the product matrix to the required water content. This is of course a rather costly solution as one loses anything from 1% to about 10% in product weight. In addition, rendering a product extremely dry may alter numerous product characteristics like texture, palatability, and solubility. Another strategy to protect probiotic microorganisms against the effects of moisture is by encapsulation of the dry biomass in materials, which form a barrier towards water. Although a solution for a number of cases, the protection against moisture by coatings of films consisting of food materials is only transient because even hydrophobic food materials like lipids have appreciable rates of moisture migration (Table 1). Even though protection against moisture strongly enhances probiotic viability during storage, the reasons for this improvement are not clear and could be related to the moisture dependence of various reactions, including oxidation, or to the effects of water on the conformations of biological macromolecules. If oxidation processes turn out to appreciably influence the microbial stability (Sigler, Chaloupka, Brozmanova, Stadler, & Ho¨fer, 1999) antioxidants may be applied under conditions where water activity control does not lead to the desired protection. Alternatively, the impact of oxidation reactions on probiotic stability may be limited by encapsulation of the microorganisms in a material with high oxygen barrier properties, such amorphous glassy carbohydrates.
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A specific technology may also have multiple associated functionalities. For instance, glassy carbohydrates are also thought to play an important role in the stabilization of fragile biological structures such as lipid membranes, proteins and nucleic acids by forming a physical stable matrix interacting with the biological structure via hydrogen bonding (Crowe, Carpenter, & Crowe, 1998; Leslie, Israeli, Lighthart, Crowe, & Crowe, 1995). Stabilization strategies complementary to the physicochemical strategies discussed above can be found in microbiology. Strain selection is a first option, but stress adaptation (Beales, 2003), has also become an important strategy to adjust probiotic viability to an application of interest. Stress adaptation consists in the application of a sublethal or gradually increasing doses of stress in order to stimulate an adaptive cellular response that enables the microorganism to resist a similar, but more intense stress at a later stage (Crawford & Davies, 1994). For example, Talwalkar and Kailasapathy (2004) were able to stress adapt several probiotic strains to the high dissolved oxygen present in yogurt by passing cells through gradually increasing concentrations of dissolved oxygen in yogurt. Such adaptive processes are usually of a complex biological nature and may for instance be controlled by either stress specific proteins, such as a group of heat shock proteins (HSPS), which confer stability by promoting the assembly of protein complexes, or by general stress proteins (GSP). Genetic tools, including bioinformatics of entire genome sequences, are increasingly being used to increase the understanding of molecular mechanisms of microbial adaptation and protection (De Angelis & Gobbetti, 2004). Although the issue of probiotic stability in food products is not yet settled, we expect that a structured analysis based on the ‘retro-design’ concept is useful as it offers a rational way to disentangle the various physico-chemical and biological factors determining probiotic stability. The final processing strategy will therefore not be optimal regarding a single process step or ingredient but rather the best compromise in terms of the target product and the requested product specifications.
Role of materials science in establishing encapsulation functionality The desired functional properties of delivery systems are generally based on the need to compensate the incompatibility between an active ingredient and the matrix of application. The most important functions for such systems are summarized in Table 1, pointing out the relation between principle of the delivery function and the potential technology by which the function may be realized. From Table 1, it is immediately obvious that many technologies are applicable only under a limited range of conditions. In addition, for numerous functionalities, no suitable technology may be available yet. In particular, barriers to locally reduce the rate of moisture migration in food systems have
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proven difficult to realize using food-grade materials (Krochta, Baldwin, & Nisperos-Carriedo, 1994). In the functionalities listed in Table 1, the material properties of food materials occupy a central role. Key functionalities like barrier properties, formation of structures physically stabilizing complex biological macromolecules require detailed knowledge on the physical properties of food-grade materials like lipids, proteins, carbohydrates and their mixtures or complexes. Over the last few decades, the materials science of foods has witnessed significant advances (Roos, 1995). Currently, major emphasis is on the elucidation of molecular and nanoscale properties of food materials (Mezzenga, Schurtenberger, Burbidge, & Michel, 2005; Kilburn, Claude, Schweizer, Alam, & Ubbink, 2005). It is expected that this will contribute to an improved understanding of molecular mechanisms underlying
Fig. 2. Materials science of amorphous carbohydrate matrices: (a) in food matrices, the molecular mobility and the rate of reactions depend exponentially on both temperature and water content. Consequently, food properties need investigation as a function of two independent parameters; (b) the glass transition of an amorphous carbohydrate matrix decreases strongly with increasing water content or water activity. An increase of the average molecular weight leads to an increase of the Tg at the constant water content or water activity; (c) a molecular interpretation of the interaction of water with amorphous carbohydrates. In dry state, the hydroxyl groups on the sugar residues form hydrogen bonds (left diagram). Water absorbed into the system leads to plasticization both by interference with the hydrogen bonding between the carbohydrate chains and by creation of additional free volume (right diagram).
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physical phenomena such as barrier properties, diffusion, phase transitions and structural rearrangements. Properties of food materials and food systems generally need to be studied as a function of two independent parameters, the temperature and the water content (Fig. 2a), for reasons outlined below. In the following, we will illustrate this development towards the molecular and nanolevels on the hand of one class of food materials, amorphous carbohydrates in the glassy state. As extensively discussed above, these materials are of importance both as a food matrix (like in milk powder, infant formula, instant coffee, confectionary) and as material for the encapsulation and protection of active ingredients such as flavors, vitamins and polyunsaturated fatty acids (Risch & Reineccius, 1995; Ubbink & Schoonman, 2003). An important concept in the material science of amorphous carbohydrates is the glass transition, which is a well-established concept used to predict the processing and stability of carbohydrate-based food products. The glass transition temperature (Tg), which separates the rubbery from the glassy state (Franks, 1985, chapter 3; Roos, 1995;), is indicative of the degree of molecular mobility of the amorphous matrix. In the rubbery state, translational and rotational motion of the matrix molecules is still possible, but in the glassy state large-scale molecular motion is effectively inhibited (Angell, 1988). The Tg of an amorphous matrix is dependent on its composition, with low molecular weight compounds generally having a lower glass transition temperature than high molecular weight compounds (Levine & Slade, 1986; Roos & Karel, 1991a,b; Slade & Levine, 1995) (Fig. 2b). Of particular importance for amorphous carbohydrates is that the glass transition temperature strongly decreases with increasing water content or water activity (Mateev, Grinberg, & Tolstoguzov, 2000; Roos & Karel, 1991a,b), as illustrated in Fig. 2a. Although large-scale motion of the matrix carbohydrates are effectively blocked in the glassy state, small-scale reorganizations within the matrix are still possible, as is witnessed for instance by the mobility of small molecules such as water (Tromp, Parker, & Ring, 1997), gases (Schoonman, Ubbink, Bisperink, Le Meste, & Karel, 2002; Whitcombe, Parker, & Ring, 2005) and small organic molecules (Goubet, Le Quere, & Voilley, 1998; Ubbink & Reineccius, 2002) in the glassy state. In addition, using spectroscopic techniques such as neutron scattering (Cicerone & Soles, 2004), electron paramagnetic resonance (Van den Dries, Van Dusschoten, Hemminga, & Van der Linden, 2000) and positron annihilation lifetime spectroscopy (Kilburn et al., 2004), evidence is accumulating that temperature changes and changes in water content of an amorphous carbohydrate matrix have profound effect on the local structure and dynamics of a carbohydrate matrix even in the glassy state (Fig. 2c). The structure and dynamics at nanometer and sub-nanometer length scales are of prime importance in understanding the barrier properties of glassy
carbohydrates (Kilburn et al., 2005) and the protective effects of carbohydrates for sensitive biological materials such as proteins (Cicerone & Soles, 2004). Consequently, understanding of processes occurring at the molecular and nanoscales are relevant for optimizing barrier properties of food materials. Such knowledge is of importance for encapsulation systems prepared by a variety of processing techniques such as spray-drying, fluidized-bed drying and melt extrusion, as discussed for instance by Bencze´di and Blake (1999), Gibbs et al. (1999), Gouin (2004), Risch and Reineccius (1995) and Ubbink and Schoonman (2003). Concluding remarks Current demands posed on food developers to satisfy often conflicting requirements with respect to the incorporation of sensitive active ingredients in food products of increasing complexity have provoked a growing interest in the development and application of delivery systems for foodstuffs. In many cases, however, it is difficult to apply established encapsulation technologies without modification to an application of interest, principally because the physical and chemical behavior of the active ingredients and the functionality of the delivery system are poorly understood. In the present article, an alternative approach is proposed which focuses on the desired functionality of the active ingredient in the food application and works then back in a systematic fashion to the various technologies, such as encapsulation technology, which may be utilized to achieve the target product. The advantage of such an approach is that the final product application remains at the center of attention, and not a specific technology itself and it postpones the selection of a technology to a later stage in order to allow matching the different factors determining product quality and stability. In addition, because of the systematic evaluation of various aspects of the delivery issue, gaps in the scientific knowledge become immediately apparent and results from emerging research are more easily adopted. We anticipate that future efforts on the delivery of active ingredients in foodstuffs will increasingly incorporate interdisciplinary scientific developments at the interfaces between biomaterials science, physical chemistry, biophysics and encapsulation technology. References Angell, C. A. (1988). Perspectives on the glass transition. Journal of Physics and Chemistry of Solids, 49, 863–870. Beales, N. (2003). Adaptation of microorganisms to cold temperatures, weak acid preservatives, low pH, and osmotic stress: A review. Comprehensive Reviews in Food Science and Food Safety, 3, 1–20. Bencze´di, D., & Blake, A. (1999). Encapsulation and the controlled release of flavours. Leatherhead Food RA Food Industry Journal, 2. Cicerone, M. T., & Soles, C. L. (2004). Fast dynamics and stabilization of proteins: Binary glasses of trehalose and glycerol. Biophysical Journal, 86, 3836–3845. Cohen, M. H., & Turnbull, D. (1959). Molecular transport in liquids and glasses. Journal of Chemical Physics, 31, 1164–1165.
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