Bananas: Cultivation, Consumption and Crop

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Bananas: Cultivation, Consumption and Crop Diseases

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Editors: Valerie A. Pearson Book Description: Bananas arise as one of the most popular fruits consumed all around the world. Banana belongs to the genus Musa from the family Musaceae. It is original from tropical regions and presents a strong ability to protect itself from the oxidative stress caused by extreme climatic conditions such as intense sunshine and high temperature. For this protection, bananas increase the production of bioactive compounds with antioxidant activity, which protect the fruit from the oxidative damage. This book provides current research on the cultivation, consumption and crop diseases of bananas. Chapter One addresses the biochemical characterization of Musa spp. genotypes, with emphasis on bioactive secondary metabolites of interest to human nutrition. Chapter Two focuses on the composition of bananas, including macronutrients, micronutrients and bioactive compounds, as well as the effect of postharvest treatments and storage conditions in the quality of bananas. Chapter Three reviews sustainable management of banana waste through renewable energy and bio fertilizer generation. Chapter Four reviews some of the

Special Focus Titles 01. Surgery of the Orbital Cavity: “NoMan'sLand” 02. Public Health: Some International Aspects 03. Diets and Diseases: Causes and Prevention 04. Strategic Intervention: What to Do When Things Go Wrong 05. Lifelong Learning: Concepts, Benefits and Challenges 06. Ageing Disgracefully, with Grace: Enjoying Growing Older 07. Information Literacy Education in Japanese Libraries for Lifelong Learning 08. Music Therapy in the Management of Medical Conditions 09. Cultural Theory for the Humanities 10. Grape Seeds: Nutrient Content, Antioxidant Properties and Health Benefits 11. Ecological Restoration: Global Challenges, Social Aspects and Environmental Benefits 12. Comparative Effectiveness Research (CER): New Methods, Challenges and Health Implications

recently reported valuable uses of banana pseudstem sap (BPS), for growth of sustainable agricultural process, food technology and valueadded medicinal products, and in textiles for improving certain functional attributes. The final chapter examines banana as an important food allergen source. (Imprint: Novinka)

Table of Contents: Preface

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Altering of Fatty Acids 09. Food Processing and Engineering Topics 10. Mycotoxicoses in Animals Economically Important

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Chapter 1 Musa spp. Functional Properties, Biofortification, and Bioavailability (C.V. Borges and M. Maraschin, Agronomist Engineer, M.Sc, Ph.D student – State University of São Paulo, Campus Botucatu – PostGraduation Agronomy, Brazil, and others)

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Chapter 2 Chemical Composition and Bioactive Compounds in Bananas and Postharvest Alterations (Raquel P. F. Guiné and Daniela V. T. A. Costa, CI&DETS Research Centre, Polytechnic Institute of Viseu, Viseu, Portugal, and others) Chapter 3 Sustainable Management of Banana Waste through Renewable Energy and BioFertilizer Generation (Chao Li, Ivo Achu Nges and Jun Fang, Department of Biotechnology, Lund University, Lund, Sweden, and others) Chapter 4 Banana Pseudostem Sap: Source of Valuable Plant Bio Molecules from Waste (S Basak, S K Chattopadhyay and Kartick K Samanta, Central Institute for Research on Cotton Technology (CIRCOT), Indian Council of Agricultural Research (ICAR), Matunga, Mumbai, India, and others) Chapter 5 Banana as a Food Allergen Source (Jasna Nikolić, Milica Popović, and Marija Gavrović Jankulović, Faculty of Chemistry University of Belgrade, Department of Biochemistry, Belgrade, Serbia) Index

Series: Agriculture Issues and Policies Binding: ebook Pub. Date: 2016 Pages: 6x9 (NBCR) ISBN: 9781634854290 Status: AN

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Chapter

BANANA AS A FOOD ALLERGEN SOURCE Jasna Nikolić, Milica Popović and Marija Gavrović-Jankulović

*

Faculty of Chemistry University of Belgrade, Department of Biochemistry, Belgrade, Serbia

ABSTRACT Banana is a perennial herbaceous plant widely cultivated in the tropical and subtropical regions. The pulp of the fruit is a rich source of minerals, vitamins, antioxidants, low glycemic carbohydrates, and fiber, and thereby its consumption has beneficial effects on human health. These nutritional values and its pleasant taste induced the introduction of banana fruit into human diet in infancy and also during convalescence. However, in spite of positive health effects, banana fruit has been recognized as an important food allergen source. The clinical manifestations of banana allergy have usually been associated with mild, local symptoms denoted as oral allergy syndrome. However, more severe reactions, as well as cases of anaphylactic reactions to banana fruit have been registered. IgE reactivity of banana is associated with different proteins, and, so far, only six allergens have been identified and characterized: profilin - actin binding protein (Mus a 1), a class 1 chitinase (Mus a 2), non-specific lipid transfer protein (Mus a 3), thaumatin-like protein (Mus a 4), beta-1,3-glucanase (Mus a 5), and recently registered ascorbate peroxidase (Mus a 6). In this review, we will *

Corresponding author: [email protected].

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Jasna Nikolić, Milica Popović and Marija Gavrović-Jankulović* address the structural features of identified banana allergens and correlate in vitro and in vivo clinical reactivity with their structural homologs from other allergen sources.

INTRODUCTION Bananas are a major food crop globally and they are grown and consumed in more than 100 countries throughout the tropic and sub-tropic regions (INIBAP, 2000). In developing countries, they are the fourth most important food crop after rice, wheat and maize. The genus Musa is thought to be derived from the Arabic name for the plant (mouz) (Hyam and Pankhurst, 1995). Banana fruit, besides its pleasant taste, contains a variety of beneficial nutrients: high levels of the biogenic amines (dopamine and serotonin), antioxidants (vitamin C, vitamin E, beta carotene), flavonoids (catechins, indole alkaloids) and vitamin K. Banana is a good source of vitamin B6 (pyridoxine) which is an important B-complex vitamin that embrace the metabolism of proteins, carbohydrates, and lipids and has a beneficial role in the treatment of several pathophysiological conditions. The fruit is also a moderate source of vitamin C. The consumption of fresh bananas provides minerals like copper, magnesium, and manganese which are important for various metabolic activities in the human body. Magnesium is important for bone mineralization and has a cardio-protective role. Manganese is a co-factor for more than 300 enzymes in the body, including the antioxidant enzyme superoxide dismutase. Copper plays an important role in iron metabolism and is required for the biological activity of ceruloplasmin, a blood constituent involved in the production of erythrocytes. Fresh banana is a very rich source of potassium, an important component of cell and body fluids that helps control blood pressure. However, the biochemical composition of banana fruits depends on the cultivar, abiotic factors such as climate, cultivation method and nature of the soil (del Mar Verde Mendez et al., 2003). Plant cell-wall polysaccharides (pectins, celluloses, hemicelluloses) are an extremely diverse set of biopolymers, which play a very important role as structural elements (Emaga et al., 2008). Dietary fiber mainly consists of soluble (pectins, gums etc.) and insoluble (cellulose, lignin, hemicelluloses, etc.) fiber fractions (Thebaudin and Lefebvre, 1997). Soluble fibers are well known to lower serum cholesterol and to help reduce the risk of colon cancer (Burkitt et al., 1974; Kelsey, 1978). Although pectin is interesting for its

Banana as a Food Allergen Source

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dietary fiber status, its main use depends on its techno-functional properties (gel-forming agents, stabilizers, or emulsifiers). For this reason, pectins are widely used in food, and pharmaceutical and cosmetic industries (Pilnik, 1990). According to the recommendations of USDA Special Supplemental Nutrition Program for Women, Infants, and Children (USDA, 2009) solid foods should be introduced between 4 and 6 months of age and banana is among the first raw foods to be consumed. Despite a range of beneficial effects on human health, the adverse reactions to banana have also been reported. These include allergies to banana and other forms of intolerance due to the amines present in the banana fruit (Maintz and Novak, 2007). Banana allergens are IgE reactive proteins or glycoproteins identified in the proteome of banana extract, which reveal diverse structure and biological activity. This study will give an overview on the banana proteome; discuss some obstacles for the isolation of total banana proteins, as well as banana proteins responsible for allergies. A differential expression of proteins during fruit ripening influences qualitative and quantitative profile of allergen extracts employed in diagnostic protocols. On the other hand, one of the major goals in allergy research is the development of accurate protocols for diagnosis in order to design a patient-tailored specific immunotherapy.

PROTEOMIC ANALYSIS For the understanding and identifying of regulatory mechanisms responsible for the accumulation of nutrients in the fruit, the discovery of protein content by proteomic studies is often performed (Toledo et al., 2007; Balbuena et al., 2011). Such kind of analysis contributes to the identification of the proteins which have essential role in cell metabolism and maintaining the cell structure, as well as the functioning and regulation of many physiological processes. It is possible to identify banana proteins which are affected by ripening, those important for the quality of fruits, or those responsible for developing allergies. These analyses have become essential in food sciences so, during past decades, many research groups focused their research on investigating proteins from this important crop. Many of the banana proteins identified and characterized so far have been deposited in various protein sequence databases. The Uniprot (http://www.uniprot.org/) database contains more than 800 registered proteins from M. acuminata species. Most of those proteins are enzymes involved in

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Jasna Nikolić, Milica Popović and Marija Gavrović-Jankulović*

development, maturation, protection from pathogens, and remodeling processes in banana fruit pulp. The level of protein occurrence depends of the developmental stages of the fruit. While databases like Uniprot provide data on all of the identified proteins, there are several databases which register novel allergens, such as a database of the International Union of Immunological Societies (www.allergen.org), AllergenOnline (www.allergen online.org) within the Food Allergy Research and Resource Program at the University of Nebraska, or Allergome (www.allergome.org). The analyses of protein maps from banana pulp at different developmental stages of maturation, using 2 dimensional - difference in-gel electrophoresis (2D-DIGE) technology, provided valuable information on the ripeningassociated changes in the tissue. This kind of analyses may also provide information on differences that occur in proteins involved in banana flavor, texture, defense, synthesis of ethylene, and regulation of expression. Fruit ripening is followed by an ethylene production, a conversion of starch into oligosaccharides, and a high respiratory activity. These changes require de novo protein synthesis (Areas et al., 1988). It was also shown that during maturation of banana fruit, the activities of the enzymes involved in starch biosynthesis were decreasing, unlike enzymes involved in starch degradation (Wu et al., 1989; Agravante et al., 1990; Iyare and Ekwukoma, 1992; Cordenunsi and Lajolo, 1995; Hill and ap Rees, 1995a, 1995b; Mugugaiyan, 1993; Dominguez-Puigjaner et al., 1992). In addition, messenger RNA (mRNA) produced intensively during fruit ripening, were mostly involved in pathogenesis, deterioration, or stress response, which explains again a rise of proteins synthesis (Clendennnen et al., 1997). The proteomic analysis of banana fruit is especially challenging due to the high abundance of interfering substances in the food matrix (pectins, starch, polyphenols, etc.). Many different methods on protein extraction from banana have been developed (Amoako-Andoh et al., 2014). In some cases (e.g., 2D PAGE) it is important to retrieve total banana protein extract and then analyze them in denaturing conditions. High water and low protein content in banana tissue contribute to low protein recovery during extraction (Amoako-Andoh et al., 2014). The proteomic studies of edible fruit are often performed to help understand regulatory mechanisms responsible for the accumulation of nutrients in the source material (Toledo et al., 2012; Balbuena et al., 2011). These kinds of studies also contribute to the identification of the proteins which are affected by ripening and allow detection of post-translational


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modification of proteins which are involved in the regulation of many enzymefunction and regulatory proteins.

BANANA PROTEINS RELATED TO PATHOGENESIS Pathogenesis related (PR) proteins represent a set of different polypeptides synthesized by plants in response to various biotic and abiotic stress factors. Many of the plant proteins, considered as potential food allergens, are related to different families of PR proteins (Hoffmann-Sommergruber, 2002). In the course of banana fruit development, an inactive homolog of class III chitinase is intensively synthesized (up to 40% (w/v) of the total proteins. During further stages which are related to the ripening of the fruit, the amount of this chitinase-related protein (CRP) progressively decreases, while several novel proteins appear. It was shown that thaumatin-like protein (TLP), beta1,3-glucanase, class I chitinase, and a mannose-binding lectin are the most abundant ripening-associated proteins (Peumans et al., 2002). Proteomic analysis revealed that chitinases (EC 3.2.1.14) are the most abundant proteins in the pulp of unripe bananas, and 11 isoforms of the class III acidic chitinase of 30-kDa were identified (Toledo et al., 2012). The main function of chitinase is in the plant defense against pathogens by hydrolyzing a glycosidic bond in chitin, which is also related to growth and development processes (Toledo et al., 2012; Kasprzewska et al., 2003). Chitinases are common in different fruit tissues and they are classified within the PR-3 protein family. Class III chitinase is the most abundant banana fruit protein in unripe fruit tissue. During the ripening process, the production of this class of chitinase is downregulated, while the amount of class I chitinase (31 kDa) increases, particularly in the pulp, (Peumans et al., 2002; Clendennen et al., 1997). Class I chitinases have an N-terminal hevein-like domain (Collinge et al., 1993), which is structurally similar with hevein and prohevein, the major latex allergens (Alenius et al., 1995). Beside the presence of the N-terminal cysteine-rich lectin or 'hevein' (chitin-binding) domain, they also contain a highly conserved catalytic domain (Clendennen et al., 1997). It was shown that there are at least 2 isoforms of class I chitinase in banana fruit pulp, which share 80% homology in sequence with hevein, in the first 16 amino acids (Mikkola et al., 1998). Endo β-1,3-glucanase (Mw 33 kDa, EC 3.2.1.39) represents one of the most abundant protein in banana pulp (Peumans et al., 2002). This enzyme belongs to the PR-2 family, whose members are very common in many fruits.

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Similar to many other plant endoβ-1,3-glucanases, the banana glucanase exhibits allergenic properties because of the occurrence of well conserved IgEbinding epitopes on the enzyme surface (Shearer et al., 2004). This banana protein also shares high homology with Hev b 2 from the rubber tree latex, and it is considered as the molecular basis for the development of IgE crossreactivity in patients allergic both to latex and banana fruit. It has also been identified as a potent allergen, recognized by IgE from sera of atopic patients allergic to latex, olive pollen, or tomato (Mikkolo et al., 1998; ReceveurBrechot et al., 2006). Molecular modeling performed using β-1,3 linked glycan trisaccharide molecule as a substrate for this enzyme, confirmed that the banana enzyme catalyzes cleaving of β-1,3-glycosidic linkage (ReceveurBrechot et al., 2006). Beside cleaving β-1,3-glycosidic linkage, enzyme assays designed by using pustulan as a substrate, showed that the banana glucanase also cleaves β-1,6-glucans. Very abundant banana fruit enzyme which also displays the β-1,3glucanase activity, is thaumatin like protein (Ban-TLP). As it is shown for TLPs from other sources, biological activity and physiological role of BanTLP is related to antifungal properties. This protein belongs to the PR-5 protein family. The structure of this 200 amino acids (Mw 21kDa) allergen with antifungal properties was defined by X-ray crystallography by Leone and coworkers (Leone et al., 2006). Three distinct domains are thus shown, stabilized by well conserved eight disulphide bridges. Due to the presence of the electronegative cleft, Ban-TLP has a strong local electronegative characteristic, correlated with an antifungal activity. The structural analysis showed the presence of conserved residues. The characteristic acidic cleft located between domains I and II represents a possible explanation for the relatively low endo β-1,3-glucanase activity of Ban-TLP and it seems that it has no biological relevance (Menu-Bouaouiche et al., 2003; Leone et al., 2006). The possible mechanism of biological activity for this enzyme is its insertion into the lipid bilayer of the pathogen membrane, thus making a transmembrane pore which changes the cell permeability (Vigers et al., 1991). Non-specific lipid transfer proteins (Mw 9 kDa, nsLTPs) belong to PR14 protein family. PR-14 family is characterized by a common fold of four αhelices stabilized by four disulfide bonds that form a central hydrophobic tunnel interacting with lipid molecules (Houser et al., 2010). nsLTPs were originally named after their ability to bind and enhance the transfer of a multitude of different types of lipid molecules across cell membranes (Sinha et al., 2014; Silverstein et al., 2007). nsLTPs are widely distributed in plants and have several known roles in fruit metabolism. It seems that nsLTPs have a role


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in the transport of cutin and suberin monomers to the outer layer of plant organs (Douliez et al., 2000). LTPs are also involved in cutin formation, where they act as carriers of acyl monomers and in the process of cell wall extension (Nieuwland et al., 2005). In most cases nsLTPs are located in the peel of fruits rather than in the pulp (Fernandez-Rivas et al., 1999; Marzban et al., 2005). These proteins are present in significant amounts in vascular tissue and in the outer cell layers of plants. They are involved in plant defense against bacterial and fungal pathogeneses as well as different environmental stresses such as drought, heat, cold, or salt (Zilversmit et al., 1984; Kader at al., 1996). nsLTPs perhaps play a role in plant growth and development, including embryogenesis, germination, and pollen-pistil interaction (Salcedo et al., 2007).

BANANA PROFILIN AND PROTEINS OF OXIDATIVE PROCESSES Banana profilin (Mw 14 kDa) is an actin-binding protein involved in the dynamic turnover and restructuring of the actin cytoskeleton, sharing high homology with other fruit and pollen profilins. This highly conserved molecules share sequence identities of more than 75% between members of distantly related organisms. The conservation of amino acid sequence is reflected in highly similar structures and biological function (Hauser et al., 2008). Profilin function is to bind to actin and affect the structure of the cytoskeleton. If the profilin is present in high concentrations, then it prevents the polymerization of actin, while if it is present at low concentrations, it enhances actin polymerization. This protein plays an important role in cytokinesis, cytoplasmatic streaming, cell elongation, growth of pollen tubes and root hairs, membrane trafficking and organization, as well as signaling pathways (Ramachandran et al., 2000; Valster et al., 1997; Vitke et al., 2004; Gibbon et al., 1998). Three-dimensional fold common to profilins is presented by two α-helices and a five-stranded anti-parallel β-sheet (Hauser et al., 2008). Unlike nsLTP, profilins are sensitive to heat denaturation and gastric digestion, so it is considered that they cannot cause allergic sensitization via the gastrointestinal tract (Breitenederet al., 2004; Rodriguez-Perez et al., 2003).

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During the fruit ripening, oxidative processes that occur inside banana tissue are accompanied by the activity of a number of enzyme systems, such as those related to the regulation of reactive oxidative species (ROS). ROSscavenging enzymes include superoxide dismutase, ascorbate peroxidase, glutathione reductase and catalase (Scandalios, 2002; Mittler et al., 2004). Ascorbate peroxidase represents the key enzyme of the major hydrogen peroxide detoxifying system in plant chloroplasts and cytosol, known as the ascorbate-glutathione cycle (Asada, 1992). The systematic name of this enzyme class is L-ascorbate: hydrogen-peroxide oxidoreductase. Hydrogen peroxide is produced inside banana tissue as a by-product during normal metabolic processes, as well as during stress conditions (oxidative stress, pathogen attacks, extreme temperatures, drought, ozone wounding, and senescence) (Blokhina et al., 2003). It was also shown that the treatment with ethylene increases the level of hydrogen peroxide content in the banana fruit during softening (Yang et al., 2008). The mechanism of action relies on the usage of ascorbate as an electron donor to convert the hydrogen peroxide into water. The banana fruit ascorbate peroxidase (Mw 12. 7 kDa, EC 1.11.1. 11) active-site includes amino acid sequence 33APLMLPLAWHSA44, with the proximal heme-li-Band located between residues 155DIVALSGGH165 (Bairoch, 1991). This enzyme has been identified in many higher plants, with isozymes distributed in different cellular compartments, including the cytosol, chloroplasts, mitochondria and peroxisomes (Shigeoka et al., 2002). Wang and associates (Wang et al., 2012) showed that ascorbate peroxidase gene from banana is predicted to code a cytosolic isoform, since the analysis of the amino acid sequence encoded by its cDNA revealed only a common core catalytic region without organelle-specific N-terminus transit peptide sequences or the C-terminus trans-membranous region found in membrane bound isoforms of this enzyme from other sources (Wang et al., 2012).

ALLERGY TO BANANA In one of the earliest reports of adverse reactions to banana the population of 2,067 allergic persons was studied in 1968-1969, 36 patients complained on various symptoms after eating banana. Among these symptoms were: itching throat, ‘gassiness’ and indigestion, cramps, diarrhea, vomiting, sore mouth or tongue, ‘canker sores,’ swollen lips, wheezing, hoarseness, urticaria and other rashes, and angioedema (Anderson et al., 1970).


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The first publication dealing with an allergen present in various fruits including banana, was that of a 30 kDa protein (Wadee et al., 1990). This report describes a patient with rhinitis and OAS after ingestion of banana and other fruits (Wadee et al., 1990) and confirms the finding of Anderson et al., (Anderson et al., 1970) of an association between ragweed pollinosis and banana. Over the years, clinical manifestations of banana consumption have been usually associated with mild symptoms; however more severe reactions have been described, among which even cases of anaphylactic reactions (Hauswirth and Burks, 2005; Saraswat and Kumar, 2005). Besides primary sensitization to banana fruit, cross-reactivity with latex and some pollen has also been reported (Grob et al., 2002; Lavaud et al., 1995). Latex-fruit syndrome was first reported by M’Raihi et al., (M'Raihi et al., 1991), wherein an allergic reaction to banana was observed in a latexallergic patient. Soon thereafter, cross-reactivity between latex and various fruits was demonstrated, and generally, this immunological status was termed latex-fruit syndrome (Blanco et al., 1994). The association between pollen sensitization and banana allergy was first described in 1970 by Anderson et al., (Anderson et al., 1970) but their work focused on adult population as well as the subsequent work by Grob et al., and Reindl et al., (Grob et al., 2002; Reindl et al., 2002). Anderson et al., associated banana cross-reactivity with ragweed pollinosis, while Grob et al., and Reindl et al. describe crossreactivity with birch pollen. In case of pediatric patients there have been several case reports (Ito et al., 2006; Moreno-Ancillo et al., 2004), but first detailed study describing banana and pollen cross-reactivity was published by Palacin et al. in 2011 (Palacin et al., 2011). So it has become evident that allergic reactions to banana fruit occur in two different forms. One type of allergic reaction is related to an allergy to tree pollen such as birch (Informall 2007) and results in the oral allergy syndrome; symptoms include itching and swelling of the mouth and throat, usually within one hour of ingestion. Molecular basis for the allergic reactions are due to the allergen profilin denoted as Mus a 1. Profilin is an important mediator of IgE cross- reactivity of antigens from different sources; cross reactivity between the banana profilin and birch profilin, Bet v 2 and the latex profilin Heb b 8 have been demonstrated (Grob et al., 2002). As a result of the widespread IgE cross-reactivity, this has led to the description of profilins as pan-allergens (Wagner and Breiteneder 2002). A second type of allergic reaction to banana fruit is associated with a latex allergy. This type of allergy causes urticaria (severely itchy skin) and gastrointestinal symptoms. Anaphylaxis and recurrent loss of consciousness

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have been reported in severe cases (Cinquetti et al., 1995; Woltsche-Kahr and Kranke 1997). People with latex allergy often show an allergy to other fruits (avocado, mango and kiwi fruit), and common IgE epitopes in latex, banana and avocado extract have been identified (Moller et al., 1998). Two of the major allergens of banana involved in the fruit-latex syndrome are the 32-33 and 34-37 kD class I chitinases, respectively. Palomares et al. (Palomares et al., 2005) reported on molecular basis of pollen-latex-fruit syndrome which was based on 1,3-β-glucanases, enzymes widely distributed among higher plants.

ALLERGENICITY OF BANANA PROTEINS According to WHO/IUIS Allergen subcommittee database, allergy to banana is attributed to six different allergens (www.allergen.org): Mus a 1 banana profilin, Mus a 2 - class I chitinase, Mus a 3 - non-specific lipid transfer protein type 1 nsLTP-1, Mus a 4 - thaumatin-like protein TLP, Mus a 5 – beta-1,3-glucanase and Mus a 6 – ascorbate peroxidase (Table 1). Mus a 1 was registered as a banana allergen in 2002 (Reindl et al., 2002). Reindl et al. showed that 44% of the tested banana allergic patients reacted to recombinant banana profilin in immunoblot (Reindl et al., 2002). Inhibition experiments indicated similar IgE reactivity of natural and recombinant allergens. In addition, high cross-reactivity to birch pollen profilin Bet v 2 and latex profilin Hev b 8 was demonstrated by immunoblot inhibition as well as in EAST inhibition experiments (Reindl et al., 2002). Due to high crossreactivity with birch pollen profilin (of about 40%), in a group of banana allergic children, sensitization to profilin was confirmed by using palm pollen profilin Pho d 2 as a marker (Palacin et al., 2011). Positive responses to Pho d 2 were found in 40–50% of the subjects by both ELISA and skin prick test (Palacin et al., 2011). Mus a 1 is regarded as an important mediator of IgE cross-reactivity between pollen and exotic fruits. Mus a 2, is a class 1 chitinase. It was first identified as a major banana allergen by Sanchez-Monge et al. (Sanchez-Monge et al., 1999). They have isolated and identified two isoforms both with hevein-like domains. The isolated allergen isoforms reached inhibition values higher than 90% in CAP inhibition assays, and fully inhibited the IgE-binding by the crude banana extract when tested by an immunoblot inhibition method. The two purified allergens provoked skin prick test responses in more than 50% of the bananaallergic patients (Sanchez-Monge et al., 1999). The authors of this study


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postulated that the presence of chitinases with hevein-like domains in other fruits and nuts, such as avocado and chestnut, could explain the crosssensitization among these foods. In recent years using a new purification protocol Nikolic et al. were able to obtain high yield of purified Mus a 2 and to show that, by using purified allergens, higher sensitivity in IgE detection was achieved when compared to the routinely used extracts (Nikolic et al., 2014). However, a more detailed study conducted in 2011 by Radauer et al. (Radauer et al., 2011) reported no correlation between latex-associated plant food allergy and sensitization to hevein or hevein-like domains. Authors considered hevein and hevein-like domains form latex, banana and avocado chitinases. They have for the first time examined sensitization profiles to hevein and hevein-like domains in a representative population of latex-allergic patients which were not preselected for sensitization to certain allergens or the existence of plant food allergy. Their findings indicated that hevein-like domains do not play a specific role in latex allergy without sensitization to fruits (Radauer et al., 2011). Mus a 3 is a non-specific lipid transfer protein (ns-LTP). It is a homologue of Pru p 3 form peach (40% N-terminal amino acid sequence identity). The putative prevalence of Mus a 3 has been estimated by IgE immune-detection in banana fruit extract using individual sera from banana allergic patients (n = 36). The recognition level of Mus a 3 was around 20% of the sera tested. However, the low amount of Mus a 3 in the banana fruit extract strongly suggests revising this estimated prevalence (Palacin et al., 2011). On the other hand, no banana reactivity has been found in adult LTP-allergic patients from Italy, and this fruit has been consequently proposed as a safe food for LTPallergic subjects (Asero et al., 2007; Asero et al., 2002). Thus, cross-reactivity between Pru p 3 and Mus a 3, as well as the putative role of the latter as the primary sensitizer in banana allergy, remain to be clarified. Thaumatine-like protein (TLP) from banana is designated as Mus a 4 by the IUIS Allergen Nomenclature Sub-Committee (Palacin et al., 2011). Contrary to other allergenic TLPs, Mus a 4 is not glycosylated, such as those from kiwi fruit and apple (Gavrovic-Jankulovic et al., 2002; Oberhuber et al., 2008). Mus a 4 is a major banana allergen in the pediatric population investigated by Palacin et al. (Palacin et al., 2011), as indicated by in vitro (72% of sera with specific IgE to) and in vivo (50% of positive SPT responses) reactivity, respectively. A more recent study using a representative panel of 16 purified TLPs on a microarray indicated that TLPs, including that from banana, are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy (Palacin et al., 2012). A

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novel purification procedure by Nikolic et al. (Nikolic et al., 2014), as in the case of Mus a 2, lead to the isolation of both IgE and IgG immune-reactive allergen. Mus a 5 is a beta-1,3-glucanase. It was first identified as an allergen in 2002 by Grob et al. (Grob et al., 2002). In this study, the purified Mus a 5 was probed with sera from 51 banana allergic patients and 84% of sera showed IgE reactivity. It is a homolog of an important latex allergen β-1,3-glucanase (Hev b 2) from Hevea brasilianis (Grob et al., 2002) and shows significant homology with the Ole e 9 allergen from olive pollen (Barre et al., 2009). There are conflicting data concerning glycosylation of Mus a 5 and its potential to provoke the symptoms of allergy in banana sensitized individuals. Palacin et al. found the molecule to be glycosylated and according to their study this glycosylation is the reason why there is a significant difference between its in vitro (specific IgE detected in 84% of sera from banana-allergic infants) and in vivo (8% of positive SPT responses) reactivity (Palacin et al., 2011). A more recent study by Aleksic et al., however, did not detect the presence of glycosylation on purified Mus a 5. The authors were able to demonstrate capability of Mus a 5 to induce clinical reactivity with upregulation of CD63 and CD203c molecules in a concentration-dependent manner in the patient monosensitized to banana (Aleksic et al., 2012). Recombinant Mus a 5 has also been produced as a novel reagent suitable for the component-resolved allergy diagnosis of banana allergy (Abughren et al., 2012, Mrkic et al., 2013). Table 1. Banana allergens Allergen Mus a 1 Mus a 2 Mus a 3 Mus a 4 Mus a 5 Mus a 6

Biochemical name Profilin Class I chitinase Non-Specific LTP type 1 Thaumatin-like protein Beta 1,3- glucanase Ascorbate peroxidase

MW (SDS-PAGE) 15 kDa 33 kDa 9 kDa 20 kDa 30 kDa 27 kDa

Food Allergen Yes Yes Yes Yes Yes Yes

Mus a 6 is the latest reported banana allergen. It is an ascorbate peroxidase of 27 kDa. According to IUIS database (www.allergome.org) both natural and recombinant Mus a 6 show IgE reactivity (10 of 11 positive by IgE to natural protein in reducing immunoblot; 7 of 11 positive to recombinant protein (E. coli) in immunoblot in the group of patients from Thailand.


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Although six IgE reactive proteins have been identified in the banana fruit, there are still undiscovered allergen forms in this allergen source which should be evaluated in terms of their clinical relevance and included in allergen microarrays for more refined food allergy diagnosis.

CONCLUSION Because of its nutritive value and pleasant taste, banana fruit is very popular and it is among the first raw foods introduced in the human diet. However, besides its beneficial effects on human health, banana can induce severe, even life-threatening allergic reactions in genetically predisposed (atopic) persons. Identification and clinical validation of IgE reactive proteins from banana fruit should provide more refined allergy diagnosis, which is the first step in the decision tree for a therapeutic approach. Understanding the mechanisms of the sensitization phase of allergic reaction and particular contribution of individual allergens to this process should provide novel therapeutic concepts for food allergy in the future.

ACKNOWLEDGMENTS The work was supported by the Ministry of Education, Science and Technological Development, Republic of Serbia, Grant No. 172049.

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