Although nanotechnology has been applied in a diverse number of disciplines to introduce different properties and characteristics to various materials, the strategies to apply nanotechnology to the food industry differ markedly from those in other areas, due to the fact that food processing involves a diverse variety of raw materials, demands high biosafety requirements, and follows a well‐regulated technological process. Food production and processing is currently employing nanotechnology in various areas, including the development of new functional materials, microscale and nanoscale food processing, food product/ingredient development processes, and also in the design of methods and instrumentation to produce improved food safety and biosecurity. There are, however, serious health concerns with regard to the incorporation of nanomaterials in foods. Certain countries and multinational/global organizations responsible for health have imposed strict regulations on the production and application of nano‐sized materials in foods. Transgressing these regulations and guidelines has serious legal implications to the manufacturers and distributors of food items, which violates the threshold or failure to meet labeling requirements.
There has been an increase in the tendency for the food industry to commercialize nanofoods. This has to date had unknown effects on consumers, even though it may mean much economically. Several countries have come up with regulations for nanofoods and from these regulations, transgressors who are after profit may face legal actions for their behavior, which may include improper or false labeling. By definition, nanofoods/feed refers to any food substance created by the employment of nano technological techniques in any part of the food chain – cultivation, production, processing, or packaging – not just in the food itself. In other words, food is regarded as “nanofood” only when nanoparticles, or nanotechnology techniques or other tools, are used during cultivation, production, processing, or packaging of that food. Food does not become nanofood in the sense that it is an atomically modified food or it has been produced using nano devices or nano machines.
The advent of nanotechnology has greatly influenced and revolutionized not only the industrial sector, but also agriculture and feed/food production sectors, where the share of nano‐based products on the market has been on the rise (Euractiv.com, 2010; Project on Emerging Nanotechnologies, 2010).
Nanotechnology has shown the potential for a positive impact on food production, processing, and in agricultural/animal husbandry practices that are directed at the production of food/feed. Currently, numerous scientific and technical publications have reported on the potential of the application of nanotechnology to boost food security, disease treatment delivery methods, and new materials for crops and food crop pathogen detection (Ozimek et al., 2010). For example, nano sensors have been devised for application in the detection of food pathogens, as well as other food contaminants and thereby increasing security of food manufacturing, food processing, and shipping of food products. Nanomaterials in foods have also played an important role in preserving the integrity of foods as well as materials in the encapsulation and functional food ingredient delivery systems that carry, protect, and deliver functional food ingredients to their specific site of action (Ozimek et al., 2010).
Foods in their natural state are composed of organic molecules such as protein, carbohydrate, fat, and lipids, with varying size and molecular weights, but they range from large complex polymers to simple molecules with sizes within the nano range. Nanotechnology has the potential to pioneer new functional food ingredients that may contain nanomaterials suitable for specific purposes, such as the encapsulation of nutrients that may play a vital function in increasing nutrient bioavailability, while some may play a vital role to enhance taste, texture, and consistency of foodstuffs, or mask an undesirable taste or odor.
Several nano‐based agro/feed/food products, such as nano‐encapsulated nutrients, antimicrobial nanoparticles, and active and intelligent food packaging, are already being produced and have already been commercialized. Other nano‐based food/feed/agro products are currently under research and development, and thus have the potential for commercialization in the future. However, producers/manufacturers of these nanofoods are obliged to prove the safe use of their products and to the environment before obtaining a license to distribute them to customers/consumers. For this reason, a number of national authorities in some countries have enforced regulatory mechanisms specifically for the application of nanotechnologies in foods/feed and agroproducts. Due to the regulatory enforcement, manufacturers and distributors of nano‐based foods are obliged to include in the labeling of their products all information about the nanomaterials that are included (Cairns, 2006; Euractiv.com, 2010; FAO/WHO, 2009; International Center for Technology Assessment (CTA), 2008; Sandoval, 2009).
There are two types of approach that are normally employed in the nanomaterial manufacturing processes:
A number of functional nanostructures are known to be used as building blocks in the process to create novel structures and introduce new functionalities in foods that are associated with the enhancement/improvement of certain categories of food properties. These functional nanostructures include food grade nanolaminate films, food grade nanoemulsions/microemulsions, nano‐liposomes/liposomes, nanoemulsions, nanoparticles, nanofibers, monolayers, etc.
Food grade novel laminate films and coatings are nanotechnologically created materials that consist of two or more layers of material with nanometer dimensions and are either physically or chemically bonded to each other. Their functional properties are governed by the characteristics of the 32 film‐forming or coat materials used during their preparation. However, physico‐chemical attributes such as composition, thickness, structure, and properties of the multi‐layered laminate or coats created around them can be controlled (Ozimek et al., 2010). For example, it is possible to change the type of adsorbing substances in the dipping solutions, vary the total number of the required/needed dipping steps, change the order that the object is introduced into the various dipping solutions, change the solution and environmental/experimental conditions such as the pH, ionic strength, dielectric constant, temperature, etc. and in all these possibilities, the properties of the resultant laminate or coat will be different from those in another set (Ozimek et al., 2010). Different types of mechanistic driving forces for adsorption of a substance to a surface can be involved, for example electrostatic, hydrogen bonding, hydrophobic interactive, thermodynamically incompatible, etc. But they are all highly dependent on the chemistry of the surface as well as that of the adsorbing substance.
Edible coatings and edible films find application in different types of foods, such as fruits, vegetables, meats, chocolate, candies, bakery products, French fries, etc., where they play an important role as moisture, lipid, and gas barriers (Cagri et al., 2004; Morillon et al., 2002). They are also useful in other functions such as to improve the textural properties of foods or as carriers of functional agents such as colors, flavors, antioxidants, nutrients, and antimicrobials (Ozimek et al., 2010).
Microemulsions refer to a spontaneously formed three‐component homogeneous system that is a clear and thermodynamically stable kind of dispersion, composed of different ratios of oil, surfactant, co‐surfactant, and water with dimensions not exceeding 100 nm (Feng et al., 2009; Radomska and Dobrucki, 2000).
The small droplet size imparts to nanoemulsions unique rheological and textural properties, which make them transparent and pleasant to the touch and thus improve the quality of food (Sonneville‐Aubrun et al., 2004). In addition, nanoemulsions in food products (e.g. low fat nanostructured mayonnaise, spreads, and ice creams) play an important role in facilitating the use of less fat without a compromise in creaminess (Chaudhry et al., 2008). Also nanoemulsions provide added stability to foods, because as the size of the droplets in an emulsion is reduced, the less likely the emulsion will be to break down and separate, implying that nanoemulsification may reduce the need for the inclusion of certain stabilizers in food products (Cushen et al., 2012).
Other attractive features of microemulsions that make them suitable for incorporation as functional nanostructures in foods, and even other fields of science and technology, include their high solubilization capability of organic and inorganic components in foods, their thermodynamic stability, spontaneous formation, can easily be scaled up, large interfacial area, nano‐size droplets, isotropy, and low viscosity (Solans and Kunieda, 1997; Stubenrauch, 2009).
Despite all these attractive features, the application of nano/microemulsions in foods is still limited due to the fact that large amounts/volumes of surfactants are required to form emulsions and it may not be economically viable and also compromises environmental safety (Zhong et al., 2009). Moreover, there are not many edible surfactants that can have direct application in foodstuffs (Flanagan et al., 2006).
In the food industry, microemulsion products find application mainly for food solubilization, in that they increase the water solubility of nutrients and vitamins, also for improving reaction efficiencies such as inter‐esterification, hydrogenation, and for fortification of foods.
With the current trend in the application of nanotechnology in improving rheological properties of foods, it may be possible to extend the application to improve food color, an area that currently has not seen much nanotechnology. For example, it may be possible to use oil‐soluble pigment compound b‐carotene to impart pigment color into aqueous‐based foods by simply using nanoemulsion technology (Astete et al., 2009). There is also a high possibility of forming nanosized structures using alginic acid and calcium ions and this nanomaterial has the potential to open doors for application of fat‐soluble colorants where it is possible to change the concentration of the b‐carotene present in the nanomatrix and thus change food color (Cushen et al., 2012).
Liposomes are another class of useful nanostructure capable of adding functionality in food. These functional nanostructures have biological and chemical properties which can be explained as spherical bilayer membrane structures with aqueous cores. Nano‐liposomes and liposomes can be useful in applications that require the aspects to contain and deliver hydrophilic, or water‐soluble, food ingredients. One attractive feature that is characteristic to nano‐liposomes/liposomes is that their internal pH is adjustable, and therefore they can contain and maintain stability of food ingredients. The disadvantages associated with lipopsomes include the fact that they are very fragile and leakage problems may occur. Otherwise, nano‐liposome technology has found application in food technology, which involves areas such as encapsulation and controlled release of food materials, in areas where there is a need for enhancement of food/nutrient bioavailability, food stability, and improved shelf‐life of sensitive food ingredients. These nanostructures have been used in the food industry to deliver flavors and nutrients and in the immobilization of antimicrobial ingredients in order to prevent microbial spoilage of food products.
Nutraceuticals (e.g. bioactive proteins, etc.) play important functions in food, including the fact that they are nutrients themselves and also contain functional groups that improve the health of consumers, because they are bioactives in functional food systems (Chau et al., 2007). The small size of the nanoparticle bioactives is vital, due to the fact that this attribute tends to improve other properties of functional foods, such as improving the availability of the foods, improving the delivery properties of functional foods, as well as the solubility of the bioactives and hence their biological activity, since the bioactivity is a function of the bioactive’s ability to be transferred across intestinal membranes into the blood (Chen et al., 2006; Shegokar and Muller, 2010). The application of nanotechnologies is also vital in improving the stability of micronutrients such as nutraceuticals, omega‐3 fatty acids, and certain beneficial probiotic bacteria species (lycopene, vitamin D2, and beta‐carotene) during processing, storage, and distribution (Chen et al., 2006; Neethirajan and Jayas, 2011).
Nanoparticles, such as nano silver and nano zinc oxide have been applied as food additives or food supplements, where they enhance gastrointestinal uptake of metals. They are also useful in water purification works, where they play a role in the removal of contaminants or catalyzing the oxidation of certain contaminants (Ozimek, 2010). Moreover, filters with nano‐pores have been highly useful in water purification works, where they play an important function in terms of pathogen removal from water.
Encapsulation of functional food ingredients refers to the process that involves the isolation of the active food ingredient within the food product using food‐grade materials. The processes to create capsules and then encapsulation of target ingredients takes into account the structuring of the active ingredient, mainly at either molecular or nanoscale levels, using food‐grade ingredients capable of interacting with the active ingredient. Currently, there are numerous new functional food ingredients that are being integrated/encapsulated into various food matrix systems and the aim of encapsulation of these functional food ingredients is to improve the functionality of these ingredients in various food matrices. Moreover, encapsulation enhances the bioavailability as well as the ability to disperse these functional food ingredients far more than compared to their bioavailability and dispersal in their natural systems (Haruyama, 2003).
Generally, the vital components of functional foods, such as nutraceuticals, vitamins, probiotics, antimicrobials, essential oils, antioxidants, drugs, and preservatives are rarely used in their pure natural form, but they normally form part of the delivery system. The ideal delivery system for functional food ingredients has to be compatible with the food product’s attributes such as taste, texture, appearance, and shelf life of the final food product and the characteristics of the delivery system govern the efficacy of the functional food’s ingredients in the food. A delivery system for functional foods, as is the case for the delivery systems of other substances such as drugs, etc., plays several important roles, including:
The encapsulation of functional food ingredients in food is normally made possible by nano‐sized self‐assembled structured liquids (NSSL) technology, which allows the addition of insoluble compounds into food, such as a healthier version of canola oil (Ozimek, 2010). There is a diverse variety of delivery systems that are currently in use to encapsulate functional food ingredients, depending on the type of functional food to be delivered, molecular form, as well as physical form of the functional food, and they include simple solutions (nanodispersions and nanocapsules), association colloids, nanoemulsions, nanostructured multiple emulsions, biopolymeric nanoparticle matrices, etc. Each of these possess unique and specific sets of attractive features, as well as shortcomings in terms of their suitability for encapsulation, protection, and delivery of functional ingredients, regulatory status, ease of use, biodegradability, and biocompatibility (Ozimek, 2010).
By definition, colloids refers to a stable system of any substance that contains small particles that are uniformly dispersed throughout a particular matrix. In cases where there are much smaller particles, which may range in size from 5 to 100 nm and which form a transparent solution (e.g. surfactant micelles, vesicles, bilayers, reverse micelles, and liquid crystals), this system is known as an association colloid (Bilska et al., 2009; Garti and Benichou, 2004; Garti et al., 2005; Golding and Sein, 2004). Association colloids have several advantages, including the fact that they form spontaneously, they are thermodynamically favorable, and are generally transparent solutions. However, they are associated with several drawbacks, including the fact that they tend to compromise food properties such as the flavor of the ingredients, because their formation requires the use of large amounts of surfactants, which may also lead to compromising the regulations and guidelines threshold limits. Another limitation is that they have a tendency to undergo spontaneous dissociation if diluted (i.e. their formation is concentration driven).
Nanoemulsions can be defined as emulsions with droplet diameters of less than 100 to 500 nm and functional food ingredients are incorporated/encapsulated either within these nanoemulsion droplets (to facilitate the slowing down of chemical degradation processes by engineering the properties of the interfacial layer surrounding the ingredients of the functional foods), in the interfacial region, or in the continuous phase (Jasińska, 2010; McClements, 2004; McClements and Decker, 2000).
Examples of multiple emulsions include oil‐in‐water‐in‐oil (O/W/O) and water‐in‐oil‐in‐water (W/O/W) emulsions, such that the ingredients of the functional foods can be encapsulated either within the inner water phase, the oil phase, or the outer water phase, and thereby create one homogeneous, continuous, and single multiple‐functional system (Flanagan and Singh, 2006; Garti and Benichou, 2001, 2004).
By definition, active packaging materials refer to those that are capable of releasing either nanoscale antimicrobial compounds, antioxidants, and/or flavors, which can also enhance the shelf life and sensory characteristics of foods (Cushen et al., 2012). An example of food contact materials‐bound active nanomaterials include the nano‐Ag embedded baby bottles (Alfadul and Elneshwy, 2010). The success in the area of active bound food contact materials is opening the possibility for the introduction of intelligent food contact materials in which nanosensors, which have the ability to identify specific microbial and/or chemical contaminants or environmental conditions, can be incorporated into food packaging matrices (Neethirajan and Jayas, 2011). These intelligent food contact materials are also capable of changing an environment in response to a stimulus such as pH, pressure, presence of gases, liquids, or products of microbial metabolism or spoilage accelerators, such as temperature or light intensity and thereby alert consumers or distributors to the contamination (Otles and Yalcin, 2008). There is also another possibility of combining food packaging materials with active substances, in order to obtain a composite that can be useful in controlling surface microbial contamination of foods and thereby extend the food’s shelf life as well as improving its quality and safety (Vermeiren et al., 2002).
Food‐grade biopolymers such as proteins or polysaccharides can be used to produce nanometer‐sized particles (Chang and Chen 2005; Gupta and Gupta, 2005; Ritzoulis et al., 2005). Several mechanisms, including aggregative (net attraction) or segregative (net repulsion) interactions, can be employed in the separation process of smaller bio‐nanoparticles from a single biopolymer. The resultant bio‐nanoparticles are then used in the encapsulation procedures for functional food ingredients and are then released in response to distinct environmental mechanisms.
Biopolymers in this context will refer to organic substances with molecular backbones that are composed of repeating units of either sugar moieties (saccharides), nucleic acids, or amino acids and in some cases various additional chemical side chains are attached to these functional groups and thus contribute to the shaping of the polymer’s molecule. Biopolymers are thus a type of polymer produced or generated through natural means by living species. Biopolymers can also be defined as types of polymers composed of monomeric units that are covalently bonded, forming chain‐like molecules, and the prefix “bio‐” implies that these types of polymers are biodegradable and the most likely degradation products (organic by‐products) are mainly CO2 and H2O, which are safe when entering the environment (Liu et al., 2005; Muratore et al., 2005). Biopolymer nanoparticles which find application in foods are known to be highly bioactive solid particles with diameters of 100 nm or less and they may be used in foods to play various functions, for example as carriers of antimicrobial components. For example, nicin‐containing biopolymeric nanoparticles display a more enhanced potent activity against E. coli O157:H7 than those particles that do not contain nicin.
Previously, before the advent of technology (nanotechnology), the food industry relied on natural biopolymers to cater for the needs of food packaging fabrics. The biopolymers that were mostly used included carbohydrate‐derived biopolymers such as cellulose, chitosan, and agar; and protein‐derived biopolymers such as gelatin, gluten, alginate, whey protein, and collagen. In the recent past, the food industry has witnessed the booming of synthetic biopolymers that possess more improved properties as compared to the natural ones. For example, synthetic biopolymers (i.e. polylactic acid (PLA); polycaprolactone (PCL); polyglycolic acid (PGA); polyvinyl alcohol (PVA); and polybutylene succinate (PBS)) have relatively better properties in terms of durability, flexibility, high gloss, clarity, and tensile strength (Rhim et al., 2013).
There are generally three main groups of biopolymers that are normally used in the food industry, in a classification that is based on their origin, which include:
Despite all the attractive features of using biopolymers in foods, they still present some shortcomings. especially with their use as food packaging materials if they are to be compared with the conventional non‐biodegradable materials such as those derived from petroleum. The limitations include poor mechanics in the sense that they have low tensile strength and also are associated with barrier properties in the sense of high water permeability properties. Moreover, biopolymers are generally known to be brittle, have low heat distortion temperature, low resistance to extreme heat and humidity, low flexibility, and low resistance to prolonged process operations. To address these challenges, bio‐nanocomposites have been introduced as new materials for food packaging, as they offer enhanced mechanical and barrier properties of biopolymers.
The bio‐nanocomposite materials that are mostly used for food packaging applications are mainly starch and derivatives, which are considered to be safe because they are edible and this polymer is also known to be completely degradable and has the ability to stimulate biodegradability of other non‐biodegradable materials that may be present in a starch blend or composite (Heydari et al., 2013; Nafchi et al., 2013; Pan et al., 2014; Sorrentino et al., 2007; Tang et al., 2008).
Nanofillers are also known as nanophase materials, and like other nanomaterials have reduced size that makes them possess large surface area to volume ratios, and are therefore attractive for use as fillers in bio‐nanocomposites that are intended for use as food packaging materials. The food packaging biopolymers depend to a large extent on the large surface area of the nanosized phases/fillers to provide them with a large interfacial or boundary area between the matrix or biopolymer and nanofiller. Generally, the bio‐nanocomposites intended for use as food packaging are usually designed to have the ability to endure the mechanical and thermal stress during food processing, transportation, and storage. Thus, incorporation of nanophase/fillers in biopolymers is to provide the biopolymers with a large interface in order to enable the modification of molecular mobility, and the relaxation behavior besides mechanical, thermal, and barrier properties of the bio‐nanocomposites to acquire such properties (Azeredo et al., 2011: Rhim et al., 2013a,b).
There are two general groups that classify nanofillers, based on the nature of their chemistry used as food packaging materials, and they include:
The organic‐based nanofillers are further subdivided into three main groups:
The inorganic nanofillers are subdivided into two main groups:
These metal nanoparticles are vital components of food packaging materials or food storage materials/devices, where they improve barrier properties of packaging or storage materials, also serving as antimicrobial agents, or when they are created/engineered as active nanoparticles they function in foods by checking the migration out of packaging materials where they function as oxygen scavengers and prevent the growth of microbial pathogens.
Nanoparticles that have been engineered and created as complex nanostructures are useful as nanosensors in food packaging, where they serve as detection systems in cases of food deterioration.
Nanoparticles or nanofillers that are engineered as nano‐sized nutrients, nano‐sized foods, or nano encapsulates, find application in food as part of the delivery systems for functional foods and therefore are highly useful as food additives or food supplements, where they play the important function of enhancing the uptake of functional foods and are also useful in the protection of targeted delivery of functional foods. The classification of nanofillers that find application in the food industry in food delivery systems based on their structural nature, groups nanofillers into a number of classes:
Solid lipid nanoparticles (SLNs) are crystallized emulsions made up of a high‐melting point lipid and a bioactive lipophilic component. In foods, they are used as part of the composition of the delivery systems for functional foods as encapsulation components. Functional foods are normally composed of bioactive ingredients such as carotenoids, amino acids, omega‐3 fatty acids, vitamins, phytosterols, probiotics, etc., which play an important role in improvement of the health status of consumers. However, the incorporation of the ingredients of functional foods into the food matrices requires the facilitation of encapsulated matrices to serve as delivery systems that need to be designed and engineered specifically for each class of functional food ingredients. The encapsulation systems are attractive, as they are created with targeted properties such as high physical stability, ability to protect ingredients of functional foods against chemical processes such as chemical degradation, and moreover possess capability to ensure precise control over the release of encapsulated components during mastication and digestion to maximize adsorption. One form of encapsulation system, known to have the potential for use in functional foods, is solid lipid nanoparticles (SLN), which is composed of crystallized nanoemulsions with the dispersed phase composed of a solid carrier lipid–bioactive ingredient mixture.
Although nanofibers and nanotubes (diameters range in size from 10 to 1000 nm) are not composed of food‐grade substances, and thus have limited potential applications in foods, they still hold immense future potential application in the food industry. For example, it has been reported that under appropriate environmental conditions, certain globular milk proteins can self‐assemble into structures that resemble that of nanotubes (Graveland‐Bikker and de Kruif, 2006.; Graveland‐Bikker et al., 2006). Moreover, nanofibers such as cellulosic nanofibers have found application in foods, especially in the immobilization of bioactive substances such as enzymes, vitamins, and antimicrobials; as delivery systems for nutraceutical and controlled release of other functional food ingredients (i.e. they are used as delivery systems of nutraceuticals and nutrients to protect them during processing and storage or in delivery systems for transferring the components to the target site in the body); and as biosensors.
Food‐grade polymeric nanomaterials and nanocomposites can be created at the nanoscale, but in most cases it is preferable to incorporate nanoscale materials in the structures of polymer matrices (Yang et al., 2007). Such nanoscaled materials can impart to food contact materials (FCMs) useful attributes such as enhanced flexibility, gas excellent barrier properties, and also temperature control and moisture stability due to the reinforcement contribution provided by nanoscaled nanomaterials and nanocomposites, which play an important role as nanoscale fillers (Alexandre and Dubois, 2000; Giannelis, 1996; Sinha Ray et al., 2002). Examples of such food‐grade nanoscaled polymeric fillers include the clay montmorillonite, which is actually stacked silicate sheets known to have a high ratio of length to thickness (aspect ratio) and a plate‐like morphology (Rhim and Ng, 2007); laponite (a synthetically prepared clay composed of sodium magnesium lithium silicate) is another example and has been reported to provide a higher aspect ratio as compared to montmorillonite (Chung et al., 2010).
In order to establish risk assessment of nanofoods, it is required that the characterization of nanomaterials incorporated into foods be done appropriately and exhaustively, in order to establish accurate and precise data of the nanomaterial used. This is because the characterization of nanocomposites and nanomaterials used in foods is more complex, thus requiring a more detailed scope in terms of the analytical parameters to be investigated, the extent and range of properties required to generate detailed information, and in‐depth data regarding the nanomaterials used in foods. Moreover, method development has to be done to establish standard analytical procedures and protocols that are capable of detecting the presence of nanomaterials in foods, food contact materials, food packaging, etc., at below the stipulated guidelines and threshold limits (Tiede et al., 2008). Moreover, certain crucial information is required, such as the possible accumulation of particulate nanomaterials in foods, and if the food that contains nanoscaled material is consumed, what could be the effects on the body of the victim/s after ingestion (exposure and absorption) (Handy and Shaw, 2007)?
In addition to knowledge about the possible accumulation of nanoscaled materials in foods, and their fate in the body, it is also imperative to establish the target organs/tissues where these nanoscaled materials accumulate, and their elimination profiles, etc. Currently, the risk assessment on food‐grade nanomaterials is yet to be refined, as it has to overcome all these obstacles, including the challenges in the reliable methods for characterization of the nanoparticles/nanomaterials, sensitive and selective methods for their detection, and also lack of reliable information and data related to the toxicology properties of food‐grade nanomaterials (European Food Safety Authority (EFSA), 2009).
In as much as there are many potential benefits of nanofoods, nutritional‐wise and economic‐wise, there are also potential risks to human health that are associated with nanofoods (Pusztai and Bardocz, 2006; Siegrist, 2008). For example, some scientific reports have indicated that inhaled nanoparticles have the potential to accumulate in the lungs and cause chronic diseases (Chau et al., 2007; Poland et al., 2008). Generally there is still huge debate with regard to direct risks (due to direct ingestion of nano‐containing foods/water) and the debate is mostly on their bioavailability. The bioavailability phenomenon in question is the one in which nanoparticles cross cellular barriers in the body, which are normally impossible to be crossed by normal foods in their natural state, then spread and accumulate in other parts of the body to cause unknown long‐term health effects (Chun, 2009). If indeed bioavailability is enhanced with nanomaterials present in foods, then there are even more risks to human health, because this may lead to changes of the nutrient profile, greater absorption of nano‐additives, and the potential introduction of foreign substances into the blood (Buzby, 2010; Chaudhry et al., 2008). Another potential risk may originate from nano‐sized materials, such as nanoparticles intended to function as antimicrobial agents in food packaging that may contaminate by leaching, which may cause such nanoparticles to migrate into the food (Chaudhry et al., 2008). There is also a potential for bioconcentration/bioaccumulate in the biological environment and thus the potential to uncontrollably contaminate the whole of the food chain (Buzby, 2010; Chaudhry et al., 2008).
In terms of regulations on the safety of nanofoods, there are no standardized procedures or universal regulations in place that govern the production, application, or utilization of nanofoods. For example, the Food and Drug Administration (FDA) in the USA regulates nanofood products but not the technology used to produce them, while the Institute of Food Science and Technology (IFST) in the UK recommends that nanomaterials in foods must be treated as new, potentially harmful materials until rigorous testing proves their safety.
Generally, the regulation regimes for use of nanoscaled materials in foods fall within the scope of both the broad horizontal legislation as well as the specific vertical legislation. Different countries follow different sets of horizontal and vertical regulations. For example, in Europe, there is a horizontal regulation governed by the Directive 2001/95/EC known as the General Product Safety Directive (GPSD), which requires observance of the general safety of products that are being marketed and/or supplied to consumers (human safety rather than environmental safety). In addition to this, the EU came up with another piece legislation, known as the REACH legislation, in which a new labeling style for all marketed products has to include safety information and this legislation takes into account the ecotoxicity aspect of the product being sold. The EU has also imposed another regulation (Regulation (EC) No. 1272/2008) that supplements the REACH legislation and requires that consumers of potentially hazardous substances be notified of the possible risks by means of a new labeling system that includes the use of safety symbols as well as the inclusion of safety data sheets to consumers.
It should be noted that nanoscaled materials may not be directly mentioned in this legislation, but producers of edible products who incorporate nanomaterials into their products are to adhere to the legislation as well as the revision of the legislation, in order to include the use of nanomaterials in foods that have already been suggested (UBA, 2009).
As for vertical legislation, the EU has suggested to limit guideline values, labeling requirements, and risk assessment for products being marketed that include foods, and that where there is a change in the starting material used in the food production/processing or in the production method of an additive (e.g. a change of the particle size), this particular food product has to go through a new authorization process and safety evaluation. Moreover, the European Commission Directive 96/77/EC sets limiting standards to the quantity of certain impurities permitted within food additives, which must be adhered to. This directive controls the use of food additives, ensuring that manufacturers only use approved, quality grades of additives that have passed safety testing. With regard to novel food (i.e. foods and food ingredients that have not been used for human consumption to a significant degree), Regulation (EC No. 258/97) requires them to undergo a safety assessment prior to being placed on the market. Another regulation on novel foods/nanofoods, Novel Food Regulation (EC No. 258/97), is even more specific in terms of the inclusion of nanoscaled materials in foods (EC No. 258/97, 1997).
The EU has another regulation that governs Active and Intelligent Materials, which requires that if legislation limits the quantity of a substance in a food, the total quantity should not exceed that limit, regardless of the source, i.e. originally included in the foodstuff or following release of that substance from the Food Contact Material (FCM). If it happens that a substance is released into the food in this way, it is required to be included in the ingredients list. For active food substances that are not designed to be released from the packaging and have no function in the food, there is a risk that these substances may migrate into the food.
Generally, in the rest of the world, legislation on the use of nanomaterials in foods tends to be cautious towards potential risks posed by the new applications (Chau et al., 2007).
As explained above, it is expedient to have a thorough knowledge regarding the potential of the risks of nanomaterials that are incorporated into foods and therefore it is imperative to have in place appropriate analytical methods capable of quantifying these nanomaterials within a food matrix, as well as analytical procedures that can provide data on the characteristics of the very same nanomaterial. The ideal methods ought to be unambiguous and must involve only limited manipulation of the samples, in order to avoid any possible introduction of artefacts.
Generally, due to the physico‐chemical properties, nanomaterials in foods can be analyzed and characterized using different methods and techniques, which may include those based on microscopy, spectrometry (e.g. single particle inductively coupled plasma‐optical emission spectroscopy/mass spectrometry); size separation with light scattering detection (e.g. field flow fractionation with dynamic light scattering (DLS)); multi‐angle light scattering (MALS) detection; chromatography (e.g. hydrodynamic chromatography); surface characterization (e.g. small‐angle X‐ray scattering); as well as different variants and combinations of the above‐mentioned techniques. One important consideration during the analysis and characterization of nanomaterials in foods involves method validation and streamlining for detection, because of the possibility that different methods or techniques may yield different measurement data or information for a given sample.
An in‐depth discussion about the application of electron microscopy‐based methods in food forensics cases will be discussed in chapter 13. However, the application of electron microscope‐based methods, especially those that are known for their great resolving power due to their ability to make use of an electron beam with wavelengths well below the nm range for the analysis and characterization of nanomaterials in foods, enables visualization and characterization of nanosized objects (Kachlicki, 2007). Of the electron microscopy‐based techniques, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) seem to present the more likely potential application for the detection and characterization of nanomaterials in foods. The mode of operation of scanning electron microscopy involves the generation of the sample by scanning the sample surface with a low‐energy beam of electrons, normally in the range that lies between 1 and 30 keV, and then detecting the electrons that are scattered from the sample (Denk and Horstmann, 2004). On the other hand, the mode of operation of transmission electron microscopy involves the transmission of a high‐energy electron beam, normally in the range between 80 and 300 keV, across/through a very thin layer of the sample, and then detecting:
Sampling is always the first step in any analytical procedure and is crucial in the sense that it determines accuracy, precision, and the detection limit of the method. The most important aspects in the sampling regime for the analysis and/or characterization of nanomaterials in foods include obtaining a representative and homogenous sample with a uniform distribution in terms of its composition. Particular sampling protocols normally are dependent on the type of nanomaterials under investigation and also the matrix (Liu et al., 2009).
Sample preparation is essential in electron microscopy analysis of nanomaterials in foods, because electron microscopes operate under vacuum conditions and therefore wet samples (samples containing any liquid/water) cannot be introduced into the scanning electron microscope without proper preparation to dry the sample and also to ensure that the sample is able to scatter the electron‐beam. Several techniques are normally employed to dehydrate the sample and include chemical fixation, followed by either dehydration or drying and then subjecting the dehydrated sample to either conventional SEM or TEM. Another drying technique involves freezing followed by analysis using either cryo‐transmission electron microscopy or cryo‐scanning electron microscopy.
Dehydration of food sampled in the analysis of nanomaterials can also be achieved by using ethanol (Rahman et al., 2008). Despite the advantages that ethanol offers in terms of its dehydration power, it is not appropriate for use in food samples that contain high levels of saturated fats or even water‐soluble carbohydrates. Other possible problems that may come from the extreme use of chemical dehydration include the potential introduction of artefacts or even possible loss of nanomaterials that may be associated with one of the phases that is being discarded. Moreover, there may be a potential triggering of reactions between these chemicals and the nanomaterials. It is therefore highly recommended that methods used for dehydration in the analysis of nanomaterials in foods be carefully examined and proved before using them. Alternatively, it may be advisable to employ sample‐drying techniques to avoid all these potential problems (Lorenz et al., 2010; Novak et al., 2001). However, even drying methods in the analysis of nanomaterials in food samples have limitations in that they are mainly suitable if the food matrix is liquid and its density is low enough to allow the formation of a very thin film (Lorenz et al., 2010).
Other sample preparation techniques include the fixation of food samples with glutaldeyde, post‐fixation using osmium oxide and then subjecting the samples to a chemical dehydration process followed by critical‐point drying (if samples are to be subjected for scanning electron microscopy analysis/characterization or resin embedding in the case where samples are supposed to be subjected for transmission electron microscopy). These methods are suitable for food samples such as fruits, meat, or vegetables, where there may be a need to investigate their intracellular structure. These sample preparation procedures are vital for retaining the integrity of the food samples by minimizing the possibility of changes that may occur in the sample matrices and thus enabling the uncompromised observation for the real distribution and possible interaction of the nanomaterials within the cellular/histological structure in the native state of the sample (Egelandsdal et al., 1999; Kaláb et al., 1996). For food samples such as those that are either fat containing or protein containing types of foods, the sample preparation for such foods in the analysis of nanomaterials they contain may involve post‐fixing with osmium tetroxide, or using certain heavy‐metal stains such as uranyl acetate, lead citrate, etc. In addition to these, mordant reagents such as tannic acid may also be used for staining and preservation enhancement of the food’s ultrastructure (Musyanovych et al., 2008). Other staining techniques include negative staining (Maunsbach and Afzelius, 1999). Use of organic coatings on nanomaterials has also been reported for creatine and albumin coatings on gold NPs (AuNPs) (López‐Viota et al., 2009).
The main limitation of staining procedures is that the technique is unable to make distinction for nanomaterials from densely‐stained organic structures of the same size, for example between polystyrene NPs and vascular structures in cells (Mühlfeld et al., 2007). To avoid this problem, it is suggested that the analysis involves a sample that is known to contain particles with a blank sample, and the use of appropriate control samples in order to enable the distinction of samples.
Generally, drying and dehydration of food samples may result in a significant change in the structure of the food matrix. One of the methods that may be ideal, in the sense that it may not result in significant changes in terms of the structure of the food matrix, is physical fixation by freezing the sample. This is because during the sample freezing process, the ice crystals grow and only water molecules are incorporated into the ice crystals, and the specimen is thereby segregated into crystals of water and the ridges between containing enriched regions with the dissolved material (e.g. solutes and macromolecules). Formation of large ice crystals can even rupture and destroy whole cells in biological samples. As long as care is taken to prevent the possible formation of large ice crystals that may rupture the cell, the method may be the most attractive to dehydrate the sample (Bruggeller and Mayer, 1980; Cavalier et al., 2009; Dubochet, 2009; Maunsbach and Afzelius, 1999).
Food samples that are meant for SEM analyses have to be mounted either on aluminum or carbon stubs by sticking with quick‐setting glue, epoxy cement, wax, silver paint, or double‐sided sticky tape. Where necessary, the tape may also contain carbon to avoid electrical charging of the sample. The resolution magnitude of scanning electron microscopy is normally good, but depends mainly on the type of sample that is being analyzed. Certain samples, for example organic samples (including the majority of foodstuffs), require a step whereby they are covered with a layer of electrical conductive substances such as metal, carbon, or gold in order to avoid charging effects during the process of imaging. When operating scanning electron microscopes, normally the SEM machines are equipped with several detectors that are capable of selecting specific energy ranges of the scattered signal, such that low‐energy secondary electrons (SEs) can play a crucial role in providing information regarding the surface topography, while high energy backscattered electrons (BSEs) can be crucial for mapping contrast according to the differences in terms of the atomic number, Z, of elements to which the sample is composed, which makes it possible to establish a good contrast between heavy and light elements (e.g. AgNPs in cells) (Koh et al., 2008; Jóźwiak, 2007).
Among the most important observations in the analysis of nanomaterials in foods using SEM is the characteristic depth of field, as obtained from the SEM micrographs, because this data allows the analyst to deduce the effects of nanomaterials on the food structure and the same data can be used to locate nanomaterials within the food samples (Castaneda et al., 2008).
When using TEM in the analysis of foods, the resolution depends largely on the thickness of the prepared sample and also on the accelerating voltage for the electron beam, such that the higher the voltage, the better the theoretical resolution. For most biological samples as well as food samples that are composed of structures that may be prone to electron damage, the optimal accelerating voltage of TEM is normally tuned to a magnitude of up to 100 kV (Kachlicki, 2007).
In summary, since the whole issue of nanofoods is still new and is growing rapidly, the scientific and technical question required is to shed light on whether there should be a green light to proceed with the application of nanotechnology in foods or not, or to implement universal regulatory mechanisms globally that will govern the definition of safety/toxicology and environmental impact of nanotechnology application in food production, processing, packaging and utilization, economics, etc. Eventually, the factor of consumer acceptance of nanotechnology application in foods will certainly make a strong contribution to the whole success in terms of the nanotechnology/nanoscience application in foods.
Field‐flow fractionation is a useful analytical separation technique for characterization of macromolecules such as proteins and protein complexes, and saccharides, as well as viruses, derivatized nano‐ and micron‐sized beads, sub‐cellular units, and whole cell separation (Karl‐Gustav, 2013; Roda et al., 2009). This technique is mainly based on the interaction of the analytes with a perpendicularly applied field and is normally performed using open‐channel structures by a flow stream of a mobile phase of any composition such that fractionation, unlike chromatographic separation, takes place without any surface interaction of the analyte with packing or gel media, and without using degrading mobile phases. In simple terms, the FFF mechanism does not involve any interaction of the analyte being separated nor does it need a stationary phase, but rather the mechanism is dependent on the externally generated field, which is applied perpendicularly to the direction of the mobile phase flow. This makes the technique very attractive in food applications and other bio/macromolecules such as proteins, because they can be fractionated directly while preserving the integrity.
The theory and mechanism of FFF is based on several classical laws in physical chemistry, including Brownian motion, translational diffusion, laminar flow, frictional force, frictional coefficient, and drag‐induced transport by flow, i.e. flow displacement, viscous forces (Caldwell, 2000; Karl‐Gustav, 2013; Roda et al., 2009; Schure et al., 2000). In other words, FFF separation of samples takes place within either a capillary or an empty channel, in which a laminar flow of mobile phase pushes sample components down the channel. As the field is applied perpendicularly to the parabolic flow to direct analytes into different laminar flows, based on the differences in their size, density, and surface properties, this results in different retention times. According to the principle and mechanisms of FFF, it is thus expected that the retention times will be shorter for lower molar mass/size analytes and longer for higher molar mass/size. However, in cases where the analyte diffusion becomes diminished or negligible, as sometimes happens with micron particles, then the elution order under these circumstances becomes reversed, implying that the larger particles are eluted first and the smaller particles are eluted last. The elution mode in FFF is normally known as steric or hyperlayer elution. The retention in FFF depends mainly on size, shape, density, rigidity, and surface features of the analytes.
There are several variants of FFF based on the field that is being applied, which include flow field‐flow fractionation (Flow FFF or F4), also known as symmetrical F4 (SF4) (Ratanathanawongs Williams, 2000) or asymmetrical F4 (AF4) (Wahlund, 2000), in which a second flow stream is introduced as the hydrodynamic field to develop separation. Some variants of FFF involve cross‐flow by employing cylindrical, porous channels, where either polymeric or ceramic hollow‐fiber (HF) are used as fractionation channels to make a variant that is normally abbreviated as HF5 (Jönsson and Carlshaf, 1989), There is also centrifuge‐based sedimentation FFF (SdFFF), in which the channel is positioned inside a centrifuge bowl (Moon, 2000). In other variants, gravity has been applied as the sedimentation field (gravitational FFF; GrFFF) (Giddings et al., 1979). Field flow fractionation variants utilizing fields such as thermal (thermal FFF; ThFFF), electrical (electrical FFF; ElFFF), and some utilizing split‐flow thin cells (SPLITT) to provide continuous, preparative‐scale fractionation of macromolecules and particles, have already been developed and applied in bioscience research (Giddings, 1985, 1992; Lu et al., 2004).
The commonly used mobile phase systems in FFF are aqueous in nature, although other solvents may be suitable and the composition of these mobile phases varies, depending on the nature of the analyte. However, in most cases, pure water is not recommended as a mobile phase for FFF, due to the fact that any electrostatic interaction that may occur during the fractionation procedures will be long range and may result in problems in the elution profile of sample components, as well as lack of reproducibility. For example, dilute buffer systems have been recommended as suitable mobile phases for neutral nanofoods, while for ionic or charged nanofoods and their derivatives, higher ionic strengths in the mobile phase may be desirable, even though such a mobile phase system may influence the size and conformation of a sample. Buffer solutions may be the best option for proteinaceous, polysaccharide, or other macromolecule‐based nanofoods.
FFF instruments have been coupled on‐line to various detectors, such as the multi‐angle light scattering (MALS) detector, dynamic light scattering (DLS) detector, refractive index (RI) detector, and spectrophotometric detector, and has been hyphenated to mass spectrometric‐based techniques such as the ICP‐MS. The MALS detectors detect the scattered intensity at different angles in relation to the incoming light. The light scattering data obtained from the detector is normally fitted using different models such as the Debye model (Debye, 1944), Zimm model (Zimm, 1948 a,b) and Berry model (Andersson et al., 2003; Berry, 1966).
When using FFF techniques, the aim is to obtain certain molecular properties that will be used to elucidate the important information about the sample. These molecular properties are mainly dependent on the type of detectors that have been used. Some of these properties include diffusion coefficient, hydrodynamic radii (size), and molar mass of the analyte. Generally, FFF techniques have the potential to offer the possibility to obtain vital information about molecular and conformational properties, as well as functional properties of nanoscaled foods or nanomaterials in foods and their effects in foods over a wide size distribution.
Another technique that has the potential for application in the analysis and characterization of nanofoods/nanomaterials in foods is hydrodynamic chromatography (HDC), due to its ability to provide reliable size separation that is independent of the matrix. The mode of separation in HDC is based on different samplings of the flow velocity profile caused by differences in the effective diameter (Striegel and Brewer, 2012).
Size separation can be achieved using specific hydrodynamic columns such as PLPSDA. The detectors used with HDC are traditionally differential refractometers (Penlidis et al., 1983) or UV detectors (Striegel and Brewer, 2012; Williams et al., 2002). Other detection methods include particle‐counting detection using laser scattering (Zarrin and Dovichi, 1985), MALS, DLS and viscosimetry (Brewer and Striegel, 2009); and fluorescence and inductively coupled plasma mass spectrometry (ICP‐MS) detectors (Philippe and Gabriele, 2014).
Small‐angle X‐ray scattering is a technique for evaluating the X‐ray scattering pattern of either nanoparticles or macromolecules at small angles, for the purpose of gaining information about their particle structure. With SAXS, the measurements are preferred to be done at small angles, because the larger the structures, the smaller the scattering angle. The parameters that can be looked at from SAXS measurements include size, shape, internal structures, crystallinity, orientation, porosity, etc. Therefore, SAXS may find application in the analysis and characterization of nanocrystals, emulsions, food macromolecules (proteins, enzymes, etc.), nanocomposites, and nanostructures surfaces, etc., where it can provide vital information about particle size, particle size distribution, and particle shape; size stability studies, structural studies of biomacromolecules, both in solution as well as in solid state, in their native state; can provide 3‐D shape models, etc. (Burger et al., 2008; Cedola et al., 2006; Mollenhauer et al., 2003; Sasaki and Odajima, 1996a,b). In SAXS, the scattering of X‐rays arises from differences in the electronic structure of the atoms (Lindner and Zemb, 1991).
Neutron scattering techniques encompass a range of techniques in which neutrons are used as probes in the analysis of structural and dynamic properties of materials, by measuring their change in direction and energy after interacting with a sample (Pynn, 1990). The scattering of neutrons is highly dependent on the nuclear structure of the atom (Lindner and Zemb, 1991). The atom of any element is composed of protons, electrons, and neutrons and the number of protons in an atomic nucleus defines the elemental type, while the number of neutrons defines the elemental isotope. Since neutrons are scattered by the atomic nucleus, this means that the scattering from different isotopes can differ significantly (Sears, 1992).
Neutron scattering techniques can be classified into two main groups, namely elastic neutron scattering and inelastic neutron scattering techniques. The elastic neutron scattering techniques involve a process in which the energy, or equivalently, wavelength of the neutron does not change as a result of the scattering event with nuclei in the target sample. Examples of elastic‐based techniques include:
The elastic scattering techniques are useful in providing information about the structure, ranging from the sub‐Angstrom (<10_10 m) to supra‐micron size range (>10_5 m), such as an ordered structure of a fiber, where neutron diffraction can be used for analysis; analysis of the structure of a casein micelle, where SANS can be used; the conformation of a protein at an interface, where neutron reflectometry technique can be used; and analysis of the arrangement of droplets in an emulsion, where ultra‐SANS can be used. The spin echo small‐angle neutron scattering (SESANS) technique employs the spin in its mode of operation.
The inelastic neutron scattering involves an energy change emanating from a scattering event, in which the neutron may either lose or gain energy by imparting energy to or from the sample through diffusion controlled processes. These inelastic scattering techniques are useful in generating information about dynamics across a broad temporal range with vibrational spectroscopy through to quasi‐elastic neutron scattering and spin echo spectroscopy. Another attractive feature of inelastic techniques is that they are capable of providing simultaneous spatial information if angular dependent information is collected (Byron and Gilbert, 2000).
Neutron scattering techniques are important in the investigation of the properties and their effects on the final characteristics of the food for the purpose of preserving the integrity of foods as they are in the original setting.
Generally, unlike electron‐based microscopic techniques, neutron scattering techniques provide bulk information, with the scattering representative of the whole food (Jacrot, 1976; Lu et al., 2007; Paciaroni et al., 2005; Porcar et al., 2004).
In the case of small‐angle neutron scattering (SANS), it is possible to use this technique to probe structures over a size range from approximately 1 nm to several hundreds of nm present in food systems and as such it finds application in the elucidation of the quaternary structure of a protein, the conformation of a polysaccharide chain, and the lamellar structure in granular starches (Jacrot, 1976; May, 2002; Wignall, 1993).
The application of nanomaterials, novel/intelligent materials in foods, and food packaging has gained momentum and is now a reality. Regulations on their use vary from country to country. There is a need for all laboratories to be well equipped for the analysis and uncovering of any violations that involve the incorporation of these materials in packaging, or any steps involved in food processing.