A growing attention is currently being paid to the “nano-world”, which is represented by entities, called nanomaterials (NMs), with at least one dimension lower than 100 nm, and with interesting physicochemical properties applicable to a multitude of fields such as cosmetic, electronic, environmental, industrial food and biomedicine [1]. This limiting size is accepted as the separation border between the nano- and micro-world. Science and technology applied to materials have been so far consubstantial with the development and welfare of society; hence, nanoscience & nanotechnology currently emerges as an increasingly important trend within it. This special interest in NMs arises from the improvement of some nanoscale properties with respect to the properties of analogous bulk materials [2]. Such behaviour is due to the appearance of the “quantum size effect” from the electronic wavefunction, to the limited physical dimensions of the nanoparticle (NP), being this effect just limited to the nanoscale [3, 4]. Owing to this confinement at the nanoscale, the phonons on which thermal, optical, and conductive properties depend, will impart distinctive characteristics to the NMs compared to the unconfined and macroscopic counterparts, with a like-photons behaviour. Under this situation, a new set of discrete quantum energetic states will be established with further energetic available transitions (and hence new or potentialized properties) [3].

Additionally, there is a significant rise in the surface-volume ratio, which entails a higher number of atoms on the surface and, thus, a common increase of reactivity because most of the molecules, ions, or other components appear on the NP surfaces. As a result, properties such as colour, solubility, conductivity, and reactivity among others become completely different in the nano-world with respect to the micro/macro-world [5–7].

On the other hand, other relevant properties of some metallic NPs like luminescence and size-dependent colour come from photon-photon interactions at nanoscale range, which are in turn related to localized plasmon resonance effects [7]. Thus, when the particle size is decreased, it affects the already existing gap energy between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) levels, changing in this way the energy and frequencies emitted by the NP photons. In turn, owing to interactions between NPs and the surrounding particles in diverse media, it is possible to observe different colours for such NPs with both different sizes and environments [6, 7].

Nanotechnology is besides a multidisciplinary area as Feynman’s statement confirmed (1959), where he expressed that some scientific disciplines such as chemistry and biology would be more deeply studied/researched if they could be manipulated and observed at the atomic scale [8]. Thus, this review tries to give a critical vision of the issues related to the implementation of NMs in food field, also providing a systematic assessment of their potential risks as well as taking a step forward by including the most widely used applications and characterization techniques within this field. With this aim Figure 1 tries to display an informative table of contents (TOC) diagram about all the addressed items in this review.

Undoubtedly, an amazing number of nanotechnology applications in food science are revolutionizing the food industry, highlighting the following topics [9–11]:

(1) Increasing the self-life of food and improving its quality.

(2) Bioactive fortification with controlled release by means of nanocarrier systems.

(3) Colouring, flavourings, adding nutritional additives, and modification of food structures and textures.

(4) Antimicrobial agents and detection of chemical alterations by using intelligent packaging systems.

The food field is, of course, one of the important areas where nanoscience and nanotechnology have a wide incidence. Thus, there are a great number of current applications of both inorganic and organic NMs in food, food additives, and food-contact materials. It summarized in Table 1 the most used NMs in food field so far and where they are applied.

However, other important aspects as their toxicity and environmental damage must be also considered, since derived from their unpredictable behaviour as NMs are able to cross biological membranes, tissues, and organs that their analogues at the macro-scale normally cannot. Thus, NMs can reach the bloodstream through several pathways, and once there, they can be carried around the body reaching organs and tissues, e.g., brain, heart, liver, kidneys, spleen, nervous system, bone marrow, etc., which may entail a rational inherent concern [30–32].

Considering the applications previously cited, the present and future of NMs research should also focus on the interaction between the NPs and living beings. In this context, it is necessary to develop accurate and robust methodologies for the characterization of physiochemical properties of NPs as well as for their quantification in vivo conditions. However, there is almost no global standardized methodology able to assess the toxicological effects of NMs in commercial applications [33, 34].

Thus, although there is no doubt about the great benefits that NMs bring to food and food industry (as Table 1 displays), they also involved new threats to food sector. As before mentioned, their risks are associated with the potential toxicity to human health and to environmental damages, thereupon the safety of the NMs has attracted attention in line with their increasing use [33]. As with any other new discipline, it is quite unregulated, so it is necessary for information-gathering (studies about toxicity, legislation, customer information, etc.) lacking the legal and scientific tools, as well as resources to oversee the exponential market growth of nanotechnology [35].

To ensure the appropriate balance between benefits (competitiveness for the agri-food sector) and risks is necessary to have reliable analytical information on the presence and amount of NMs in food samples. With this aim, validated analytical methodologies for monitoring NMs in food are needed. So, from a broader point of view, the role of analytical science associated with NMs-foods can be summarized in the following outstanding points:

(1) Characterization of NMs used in the food industry (synthetic or natural).

(2) Quality control of products and foods containing NMs with nutritional or healthy interests.

(3) Detection/determination of NMs in food because of their potential toxicity.

(4) Production of reliable working standards involved in the corresponding analytical methods.

To begin these tasks, two fundamental goals within analytical nanoscience and nanotechnology can be distinguished. The first one is related to the characterization of NMs because they will be used for other purposes. The second one is the identification/determination of NMs in particular samples. Both aspects will be next discussed in this review.

There is a vast amount of information related to NMs, for instance, size, size distribution, agglomeration, shape, structure, composition, surface charge, and concentration when they are present in samples. So, it is important to know these parameters about the NMs-based formulations used in food industry, as well as the risks associated with them through accurate and appropriate analytical techniques to provide a proper and exhaustive analytical characterization. From an analytical point of view, there are many instrumental techniques to supply this information, primarily microscopic, diffraction, spectroscopic, and instrumental separation techniques. Therefore, it is important to match the type of needed information with specific technique(s) [36].

Currently, it does not exist a single analytical tool able to provide all meaningful information about NPs, since depending on the NPs’ nature, we should use a combination of different analytical techniques [36].

The Table 2 provides detailed and specific referenced information about the huge number of analytical tools commonly used for the analytical physic-chemical characterization of NPs. They have been there classified into microscopic [37–48], spectroscopic [37, 38, 40–42, 44, 49–54], and mass spectrometry techniques [40, 42, 55–57], although these last ones are usually coupled with those based on size-separation criteria [42, 58, 59] or either to other ionization tools such as electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), laser desorption/ionization (LDI) and inductively coupled plasma (ICP) [42, 55] (all these references are also displayed in Table 2). Other emerging technologies for the analytical characterization of NMs such as microfluidics and size separation techniques will be commented on later in the last section of this review, which is relative to future perspectives and challenges. On the other hand, as an illustrative example, Figure 2 shows a full physic-chemical characterization of NOMs, specifically based on quercetin-nanoemulsions (Q-NEs), implemented as nutraceutical supplement [60] by means of diverse spectroscopic and microscopic techniques.

In addition to the analytes of interest, the sample matrix can contain other substances that could shift out of range the selected analytical signals and some of which may show up naturally as NMs. This may occur due to the existence of assorted thermodynamic equilibria, generation of diverse nature corona around NPs, reaction of some redox-sensitive particles, or either the presence of natural NPs. So, the knowledge of multiple factors may be required to optimize, develop, and validate robust methodologies to characterize NPs without altering their native features in any way [90–93].

The second side is the identification/detection/determination of NMs in specific samples, and this goal is frequently referred to as analytical nanometrology (ANM) [94]. Different analytical techniques, already included in Table 2, can be used. In many cases, the objective is NPs of inorganic nature (some of them synthetic NMs, ENMs), although growing attention is being paid to organic NMs (especially in food and biomedical fields). Obviously, food safety is indeed a contemporary issue within the implementation of NPs in food. Consequently, it is acknowledged that ingredients that are typically regarded as safe at the macro level may not be safe at the nanoscale. When NMs reach the human body, they may be recognized by the immune system, leading to unwanted biological responses. This is due to the ability of NPs to interact with cells, proteins, and even DNA, eventually causing genetic mutations. This genotoxicity may be caused by direct damage (direct interaction with DNA) or by indirect damage by reactive oxygen species (ROS). The generation of ROS has been also studied in “in vivo” conditions [33], where these oxidation processes of biomolecules have driven tissue degradation, which in turn led to carcinogenesis events among others [95, 96].

Nevertheless, the host response will depend on the NM’s interaction with any component of the body (e.g., cells, lipids, and proteins) and the NMs’ features. Therefore, appropriate testing methodologies are needed for the safety control of food containing (or potentially containing) NPs. Regarding this, it must be recognized the disproportion between the investment devoted to the development of new products (e.g., NMs) versus that one spent on researching the safety of these products.

Finally, the toxicity of the NMs greatly depends on the level of human exposure to them, which is closely related to the bare specific area and the concentration usage. Thus, Table 3 summarizes ENMs used in the food field, their discrete reported toxicity from diverse “in vivo” studies, as well the appropriate European legislation to control their application.

Thus, silver is a blatant example of a regulated additive in Europe (E174), as already cited in Table 3. Sometimes, it is occasionally employed as NPs (Ag NPs), which enhance the quality, safety, and shelf-life of packaged food and drinks due to their antibacterial capabilities, but they may have potential health hazards which are not yet fully understood and not entirely regulated as a result. Hence, it is crucial to monitor their presence in dietary samples (silver-coloured pearls, used as decoration in pastry for instance). Recent research is based on the chemiluminescence of luminol/Ag⁺ in alkaline medium induced by the presence of Ag NPs. This method involves easy monitoring, since is a straightforward qualitative screening for Ag NPs in pastry manufacturing, whereas asymmetric-flow FFF (AF4) coupled with inductively coupled plasma-mass spectrometry (ICP-MS) was used to confirm the findings [109]. This approach has been also applied to control the migration of Ag NPs from food containers into food simulants [110].

Another noteworthy example in this area is SiO₂ NPs. According to the specific matrix, there may be different maximal allowed levels for silica, an EU-authorized food additive (E551). Thus, 50 g kg^–1 for dry powdered emulsions and flavouring preparations or 10 g kg^–1 in nutrient preparations for infants feeding and young children. It is also used for beer and wine clearance, and as an additive in thickened pastes and salt samples. The main issue associated with its use as NPs lies in what performed studies reporting about some potential real hazards (pregnancy and coagulopathy difficulties), but the current legislation does not distinguish between macro- and nano-silica yet. This highlights the importance of monitoring SiO₂ NPs in potentially containing meals. With this aim, CE coupled to an evaporative light scattering detector (CE-ELSD) was used to develop an approach for controlling their presence in salt samples, a very common ingredient in a wide variety of foods [88]. The electropherogram obtained for a blend of nano- and micro- SiO₂ particles of different sizes together with their TEM confirmation is displayed in Figure 3.

The study of sugary meals containing TiO₂ NPs is another meaningful topic in food industry. These NPs have demonstrated potential risks to humans. TiO₂ NPs enter the blood and lymph after ingestion where they can reach sensitive target areas (bones, brain, liver, and heart). As NPs present cytotoxic, genotoxic, and carcinogenic effects. However, TiO₂ is widely used in a variety of areas such as cosmetics, ceramics, catalysts, etc., including food field, where it is regarded as a food-grade additive (E171 in EU). The legislation does not distinguish again between micro- and nano-forms, requiring the development of accurate analytical methodologies to identify and quantify TiO₂ NPs in foods. A feasible screening-confirmation strategy has been recently developed for the analysis of TiO₂ NPs in sugar and sugar-containing products. Raman spectroscopy was employed to perform a direct screening of samples to determine whether TiO₂ NPs were present or not. Then, positive samples were examined to know about the crystallographic structure of TiO₂ (anatase or rutile), which were next confirmed by CE, reporting their nano- or micro-size too [89]. This strategy is summarized in Figure 4. Thus, sugar samples were initially checked using Raman spectroscopy to determine the presence of TiO₂, being also able to discriminate between its two crystallographic forms through their distinctive scattering bands. When there was a positive result for TiO₂ in sugar samples, it was required to confirm its presence (total or partially) as TiO₂ NPs. This last step was carried out by CE since the electropherogram allows us to discern between nano- and micro-forms of this compound.

ZnO NPs characterization is also relevant for the food field. It can be used in the packaging linings of food cans for meat, fish, maize, and peas to preserve colors and prevent rotting. Additionally, they can be added as food additive as a source of zinc used to fortify cereal-based foods. According to reported research, large concentrations of ZnO are harmless, but when they are replaced by NPs of the same material (ZnO NPs), they penetrate cells, being intracellularly dissolved and triggering an acute cytotoxic process, displaying a molecular mechanism whose chemical basis is thoroughly outlined [111]. Hence the interest for their analytical control in food. Regarding it, single particle ICP-MS (sp-ICP-MS) has proven to be a highly effective technique to detect and characterize the presence of ZnO NPs in cereal-based foods [112].

Recently, NOMs like nanoemulsions, nanoliposomes, and nanomicelles among others, are emerging as very helpful nanotools for encapsulating lipophilic systems or acting as nanocarriers for biologically active compounds. Nutraceuticals are food ingredients that provide benefits since they are thought to prevent chronic diseases and enhance human health. However, their usefulness in food industry is constrained due to their weak water solubility, low bioavailability, pH sensitivity, and easy degradation in adverse media such as environmental gastric conditions. Many nanotechnological approaches have arisen to overcome all these limitations, such as the already mentioned nanosystems with the aim to exploit their therapeutical effects. An extensive revision was published on the analytical control of nanodelivery lipid-based systems for the encapsulation of nutraceuticals [113]. In addition to the intrinsic difficulty in characterizing NOMs, especially when they are present in complex matrices such as food, the authors noted the major challenges from an analytical point of view: sampling and sample treatments (separation procedures), the need for reference materials, and the lack of trustworthy methods (validated methodologies barely exist today); but the regulatory issues also present great significance. Thus, there is not a widely acknowledged international regulatory framework for food safety that would standardize the management of risks associated with the use and application of such nanotechnology in the food field [114]. Most likely, EU has one of the most restrictive legislations in this respect, with regulations that implicitly/explicitly or either partially/fully address the standardization of NOMs in food. Such are the cases of Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) 2283/2015, 1332/2008, 1925/2006, 609/2013, 1924/2006 regulations, the directive 2002/46/CE (food supplements), and the “Guide to assess the risk about the application of nanoscience and nanotechnology in food and in the food chain” [Scientific Committee European Food Safety Authority (EFSA), 2011] [115–117].

It is obvious that incorporation of NOMs to food is an impressive way to enhance the bioavailability and bioaccessibility of food containing nutraceuticals. However, to know the nature, structure, properties, and risks associated with the implementation of these delivery systems is critical to consider the detection, identification, and characterization of these NOMs by using reliable and validated methodologies.

Regarding these NOMs analyses, it is an interesting task the discrimination between curcumin and nanocurcumin. Curcumin is a lipophilic bioactive compound with substantial health benefits due to its pharmacological actions and chemoprotective qualities for treating several diseases. It is characterized by its poor aqueous solubility that causes low bioavailability, high instability, or even degradation at basic aqueous media and under light radiation, limiting so its distinctive benefits. Food technology has achieved solutions to overcome such disadvantages by encapsulating curcumin. Currently, nanocurcumin is found in many nutritional supplements. However, these nanoformulations can be the object of significant doubts relative to food safety considerations due to the use of surfactants and other stabilizing chemicals also employed to prevent agglomeration and to hasten the delivery of their active ingredients. Unfortunately, few toxicological data about these nanosized curcumin formulations have been evaluated so far. Many of these nanosized particles are likely to display adverse effects owing to their small size and rapid diffusion through the entire body, specifically above a certain dosage, being of great concern the requirement of upcoming food sample stability studies and its associated nanotoxicological research. Thus, monitoring and quantifying the production and stabilization of such nanosized curcumin in food and supplementary matrices is crucial to ensure consumer safety. However, most of the research works reported so far have been just focused on the determination of free curcumin in different kinds of samples whereas only a few works have approached the detection of diverse curcumin nanoformulations, but their quantification always involved the breakup of curcumin-containing nanosystems, altering so its native nanostructure. Thus, it has been recently reported the quantification and discrimination of nanocurcumin (via micelle encapsulation) from free curcumin using a selective fluorescent probe based on graphene quantum dots (QDs) [118]. The presence of both analytes produces a gradual quenching in the emission response of the nanosensor; however, the presence of free curcumin causes a red shifting in the maximum wavelength of the graphene NPs response while the presence of nanocurcumin keeps it constant. This difference allows the determination and discrimination of both compounds in different types of food samples.

Another illustrative example lies in the antioxidant quercetin, which is a natural phenolic bioactive mostly found in fruits and vegetables among others. Furthermore, its intake is associated with numerous beneficial therapeutic effects against a wide range of illnesses. However, two factors prevent its therapeutic activity exclusively through diet: (1) food preparation and processing, since the quercetin concentrations considerably drop or perhaps entirely disappear; and (2) its lipophilic nature, which origins low bioavailability in the body. To overcome these limitations, quercetin dietary supplements were manufactured in the food industry. Thus, a nanometric carrier nanoemulsions-based has been recently developed to encapsulate it. Hence the helpfulness to propose analytical strategies for the discrimination between quercetin and nanoencapsulated quercetin. With this aim, it has been designed a liquid-state quantitative surface enhanced raman spectroscopy (SERS) sensing platform for the identification and quantification of Q-NEs using Au NPs like plasmonic arrays [119]. The differences in the Raman spectra for free and encapsulated quercetin allow its easy discrimination, with good sensitivity.