**2. Allergenic proteins**

etc. It is the most severe reaction, especially to food containing proteins, and (ii) the non-IgE-mediated allergy such as that of gluten or celiac disease, where the allergic symptom is triggered by ingestion of gluten from cereals namely wheat, rye and barley in their diets. In recent years, food allergies have caused a major health alarm affecting nearly 1% of adult population in the world and from 6 to 8% of children [2–4]. Thus, the prevalence of food allergies has increased in several regions all over the world. Further, more than 170 types of foods have been identified as potentially allergenic [5]. The animal-derived sources include eggs, milk, fish and crustacean shellfish; whereas the vegetable-derived sources include wheat, soy and nuts. The former group of proteins are responsible for causing more than 90% of allergic reactions [5, 6]. Most food allergens are stable molecules that resist the effect of food processing, cooking and the digestive process. These glycoproteins are characterized by their ability to induce a pathogenic IgE response in susceptible individuals [7]. Usually, food allergens are formed by divalent or multivalent molecules with two or more antibodies–binding sites calls epitopes, which are responsible for interacting with immune effector molecules such as the IgE antibodies [2–4]. Moreover, many food proteins especially those derived from ani-

Currently, there is a worldwide search for new materials of natural origin that confers the physical, chemical and sensory characteristics to food products similar to those of synthetic additives applied on a daily basis. These synthetic compounds have been considered as potentially toxic in hypersensitive people, leading to health problems, causing allergies,

Proteins are macromolecules considered as emergents, these are versatile compounds having a good biocompatibility, biodegradability, high nutritional value, amphiphilic properties, and exhibit a strong interaction with several types of active compounds via hydrogen bonds, and electrostatic interactions [12]. Further, proteins are also able to function as emulsifiers, foaming and gelling agents [13–16]. Their chemical and structural versatility makes them suitable candidates for the delivery of bioactive hydrophobic and hydrophilic ingredients from a wide

One of the emerging and promising uses of proteins is in the microencapsulation tecnology of different compounds in the pharmaceutical, food and cosmetic fields. This technology is defined as a mechanical, chemical or physico-chemical process that isolates and protects the potentially sensitive active ingredients (i.e., liquid, solid or gas) from the damaging environment. In most cases, spherically-shaped products are obtained and the resulting particles could be classified according to their size as capsules (1–1000 μm), microcapsules (100–1000 nm) or nanocapsules (1–100 nm). In this process, the active ingredient is protected from the environment by a membrane, which in turn is named as the wall or coating material. This membrane controls the release and stability of the core material [17]. Nonetheless, to date, only a few proteins have been considered to be effective coating materials for the encapsulation of several core compounds such as vitamins, minerals, microorganisms, oils, phenolic compounds, among others. These proteins are mainly obtained from animal sources rather than plant sources. Proteins derived from milk, wheat, soy and cereals are the most widely studied for this aplication, but are considered as allergenic. For this reason, research has focused on the search for new sources of nonallergenic proteins that allows for the modification of their

range of platforms such as particles, fibers, films and hydrogels [16].

mal sources could act as antigens in humans.

hyperactivity, and cancer [8–11].

44 Allergen

Proteins are macromolecules derived from plants or animals that range in size from several thousand to several million Daltons [18, 19]. In general, proteins are composed of amino acids linked through peptide bonds resulting in chain lengths ranging from ~50 to >100,000 amino acids [19]. This sequence creates a characteristic three-dimensional organization (or folding) which in turn, can be organized into four structural levels.

The primary structure of proteins is formed by the linear sequence of bound amino acids. The secondary structure is due to the formation of hydrogen bonds between the carbonyl (-CO-) and amino (-NH) groups forming a folded structure. The latter describes the path that the polypeptide backbone of the protein follows in the three dimentions. The alpha and beta helices are the most important conformations of the secondary structure. Further, the tertiary structure describes the three-dimensional organization of all atoms in the polypeptide chain, including the side groups, as well as the polypeptide backbone. The quaternary structure of the protein is formed by the association between two or more proteins that exhibit a tertiary structure, resulting in a typical functionality [19, 20].

Amino acids are amphoteric in nature and thus, react with acids or bases due to the presence of alkaline (NH2 ) and acidic (COOH) groups. Currently, there are twenty common amino acids having side chains of different size, shape, charge and chemical reactivity [19, 20]. The degree of hydrophobicity and hydrophilicity of amino acids is one of the main determinants of the three-dimensional structure of proteins. Amino acids such as glycine, alanine, valine, leucine, isoleucine, methionine and proline have non-polar aliphatic side chains; whereas phenylalanine and tryptophan have non-polar aromatic side groups. These hydrophobic amino acids are generally found within proteins, forming the so called hydrophobic core [19]. Other amino acids have ionizable side chains such as arginine, aspartic acid, glutamic acid, cysteine, histidine, lysine, and tyrosine; whereas asparagine, glutamine, serine, and threonine contain non-ionic polar groups, which are often found on the surface of the protein allowing for a strong interaction with the aqueous ambient [19].

Proteins are mainly obtained from: (i) oilseeds and protein crops such as soybeans, rapeseed, peas, lentils, broad beans and sunflowers; (ii) cereal sources such as wheat, oats, rye, barley, maize, rice, sorghum, millet and quinoa; and (iii) algae [21]. Vegetable proteins also possess several advantages such as a good biodegradability and biocompatibility, have a low cost and a high availability, and pose no health risks [22, 23].

Proteins can be classified according to their sedimentation coefficient (S), which is defined as the rate of sedimentation per unit of acceleration of the particle in a medium. This property varies according to the molecular weight, conformational space and behavior of the protein in the environment. However, there are four large families of plant proteins that are classified according to their solubility into globulins, albumins, prolamins and glutelins. Albumin and globulins are the main constituents of plant-derived proteins [19].

Albumins are highly soluble in water and have a molecular weight ranging from 10 to 100 kDa. This group includes mostly proteins that present structural and storage functions [24]. In general, albumins contain high levels of lysine and sulfur amino acids, such as methionine and cysteine, and a high amount of disulfide bridges that favor the resistance against thermal denaturation [19, 20].

On the other hand, globulins are the main proteins obtained from vegetable sources. They are soluble in aqueous saline solutions and are mainly composed of two fractions having a sedimentation coefficient of 7S and 11S, respectively, which in turn depend on the plant source and culture conditions. Globulins are an important part of storage proteins, and have a molecular weight of more than 100 kDa. They are rich in arginine, aspartic acid, glutamic acid and their amides [19, 20].

Glutelins and prolamins are proteins soluble in basic aqueous solutions and hydroalcoholic mixtures, respectively. Their structure has been poorly studied, but these fractions represent agglomerates of globulins bound together by disulfide bonds and hydrophobic interactions, resulting in a complex morphology [21].

Currently, there are 16,712 recognized protein families in the Pfam database, but only 255 (~1.13%) of them are considered as allergens. Further, only 0.16% of the top 20 families account for ~80% of all reported cases of food allergenicity (**Table 1**) [25].

Further, it has been observed that the structural properties of proteins significantly affect their functionality, including the allergenic potential [25]. A survey conducted on common protein allergens reveals that they possess a wide range of physical characteristics, and none of them is unique to a class of protein allergens. Nevertheless, one report suggested that allergens tend to be ovoid in shape, although it is unclear why this should contribute


**Table 1.** Classification of allergenic proteins according to their family and source.

to allergenicity [26]. Further, the fact of having repetitive motifs also contribute to allergenicity [26]. Many of the important allergens are exceptionally heat stable and retain their allergenicity after heating [26]. However, there are proteins not considered as allergens that possess any of the properties described previously [26].

Particular claims have been made regarding the contribution of intramolecular disulfide bonds to the allergenicity of proteins. Thus, proteins from house dust mites could induce allergenicity if disulfide bonding through site-directed mutagenesis occurs. Otherwise, allergenicity prevents making proteins unable to bind IgE. Further, the loss of disulfide bonds may cause allergens to lose their immunologic identities (i.e., the ability to bind pre-existing specific IgE antibody) and hence, the ability to initiate the novo IgE production [26]. Further, the induction of IgE antibody by wheat agglutinin could be lessened by reducing disulfide bonds with thioredoxin [26]. However, the ability of disulfide bonds to have a qualitative contribution to the allergenicity of wheat agglutinin is still unknown. A quantitative reduction of the antigenicity of a protein could result in a reduction of the allergic response to a protein without altering its intrinsic allergenicity, only if the vigor of the immune response is reduced. In order to determine whether the intrinsic allergenicity of a protein has been altered, it is necessary to assess independently its allergenicity and its antigenicity. This may be achieved experimentally by simultaneously measuring both the IgE (allergenic) and IgG (antigenic) response upon exposure. Further, it seems likely that disulfide bonds influence allergenicity, in unpredictable ways. Their presence can profoundly affect the processing and stability of antigens, and hence the release or destruction of T-cell epitopes [26]. The presence of multiple intramolecular disulfide bonds *per se* does not make a protein as an allergen, nor does their absence preclude allergenicity. For instance, ovalbumin is considered as an allergen despite of having only a single intramolecular disulfide bond that contributes little to its stability, whereas bovine serum albumin (BSA) having 17 intramolecular disulfide bonds is much less allergenic [26].

### **2.1. Allergenic animal proteins**

classified according to their solubility into globulins, albumins, prolamins and glutelins.

Albumins are highly soluble in water and have a molecular weight ranging from 10 to 100 kDa. This group includes mostly proteins that present structural and storage functions [24]. In general, albumins contain high levels of lysine and sulfur amino acids, such as methionine and cysteine, and a high amount of disulfide bridges that favor the resistance against thermal

On the other hand, globulins are the main proteins obtained from vegetable sources. They are soluble in aqueous saline solutions and are mainly composed of two fractions having a sedimentation coefficient of 7S and 11S, respectively, which in turn depend on the plant source and culture conditions. Globulins are an important part of storage proteins, and have a molecular weight of more than 100 kDa. They are rich in arginine, aspartic acid, glutamic

Glutelins and prolamins are proteins soluble in basic aqueous solutions and hydroalcoholic mixtures, respectively. Their structure has been poorly studied, but these fractions represent agglomerates of globulins bound together by disulfide bonds and hydrophobic interactions,

Currently, there are 16,712 recognized protein families in the Pfam database, but only 255 (~1.13%) of them are considered as allergens. Further, only 0.16% of the top 20 families

Further, it has been observed that the structural properties of proteins significantly affect their functionality, including the allergenic potential [25]. A survey conducted on common protein allergens reveals that they possess a wide range of physical characteristics, and none of them is unique to a class of protein allergens. Nevertheless, one report suggested that allergens tend to be ovoid in shape, although it is unclear why this should contribute

Tropomyosin Animal Lipocalin Animal: arthropod and mammalian

Alpha/beta-caseins Mammal Trypsin-like serine proteases Animal: arthropod and mammalian

account for ~80% of all reported cases of food allergenicity (**Table 1**) [25].

**Family Source Family Source** Prolamin superfamily Plant Oleosins Plant

Cupin superfamily Plant Beta-1,3-glucanase Plant Profilin Plant Papain-like cysteine protease Plant EF-hand domain Plant, animal Thaumatin-like protein Plant

**Table 1.** Classification of allergenic proteins according to their family and source.

PR-10 Plant Expansin, C-term Plant: all grasses

Hevein-like domain Plant Enolase Fungi and plants

Class I chitinases Plant Expansin, N-term Plant: all grasses except 1

Albumin and globulins are the main constituents of plant-derived proteins [19].

denaturation [19, 20].

46 Allergen

acid and their amides [19, 20].

resulting in a complex morphology [21].

Animal food allergens are classified into three general families such as tropomyosins, EF-hand proteins, and caseins [27]. However, regardless of their family the ability to act as an allergen appears to be related to relative identity with human homologues. Thus, if the protein has a sequence identity from 54 to 63% with respect to human homologues, then it is considered as allergenic. Nevertheless, a higher identity does not implies allergenicity [27, 28].

The family of tropomyosins is divided into four types of muscle proteins, where none of them causes an IgE response in humans [25]. In fact, no human IgE response to tropomyosin from birds or fish has been identified. This is expected since these sequences have an identity with human tropomyosins greater than 63%, which is translated in the absence of allergenic activity [27].

EF proteins form the second family of animal-derived food allergens, and they are composed of parvalbumin [27]. Parvalbumins are divided into α- and β-parvalbumin. Theα-parvalbumin is considered to be non-allergenic, whereas β-parvalbumin is found in a variety of fish species, retaining the allergenic potential and is absent in human muscle [28].

Out of the caseins that have been shown to elicit an IgE response in humans, the rule of thumb is the closer the sequence to the human equivalent is, the less likely an IgE response will occur. Conversely, sensitization to BSA is the main predictive marker for the cross-reactivity to cow's milk that is present in 73–93% of patients with beef allergies, despite of the fact that BSA shares 76% identity to its human homolog [27, 29].

Cow's milk and egg allergies are some of the most common food allergies found in young children. It is estimated that ~3.8 and 2% of children younger than 5 years old have cow's milk and egg allergies, respectively. These allergens are commonly found in a variety of foods due to their technological and nutritional importance [6]. Thus, lactoglobulin is the major allergen in cow´s milk. Milk contains more than 20 protein fractions. In the curd, four caseins account for ~80% of milk proteins. The remaining 20% of the proteins, are globular proteins (e.g., lactalbumin, lactoglobulin, and bovine serum albumin), and are found in whey and egg proteins from both components (i.e., yolk and egg white) causing sensitization [30].

Parvalbumin represents the major clinical cross-reactive fish allergen. It contains heat-resistant linear epitopes that are s sensitizing by the interaction of metal-binding domains. In addition, other fish allergens are found in collagen and gelatin isolated from skin and muscle tissues. On the other hand, in shellfish, crustaceans and mollusks, tropomyosin is the major allergen that triggers allergic reactions [31].

#### **2.2. Allergenic vegetable proteins**

About 65% of plant food allergens belong to the group of prolamins, termites and the group of proteins related to pathogenesis (PR-10) [25]. The prolamin family include storage proteins, lipid transfer (LTPs), alpha-amylase/protein inhibitors and albumins from most cereal seeds. Their 3D structure consists of a compact structure with four alpha-helices stabilized by disulfide bridges and a central cavity used for lipid binding. However, similarity in their third-party structures does not indicate a similarity in their amino acid sequence between proteins in the same group. Further, prolamins from wheat are known to cause baker's asthma and celiac disease in humans. Alpha-amylase/trypsin inhibitors from various cereals, such as wheat, barley and rice, have also been involved in allergies. Some of the clinically reported allergenic plant proteins belongs to the family of prolamins such as Ara h 2, Ara h 6, Sin a 1, Ber e 1, Ses i 2 and Jug v 1. Non-specific LTPs are known to be the main food allergens in the fruits of the *Rosaceae* family. The presence of specific IgE in LTP is considered a significant risk factor for allergy and may serve as a diagnostic marker [25]. The most relevant termite-type allergens are the 7S and 11S globulins. 7S globulins include Arah1, Jugr2 and Sesi3, whereas the 11S globulins include the Arah3, soy glycinins, Bere2, Cora9 and Fage1 [32]. The third structure of the proteins belonging to the termite group consists of a series of antiparallel β leaves associated with an α-helix forming a cavity [32]. This structure is also found in several lipocalin-like proteins involved in the transport of hydrophobic ligands, including milk β-lactoglobulin [32]. Further, plant prophylines share about 70% of the amino acid sequence homology [32–34].

All gliadin and glutenin protein fractions have been described as wheat grain allergens. The main properties of these proteins is to form a continuous viscoelastic network when flour is mixed with water to form a dough to be used in products such as bread, pastries, pasta, and crackers. However, wheat gluten exhibits a low solubility in aqueous solution. This fact limits the applications of wheat gluten in various types of food, since a good solubility is the main requirement for use in liquid foods and beverages. Furthermore, solubility is closely related to other functional properties of proteins such as the foaming, emulsifying, and gelling ability [35]. In soybean there are eight registered proteins which could trigger an allergic response. However, two of them named as β-conglycinin and glycinin represent ~70% of the soybean proteins. As a result, the incidence of soy protein allergy is much lower than other food allergens, such as milk or peanut proteins [36].

There are over 10 allergenic proteins identified in peanuts of which the Ara h 1, Ara h 2, and Ara h 3 are the most abundant peanut allergens and cumulatively represent approximately half of the total protein content [37]. Furthermore, the prevalence of allergy to tree nuts is estimated to be about half of that of peanut allergy. Tree nut allergic reactions tend to be severe and accidental exposures are common. Walnut, cashew, almond, pecan, Brazil nut, hazelnut, macadamia nut, pistachio, and pine nut are the most common tree nuts responsible for allergy cases in the USA and Europe. Moreover, from 20 to 50% of peanut allergic patients are also allergic to tree nuts [38].
