**4. Proteins used as coating materials: an emergent trend**

of tolerance. Alterations in these regulatory mechanisms alter the induction of tolerance, resulting in food allergy [30]. An allergy reaction require a complex interaction between the protein and the immune system [26]. The National Institute of Allergy and Infectious Diseases of the United States, identified four categories of immune-mediated adverse food reactions such as IgE-mediated, non–IgE-mediated, mixed, and cell-mediated reactions. The most prevalent non-IgE-mediated reactions are eosinophilic esophagitis (EoE), the food-protein induced enterocolitis syndrome, proctocolitis, entheropathy and celiac disease [41]. The IgE-mediated reaction is by far, the most well established mechanism, where the antibodies bind to the high affinity receptors of mast cells and basophils, and to the low affinity receptors on macrophages, monocytes, lymphocytes and platelets. Thus, IgE are able to bind a specific receptor on the surface of mast cells and basophils, when two or more of these captive IgE molecules bind to their specific antigen, becoming cross-linked on the surface of the cell [26]. Once the allergens penetrate the mucosal barriers and bind to the IgE of mast cells and basophils, these cells release mediators that cause vasodilatation, smooth muscle contraction and mucosal secretion, giving

In order to crosslinking to takes place, at least two antibody molecules must bind to the inducing allergen. An allergen must therefore contain at least two IgE binding sites, each one contains at least 15 amino acid residues. This implies a lower size limit for protein allergens of approximately 30 amino acid residues [26]. The IgE-mediated allergic immune response can be divided into three phases: (i) the sensitization phase in which B lymphocytes switch to the production of specific IgE, (ii) the effector phase consisting of an acute reaction and a facultative late-phase reaction; (iii) a chronic phase that may be the result of repetitive late phase reactions. The acute reaction causes activation of mast cells and basophils releasing histamine, leukotrienes, and other mediators known to be responsible for the wheal and flare reaction occurring in the skin and at various mucosal sites including the eye, nose, lung, and gastrointestinal tract [7]. The IgE-mediated reaction occur immediately or within 1–2 h of ingestion, whereas non-IgE–mediated reactions

A limited number of foods are responsible for the majority of reactions in IgE-mediated food allergy. For instance, allergy to cow milk, eggs, wheat, and soy are more common

Receptor of Ig-E

Histamines released (degranulation)

Symptoms of an immediate hypersensitivity

rise to the typical symptoms of immediate hypersensitivity (**Annex 1**) [30].

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generally have a delayed onset beyond 2 h of ingestion [42].

Ig-E

Bovine β-lactoglobulin (allergen protein)

**Annex 1.** IgE-mediated protein hypersensitivity.

There are many coating materials considered as "generally regarded as safe (GRAS)" which are used in the encapsulation process. Most of them are derived from natural sources such as natural gels (e.g., gum arabic, alginates, carrageenan and mesquite gum), modified starches, maltodextrin and proteins (e.g., whey proteins, gelatin, soy, rice, sunflower and peas) [13, 18, 43–49].

Animal-derived proteins are the most widely used coating materials, either alone or in complexes with polysaccharides applying techniques such as coacervation and spray drying. For instance, Chi and City [50] used whey protein to encapsulate rambutan oil by spray drying. They obtained an EE and yield of 69.9 and 28.5%, respectively using gelatin as a coating material; whereas whey proteins rendered an EE of 73.9% and yield of 58.6%. The best yield and EE was achieved with milk proteins instead of proteins obtained from tissues. Likewise, several researchers have evaluated the encapsulation activity of proteins such as gelatin [19, 45, 51], sodium caseinate in combination with lactose, or maltodextrin and other proteincarbohydrate complexes [19]. Remarkably, Rubio and his team [52] patented the process to obtain microcapsules and nanocapsules based on free whey proteins or complex polysaccharides employing "Blow-spinning," "blow-spraying," "electrospinning" and "electrospraying" as encapsulation techniques. In general, animal proteins tend to be more soluble, flexible and smaller in size at a broader pH range than proteins obtained from plant sources. For instance, casein proteins have a molecular mass of 20 kDa, whereas the molecular mass of soy proteins is 350 kDa. Further, animal proteins have a much faster diffusion kinetics at the interfaces and present a greater stability in emulsions, which is the key in many encapsulation processes [12, 53]. On the other hand, the large globular nature of vegetable proteins presents a greater challenge at stabilizing the interface in an emulsion and these proteins render a low EE. These emulsions are stabilized by increasing the viscosity of the continuous phase instead of acting on the interface, leading to a lower entrapment efficiency of the dispersing compound resulting in a higher susceptibility towards oxidative and degradation reactions. However, plant proteins exhibit several advantages that make them highly attractive in the pharmaceutical, cosmetic and food fields [12, 53]. For instance, plant proteins reflect the current "green" and "clean" label trends, and they are not considered allergenic as compared to the animal-derived proteins. In addition, the niche marketing restrict the use of animal proteins in the diet and increase their cost promoting plant proteins as ideal coating materials from more abundant sources [12, 53].

In order to replace synthetic polymers and animal-derived products, there is a growing interest in the industrial use of renewable resources from natural origin having unexplored applications. Thus, natural macromolecules such as plant proteins have drawn considerable attention due to their availability, biodegradability, renewable character and various physicochemical properties that make them able to form films. The film forming ability of proteins is based on the unfolding characteristics of the protein structure in a solvent. This unfolding is favored by pH changes, addition of electrolytes, heat treatments, or solvent removal [13, 19, 20, 43, 54–56]. The solvents used to prepare the protein solutions are mostly water, ethanol, and rarely acetone. Proteins must be in an open or extended form in order to allow the molecular interactions for the formation of the film to take place. Further, these interactions depend on the protein structure (degree of extension) and the respective sequence of hydrophobic and hydrophilic amino acids in the protein. Therefore, vegetable proteins are very suitable materials for the encapsulation process of active ingredients in the food, pharmaceutical and cosmetic fields [12, 46, 53].

#### **4.1. Encapsulation methods with proteins**

Since the encapsulation methods are diverse and complex, they are classified according to the type of organic solvent, the energy expenditure, or application field. However, the most common classification method is related to the production process. In this case, it could be classified as physical, chemical or physicochemical processes [46, 57]. In general, the encapsulation process involves three steps: (i) the formation of a wall around the core material; (ii) complete closure of the wall, so any possible leakage of the core is avoided; (iii) generation of a capsule, either by chemical reactions or physical treatments [58]. The particle shape thus obtained depends on the physicochemical properties of the core, the coating material and the technique implemented (**Figure 1**) [21].

The morphology (shape and structure) of the microparticles is in turn, classified into two categories: capsules and spheres [18]. In the first case, the capsule is composed of a cluster of particles having a liquid or solid core surrounded by a continuous solid coating, which is generated only by chemical methods. In the second type, the sphere is formed mechanically, either by a process of atomization or milling process. Whereby, the active ingredients are finely dispersed as fine solid particles or liquid droplets within the matrix. A third category may rise comprising more complex structures, such as multilayer capsules or multilayer spheres. Further, both capsule and spherical morphologies should not have defects or pinholes to ensure a greater stability. Therefore, the presence of defects may increase the rate of oxidation or hydrolytic degradation (**Figure 1**) [18, 53]. Further, microparticles might exhibit from irregular to spherical shape. In fact, the coating material is generally adapted to the outline of the particle having a wide variety of shapes [21].

The most common encapsulation techniques include spray drying, extrusion, coacervation, liposome formation, fluid bed coating, inclusion complexes, ionic gelation, lyophilization, cocrystallization and emulsification. As mentioned previously, their morphology is classified into two major categories named as microspheres and microcapsules. Microspheres are typically

of animal proteins in the diet and increase their cost promoting plant proteins as ideal coating

In order to replace synthetic polymers and animal-derived products, there is a growing interest in the industrial use of renewable resources from natural origin having unexplored applications. Thus, natural macromolecules such as plant proteins have drawn considerable attention due to their availability, biodegradability, renewable character and various physicochemical properties that make them able to form films. The film forming ability of proteins is based on the unfolding characteristics of the protein structure in a solvent. This unfolding is favored by pH changes, addition of electrolytes, heat treatments, or solvent removal [13, 19, 20, 43, 54–56]. The solvents used to prepare the protein solutions are mostly water, ethanol, and rarely acetone. Proteins must be in an open or extended form in order to allow the molecular interactions for the formation of the film to take place. Further, these interactions depend on the protein structure (degree of extension) and the respective sequence of hydrophobic and hydrophilic amino acids in the protein. Therefore, vegetable proteins are very suitable materials for the encapsulation process of active ingredients in the food, phar-

Since the encapsulation methods are diverse and complex, they are classified according to the type of organic solvent, the energy expenditure, or application field. However, the most common classification method is related to the production process. In this case, it could be classified as physical, chemical or physicochemical processes [46, 57]. In general, the encapsulation process involves three steps: (i) the formation of a wall around the core material; (ii) complete closure of the wall, so any possible leakage of the core is avoided; (iii) generation of a capsule, either by chemical reactions or physical treatments [58]. The particle shape thus obtained depends on the physicochemical properties of the core, the coating material

The morphology (shape and structure) of the microparticles is in turn, classified into two categories: capsules and spheres [18]. In the first case, the capsule is composed of a cluster of particles having a liquid or solid core surrounded by a continuous solid coating, which is generated only by chemical methods. In the second type, the sphere is formed mechanically, either by a process of atomization or milling process. Whereby, the active ingredients are finely dispersed as fine solid particles or liquid droplets within the matrix. A third category may rise comprising more complex structures, such as multilayer capsules or multilayer spheres. Further, both capsule and spherical morphologies should not have defects or pinholes to ensure a greater stability. Therefore, the presence of defects may increase the rate of oxidation or hydrolytic degradation (**Figure 1**) [18, 53]. Further, microparticles might exhibit from irregular to spherical shape. In fact, the coating material is generally adapted

The most common encapsulation techniques include spray drying, extrusion, coacervation, liposome formation, fluid bed coating, inclusion complexes, ionic gelation, lyophilization, cocrystallization and emulsification. As mentioned previously, their morphology is classified into two major categories named as microspheres and microcapsules. Microspheres are typically

materials from more abundant sources [12, 53].

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maceutical and cosmetic fields [12, 46, 53].

**4.1. Encapsulation methods with proteins**

and the technique implemented (**Figure 1**) [21].

to the outline of the particle having a wide variety of shapes [21].

**Figure 1.** Morphology of the particles obtained by microencapsulation: (a) single microcapsule, (b) microsphere, (c) multilayer microcapsule, (d) multilayer microspheres, and (e) irregular microcapsule.

formed by a physical process such as spray drying, fluid bed coating, extrusion, and multiple emulsification [18, 59, 60]. On the other hand, the chemical process associated to the capsule formation include phase separation, ionic gelation, coacervation and liposome formation [18, 44, 57]. The different methods used for microencapsulation and the resulting main features are presented in **Table 2**.

The choice of the encapsulation technique for a particular process depends on: (i) the desirable size, biocompatibility and biodegradability of the particles; (ii) the physicochemical properties of the core and the coating materials; (iii) the intended use; (iv) the desired release mechanism from the core; and (v) the production costs. Some of the most commonly used encapsulation techniques which employ proteins as the coating material are described as follows:


**Table 2.** Properties of the microcapsules produced by different techniques.

#### *4.1.1. Emulsification*

The emulsification process involves the formation of colloidal systems formed by two immiscible liquids in which the dispersed phase is in the form of small droplets (i.e., between 0.1 and 10 μm) distributed in a continuous or dispersing phase. Emulsions are unstable if they are allowed to stand for some time. As a result, the molecules of the dispersed phase are redistributed forming a layer that can precipitate or migrate to the surface depending on the density gradient between the two phases [21]. In general, emulsions are classified according to the continuous phase when one liquid is dispersed into another. For instance, an O/W emulsion is formed when a hydrophobic liquid is dispersed in water or in a water-soluble liquid. On the contrary, a W/O emulsion is formed when a water-soluble liquid is dispersed in a hydrophobic solvent [57]. Moreover, the stabilization process of the emulsions is achieved by three mechanisms:


One of the mostly widely used strategies to achieve the stabilization of emulsions is by the incorporation of surfactants in the system. These compounds have amphiphilic (i.e., hydrophilic and hydrophobic) regions in their structure and hence, they have the ability to reduce the interfacial tension between the phases of the emulsion system leading to a better stability [62]. Polysaccharides are the most commonly used emulsifiers and are mainly represented by native and denatured starches, phospholipids and proteins. Plant protein properties such as water solubility, amphiphilicity, the ability to self-associate and interact with a variety of substances, a high molecular weight and flexibility make them ideal for encapsulation by emulsification followed by techniques such as spray drying, coacervation, ionic gelation and solvent evaporation [21].

The absorption and emulsifying properties of the proteins at the interface of the colloidal system depend on many factors such as protein structure, state of aggregation, pH, and ionic strength [63]. For instance, caseins, which have a random spiral conformation, tend to form an interweaved layer; whereas whey proteins that are globular in shape usually form aggregates at the interface of the colloidal system [64]. Further, bovine serum albumin, whey protein and proteins isolated from plant sources such as soybean, pumpkin seed, quinoa and peas have been used as emulsifiers in many encapsulation systems, including W/O and W1/O/ W2 double emulsions [65–67]. Proteins could also be combined with other emulsifiers to stabilize emulsions. In fact, protein-polysaccharide complexes having electrostatic interactions between molecules of opposite charge at a certain pH and ionic strength range could stabilize emulsions. For instance, the sodium caseinate/soybean lecithin complex is an stable emulsion which is used to encapsulate phenolic compounds, simultaneously [68].

#### *4.1.2. Spray drying*

*4.1.1. Emulsification*

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by three mechanisms:

pended liquid;

culation rate is reduced.

and solvent evaporation [21].

The emulsification process involves the formation of colloidal systems formed by two immiscible liquids in which the dispersed phase is in the form of small droplets (i.e., between 0.1 and 10 μm) distributed in a continuous or dispersing phase. Emulsions are unstable if they are allowed to stand for some time. As a result, the molecules of the dispersed phase are redistributed forming a layer that can precipitate or migrate to the surface depending on the density gradient between the two phases [21]. In general, emulsions are classified according to the continuous phase when one liquid is dispersed into another. For instance, an O/W emulsion is formed when a hydrophobic liquid is dispersed in water or in a water-soluble liquid. On the contrary, a W/O emulsion is formed when a water-soluble liquid is dispersed in a hydrophobic solvent [57]. Moreover, the stabilization process of the emulsions is achieved

**Table 2.** Properties of the microcapsules produced by different techniques.

**Encapsulation method Particle size (μm) Max. load (%) Type of process Reference** Simple coacervation 20–200 <60 Chemical [61] Complex coacervation 5–200 70–90 Chemical [61] Co-current spray drying 1–100 <40 Physical [21] Counter-current spray drying 50–200 10–20 Physical [21]

i. Formation of a strong emulsifier layer or film around the individual droplets of the sus-

ii. Formation of an electrostatically charged layer on the surface of the individual droplets; iii. Viscosity increase of the dispersant medium. Thus, as the viscosity of the dispersant liquid increases, the Brownian motion slows down decreasing the probability of particles to agglomerate. As a result, the sedimentation rate of the particles or their possible floc-

One of the mostly widely used strategies to achieve the stabilization of emulsions is by the incorporation of surfactants in the system. These compounds have amphiphilic (i.e., hydrophilic and hydrophobic) regions in their structure and hence, they have the ability to reduce the interfacial tension between the phases of the emulsion system leading to a better stability [62]. Polysaccharides are the most commonly used emulsifiers and are mainly represented by native and denatured starches, phospholipids and proteins. Plant protein properties such as water solubility, amphiphilicity, the ability to self-associate and interact with a variety of substances, a high molecular weight and flexibility make them ideal for encapsulation by emulsification followed by techniques such as spray drying, coacervation, ionic gelation

The absorption and emulsifying properties of the proteins at the interface of the colloidal system depend on many factors such as protein structure, state of aggregation, pH, and ionic Microencapsulation by spray drying is the most widely known technique used to encapsulate food ingredients such as vitamins (C and E), fragrances, probiotic bacteria, lipids, vegetable oils, minerals (i.e., iron), anthocyanin pigments, milk, and foodstuffs [45, 47, 48, 60, 69]. The technique of microencapsulation by spray drying has been used since the 1950s and is currently applied at the industrial and academic level due to its rapid speed, economy and simplicity. It involves the dispersion of the active ingredient with the encapsulation material that is pumped into a spray chamber followed by dehydration with circulating hot air at a temperature between 150 and 200°C [45, 47, 48, 60, 69]. Spray drying provides a relatively high EE as compared to other methods. The highest EE achieved with spray drying is between 96.0 and 100.0%, which is much higher than those obtained with alternative methods [18]. Several parameters need to be controlled during the spray drying process. The inlet and outlet drying air temperatures, the feed flow of the product, the residence time and the characteristics of the raw material are the most important factors [18]. The initial mixture needs to be in a form of a dispersion, a solution, or an O/W emulsion having a low viscosity. The process is divided into different stages:


The ideal materials for spray drying should have a low viscosity at high concentrations, a high solubility, have a good emulsifying and film forming ability, and hold efficient thermal properties (i.e., low effective diffusivity and low conductivity) to protect the encapsulated material during the drying process [21]. Thus, the drying process can be carried out following three different patterns: (i) co-current, if the solution is atomized in the same direction than the hot air flow (**Figure 2a**); (ii) countercurrent, if the sprayed microdroplets move in the opposite path than the flow of hot air (**Figure 2b**); and (iii) mixed, if the spray droplets move without a defined trajectory and at a high entropy. Thus, the air flow moves in opposite and/or concurrent directions (**Figure 2c**).

**Figure 2.** Spray-drying pattern types: (a) co-current, (b) counter-current, and (c) mixed.

The average particle size obtained by the atomization process varies between 1 and 100 μm for the co-current and between 50 and 200 μm for the counter-current drying pattern [21].

#### *4.1.3. Coacervation*

Coacervation is a chemical method of phase separation. The term "coacervation" was introduced in the colloid chemistry field by Bungenberg de Jong and Kruyt in 1929 to describe the spontaneous separation of the liquid/liquid phases that can occur when polyelectrolytes of opposing charges are mixed in an aqueous medium [17, 44, 70–73]. Coacervation is defined by the International Union of Pure and Applied Chemistry (IUPAC) as the separation of a colloidal system into two liquid phases. Thus, coacervation is an intermediate state between dissolution and precipitation and hence, it leads to a partial desolvation in opposition to the exhaustive solvation associated to the process of precipitation. Therefore, any factor that involves polymer desolvation causes the coacervation phenomenon [53]. The coacervation-triggering phenomena include temperature changes, pH modification and the addition of an incompatible anti-solvent, salt or another polymer [18, 53]. The coacervation procedure can be classified into two types: (i) simple or (ii) complex coacervation, depending on the number of polymers involved [18]. Complex coacervation mainly occurs by electrostatic interactions between two or more solutions of opposite-charged polymers producing two immiscible liquid phases: (i) one is the continuous phase having a low polymer concentration, whereas, the second one is composed of the polymer rich dense phase, also named as the coacervate phase, which in turn is used to coat a variety of active core ingredients. Usually, the coacervate complexes possess the combined functional properties of each polymer involved [21]. On the other hand, simple coacervation only implies one polymer, and thus, it is not very popular in the food and pharmaceutical fields [18, 43]. In general, the process of coacervation involves the following steps:


#### **4.2. Structural changes in proteins: a strategy to enhance their functionality**

The average particle size obtained by the atomization process varies between 1 and 100 μm for the co-current and between 50 and 200 μm for the counter-current drying pattern [21].

**Figure 2.** Spray-drying pattern types: (a) co-current, (b) counter-current, and (c) mixed.

Coacervation is a chemical method of phase separation. The term "coacervation" was introduced in the colloid chemistry field by Bungenberg de Jong and Kruyt in 1929 to describe the spontaneous separation of the liquid/liquid phases that can occur when polyelectrolytes of opposing charges are mixed in an aqueous medium [17, 44, 70–73]. Coacervation is defined by the International Union of Pure and Applied Chemistry (IUPAC) as the separation of a colloidal system into two liquid phases. Thus, coacervation is an intermediate state between dissolution and precipitation and hence, it leads to a partial desolvation in opposition to the exhaustive solvation associated to the process of precipitation. Therefore, any factor that involves polymer desolvation causes the coacervation phenomenon [53]. The coacervation-triggering phenomena include temperature changes, pH modification and the addition of an incompatible anti-solvent, salt or another polymer [18, 53]. The coacervation procedure can be classified into two types: (i) simple or (ii) complex coacervation, depending on the number of polymers involved [18]. Complex coacervation mainly occurs by electrostatic interactions between two or more solutions of opposite-charged polymers producing two immiscible liquid phases: (i) one is the continuous phase having a low polymer concentration, whereas, the second one is composed of the polymer rich dense phase, also named as the coacervate phase, which in turn is used to coat a variety of active core ingredients. Usually, the coacervate complexes possess the combined functional properties of each polymer involved [21]. On the other hand, simple coacervation only implies one polymer, and thus, it is not very popular in the food and pharmaceutical fields [18, 43]. In general, the process of coacervation

i. Dispersion: A vigorous stirring of the active ingredient (liquid or solid particles) is carried out in a solution of the polymer, or a mixture of polymers that will form the wall material;

ii. Induction: Coacervation is induced by one of the previously described phenomena. The system of the solution becomes transparent and under the microscope the coacervate

droplets have an appearance similar to that of an emulsion;

*4.1.3. Coacervation*

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involves the following steps:

In recent years, the demand for multifunctional products has increased, and researchers need to develop or modify techniques to improve the functionality of proteins since most ofthem show no variability in their desirable functional properties [12, 19, 21]. Even though proteins are versatile materials with interesting properties, it may be necessary to modify the inherent properties such as solubility, hydrophobicity, hydrophilicity, the gelling, emulsifying, foaming and allergenicity. As a result, a versatile material is obtained, with less allergenicity and a wide variety of applications in different fields [12, 19, 21]. Several modifications can be conducted in proteins since amino acids have side chains of different sizes, shapes, charges and chemical reactivity. The reactivity of a protein, in terms of its ability to be chemically modified will be largely determined by the composition and location of the amino acids on its three-dimensional structure. These modifications can be made by physical (i.e., pressure and temperature), chemical or enzymatic methods [19]. Some of the feasible modifications of proteins are illustrated in **Annex 2**.

The term "crosslinking" is commonly used to describe the intra or inter-covalent bonding of a protein. As a result, the molecular size and shape, and the functional properties may be affected by crosslinking. Crosslinking can also be used for the stabilization and subsequent modulation of the release properties of the protein-based controlled release systems. Different methods can be used for crosslinking purposes, ranging from physical to enzymatic and chemical modifications [46, 74]. Crosslinking can be controlled by the proper reaction mechanism, reactive groups of the protein, and type of crosslinking reagent. The number of reactive groups per protein chain, types of functional groups, and spacer length of the crosslinking agent determine the resulting crosslinking density. The most relevant physical modifications of proteins imply variations in temperature and pressure that lead to structural changes or their denaturation. The main disadvantage of the heat treatment as a crosslinking method is that the reaction is difficult to control. The denaturation process of proteins begins when the structure itself becomes an intermediate product. As the reaction progress, the structure is altered, but the secondary structure remains unchanged. Once the chains unfold completely, the denaturation process is concluded. The magnitude of these changes depends on the protein source and environmental conditions (e.g., pH, solvent, presence of salts, surfactants, etc.) [75]. The more reactive the reducing sugar is the stronger and darker the gel becomes [12, 43, 46, 56, 76].

The enzymatic modification of proteins is another useful method. Various enzymes have the ability to crosslink proteins. Examples thereof include transglutaminase (TG), disulphide

**Annex 2.** Schematic representation for the typical chemical modifications of proteins.

isomerase, peroxidase, lipoxygenase, and catechol oxidase. In a recent overview on enzymatic crosslinking, microbial TG, lactoperoxidase and glucose oxidase are highlighted as the enzymes available on a sufficient large scale for industrial applications [19]. TG is an acyltransferase that catalyzes the introduction of c-(y-glutamyl)-lysine crosslinks into proteins, making TG an interesting enzyme for food grade protein crosslinking. TG has been used to crosslink several proteins [19]. In general, caseins appear to be more susceptible to TG-induced crosslinking than whey-proteins, possibly due to the predominantly random structure of caseins, in contrast to the globular structure of whey proteins [19]. A less known type of crosslinking involves the peroxidase-catalyzed reaction between the side chains of two tyrosines, resulting in a C-C bond between the two carbons in an ortho position with respect to the phenol group [19]. This type of crosslinking is very strong due to the intermolecular formation of bonds. The use of co-substrates to enhance the crosslinking efficiency of peroxidases reduces the amount of enzyme needed and therefore increases the cost effectiveness. The co-substrates are phenolic compounds such as mono- and di-hydroxy benzene derivatives (e.g., catechol, ferulic acid, and *p*-hydroxybenzoic acid) and probably act as a spacer between the protein molecules [19].

On the other hand, the chemical modification of proteins has been the most widely studied method among scientists. These reactions are mainly represented by deamidation, acylation, chemical hydrolysis, and cationization reactions. There is a plethora of research conducted on the use of chemical agents for protein crosslinking [19]. The most widely used reagents are bifunctional having two reactive groups that can be used to introduce inter- and/or intramolecular bonds into proteins [19]. These reagents in turn, can be classified as homobifunctional or heterobifuntional. Homobifunctional reagents (i.e., glutaraldehyde) have two identical functional groups, whereas heterobifunctional reagents have two different functional groups. It is difficult to control the reaction conditions (i.e., pH, ionic strength and protein:reagent ratio) of homobifunctional reagents to ensure an intra- or intermolecular crosslinking. On the other hand, heterobifunctional reagents can be used in a more discriminating way. In this case, crosslinking occur in separate sequential steps, and the formation of intermolecular crosslinks can be avoided or stimulated. Tannins are complex polyphenolic substances that can be derived from galls, but also from fruits (e.g., pomegranate) and tea. Tannic acid (TA) is capable of complexing or crosslink proteins by forming a multiple hydrogen bonds network [19]. Proteins can hereby be physically crosslinked and thus, become more resistant to enzymatic degradation. This type of crosslinking is partially reversible. Further, some metal ions can oxidize TA and proteins, whereas other metals could inhibit the formation of hydrogen bonds.

All of these reactions modify the secondary or tertiary structure of the proteins using different compounds that could form linear biopolymers, biopolymers with hydrophobic linkage aggregates outside the structure, cationic or anionic biopolymers, or biopolymers in which certain specific amino acids are exposed. All of these modifications allow for an effective interaction of protein derivatives at the interface of emulsions, or with the active compound in the core, resulting in an improved EE. On the other hand, a protein could loss its original conformation or bonding which is responsible for its allergenicity, e.g., formation of disulfide bonds, which are responsible for binding the IgE and hence, triggering allergenicity.

isomerase, peroxidase, lipoxygenase, and catechol oxidase. In a recent overview on enzymatic crosslinking, microbial TG, lactoperoxidase and glucose oxidase are highlighted as the enzymes available on a sufficient large scale for industrial applications [19]. TG is an acyltransferase that catalyzes the introduction of c-(y-glutamyl)-lysine crosslinks into proteins, making TG an interesting enzyme for food grade protein crosslinking. TG has been used to crosslink several proteins [19]. In general, caseins appear to be more susceptible to TG-induced crosslinking than whey-proteins, possibly due to the predominantly random structure of caseins, in contrast to the globular structure of whey proteins [19]. A less known type of crosslinking involves the peroxidase-catalyzed reaction between the side chains of two tyrosines, resulting in a C-C bond between the two carbons in an ortho position with respect to the phenol group [19]. This type of crosslinking is very strong due to the intermolecular formation of bonds. The use of co-substrates to enhance the crosslinking efficiency of peroxidases reduces the amount of enzyme needed and therefore increases the cost effectiveness. The co-substrates are phenolic compounds such as mono- and di-hydroxy benzene derivatives (e.g., catechol, ferulic acid, and *p*-hydroxybenzoic acid) and probably act as a spacer

**Annex 2.** Schematic representation for the typical chemical modifications of proteins.

On the other hand, the chemical modification of proteins has been the most widely studied method among scientists. These reactions are mainly represented by deamidation, acylation, chemical hydrolysis, and cationization reactions. There is a plethora of research conducted

between the protein molecules [19].

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Currently, many researchers are searching for alternatives to improve the encapsulation properties of vegetable proteins isolated from legumes (e.g., soybeans, peas, chickpeas and lentils), sunflower seeds and cereals (e.g., oats, wheat, barley and corn) [19–21, 44, 71, 77]. The chemical and enzymatic hydrolyses, the acylation [21], cationization [21, 78] and Maillard reactions [12, 19, 77] are the best chemical treatments to improve their functionality [12, 43]. For instance, Nesterenko et al. [46] studied native and modified soybean and sunflower seed proteins to encapsulate α-tocopherol by spray drying. Likewise, the EE increased from 82.6% for native soy protein to 94.8% for the modified product. Moreover, the EE of the native sunflower seed protein was 79.7%, whereas that of the acylated proteins was 99.5%. They concluded that the structural modification of the proteins increased the affinity between the active ingredient and the coating material, improving the encapsulation process, hydration and net protein load. Similar results were obtained by Chen Lia et al. 2015 [76], who encapsulated oleoresin obtained from tomato using soybean proteins conjugated with gum arabic as the coating material. These chemical modifications produced by Maillard reactions were conducted by heating at 60°C and at a relative humidity of 79% for 3, 6 and 9 days. They obtained EE of 69.25, 76.47, 80.91 and 84.69% for the native and modified proteins treated for 3, 6 and 9 days, respectively. The 3D change in the protein structure favors the stabilization of the emulsions, the encapsulation process and improves their biocompatibility.
