Nanostructures of whey proteins for encapsulation of food ingredients

Nanostructures of whey proteins for encapsulation of food ingredients

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Oscar L. Ramos*,†, Ricardo N. Pereira*, Lı´via S. Simo˜es*, Daniel A. Madalena*, Rui M. Rodrigues*, Jos
e A. Teixeira*, Anto´nio A. Vicente* *Centre of Biological Engineering, University of Minho, Braga, Portugal, †Universidade Cato´lica Portuguesa, CBQF - Centro de Biotecnologia e Quı´mica Fina – Laborato´rio Associado, Escola Superior de Biotecnologia, Porto, Portugal

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Whey protein-based nanostructures

Proteins are macromolecules which are complex to study due to their dynamic behavior and ability to assemble into a range of different structures. High moisture three-dimensional networks of interconnected protein particles can be produced and dispersed in a continuous liquid phase providing desirable texture properties to the food matrix (Bhopatkar, Hamaker, & Campanella, 2012) or improving delivery of value-added bioactive ingredients through micro- and nanoencapsulation techniques (Jones, Decker, & McClements, 2009; Jones, Lesmes, Dubin, & McClements, 2010). Several food proteins can be used as building blocks for development of these functional molecular architectures, but among them, whey proteins from milk are gaining increased attention (Assadpour, Jafari, & Maghsoudlou, 2017; Faridi Esfanjani, Jafari, & Assadpour, 2017). Whey proteins are the soluble protein fraction in milk mostly encompassed of globular proteins, such as β-lactoglobulin (β-lg) and α-lactalbumin (α-la) which comprise almost 50% and 20% of the total protein content, respectively. These proteins are now considered valuable by-products from the cheese industry, relatively inexpensive, classified as GRAS (generally recognized as safe) ingredients, being eventually the most studied globular proteins (Mohammadi, Jafari, Assadpour, & FARIDI Esfanjani, 2016; Nicolai & Durand, 2013). Solutions of native whey proteins have the ability to form rigid irreversible aggregates when heated at temperatures above their denaturation temperature (60°C). These protein aggregates are currently used in several food formulations due to their functional, nutritional, and biological properties (i.e., amino acid composition, digestibility behavior, and excellent sensory characteristics) (Bryant & McClements, 1998; Ramos et al., 2017). Aspects such as protein-protein interactions, aggregation pathways, aggregates’ morphologies, and processing techniques determine much about the functionality and ability to form protein gels and nanohydrogels (Abaee, Mohammadian, & Jafari, 2017). Thus these subjects will be addressed later

Biopolymer Nanostructures for Food Encapsulation Purposes. https://doi.org/10.1016/B978-0-12-815663-6.00003-3 © 2019 Elsevier Inc. All rights reserved.

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Biopolymer Nanostructures for Food Encapsulation Purposes

in a critical manner to understand how proteins can be transformed into building units that will allow the development of protein-based aggregates.

1.1 Aggregation pathways Production of whey protein aggregates requires unfolding of the native structure of the protein and the most common method to achieve this is through thermal denaturation (Foegeding, 2006; Nicolai, Britten, & Schmitt, 2011; Pereira et al., 2016). The level of protein denaturation under heat effect is strictly dependent on solution properties (i.e., protein concentration, pH, and ionic strength) and extrinsic factors related with heating conditions (i.e., temperature, heating rate, treatment time, and type of heating method). Nevertheless, the development of protein aggregates can also be achieved through other physical methods (e.g., pressure) but also chemical (i.e., acid, ionic) or biological (i.e., enzymatic) methods—or even by combination of both (Ryan, Zhong, & Foegeding, 2013; Stokes, 2012). The way how thermodynamic stability of proteins is affected and the balance between attractive and repulsive forces, between unfolded protein molecules, can give rise to different aggregate morphologies.

1.1.1 Heating As aforementioned, heating above denaturation temperature may cause the aggregation of thermo-labile globular proteins, such as the case of β-lg and α-la (Nicolai & Durand, 2013; Pereira, Teixeira, & Vicente, 2011). In general, thermal denaturation consists of several sequential steps, starting by a first destabilization and unfolding of protein quaternary structure. This is followed by inter- and intramolecular interactions between unfolded protein molecules that results on development of primary aggregates (the building blocks) and secondary aggregates (association of primary aggregates), which give rise to protein 3D network above a critical point of aggregation (Rodrigues et al., 2015). The rate of aggregation and the size of protein aggregates depend much on the heating load, heating rate, and type of heating method, that is, direct or indirect heating. Indeed, uniform heating and the nonexistence of hot surfaces can produce clear differences in protein denaturation kinetics at similar heating rates and holding times. Ohmic Heating (OH) technology is an outstanding example about the way how the heating method can influence aggregation properties of whey proteins. OH is a food processing process where an electric current is passed through a semiconductive material generating an internal heat in agreement with Joule’s law. Given its volumetric and fast heating, OH imposes changes on protein denaturation kinetics that impairs extensive aggregation (Pereira et al., 2011). In addition, nonthermal effects in combination with a moderate electric field seem to offer an opportunity to modulate or control the size of protein aggregates (Pereira et al., 2016).

1.1.2 Acid Acid-induced gelation of whey protein isolate (WPI) through the use of glucono-d-lactone (GDL) has been extensively reported (Cavallieri & da Cunha, 2008; Rabiey & Britten, 2009; Sadeghi, Madadlou, Khosrowshahi, & Mohammadifar, 2014). GDL hydrolyses

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to gluconic acid in aqueous environment allowing a progressive and controlled acidification of the protein solutions, being the rate of acidification intrinsically dependent on factors such as temperature, protein concentration, and ratio of protein and GDL (de Kruif, 1997; Kharlamova, Chassenieux, & Nicolai, 2018). Acid gelation may often require a physical disruption (e.g., achieved by heating or pressure) of the native structure of the protein molecule in order to expose it to further reactions. Milk proteins, such as whey and casein fraction, present an isoelectric point (pI) and zero net charge at acid pH (ranging from 4.5 to 5.5, depending on the type of protein). This means that acid changes toward pI (i.e., induced by fermentation processes or addition of acids) allow minimizing repulsive forces, thus favoring noncovalent interactions and fractal aggregation between protein molecules.

1.1.3 Ionic Increase of ionic strength of a given protein solution is easily achieved through addition of common salts such as calcium and sodium. Depending on the balance between protein concentration and ionic strength, the added ions will shield electrostatic charges of the molecules, thus increasing the rate of protein aggregation during the thermal denaturation process. Aggregation and gelation can take place during heating, when electrostatic shielding and ion/hydrophobic interactions are highly favored or can occur at room temperature (as acid gelation)—commonly called as cold gelation. In general, cold gelation induced by salt involves two steps: (1) predenaturation of proteins, in which proteins are unfolded and primary aggregates produced; and (2) addition at room temperature of an excess of cations (ferrous, calcium, or barium salts) to the denatured protein solution (Marangoni, Barbut, Mcgauley, Marcone, & Narine, 2000; Pereira et al., 2017). This cold gelation approach can be particularly interesting when the intention is to produce a protein gel with certain rheological properties, or even to associate heat-sensitive bioactive compounds such as vitamins to a given protein network.

1.1.4 Enzymatic Whey protein aggregation and even gelation can also be promoted through enzymatic reactions, which can be complementary to heat-induced gelation. Microbial transglutaminase (TG: EC 2.3.2.13) that catalyzes covalent cross-linking of whey proteins and other proteins (e.g., soy and casein), through an acyl transferase, has been extensively described (Otte & Qvist, 1998; Stender et al., 2018). Peroxidase (POD: EC 1.4.3.13) can also induce cross-linking of whey proteins, such as β-lg, α-la, and bovine serum albumin (BSA), resulting in aggregates of different sizes (Saricay, Dhayal, Wierenga, & de Vries, 2012; Stahmann, Spencer, & Honold, 1977). Bacillus licheniformis protease (BLP) can also be used to produce whey protein aggregates through cleavage of peptide bonds at the C-terminal side of glutamate and aspartate residues, thus favoring noncovalent interactions between the peptides (Spotti, Tarhan, Schaffter, Corvalan, & Campanella, 2017). Enzymatic aggregation has the potential to change important structural properties of proteins (e.g., size and conformation), thus bringing new functional aspects regarding gelation, emulsion, and thermal stability (Tang & Ma, 2007).

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Biopolymer Nanostructures for Food Encapsulation Purposes

1.2 Molecular interactions A comprehensive view of molecular interactions that rules protein stability is essential to the development of processing strategies that control the aggregation of globular proteins. Physical and chemical processes induce structural changes in globular proteins making their peptide chain more available for further reactions. As a consequence, unfolded molecules may interact with each other leading to extended aggregation and even gelation (Nicolai et al., 2011). Molecular interactions in globular proteins can be covalent or noncovalent, repulsive or attractive, long range or short range, and also temperature dependent. The role of the different types of interaction on protein aggregation processes still needs to be more elucidated. It is generally accepted that covalent interactions between free SH groups, establishing disulfide bonds, is crucial for initiation of aggregation of globular proteins such as whey (Shimada & Cheftel, 1989; Wijayanti, Bansal, & Deeth, 2014). On the other hand, noncovalent interactions seem to play an important role in propagation step of protein aggregation, being even more important when pH values are close to the pI or at higher salt concentrations (Hoffmann & van Mil, 1997; Ramos et al., 2017). Processing by heat or pressure may promote alterations in the protein’s conformational structure, exposing their free SH groups initially buried within the native protein structure, which are important to initiate the aggregation process. Several kinds of noncovalent interactions can be distinguished such as electrostatic, hydrogen, hydrophobic, hydration, and van der Waals. Electrostatic interactions (repulsive and attractive) are extremely dependent on pH and ionic strength of surrounding aqueous environment. At their pI, globular proteins have no net charge, above or below they are negatively and positively charged, respectively. Addition of salts and counter ions promotes electrostatic screening of protein charges, changing the balance between these repulsive and attractive forces. Electrostatic interactions established among charged proteins strengthen with temperature due to their entropic origin. Hydrogen bonding mainly stabilizes globular protein aggregates (Croguennec, O’Kennedy, & Mehra, 2004; Ramos et al., 2014). Intermolecular hydrophobic interactions are temperature dependent; they tend to increase in strength with the increasing of temperature (De Wit, 1990). Due to unfolding of protein molecules, apolar residues become accessible to solvent molecules and further reactions occur. Independently of the protein state (folded or unfolded), van der Waals interactions seem to play a minor role for aggregation mechanisms. However, they can be of great importance when protein molecules are large enough to act as a colloidal particle, promoting strong aggregation between biopolymer molecules (Bryant & McClements, 1998).

1.3 Protein aggregates’ morphology Globular protein aggregates can be composed of spherical or fibrillar conformation, as well as be distinguished by their particle size (micro or nanoscale). Designing protein networks’ size from micro- to nanometers allows their incorporation in food without affecting food sensory properties, and more recently, the development of novel protein nanoparticles with improved functionalities (Augustin, 2003; Chen, Remondetto, & Subirade, 2006). By increasing protein concentration, primary aggregates can link

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into a higher extent forming larger polydisperse self-similar aggregates, thus leading to precipitation (at low protein concentration), or to gelation (at high protein concentration) (Nicolai et al., 2011). Depending on pH values, different types of aggregation mechanisms and globular β-lg protein aggregates can occur as recently proposed (Loveday, Rao, & Singh, 2012; Nicolai et al., 2011; Nicolai & Durand, 2013). Between pH 4 and 6, hydrogels can be characterized by spherical aggregates binding together to form the threads of the network (Remondetto, Paquin, & Subirade, 2002). By shifting pH toward the pI, or increasing ionic strength, the aggregate networks become coarser, and their size can increase from nano- to micrometers (Ikeda & Morris, 2002; Phan-Xuan et al., 2013). Rheological studies of β-lg gelation performed by Hermansson’s group (Ikeda & Morris, 2002; Phan-Xuan et al., 2013; Stading & Hermansson, 1990, 1991; Stading, Langton, & Hermansson, 1992) have shown the formation of protein networks at narrow pH intervals ranging between 4 and 6, as follows: (i) regular, in the pH range 5–6, showing uniform distributions of particle size; (ii) irregular, at pH 4.5, where there are particulate uneven aggregates; and (iii) a mixture of network types at pH 4 with clusters of particles embedded in a fine-stranded network. In salt-free solutions at pH values far from the pI (i.e., above pH 6 or below pH 4), protein solutions are transparent or translucent, being composed of flexible linear or fibrillar strands. These aggregates have a diameter in the order of nanometers and can give rise to protein gels called “fine stranded” (Aymard, Nicolai, Durand, & Clark, 1999; Ikeda & Morris, 2002). Strands increase in width and length when pH is lowered toward more acidic values or increased above pH 6. At acidic pH the hydrogels produced have a more brittle nature, once intermolecular disulfide covalent bonding is unlikely to occur (Stading & Hermansson, 1991).

1.4 Nanohydrogels Protein hydrogels present interesting functional properties, showing several potential applications in food biotechnology. Their main feature is the ability to entrap a large amount of water (ca. 30 times their size) without destabilizing the inherent internal structure (Caccavo, Cascone, Lamberti, & Barba, 2018; Cerqueira et al., 2017b; Chen et al., 2006; Qiu & Park, 2012; Stading & Hermansson, 1991). The swelling capacity (and thus the soft and pliable properties of hydrogels) is established by hydrophilic character of constituent chemical entities such as the hydroxyl, carboxyl, ether, amines, and sulfate groups in the protein structure (Ramos et al., 2017). These systems are super absorbent, having the ability to contain over 99% of water, thus owning a degree of flexibility very similar to natural tissues. These protein network systems can be used to provide desirable texture properties to the food matrix (Bhopatkar et al., 2012) and also to protect and improve delivery of value-added bioactive ingredients through nanoencapsulation techniques (Jones et al., 2009, 2010). Hydrogels can react to external stimuli (i.e., pH, temperature, concentration of metabolites) and release a given active compound entrapped in response to such a change (Banerjee & Bhattacharya, 2012). Globular protein aggregates can be formed into gel particles that range from the nano-to-millimeter length scale, thus presenting different functionalities. The designation nanohydrogel implies that, independently of size distribution (at the nanoscale, in this case), the nanoparticles are a cross-linked

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Biopolymer Nanostructures for Food Encapsulation Purposes

network able to entrap a large fraction of solvent. A transmission electron image of β-lg nanohydrogel, formed by gelation induced by thermal heating, is presented, as an example, in Fig. 1A.

Fig. 1 Transmission electron microscopy images (scale bar 100 nm) obtained by using the negatively stained method of (A) nanohydrogels and (B) nanofibrils made from bovine β-lg (10 g L 1) after thermal treatment at 80°C for 20 min at pH 6.0 and 4.0, respectively; and of (C) nanotubes formed from bovine α-la (30 g L 1) partially hydrolyzed with serine endoprotease from Bacillus licheniformis (BLP) (4%, w/w), by heating at 50°C for 24 h at pH 7.5, in the presence of manganese.

Nanostructures of whey proteins for encapsulation of food ingredients

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Final characteristics of these nanohydrogels can be tailored by small changes in the temperature of gelation, pH, protein type, and salt concentration during thermal aggregation (Lefe`vre & Subirade, 2000; Mulvihill & Kinsella, 1988; Ramos et al., 2017; Stading & Hermansson, 1991). Particular enhanced functionalities can be found in different types of gel networks at nanoscale such as the case of nanotubes or nanofibrils (Fig. 1). At low pH (<2.0) and ionic strength, nanohydrogels can be composed of long rigid fibrils up to several microns long and an average diameter between 4 and 5 nm (Fig. 1B) (Nicolai et al., 2011). This seems to be a general property of the globular proteins once these structures are seen with β-lg, BSA, and ovalbumin (Foegeding, 2006; Gosal, Clark, Pudney, & Ross-Murphy, 2002; Gosal, Clark, & Ross-Murphy, 2004; Sagis, Veerman, & van der Linden, 2004). Because of the high ratio of length (micrometer) to width (nanometer), fibril solutions can entangle to form physical gels at relatively low protein concentrations (Loveday et al., 2012). Self-assembly of α-la after partial hydrolysis by a serine endoprotease (known as BLP) from Bacillus licheniformis can result in formation of hydrogels composed of hollow tubes of about 20 nm in outer diameter, 7–8 nm of cavity diameter, and several hundreds of nanometers (or even micrometers) long (Fig. 1C); in this process, the presence of calcium (Ca2 + ) is essential. Calcium allows a spatially restricted creation of ionic bonds between carboxyl acid groups on peptide fragments resulting from the action of BLP (Ipsen, Otte, & Qvist, 2001). Nanotubes are usually formed at α-la concentrations of 30 g L 1 and a Ca2+/α-la molar ratio >1.5 (Ipsen & Otte, 2007), while linear fibrils are formed at α-la concentrations of <30 g L 1. In general, nanotubes gels can be formed at 50°C and pH between 7.5 and 8.0, which are the optimal activity conditions for BLP (Birktoft & Breddam, 1994; Ipsen & Otte, 2007; Svendsen, Jensen, & Breddam, 1991). Between Ca2+/α-la molar ratio of 1.5 and 6, fine stranded networks of tubules with a translucent appearance can be produced, while higher Ca2+ concentrations can have negative effect on the tubule formation, resulting in amorphous structures (Graveland-Bikker, Ipsen, Otte, & DE Kruif, 2004). On the other hand, Esmaeilzadeh (Esmaeilzadeh, Fakhroueian, Esmaeilzadeh, & Mohammadi, 2013) produced α-la nanotubes with 3–8 nm in outer diameter by using an innovative, yet low cost, approach based on acid hydrolysis through specific agents such as surfactants, pH reagent, Tris-HCl buffer, and polar solvents. In this strategy, α-la was chemically hydrolyzed in the same cleavage sites used by BLP, being the addition enzymes or the application of high temperatures not required (Esmaeilzadeh et al., 2013).

1.5 Food applications Nanohydrogels are an ideal candidate for use in encapsulation techniques and controlled delivery of bioactive ingredients (Abaee et al., 2017; Mokhtari, Jafari, & Assadpour, 2017). They present a reduced size (subcellular) associated with their large surface area and environment similar to that of a biological tissue (due to their high water content), making them relatively biocompatible. They also have other properties which make them a viable choice ideal material to be used for delivery of proteins and peptides, for instance: improved solubility and bioavailability

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Biopolymer Nanostructures for Food Encapsulation Purposes

(especially for those ingredients with poor solubility in aqueous matrices or with poor absorption rates), soft and elastic properties that reduce the irritation upon in vivo implantation, prevention of cell adhesion and protein absorption and due to the low interfacial tension between water and hydrogels, different hydrophilicities and molecular sizes which allow wide acceptability for individual drugs, associated tissues, beneficial action on the gastrointestinal (GI) mucosa, possibility to specifically tailor cross-linking density and swelling for release of incorporated bioactive ingredients. Hydrogels made from whey globular proteins show the ability to entrap both lipophilic and hydrophilic nutrients without significantly affecting their activity, which can contribute to the development of innovative functional foods (Sharma, 2012a, 2012b). In recent years, the ability of globular proteins to form gel networks under cold conditions is attracting attention due to the possibility of application of these systems in novel food and nonfood areas. At lower iron concentrations, linear aggregates allow the development of filamentous forms due to hydrophobic interactions, whereas at high iron concentration a particulate gel is produced by random aggregation of spherical aggregates or larger size, where van der Waals forces are predominant (Pereira et al., 2017; Remondetto et al., 2002). Filamentous nanohydrogels may constitute an excellent matrix for transporting iron and promoting its absorption, thus allowing the development of innovative functional foods while addressing a mineral deficiency that concerns a large number of people all over the world.

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Nanoencapsulation of food ingredients

Besides nutrition, food ingredients are increasingly being used to develop novel and functional food products, as an opportunity to face the consumer demands for safer and healthier foods, thus contributing to improve human health by reducing the risk of development of nutrition-related diseases ( Jafari and McClements, 2017; Sabliov, Chen, & Yada, 2015). Nonetheless, some biological properties of food ingredients are often lost as a result of their poor solubility in foods, instability during food processing conditions (i.e., temperature, light, presence of oxygen or interaction with other food compounds), and in the GI tract (i.e., pH change, presence of enzymes and other agents)—all these factors limit the potential human health benefits and activity of food ingredients (Cerqueira et al., 2017a; Cruz, Garcı´a-Estrada, Olabarrieta, & Rainieri, 2015). In this regard, extensive strategies for safe delivery of food ingredients have been explored to protect their functionality and enable a release in targeted sites where absorption is desired (Akhavan, Assadpour, Katouzian, & Jafari, 2018; Assadpour & Jafari, 2018; Benshitrit, Levi, Tal, Shimoni, & Lesmes, 2012). Whey protein-based nanostructures, and their fractions, have been developed and applied as suitable carriers for a range of food ingredients including antioxidants, antimicrobials, flavors/ odors, fatty acids, minerals, or bioactive peptides (Faridi Esfanjani & Jafari, 2016; Loveday et al., 2012). Some examples of whey protein-based nanostructures, including the bio-based material entrapped bioactive ingredient, activity, nanostructured size, and encapsulation efficiency, are summarized in Table 1.

Table 1 Examples of whey protein-based nanostructures including the bio-based materials, entrapped bioactive ingredients, activity, nanostructured size, and encapsulation efficiency Food ingredients

WPC-based material

Bioactive compounds

Nanostructured size (nm)

Encapsulation efficiency (%)

Antioxidants/ phenolic compounds

β-lg α-la β-lg β-lg/alginate Zein/β-lg

Curcumin Curcumin Vitamin B2 Quercetin Tangeretin

164.4  8.5 63.5  1.2 172.0  1.0 225.2 263.0  13

98.2  1.4 97.5  2.1 26.1  1.0 95.1 73.0

β-lg/dextran

β-Carotene

68.2  2.9

98.4  1.2

WPI/pectin β-lg β-lg/chlorogenic acid WPC β-lg

Anthocyanin EGCG EGCG Phytosterols EGCG

203.6  3.6 9.7  0.10 105.8 171.3  0.5 30.0  0.5

55.0  6.0 77.9  0.46 73.5 90.5  3.3 94.0  8

WPI

Date palm pit extract D-Limonene

78.3  2.3

92.9  17.3

160.0

88.0

225.0 100.0

24.0 64.0  10.0

WPI/lauric acid

Ethyl hexanoate ω-3 Polyunsaturated Echium oil

150.0

78.0

WPI

Zinc

NA

94.0  1.0

Antimicrobials

Flavors/odors

WPC/pectin

Fatty acids

WPI β-lg/pectin

Minerals

Reference Teng, Li, and Wang (2014) Yi et al. (2016) Madalena et al. (2016) Mirpoor, Hosseini, and Nekoei (2017) Chen, Zheng, McClements, and Xiao (2014) Yi, Lam, Yokoyama, Cheng, and Zhong (2014) Arroyo-Maya and McClements (2015) Li, Du, Jin, and Du (2012) Fan, Zhang, Yokoyama, and Yi (2017) Cao, Ou, Lin, and Tang (2016) Lestringant, Guri, G€ ulseren, Relkin, and Corredig (2014) Bagheri, Madadlou, Yarmand, and Mousavi (2013) Ghasemi, Jafari, Assadpour, and Khomeiri (2018) Giroux and Britten (2011) Zimet and Livney (2009) Azizi, Kierulf, Connie Lee, and Abbaspourrad (2018) G€ ulseren, Fang, and Corredig (2012)

Note: NA, information not available; EGCG, epigallocatechin-3-gallate; β-lg, β-lactoglobulin; α-la, α-lactoalbumin; WPI, whey protein isolate; WPC, whey protein concentrate.

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Biopolymer Nanostructures for Food Encapsulation Purposes

2.1 Antioxidants The oxidation of food ingredients and subsequent reactions that can occur during storage and transportation may deteriorate the safety and quality of food products, thus compromising the shelf life of food and consequently consumers’ acceptability (Taghvaei & Jafari, 2015; Vavrusova et al., 2015). Additionally, the presence of free radicals may perform undesired biological functions in the human body; for instance, they may cause cellular damage and aging or promote the development of some illnesses such as cardiovascular or neurological disorders and Alzheimer’s disease (Brandelli, Daroit, & Corr^ea, 2015). There are a wide range of bioactive ingredients that are increasingly being used by the food industry, which are essential due to their antioxidant activity and natural origin (i.e., β-carotene, quercetin, and curcumin). Curcumin is a natural polyphenol found in turmeric root, which has a wide spectrum of biological activities including antiinflammatory, antiproliferative, and antiangiogenic, being particularly relevant as a natural antioxidant agent (David, Zagury, & Livney, 2015). The major challenge associated to the use of curcumin as food ingredient is related to its low solubility in aqueous solution and, as a consequence, its low bioavailability (Li, Cui, Ngadi, & Ma, 2015). It is well reported that curcumin has remarkable properties as bioactive therapeutic agent to help treating several human diseases (Yi et al., 2016). Therefore many efforts have been done to overcome the issues associated to its use and thus emergent approaches such as the use of nanostructured systems made from bio-based materials have been explored (Rafiee, Nejatian, Daeihamed, & Jafari, 2018). Fractions of whey proteins (e.g., β-lg, α-la) isolated or combined with various bio-based materials can be used as promising ingredients to develop innovative delivery systems. According to the available literature (Teng et al., 2014), protein nanostructures composed by β-lg were able to improve the curcumin stability and aqueous solubility. These authors also demonstrated that the nanostructures maintained their integrity in simulated gastric fluid at pH 5 after oral intake, which indicates the ability of these structures as carriers of functional ingredients (Teng et al., 2014). Besides this, nanostructures were also produced from α-la isolated and α-la/dextran conjugates for encapsulation and delivery of curcumin (Yi et al., 2016). The authors evaluated the morphology, physicochemical stability, particle size, and antioxidant activity of such nanostructures loaded with curcumin under different environmental conditions (i.e., pH, temperature, and salt concentration). Results showed that α-la and α-la/dextran conjugates were able to encapsulate curcumin into spherical shape structures with particle size around 60 and 70 nm, respectively, thus protecting curcumin from oxidation, when compared with the results found for free curcumin. Moreover, dextran did not affect the encapsulation efficiency and loading capacity of α-la structures, instead, α-la/dextran conjugates improved the curcumin stability under different environmental conditions (in particular for heating at 95°C) when compared to α-la structures. These findings suggest that α-la can be used as a suitable nanostructure to enhance the solubility, stability, and biological properties of curcumin for food applications.

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2.2 Antimicrobials Antimicrobials are bioactive ingredients frequently used to control microbial contamination in food, inhibit growth of pathogenic microorganisms or delay the microbial spoilage, thus improving the safety and quality of food products, while enlarging their shelf life (Fu, Sarkar, Bhunia, & Yao, 2016; Tajkarimi, Ibrahim, & Cliver, 2010). Food antimicrobials are classified as synthetic and naturally occurring, but due to the growing consumer demand for “green” products, there is an increasing trend for not consuming food products containing synthetic additives and as a consequence, a reduction of their use by food industry (Fu et al., 2016). Epigallocatechin-3-gallate (EGCG) is a biologically active compound present in green tea (Camellia sinensis) and among its antioxidant, antiinflammatory, and antiobesity potential activities, it has been increasingly used due to its recognized antibacterial and antiviral properties (Faridi Esfanjani, Assadpour, & Jafari, 2018; Li et al., 2012). Lestringant et al. (2014) developed β-lg nanostructures for encapsulation of EGCG that showed particle size of 30.0  0.5 nm and binding affinity of 94.0  8.0% (Table 1). In addition, results demonstrated that these systems significantly protect EGCG against oxidation (thus enhancing its storage stability) and exhibited an antiproliferative cellular effect—assessed through in vitro Caco-2 cell monolayer experiments (Lestringant et al., 2014). In another study conducted by Bagheri et al. (2013), date palm pit extract, which is a rich source in phenolic compounds displaying antimicrobial activity, was successfully entrapped into WPI nanostructures, but without the establishment of covalent interactions (as indicated by FTIR assays). Data also showed the formation of spherical particles with 92.9  17.3 nm and that date palm pit extract was efficiently encapsulated, that is, 78.3  2.3% in WPI nanostructures (Bagheri et al., 2013)—see Table 1.

2.3 Flavors/odors Flavors and odors are very relevant features in food quality, often determining consumers’ preference (Mao, Roos, Biliaderis, & Miao, 2015). Whey proteins and their fractions have been successfully used as base material to encapsulate flavors, but different proteins exhibited distinct binding capacities: BSA showed the highest affinity for 2-nonanone flavor, followed by β-lg, while α-la displayed the weakest binding capacity (K€ uhn, Zhu, Considine, & Singh, 2007). Recently, Ghasemi et al. (2018) developed a nanocomplex (formed between positively charged WPC and negative charged pectin through electrostatic interactions) to encapsulate and protect D-limonene from oxidation. The D-limonene-loaded nanocomplex showed a particle size of ca. 160 nm and an encapsulation efficiency of ca. 88.0% (Ghasemi et al., 2018). On the other hand, Giroux and Britten (2011) developed a WPI nanostructure by cross-linking denatured whey protein through pH-cycling method for the encapsulation of ethyl hexanoate as flavoring agents. The WPI nanoparticles formed at pH 5.0 and 5.5, in the absence of calcium, exhibited particle sizes lower than 300 nm,

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Biopolymer Nanostructures for Food Encapsulation Purposes

displaying a more porous and less compact inner, which may promote aroma retention (Giroux & Britten, 2011). In this sense, these nanoparticles have a great potential to control the aroma release in food and beverage products.

2.4 Fatty acids Fatty acids are nutritionally essential due to their recognized beneficial effects on human health enhancement and their consumption may reduce the incidence of diseases such as cancer, diabetes, and cardiovascular problems (Cruz et al., 2015; Tokle, Mao, & McClements, 2013). However, the functional foods fortified with fatty acids are highly susceptible to oxidative deterioration, which may result in the production of unpleasant and unacceptable flavors and odors (Carneiro, Tonon, Grosso, & Hubinger, 2013). In this sense, recent research is trying to overcome these challenges by encapsulating fatty acids, and thus a few studies were highlighted in this regard (Ghorbanzade, Jafari, Akhavan, & Hadavi, 2017). Zimet and Livney (2009) developed promising nanostructures, based in the mixture of proteins and polysaccharides through electrostatic interaction strategy, for encapsulation and controlled delivery of ω-3 poly-unsaturated fatty acids (DHA). Results showed that such nanostructures, displaying a particle size of ca. 100.0 nm, have protected DHA from degradation during heating at 40°C for 100 h, while maintained the colloidal stability. Azizi et al. (2018) investigated the combination of WPI with different lipid-base materials (i.e., lauric, palmitic, and stearic acids), for echium oil encapsulation. Results revealed that lauric acid enhanced the physical stability of colloidal delivery system by reducing the number of surface pores, thus exhibiting a spherical shape with droplets size of 150.0 nm. Also, lauric acid improved the echium oil chemical stability (i.e., prevent oxidation) and showed the highest encapsulation efficiency of the oil, when compared with structures made from palmitic and stearic acids (Azizi et al., 2018).

2.5 Minerals Although mineral elements are needed in reduced amounts by the human body, they are crucial and their deficiency, caused by a lack of daily intake or due to difficulties in absorbing the mineral from food, may result in serious health problems, for example, weak bones, fatigue, or a decreased immune system (Gharibzahedi & Jafari, 2017). Encapsulation may overcome these issues by preventing their interaction with other food compounds (i.e., proteins and lipids), which reduces the mineral bioavailability, thus allowing a higher absorption by human gut (Cruz et al., 2015). In this regard, G€ ulseren et al. (2012) developed nanostructures made from WPI for zinc chloride encapsulation. These nanoparticles displayed a remarkable encapsulation efficiency, that is, between 80% and 100% at pH 3, and stability (i.e., during 30 days at 22°C). These findings showed that nanostructures made from whey proteins constitute promising delivery systems of minerals in acidic food products.

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2.6 Bioactive peptides Bioactive peptides have been reported to promote human health benefits (Udenigwe & Aluko, 2012) and their encapsulation has been extensively studied by pharmaceutical and biomedical industries (Mundargi, Babu, Rangaswamy, Patel, & Aminabhavi, 2008; Pisal, Koloski, & Balu-Iyer, 2010; Sarabandi, Sadeghi Mahoonak, Hamishekar, Ghorbani, & Jafari, 2018). Despite the development of satisfactory nanostructures for encapsulation and controlled release of bioactive peptides for pharmaceutical applications, very little has been done in the food industry. This can be justified by the difficulty to meet the food legislation requirements, in particular to guarantee that all ingredients are GRAS, and that the processing is performed under the permitted conditions (Pinheiro, Gonc¸alves, Madalena, & Vicente, 2017). Indeed, the pharmaceutical knowledge must be explored for food applications in order to achieve the desired characteristics of bioactive peptides delivery systems. The principal challenge faced during the encapsulation of peptides with biological activities in protein-based systems is the chemical similarity between the matrix and the encapsulated ingredient (Mohan, Rajendran, He, Bazinet, & Udenigwe, 2015). The main recent studies encompassing the encapsulation of bioactive peptides were compiled by Tavares, Ramos, and Malcata (2017), which have developed microstructures from β-lg, through high intensity ultrasound, for encapsulation of a bioactive peptide concentrate, and by Wang et al. (Wang, Ren, Ding, Xu, & Chen, 2018), which used WPI combined with beet pectin (through enzyme cross-linking approach) to encapsulate BSA, thus achieving an encapsulation efficiency of 96.9%.

3

Characterization techniques

The general aspects comprising the characterization of whey protein-based nanostructures are similar to the employed techniques for other organic nanostructures. Parameters like size, structure, morphology, surface properties, and biological interaction are essential to define structure-function relationships and ensure the desired functionality ( Jafari & Esfanjani, 2017). Several techniques are available to perform the characterization of such parameters, but because each has its advantages and limitations, complementary techniques must be combined to attain an accurate characterization ( Jafari, Esfanjani, Katouzian, & Assadpour, 2017). In this section, the techniques commonly used to characterize whey protein-based nanostructures will be briefly presented and critically discussed considering the specificities of these systems.

3.1 Size, shape, and surface properties Size, shape, and surface properties affect the general stability, appearance, mechanical properties and ultimately are fundamental to control the delivery and interaction with the body (Pan & Zhong, 2016). As a result, the accurate control of these characteristics is key to achieve a successful nanostructured system with the intended functionality.

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3.1.1 Light scattering and other spectroscopic techniques Light scattering techniques, such as static light scattering (SLS) and dynamic light scattering (DLS), are the primary tools to characterize size distribution of nanostructures. DLS is for sure the predominating technique applied to nanoparticle suspensions, since it is a direct and nondestructive method to determine the hydrodynamic radius distribution on a significant concentration range, with accuracy from 1 nm to submicron range (Xu, 2015). Other scattering techniques, such as small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and Raman scattering, can also be used to obtain information about morphology, aggregation, and even structural features and geometric conformation of nanostructures (Svergun & Koch, 2003). Fluorescence correlation spectroscopy (FCS), consisting of a correlation analysis on the fluorescent fluctuation of particles on a confined space, can be used as alternative to DLS, once it produces similar information such as size, concentration, and diffusion coefficients estimation; additionally, it provides structural and reaction/interaction kinetic data. The requirements of the fluorophores and the limited range of analysis are some of the drawbacks of this technique (Boukari & Sackett, 2008).

3.1.2 Surface properties Surface properties, such as charge and affinity, are of major importance to ensure the right stability and functionality of nanostructures, once they define the extent of interactions between the nanostructures and their environment (Tarhini, GREIGEGerges, & Elaissari, 2017). Zeta potential defines the net electrostatic charge of the particles on a system. It is determined by the mobility of the particles under an electric field, in which higher values of zeta potential (i.e., >30 mV) correspond to sufficient electrostatic repulsion between the particles, and thus to higher colloidal stability (Crucho & Barros, 2017). Other surface properties like hydrophobicity may be determined by contact angle in solids’ surfaces and with hydrophobic fluorescent probes in suspensions. The latter case is particularly relevant because the hydrophobic interactions often define intermolecular bonds among the particles, between the particles and bioactive ingredients, and ultimately biochemical interactions (i.e., enzymes, cell membranes) (Tarhini et al., 2017). Other surface properties can also be determined such as the biological affinity, by using antigen-antibody interaction (Kleber, Krause, Illgner, & Hinrichs, 2004).

3.2 Fractionation techniques Fractionation techniques are based on the separation of macromolecules and supramolecular structures by their shape and size. Gel electrophoresis and chromatography are classical and well-implemented techniques in biotechnology (Nes, 1999). Despite being proficient methods of fractioning, they have low size range definition and quantification capacity. They are, however, relevant in the design of nanostructured protein systems, especially for multiproteins systems (i.e., whey protein-based systems), as

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they can help to understand the aggregation processes, the protein fractions involved, and to characterize polydisperse systems (Roufik, Paquin, & Britten, 2005). Centrifugation and membrane separation are also effective techniques to characterize fractions of nanostructures and molecular constituents, with the added advantage of being nonrestrictive and nonspecific to a size range (Liu, Andya, & Shire, 2006; Philo, 2009). These techniques are also useful as complementary characterization, for instance, in association with methodologies applied in the determination of encapsulation efficiency and loading capacity.

3.3 Imaging techniques Optical microscopy allows the observation of general features of structures up to a few hundred nanometers. However, detailed morphology and sizing is not possible since the technique does not have the adequate resolution for observations at nanoscale (Hartschuh, 2008). On the other hand, the development of fluorescence microscopy allowed visualizing with precision the positioning of fluorescent molecules. Using this technique allows identifying autofluorescent compounds or others that specifically bind fluorescent markers and assess, for example, if a bioactive agent is adsorbed on the surface, included on the matrix, or encapsulated inside a nanostructure (Rust, Bates, & Zhuang, 2006). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are the most common techniques to observe nanostructures. Once these techniques do not rely on light, but in electron beams, the resolution obtained is much higher than optical microscopy, allowing a definition below 1 nm. Both TEM and SEM allow to directly measure size, shape, distribution, and aggregation. Additional analyses can also be performed along with these techniques, such as energy dispersive spectroscopy. This technique performs an elemental analysis in the sample, allowing a qualitative composition assessment and due to its local resolution it may provide information on the location of some compounds (e.g., determine if a compound is included on a matrix, adsorbed on the surface, or entrapped into the interior) (Pan & Zhong, 2016; Su, 2017). TEM provides more spatial resolution and, being a transmission technique, it allows studying the interior of nanostructures. The drawback associated to electron microscopy techniques is mostly related with the significant sample preparation procedures required, especially on liquid and suspension samples. Processes of fixation, drying, staining, or coating with metal are frequently necessary, which may result in undesired changes in the intrinsic physicochemical properties of the native state of material samples (Lin, Lin, Wang, & Sridhar, 2014). Atomic force microscopy (AFM) consists of the acquisition of topographical data by measuring the force between a probe, contacting or not, and the surface being tested. This technique has a high resolution capacity and can obtain a similar information, when compared to that of electronic microscopy, with only few sample preparation procedures required and conditions closer to the physiological ones (Hoo, Starostin, West, & Mecartney, 2008). Furthermore, as AFM is based on the measurement of forces, the structural information and interactions between components can be obtained and characterized.

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Despite the advantages of the microscopy techniques, they are destructive techniques that require significant sample processing and have limited population analysis. Therefore these techniques are commonly used as complementary characterization tools. Thus other methodologies that allow the characterization and evaluation of the nanostructures in situ, without destroying them, and with a high sampling rate (in order to obtain a significant information of populations) are recommended (Lin et al., 2014; Pan & Zhong, 2016).

3.4 Structural properties and composition Microscopy and scattering techniques can also contribute to the characterization of the structure and composition of nanostructures. Electron microscopy can directly observe the interior and surface of materials and retrieve structural information, as it can be used along energy dispersive spectroscopy to obtain information about chemical signatures (Su, 2017). SANS, SAXS, and X-ray diffraction allow a more detailed characterization of structural organization of the nanomaterials, than primary dimension analysis of nanostructures (Svergun & Koch, 2003). Other techniques such as Raman scattering and Fourier-transform infrared spectroscopy (FTIR) are often used as complementary techniques to obtain additional information about the structure and interactions established within the material being characterized (Pan & Zhong, 2016). All these spectroscopic techniques are often used to provide information on the interactions established (i) between the proteins composing the matrix of the nanostructures, or (ii) between the matrix molecules and bioactive ingredients entrapped into the matrix. Other spectroscopic techniques such as the fluorescence of the aromatic amino acid pool and circular dichroism (CD) are often used because they provide information about local conformation and structural properties of proteins (Pelton & Mclean, 2000). Thermal techniques such as differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) can be used to evaluate the stability, association/ dissociation equilibrium, and structural features of nanomaterials and their interactions (Atri, Saboury, & Ahmad, 2015; Yi et al., 2014).

3.5 Characterization strategies The inherent scale specificities of the nanostructured systems give rise to particular physicochemical properties and specific challenges on their characterization. Furthermore, the production of protein-based nanostructures is usually triggered by promoting interactions between the proteins and other compounds. Thus an initial step of functionalization is frequently required, where through a physicochemical process (i.e., thermal, electric, radiation, pressure, enzymatic, chemical) (Nicolai et al., 2011) the protein conformation is changed and their reactivity is boosted. The nature and extent of these modifications dictates the interactions, governing the aggregation process, structure formation, and association of bioactive ingredients. In this sense, the characterization of protein-based systems often includes the characterization and

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control of the proteins before, after functionalization, and during intermediate steps of aggregation (Pereira et al., 2016; Rodrigues et al., 2015). Several strategies can be adopted to characterize whey protein-based nanostructures and their functionalities, depending mostly on the final state of the obtained whey protein-based systems. For structures prepared on suspensions, the primary characterization of size and surface properties occurs during the production process. Light scattering and zeta potential are the main techniques employed to evaluate the size and stability of the systems. Cao et al. (2016) developed proteinbased nanostructures from WPC to encapsulate phytosterols. They characterized the size, polydispersity, and zeta potential by DLS technique to evaluate their stability during storage; while TEM was used to assess their morphology and to confirm the characteristics evaluated by DLS. Afterwards, the nanostructures were subjected to a freeze-drying process and the resulting structures were evaluated in terms of their structural features by DSC and X-ray diffraction to perceive the impact of the encapsulating process and compound on the WPC structure (Cao et al., 2016). In another study, Fan et al. (2017) described the formation of β-lg-chlorogenic acid nanostructures for EGCG encapsulation. The resulting nanostructures were characterized by DLS, zeta potential, and TEM analyses, while the conjugate formation was confirmed by SDS-PAGE and the structural changes evaluated by CD and FTIR (Fan et al., 2017). In the case of solid or solid-like state processes such as the production of nanocoatings, nano-laminated structures, electrospraying, or nano-spray drying, the characterization of these systems requires other specificities based on the physical state of the sample. Zhong et al. (2018) produced electrospun whey protein-based fibers and evaluated their size and morphology by SEM. The data obtained were posteriorly correlated with the viscosity of the solution and with the SANS analysis, indicating a strong relation between the hydration and network formation of the solutions with the fibers’ morphology. Lo´pez-Rubio and Lagaron (2012) produced WPI capsules loaded with β-carotene through electrospraying. The morphology and size of resulting capsules were characterized by SEM, and fluorescence microscopy was used to evaluate the presence of β-carotene inside the capsules, while the inter- and intramolecular interactions and secondary structural changes of the proteins were evaluated through FTIR (Lo´pez-Rubio & Lagaron, 2012). Thus in order to ensure an accurate characterization of whey protein-based nanostructures and their functionality, a complementary set of techniques is needed. These techniques must be adequate to the nanostructure constituents, encapsulated material, and chemical and physical properties of the system. Besides the physicochemical characterization through the techniques mentioned before, the assessment of the biological properties of whey protein-based nanostructures is also essential to confirm their functionality and biocompatibility. In this sense, the evaluation of degradation and release profiles of bioactive ingredients from whey protein-based nanostructures (through an in vitro GI system), and of cytotoxicity are essential to validate their applicability to food products. Thus in the next section, the behavior of whey protein-based nanostructures and the bioavailability of bioactive ingredients will be addressed.

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Behavior of whey protein nanostructures and bioavailability of food ingredients

4.1 Gastrointestinal in vitro and in vivo assessment Whey protein nanostructures’ digestibility is a widely studied topic due to their enzymatic digestion resistance in the native state (e.g., native β-lg nanoparticles are resistant to proteolysis in the stomach) (Bohn et al., 2017; Li et al., 2015; Sarkar, Goh, Singh, & Singh, 2009), or when they are produced at certain production conditions (e.g., semiunfolded state) (Madalena et al., 2016). Nevertheless, whey protein-based nanoparticles showed to be appropriate nanosized delivery structures for the protection of bioactive ingredients that are susceptible to digestion conditions (e.g., riboflavin (Madalena et al., 2016), green tea catechins (Shpigelman, Cohen, & Livney, 2012), among others) or poorly absorbed in the small intestine due to poor water solubility (e.g., curcumin (Li et al., 2015), lutein (Eriksen, Luu, Dragsted, & Arrigoni, 2017; Zhao, Shen, & Guo, 2018), among others). Studies regarding the assessment of whey protein digestibility and bioaccessibility (i.e., the amount of bioactive compound that has been released to the aqueous medium and is ready to be absorbed) and bioavailability (i.e., the amount of bioactive compound that has been absorbed and enters the bloodstream) of food ingredients (Lucas-Gonzalez, Viuda-Martos, PerezAlvarez, & Fernandez-Lopez, 2018) can be found in the literature using in vitro and in vivo digestion models. In vitro digestion assessment experiments are currently widely used since these in vitro models are simpler, cheaper, and more ethical to use, when compared with in vivo ones (Lucas-Gonzalez et al., 2018; Pinheiro et al., 2017). In this regard, it is important to consider that the digestion process is a complex dynamic system that is very difficult to mimic in vitro. Thus several aspects can interfere with protein nanostructures during digestion (e.g., pH variations, temperature, ionic strength, enzymatic hydrolysis, mechanical grinding and mixing, among others) which can, in turn, change the bioavailability and bioaccessibility of food ingredients (Bohn et al., 2017; Lucas-Gonzalez et al., 2018; O’Neill et al., 2015; Sah, Mcainch, & Vasiljevic, 2016). The process of designing whey protein nanostructures must therefore take into consideration the changes in the environmental conditions that occur during the digestion process and the preprocessing strategies (e.g., heating temperature and time, pH, ionic strength, pressure, among others) (Franco, Perez, Conesa, Calvo, & Sanchez, 2018; Ramos et al., 2017). Environmental and production temperature is a key aspect to modulate the behavior of whey protein nanostructures under digestion conditions (Ramos et al., 2017; Sah et al., 2016). For instance, at 37°C, riboflavin is more rapidly released from whey protein hydrogels, when compared to lower temperatures (O’Neill et al., 2015). Moreover, at denaturation temperatures (i.e., temperatures above 60°C) previously inaccessible amino acids become exposed due to protein unfolding (Ramos et al., 2017) and, therefore, more susceptible to enzymatic attack by pepsin in the stomach (Keil, 2012; Sah et al., 2016). On the other hand, β-lg in its

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native form can resist to pepsin digestion under in vitro GI conditions due to its native globular folded tertiary structure that buries important peptide bonds, known as cleavage sites for pepsin hydrolysis (Reddy, Kella, & Kinsella, 1988; Sah et al., 2016). However, in the small intestine, whey proteins showed to be more susceptible to enzymatic degradation by trypsin (Martinez, Martos, Molina, & Pilosof, 2016; Yi et al., 2014) and chymotrypsin (Martinez et al., 2016). This shows that temperature is one of the most important modulation parameters for protein functionality and digestibility, since it can alter whey protein conformation, turning it either more resistant or more susceptible to enzymatic digestion, depending on the conditions applied. The pH variation that occurs during digestion and the ionic strength of the medium also define the whey proteins’ conformation and, therefore, their behavior under digestion conditions. The environmental pH has a major effect over proteins’ inter- and intramolecular interactions by controlling their folding behavior and protein-protein bonding (Ramos et al., 2017). It was previously observed that heated WPI was more susceptible to enzymatic digestion when produced at a pH above its pI. This behavior can be explained by the exposure of hydrophobic amino acid residues due to protein unfolding and aggregation (Zhang & Vardhanabhuti, 2014). Transition between the stomach and the small intestine is characterized by a drastic change in pH (from acidic to alkaline conditions), which is responsible for modifying the protein conformation, thus affecting the tertiary and quaternary structure of whey proteins (Madalena et al., 2016; Ramos et al., 2017). This can promote the enzymatic digestion by trypsin and chymotrypsin in the small intestine (Sah et al., 2016; Zhang & Vardhanabhuti, 2014). The presence of salts during digestion must also be taken into consideration, since it is well known that they change the environmental ionic strength and, consequently, interfere with the electrostatic interactions between proteins (Ramos et al., 2017). In fact, different salts, depending on which protocol is used, are present during the in vitro digestion process (e.g., K+, Na+, Cl , H2PO4, HCO3 ,CO3 2 , Mg2+, NH4 + , Ca2+) (Minekus et al., 2014), which can promote protein aggregation by electrostatic, hydrophobic, and cross-linking interactions (Ramos et al., 2017). Thus the diffusion rate of bioactive ingredients depends on the ionic strength of the digestion phase, where high salt concentration implies higher diffusion rates of bioactives (O’Neill et al., 2015). Despite the development of several in vitro digestion models to simulate the physiological conditions of the human digestive system, there are still some gaps to be filled due to the complexity of the digestion process. Consequently, it is inevitable to use in vivo models to evaluate the digestibility of controlled delivery systems in order to validate and regulate their application in food products. In this sense, different in vivo models have been used to assess the digestibility of whey proteins, such as rats (Diarrassouba et al., 2015; Kitabatake & Kinekawa, 1998), adult pigs (Bohn et al., 2017), and piglets (O’Neill et al., 2015). The studies, carried out until now, show a good in vitro-in vivo correlation regarding whey protein digestibility, where similar peptide release patterns were identified in both in vitro and in vivo studies (Bohn et al., 2017). In fact, Kitabatake and Kinekawa (1998) showed that native β-lg was resistant to in vivo

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gastric conditions, while heated β-lg was more susceptible to pepsin digestion, while both native and heated β-lg were degraded in the small intestine. This behavior was previously observed in vitro and could indicate that whey proteins, in addition to exhibiting a similar behavior in vitro and in vivo, were able to improve the bioavailability of bioactive ingredients, as it was observed by Diarrassouba et al. (2014). These authors have reported that the bioavailability of vitamin D3 was enhanced when encapsulated into β-lg structures, via in vivo studies in adult male rats. Despite the existence of promising evidence that whey protein nanostructures are suitable carriers to improve the bioavailability and bioaccessibility of bioactive ingredients, more studies are needed to better correlate in vitro-in vivo data and to understand the behavior of such delivery systems under digestion conditions.

4.2 Cellular in vitro and in vivo assessment In vitro digestion models are currently being widely used to predict the bioavailability of bioactive compounds. As aforementioned, bioavailability can be defined as the amount of bioactive compound that has been absorbed and enters the bloodstream and, as such, intestinal absorption must be simulated to measure this parameter. For this purpose, several techniques are being employed where dialysis (Bohn et al., 2017; Miller, Schricker, Rasmussen, & VAN Campen, 1981) and centrifugation followed by filtration (Bohn et al., 2017; Kulkarni, Acharya, Rajurkar, & Reddy, 2007) are the most commonly used. However, these techniques have several limitations that could lead to misleading bioavailability predictions. For instance, the prediction of heme-iron or ferritin bioavailability via dialysis would be misleading, since these ingredients are not dialyzable (Bohn et al., 2017). In this sense, cellular models, usually consisting of Caco-2 cell lines, are being used to evaluate the bioactive compound intake, bioavailability, and cytotoxicity. The human intestinal epithelium is a complex pluricellular model (i.e., presence of enterocytes, goblet, enteroendocrine, Paneth, and stem cells (Kleiveland, 2015, Lea, 2015b)) that is responsible for the absorption of digested nutrients due to its microvilli structure. To mimic the human intestinal epithelium, Caco-2 cells have been widely used since they (i) have the ability to spontaneously differentiate into microvilli-like structures with similar enzymes and transporter proteins; (ii) present highly correlated results when compared to in vivo absorption profiles; and (iii) are simple to implement and produce reproducible results, when compared to in vivo assays (Lea, 2015a, 2015b). However, Caco-2 cells are unable to produce mucins that are typically present in the human intestinal mucus. The lack of mucus results in higher permeability, consequently leading to a misleading determination of bioactive ingredients’ bioavailability (Kleiveland, 2015). For this purpose, a cocultured cell line Caco-2/HT29MTX is often used since HT29-MTX cells have the ability to produce mucus (Kleiveland, 2015; Lea, 2015b). Typically, these cellular models are cultured in a porous membrane with 0.4 μm porosity, made from polyester, polycarbonate, or polyethylene terephthalate, which are ideal for cellular transport assays due to its inert nature (Lea, 2015a). The absorption of whey protein structures and the bioavailability of bioactive compounds entrapped into those structures can thus be studied using Caco-2-based cellular models.

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Whey protein nanostructures are GRAS materials (Koh et al., 2015; Ramos et al., 2017) and thus expected to be nontoxic. In fact, evidences show the lack of toxicity of whey proteins nanoparticles in Caco-2 cell assessment (Koh et al., 2015), which makes these systems suitable to be applied in food products, since they can improve the bioavailability of bioactive ingredients (e.g., β-carotene (Yi, Lam, Yokoyama, Cheng, & Zhong, 2015) and curcumin (Li et al., 2015)). Moreover, it has been recently observed that whey protein nanodispersions improved the bioavailability of astaxanthin in Caco-2 cell models (Shen, Zhao, Lu, & Guo, 2018). The cellular uptake of whey proteins can be modulated by changing the protein concentration, heating temperature, surface charge, and particle size. In fact, increasing the heating temperature during the production process can result in an increased surface charge that may lead to stronger electrostatic repulsion between particles and cell membrane and thus, to a reduction in cellular uptake. On the other hand, higher protein concentration is expected to result in higher cellular uptake (Koh et al., 2015; Riihimaki et al., 2008). This can be explained by the fact that whey proteins, specially β-lg, are resistant to pepsin digestion, so using higher protein concentrations may result in higher levels of protein reaching the small intestine, when compared with other controlled delivery systems (e.g., soybean protein isolate (Yi et al., 2015)), thus improving its cellular uptake. Whey protein nanoparticles can permeate through the Caco-2 cell membrane via the transcellular route (Bernasconi et al., 2006; Puyol, Dolores Perez, Sanchez, Ena, & Calvo, 1995; Riihimaki et al., 2008) with degradative and transcytosis pathways (Heyman & Desjeux, 1992; Puyol et al., 1995), indicating that important peptides, resulting from trypsin digestion, can also be absorbed in the small intestine. Hence, whey protein nanoparticles can be applied to enhance the bioavailability of bioactive ingredients, since it has been observed that higher amounts of bioactives can pass through cellular membranes when they are associated. Moreover, Caco-2 cellular models could be used to observe the nontoxicity of whey protein nanostructures and to determine which peptides, from whey protein trypsin digestion, could be absorbed. Despite the potential of whey protein nanoparticles for controlled delivery applications, more studies regarding their toxicity should be made to ascertain their applicability to food products. In fact, there are some concerns regarding the use of nanotechnology in food applications since nanostructures, due to their reduced size, can permeate through cellular and subcellular barriers (Coles & Frewer, 2013; de Souza Simo˜es et al., 2017). However, this is a preliminary step toward regulating the application of these nanostructures in food products. For this purpose, correlations between in vitro (i.e., in vitro digestion and Caco-2 cell models) and in vivo (i.e., animal and human trials) tests must be established to allow a better understanding of the absorption of bioactive ingredients and, therefore, their bioavailability, in order to design efficient and optimized controlled delivery systems. Moreover, cellular models, in particular Caco-2 cell lines, are adequate simulators of the human epithelial intestinal surface, since they present similar behavior regarding nutrient absorption. On the other hand, they still present limitations, for example, lack of important metabolizing enzymes (such as cytochrome P450 enzymes) and absence of intestinal motility. Therefore efforts must be made to develop more reliable systems to better evaluate the bioavailability of bioactive ingredients.

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Concluding remarks

The use of nanotechnology for food industry applications is very recent but, as it has the capability to impact all levels of food production, it is expected that its applicability to food products, in particular in the development of innovative food-grade oral delivery systems for bioactive food ingredients, will grow fast in the incoming years (Ramos et al., 2017). Further, the design and application of whey protein-based nanostructures as drug delivery systems for pharmaceutical purposes, in particular in the nanomedicine field, has been accepted to play a crucial role in the treatment of various life-threatening diseases as it provides safe, effective, and stable therapeutic effects (Verma, Gulati, Kaul, Mukherjee, & Nagaich, 2018). Despite the great potential benefits, significant challenges related with functional aspects still need to be overcome such as degradation of bioactive ingredients when added to complex food matrices during processing and storage conditions, bioavailability and bioaccessibility of bioactive ingredients after passage through the harsh conditions of the GI tract, and the potential toxicity of whey protein-based nanostructures and of the bioactive ingredients to the human cells. These issues have been the subject of recent scientific studies and considerable advances have been done toward a better rationalization of the behavior of such whey protein-based nanostructures in the GI and cellular systems— as shown before; however, more in vitro studies are clearly needed for a full understanding of the real impacts of such nanostructures. All this data, correlated with in vivo experiments, will be essential for a more accurate assessment of the biological activity and fate of the ingested whey protein-based nanostructures and entrapped bioactive ingredients, and to ascertain the real impacts from their use to environment and human health. This information may be a useful tool to overcome unsolved issues related with the development, functionality, applicability, and safety assessment of food-related nanotechnology, thus representing a serious contribution to make nanotechnology safer and to demystify its use, promoting its widespread applicability in the coming years. Moreover, in order to be successful in such a long journey, ethical, legal, and social issues embracing food nanotechnology should also be considered, as well as the proper education of the public in order to gain consumer acceptance.

Acknowledgments Oscar L. Ramos, and Ricardo N. Pereira acknowledge their Post-Doctoral grants (SFRH/BPD/ 80766/2011 and SFRH/BPD/81887/2011, respectively) and Daniel A. Madalena and Rodrigues R. Martins acknowledge their Doctoral grants (SFRH/BD/129127/2017 and SFRH/BD/ 110723/2015, respectively) to the Fundac¸a˜o para a Ci^encia e Tecnologia (FCT, Portugal). Lı´via S. Simo˜es gratefully acknowledges her grant to CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, Brasil) from Brazil. This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/Multi/50016/2019 and UID/BIO/04469 units and COMPETE 2020 (POCI-01-0145FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.

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