Changes Induced by Pressure Processing on Immunoreactive Proteins of Tree Nuts

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Article Changes Induced by Pressure Processing on Immunoreactive Proteins of Tree Nuts

Carmen Cuadrado 1,* , Africa Sanchiz 1 , Fatima Vicente 1, Isabel Ballesteros 2 and Rosario Linacero 3

1 Departamento de Tecnología de Alimentos, SGIT-INIA, Ctra. La Coruña Km. 7.5, 28040 Madrid, Spain; [email protected] (A.S.); [email protected] (F.V.) 2 Facultad de Ingeniería y Ciencias Aplicadas, Grupo de Investigación en Biotecnología Aplicada a Biomedicina (BIOMED), Universidad de Las Américas, 72819 Quito, Ecuador; [email protected] 3 Departamento de Genética, Fisiología y Microbiología Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-91-347-6925

Academic Editor: Maksymilian Chruszcz Received: 29 January 2020; Accepted: 20 February 2020; Published: 20 February 2020

Abstract: Tree nuts confer many health benefits due to their high content of vitamins and antioxidants, and they are increasingly consumed in the last few years. Food processing is an important industrial tool to modify allergenic properties of foods, in addition to ensuring safety and enhancing organoleptic characteristics. The effect of high pressure, without and with heating, on SDS-PAGE and immunodetection profile of potential allergenic proteins (anti-11S, anti-2S and anti-LTP) of pistachio, cashew, peanut, hazelnut, almond, and chestnut was investigated. Processing based on heat and/or pressure and ultra-high pressure (HHP, 300–600 MPa) without heating was applied. After treating the six tree nuts with pressure combined with heat, a progressive diminution of proteins with potential allergenic properties was observed. Moreover, some tree nuts proteins (pistachio, cashew, and peanut) seemed to be more resistant to technological processing than others (hazelnut and chestnut). High pressure combined with heating processing markedly reduce tree nut allergenic potential as the pressure and treatment time increases. HHP do not alter hazelnut and almond immunoreactivity.

Keywords: pistachio; cashew; peanut; hazelnut; almond; chestnut; allergens; pressure processing; thermal processing

1. Introduction Food allergy affects to 1–3% in the general population, and it reaches up to 8% in children. Currently, food allergy is the main cause of anaphylactic reactions treated in hospital emergency departments in Western countries. It has been estimated that food allergy causes approximately 30,000 anaphylactic reactions and 2000 hospitalizations annually in the U.S. [1]. In the European Union, tree nuts are included in the list with the 14 most allergenic ingredients, according with this regulation, and its presence must be indicated in foods. Tree nuts are, after fruits, the most implied allergens (26%) in allergic reactions in Spain [2]. Despite these facts, tree nuts consumption is on the rise because of its recognized health benefits. Tree nuts are valuable foods rich in proteins, minerals, vitamins, antioxidants, and a considerable high content of unsaturated fatty acids, and their global production is increasing in the last years [3]. Most nut allergens are seed storage proteins such as vicilins (7S globulin subunits composed of approximately 50–60 kDa), legumins (11–13S globulin subunits composed of acidic peptides of 30–40 kDa and basic 15–20 kDa) and 2S albumin (15 kDa) [4]. Other allergens of nuts with known biological function, such as lipid transfer proteins (LTP), profilins, and proteins homologous to pathogenesis-related (PR)

Molecules 2020, 25, 954; doi:10.3390/molecules25040954 www.mdpi.com/journal/molecules Molecules 2020, 25, 954 2 of 11 proteins are considered panallergens because they contribute to the allergenicity of a large group of pollen, nuts, seeds, fruits, and other plants [5]. A common feature of the nut allergenic proteins is their resistance to proteolysis and denaturation [6]. Among nuts, cashew (Anacardium occidentale) allergy is a significant health problem and the second most allergic nut in the U.S. [7]. Clinical manifestations of allergy to cashew often are severe anaphylactic reactions, and even more severe than with peanuts [7]. Three allergenic proteins of cashew have been identified and characterized: Ana o 1 (7S vicilin, 50 kDa) [8], Ana o 2 (11S legumin, 55 kDa) [9], and Ana o 3 (2S albumin, 14 kDa) [10]. Pistachio (Pistacia vera) is another well characterized nut for its allergenic potential and cross-reactivity with cashew and mango [11]. Similar to cashew, the 5 pistachio major allergens correspond to seed storage proteins: two 11S legumins (Pis v 2 and 5, 32 and 36 kDa), a 2S albumin (Pis v 1, 7 kDa), one 7S vicilin (Pis v 3, 55 kDa), and a superoxide dismutase (Pis v 4, 25.7 kDa) [12–14]. Peanut allergy is one of the most common IgE-mediated reactivity to food because of its severity and lifelong persistence [15]. Considerable effort has been spent in characterizing peanut allergens and eleven allergenic proteins have been identified until now (Ara h 1-Ara h 11). The major peanut allergens, Ara h 1 (65 kDa, vicilin) and Ara h 2 (17 kDa, conglutin), are recognized by 70–90% of sensitized subjects [16] and Ara h 3 (11 S legumin) has been considered to play a lesser allergenic role [17]. Food allergies to hazelnut and almond represent an important health problem in industrialized countries because of their high prevalence and severity [18]. Several hazelnut allergens are well characterized being Cor a 1 (18 KDa, Bet v 1 family) the major one. Other allergenic proteins are Cor a 2 (profilin), Cor a 8 (lipid transfer protein, LPT), Cor a 9 (11S globulin), Cor a 11 (vicilin-like protein), and Cor a 12, Cor a 13, and Cor a 14 belonging to the 2S albumins [19]. So far, eight allergenic proteins have been described in almond: Pru du 1 (PR-10 protein), Pru du 2 (thaumatin-like protein), Pru du 2S albumin, Pru du 3 (lipid transfer protein), Pru du 4 (profilin), Pru du 5 (60S ribosomal protein), Pru du 6 (11S-legumin), and Pru du γ-conglutin (7S vicilin) [20]. Of these eight allergens, Pru du 3, Pru du 4, Pru du 5, and Pru du 6 are recognized as such and included in the WHO IUIS allergen − list. Allergy to chestnuts has been widely reported in the latex-fruit syndrome [21,22]. However, few studies address actual allergy to chestnuts in patients reacting primarily to this food. According to WHO-IUIS list three food allergens have been identified until the date in this tree nut, all of them from the plant defense system: Cas 5, a class I chitinase [23], Cas s 8, a non-specific type I lipid transport protein (LTP, lipid transfer protein) [24] and Cas s 9, a cytosolic class I small heat shock protein [25]. Additionally, a thaumatin like protein, Cas s TLP, has been described as chestnut food allergen [26]. Food processing, that is used in food industry for several applications, can alter the structure, function, and properties of proteins, and thereby also modify the IgE reactivity of allergens, in such a way that it has been proposed as a method to obtain food with altered allergenicity. Some daily processing methods have been shown to be effective in decreasing the content of specific allergens in certain foods, which may open a future path for hypoallergenic food development or pave the way to the use of specifically processed foods for tolerance induction. Other treatments, however, have the capacity to increase the allergenicity of certain foods [27]. Food processing, particularly heat treatment, may reduce the allergenicity of some foods, so it could be useful for the control of their allergenic risk [28]. Thermal treatment changes the structure of proteins, their immunogenic potential, and, therefore, the overall allergenicity of the food. These effects depend on the temperature, type, and duration of the treatment, as well as of the intrinsic characteristics of the protein and of the physicochemical conditions of its microenvironment [29,30]. There are no general rules about the effect of processing on the allergenicity and hence, it has the ability to generate new allergenic epitopes (neoallergens) as well as to abolish the existing reactive epitopes [31–33]. Several studies have evaluated the structural changes induced by treatments such as boiling, microwave heating and pressure-cooking and their effects on legumes and nuts allergenicity, showing that processing based on pressure and heat seems to have an important impact on reducing in vitro IgE-binding capacity [31,32,34–36]. High-hydrostatic pressure (HHP) is considered an emerging processing technology used to develop novel and high-quality foods. This novel-processing technique even renders harmless foods, which would be of considerable benefit to Molecules 2020, 25, x FOR PEER REVIEW 3 of 11

Moleculestechnique2020 even, 25, 954 renders harmless foods, which would be of considerable benefit to consumers.3 HHP of 11 treatment of foods can be used to create new products (new texture or taste) or to obtain analog products with minimal effect on flavor, color, and nutritional value and without any thermal consumers.degradation. HHP It is treatment well established of foods that can higher be used pressure to create has new a disruptive products effect (new textureon the tertiary or taste) and or toquaternary obtain analog structure products of most with globular minimal proteins, effect on with flavor, relatively color, and little nutritional influence value on the and secondary without anystructure thermal [37,38 degradation.]. Therefore, It ishigher well establishedhydrostatic thatpressure higher can pressure unfold proteins. has a disruptive The typical effect pressure on the tertiaryneeded andfor the quaternary unfolding structure is around of 500 most MPa globular but it varies proteins, from with protein relatively to protein, little in influence the range on from the secondary100 MPa structureto 1 GPa [37 or, 38 reaching]. Therefore, even higher higher hydrostatic pressures pressure in special can cases. unfold The proteins. effect ofThe HHP typical on pressureimmunoreactive needed forproteins the unfolding is being currently is around investigated 500 MPa but through it varies changes from protein in protein to protein, structure in [ the39]. rangeSuch effects from 100 have MPa been to 1studied GPa or in reaching beef [40,41 even], higherapple [37,38 pressures], celery inspecial [37], and cases. in nuts The such effect as of peanut HHP on[38 immunoreactive]. However, there proteins is scarce is being information currently on investigated the effects through of suchchanges food processing in protein techniques structure [39 on]. Suchhazelnut effects and have almond been studiedimmunoreactive in beef [40 proteins.,41], apple [37,38], celery [37], and in nuts such as peanut [38]. However,Our thereaim is is to scarce study information the effect onof high the effects pressure, of such without food processingand with heating, techniques on onSDS hazelnut-PAGE andand almondimmunodetection immunoreactive profile proteins. of potential allergenic proteins (anti-11S, anti-2S and anti-LTP) of pistachio, cashew,Our peanut, aim is to hazelnut, study the almond eﬀect, ofand high chestnut. pressure, without and with heating, on SDS-PAGE and immunodetection proﬁle of potential allergenic proteins (anti-11S, anti-2S and anti-LTP) of pistachio, cashew,2. Results peanut, and Discussion hazelnut, almond, and chestnut.

2.2.1. Results Electrophoretic and Discussion Pattern and Immunodetection Assays of Processed Cashew 2.1. ElectrophoreticThe characterization Pattern andof the Immunodetection electrophoretic Assays profile of Processedof the soluble Cashew protein extracts from untreated and thermal-treated cashew samples is shown in Figure 1. The results showed that the treatments of The characterization of the electrophoretic proﬁle of the soluble protein extracts from untreated boiling during 60 min had no major effects on the IgG-binding proteins from cashew. However, and thermal-treated cashew samples is shown in Figure1. The results showed that the treatments of cashew subjected to heat and pressure treatments (autoclave) showed less distinctive stained bands boiling during 60 min had no major eﬀects on the IgG-binding proteins from cashew. However, cashew in SDS-PAGE with an increased protein fragmentation that went along with a reduction in IgG- subjected to heat and pressure treatments (autoclave) showed less distinctive stained bands in SDS-PAGE reactive bands. with an increased protein fragmentation that went along with a reduction in IgG-reactive bands.

Figure 1. SDS-PAGE and IgG anti-11S, anti-2S, and anti-LTP immunoblots analysis of control (ST),

boiledFigure for 1. 60SDS min-PAGE (B60) and and IgG autoclaved anti-11S, atanti 121-2S◦,C and for anti 30 min-LTP (AU2) immunoblots and at 138 analysis◦C for of 30 control min (AU4) (ST), cashewboiled for samples. 60 min Molecular (B60) and weight autoclaved marker at was121 Precision°C for 30 Plusmin (P(AU+).2) and at 138 °C for 30 min (AU4) cashew samples. Molecular weight marker was Precision Plus (P+). We additionally studied the electrophoretic and IgE-binding patterns of total protein extracts from cashew,We which additionally were obtained studied by the direct electrophoretic solubilization and of untreatedIgE-binding and patterns treated cashewof total flours protein in theextracts SDS samplefrom cashew buffer, aswhich described were obtained in Materials by direct and Methods solubilization (Supplementary of untreated material. and treated Figure cashew S1). flours A band in aroundthe SDS 13 sam kDaple probably buffer as corresponding described in toM theaterials 2S albumin and Methods Ana o 3(Supplementary [10] was especially material. immunoreactive. Figure S1). TheA band IgE reactive around bands 13 kDa were probably strongly corresponding reduced in the to samplesthe 2S albumin treated Anawith o heat 3 [10 and] was pressure, especially but possibleimmunoreactive. to detect evenThe IgE in the reactive sample bands AU atwere 138 strongly◦C, 15 min. reduced in the samples treated with heat and pressure,In the but Western possible blot to with detect IgG even anti-11S, in the anti-2S, sample and AU anti-LTP at 138 °C (Figure, 15 min.1), arrows indicate the protein bandsIn detected the Western (Ana o blot 2 and with Ana IgG o 3). anti 11S-11S, is not anti a-2Sffected, and by anti boiling-LTP but(Figure is eliminated 1), arrows by indicate autoclave. the Theprotein albumin bands 2S detected is resistant (Ana after o 2 autoclave and Ana treatmento 3). 11S atis 121not ◦affectedC but is by not boiling detected but after is eliminated autoclave by at 138 ◦C. No immunoreactive bands were detected when IgG anti-LTP was applied. In the IgE Western

Molecules 2020, 25, x FOR PEER REVIEW 4 of 11 autoMoleculesclave. The2020 ,albumin25, 954 2S is resistant after autoclave treatment at 121 °C but is not detected after4 of 11 autoclave at 138 °C . No immunoreactive bands were detected when IgG anti-LTP was applied. In the IgE Western blot using a pool of sera from allergic patients, (Supplementary material. Figure S1), blot using a pool of sera from allergic patients, (Supplementary material. Figure S1), apart from other apart from other bands, we saw that a major band around 15kDa was persistent and detectable until bands, we saw that a major band around 15kDa was persistent and detectable until 121 ◦C 30 min. 121 °C 30 min. 2.2. Electrophoretic Pattern and Immunodetection Assays of Pistachio Samples 2.2. Electrophoretic Pattern and Immunodetection Assays of Pistachio Samples The protein profile, visualized by SDS-PAGE, of pistachio protein extract from untreated and boilingThe protein treated profile, samples visualiz was veryed by similar SDS-PAGE, (Figure of2 ).pistachio A low number protein ofextract bands from was untreated reduced after and the boiling treated samples was very similar (Figure 2). A low number of bands was reduced after the softest heat/pressure treatment (AU 121 ◦C, 15 min) and the rest of heat/pressure treatments (AU softest heat/pressure treatment (AU 121 °C, 15 min) and the rest of heat/pressure treatments (AU 121 121 ◦C, 30 min, AU 138 ◦C, 15 min and AU 138 ◦C, 30 min) provoked a smear due to the degradation, °C, 30rich min, in low AU molecular138 °C, 15 weightmin and proteins. AU 138 The°C, 30 most min) influenced provoked processing a smear due eff ectto the was degradation, obtained after rich harsh in low molecular weight proteins. The most influenced processing effect was obtained after harsh heat/pressure conditions (138 ◦C for 30 min) (Figure2). Western blot with IgG anti-11S, anti-2S, and heat/pressureanti LTP is conditions also analyzed. (138 Arrows°C for 30 indicate min) (Figure the protein 2). Western band detectedblot with (Pis IgG v anti 2/5 and-11S, Pis anti v- 1).2S, Thereand is anti LTP is also analyzed. Arrows indicate the protein band detected (Pis v 2/5 and Pis v 1). There is an important reduction of 11S and 2S detection after autoclave treatment at 138 ◦C. However, LTP an important reduction of 11S and 2S detection after autoclave treatment at 138 °C. However, LTP was even detected after autoclaving at 138 ◦C, 30 min. IgE reactivity reduction was of 73% after wasboiling even detected treatment after in pistachio autoclaving proteins at 138 and °C the, 30lowest min. detection IgE reactivity was at reductionthe hardest was autoclave of 73% conditions after boiling(Supplementary treatment in material pistachio Figure proteins S2). and the lowest detection was at the hardest autoclave conditions (Supplementary material Figure S2).

FigureFigure 2. SDS 2. -SDS-PAGEPAGE and andIgG IgGanti- anti-11S,11S, anti- anti-2S,2S, and andanti- anti-LTPLTP immunoblots immunoblots analysis analysis of control of control (ST), (ST), boiledboiled 30 and 30 and60 min 60 min (B30, (B30, B60) B60) and and autoclaved autoclaved at at121 121°C◦ C15 15 and and 30 30 min min (AU1, (AU1, AU2) AU2) and and 138 ◦°CC,, 15 and 30 min (AU3, AU4) pistachio samples. Molecular weight marker was Precision Plus (P ). and 30 min (AU3, AU4) pistachio samples. Molecular weight marker was Precision Plus (P+).+ 2.3. Electrophoretic Pattern and Immunodetection Assays of Processed Peanut Samples 2.3. Electrophoretic Pattern and Immunodetection Assays of Processed Peanut Samples The characterization of the electrophoretic profile of the soluble protein extracts from untreated and thermal-treatedThe characterization cashew of samples the electrophoretic is shown in Figureprofile3 .of The the results soluble showed protein that extracts the treatments from untreated of boiling andduring thermal 60-treated min had cashew no major samples effects is on shown the IgG-binding in Figure proteins3. The result froms peanut.showed However, that the treatments peanut subjected of boilingto heat during and pressure 60 min had treatments no major (autoclave) effects on showed the IgG less-binding distinctive proteins stained from bands peanut. in SDS-PAGE However, with peanutan increasedsubjected proteinto heat fragmentationand pressure treatments that went (autoclave) along with showed a reduction less indistinctive IgG-reactive stained bands. bands In the in SDSWestern-PAGE blot with with an IgG increased anti-11S, pr anti-2S,otein fragmentation and anti-LTP, arrows that went indicate along the with protein a reduction bands detected in IgG (Ara- reactiveh 3 and bands. Ara hIn 9). the 11S Western is not affected blot with by boiling IgG anti or- autoclave.11S, anti-2S The, and albumin anti-LTP, 2S is arrows resistant indicate after autoclave the protein bands detected (Ara h 3 and Ara h 9). 11S is not affected by boiling or autoclave. The albumin treatment at 121 ◦C and is weakly detected after autoclave at 138 ◦C. Some immunoreactive bands were 2S isdetected resistant when after IgG autoclave anti-LTP treatment was applied. at 121 The °C putative and is Araweakly h 9 banddetected was after not detected autoclave after at autoclave138 °C . at Some immunoreactive bands were detected when IgG anti-LTP was applied. The putative Ara h 9 138 ◦C, 30 min. band was not detected after autoclave at 138 °C , 30 min. 2.4. Electrophoretic Pattern and Immunodetection Assays of Processed Hazelnut Samples The protein proﬁle by SDS-PAGE, of hazelnut protein extract from untreated and boiling treated samples was very similar (Figure4a). A number of bands was reduced after heat /pressure treatment at 121 ◦C, 30 min) and the highest diminution was obtained after harsh heat/pressure conditions (138 ◦C for 30 min) (Figure4a). Western blot with IgG anti-11S, anti-2S, and anti LTP is also analyzed and arrows indicate the protein band detected (Cor a 9, Cor a 14 and Cor a 8). There is an important

Molecules 2020, 25, x FOR PEER REVIEW 5 of 11

Molecules 2020, 25, 954 5 of 11

reduction of 11S, 2S, and LTP detection after autoclave treatment at 138 ◦C. IgE reactivity was reduced following autoclave treatments (Supplementary material Figure S3). Molecules 2020, 25, x FOR PEER REVIEW 5 of 11

Figure 3. SDS-PAGE and IgG anti-11S, anti-2S and anti-LTP immunoblots analysis of control (ST), boiled 60 min (B60) and autoclaved at 121°C for 30 min (AU2) and 138 °C for 30 min (AU4) peanut samples. Molecular weight marker was Precision Plus (P+).

2.4. Electrophoretic Pattern and Immunodetection Assays of Processed Hazelnut Samples The protein profile by SDS-PAGE, of hazelnut protein extract from untreated and boiling treated samples was very similar (Figure 4a). A number of bands was reduced after heat/pressure treatment at 121 °C, 30 min) and the highest diminution was obtained after harsh heat/pressure conditions (138 °C for 30 min) (Figure 4a). Western blot with IgG anti-11S, anti-2S, and anti LTP is also analyzed and arrowsFigureFigure indicate 3. SDS-PAGE3. SDS the-PAGE protein and and IgGband IgG anti-11S, anti detected-11S, anti-2S anti (Cor-2S andand a 9, anti-LTPanti Cor-LTP a 14 immunoblots and Cor a 8).analysis analysis There of of iscontrol control an important (ST), (ST), reductionboiledboiled 60 of 60 min11S, min (B60)2S (B60), and and and LTP autoclaved autoclaved detection at atafter 121 121◦ autoclaveC°C for for30 30 minmin treatment (AU2)(AU2) and at 138 138 °C°C◦C. forIgE for 30 30reactivity min min (AU4) (AU4) was peanut peanut reduced followingsamples.samples. autoclave Molecular Molecular treatment weight weight marker smarker (Supplementary was was Precision Precision material Plus Plus (P(P+).+ Figure). S3).

Figure 4. (a) SDS-PAGE and IgG anti-11S, anti-2S, and anti-LTP immunoblots analysis of control (ST),

boiled 60 min (B60) and autoclaved at 121◦C at 30 min (AU2) and 138 ◦C 30 min (AU4) hazelnut samples. (b) SDS-PAGE and IgG immunoblot analysis of control and high-hydrostatic pressure (HHP) 300 to 600 MPa (300, 400, 500, 600) processed hazelnut samples Molecular weight marker was Precision Plus (P+).

Molecules 2020, 25, x FOR PEER REVIEW 6 of 11

Figure 4. (a) SDS-PAGE and IgG anti-11S, anti-2S, and anti-LTP immunoblots analysis of control (ST), boiled 60 min (B60) and autoclaved at 121°C at 30 min (AU2) and 138 °C 30 min (AU4) hazelnut samples. (b) SDS-PAGE and IgG immunoblot analysis of control and high-hydrostatic pressure Molecules(HHP2020,)25 300, 954 to 600 MPa (300, 400, 500, 600) processed hazelnut samples Molecular weight marker was6 of 11 Precision Plus (P+).

2.5.2.5. Electrophoretic Electrophoretic Pattern Pattern and and Immunodetection Immunodetection Assays Assays of of Processed Processed Almond Almond Samples Samples TheThe electrophoretic electrophoretic profile profile of of the the soluble soluble protein protein extracts extracts from from untreated untreated and and processed processed almond almond samplessamples is shownis shown in Figurein Figure5. Western 5. Western blot blot with with IgG anti-11S,IgG anti- anti-2S,11S, anti and-2S, anti and LTP anti is LTP also is analyzed also analyzed and arrowsand arrows indicate indicate the protein the protein band detected band detected (Pru du (Pru 6, Pru du du6, Pr 2S,u du and 2S Pru, and du Pru 3). du The 3). results The results showed showed that thethat treatments the treatments of boiling of boiling during 60during min had60 min no majorhad no eff majorects on effects the IgG-binding on the IgG proteins-binding from proteins almond. from However,almond. almond However, subjected almond to heat subjected and pressure to heat treatments and pressure (autoclave) treatments showed (autoclave) less distinctive showed stained less bandsdistinctive in SDS-PAGE stained band withs anin SDS increased-PAGE protein with an fragmentation increased protein that fragmentation went along with that awent reduction along with in IgG-reactivea reduction bands.in IgG- Thereactive electrophoretic bands. The migrationelectrophoretic patterns migration of high-pressure patterns of treated high-pressure flour almond treated proteinsflour almond were similar proteins to thewere control similar almond to the control as well almond as the immunodetection as well as the immunodete IgG profile.ction IgG profile.

FigureFigure 5. 5.SDS-PAGE SDS-PAGE and and IgG IgG anti-11S, anti-11S, anti-2S, anti-2S and, and anti-LTP anti-LTP immunoblots immunoblots analysis analysis of of control control (ST), (ST),

boiledboiled 60 60 min min (B60) (B60) and and autoclaved autoclaved at at 121 121◦C°C at at 30 30 min min (AU2) (AU2) and and 138 138◦C °C 30 30 min min (AU4) (AU4) and and HHP HHP treatedtreated (600 (600 MPa) MPa) almond almond samples. samples. Molecular Molecular weight weight marker marker was was Precision Precision Plus Plus (P +(P+).).

2.6.2.6. Electrophoretic Electrophoretic Pattern Pattern and and Immunodetection Immunodetection Assays Assays of of Processed Processed Chestnut Chestnut Samples Samples TheThe immunodetection immunodetection results results in chestnut in chestnut are summarized are summarized in Figure in 6Figure. SDS-PAGE 6. SDS showed-PAGE a showed similar a profilesimilar between profile untreated between untreated and boiled and samples, boiled although samples, the although immunoblot the immunoblot is slightly variable. is slightly Resistant variable. bandsResistant around bands 25 andaround 15 kDa 25 and were 15 notkDa detected were not after detected autoclave. after autoclave. IgE reactivity IgE reactivity reduction reduction was of 76% was afterof 76% boiling after treatment boiling treatment of chestnut of (Supplementary chestnut (Supplementary material Figure material S5). figure Thermal S5). treatments Thermal treatments (boiling and(boiling autoclave) and autoclave) diminished diminished protein bands’ protein number, bands especially’ number, afterespecially pressure after at pressure 138 ◦C for at 30138 min, °C andfor 30 modifiedmin, and protein modified solubility. protein Detection solubility. was Detection possible withwas allpossible antibodies with after all antibodies boiling for after 60 min boiling (Figure for6). 60 Aftermin autoclaving, (Figure 6). thereAfter is autoclaving a strong reduction, there in is the a strong immunoreactive reductionproteins’ in the immunoreactive detection. After Western proteins’ blotdetection. with anti-11S, After theWestern detection blot waswith possible anti-11S even, the atdetection autoclave was harshest possible conditions even at (Figureautoclave6). harshest conditionsIn this study, (Figure the 6 influence). of moist thermal treatments on the imunoreactivity of cashew, pistachio, peanut, hazelnut, almond, and chestnut has been analyzed. The results in Figures1–6 showed that heat and pressure treatment at the harshest conditions considered (138 ◦C, 2.56 atm, 30 min) produced a higher decrease of the IgG-binding capacity of the six tree nuts proteins than boiling without pressure or soft conditions of heat and pressure. Interestingly, although the treatments of heat and pressure seemed to affect cashew to a greater extent than pistachio allergens (Figures1 and2) both besides peanut proteins (Figure3) seemed to be more resistant to technological processing than others (hazelnut, almond and chestnut) (Figures4–6). In the specific case of cashew and pistachio, the effects of thermal processing on their allergenic properties have been addressed by a limited number of studies that used assays that evaluated IgE-binding in Western blot or ELISA [42]. In our study, we have applied thermal treatments to cashew and pistachio that included not only boiling without pressure and heat/pressure treatments at soft conditions, but also harsher conditions of heat and pressure (138 ◦C, 2.56 atm, during 15 and 30 min), which turned to be the most efficient treatment to decrease the allergenic properties of both tree nuts considered in our study. The harshest conditions of heat and pressure applied in our study produced a degradation of all the tree nuts studied (cashew, pistachio, peaenut, hazelnut, almond, Molecules 2020, 25, 954 7 of 11 and chestnut), with an increased protein fragmentation seen as an intense smear in the low molecular weight area in the SDS-PAGE. Such alteration in the electrophoretic and IgG-binding patterns after heat and pressure treatments cannot be explained by a potential loss of solubility of proteins due to the thermal treatments, since the experiments carried out with strong conditions of protein solubilization (flours directly solubilized in SDS-PAGE sample buffer) showed the same pattern of protein degradation for heat and pressure treated samples. The degradation of proteins after harsh heat/pressure treatments Moleculesobtained 2020 in, 2 our5, x FOR study PEER reminds REVIEW to the degradation produced by some enzymatic treatments [33,437 of,44 11].

FigureFigure 6. 6. SDSSDS-PAGE-PAGE and and IgG IgG anti anti-chitinase,-chitinase, anti anti-lipid-lipid transfer transfer protein protein (anti (anti-LTP),-LTP), anti anti TLP, TLP, anti anti-11S,-11S, andand anti anti-2S-2S immunoblots analysisanalysis ofof controlcontrol (ST), (ST), boiled boiled 60 60 min min (B60) (B60) and and autoclaved autoclaved at 121at 121◦C at°C 30 at min30 min(AU2) (AU2) and and 138 ◦138C 30 °C min 30 min (AU4) (AU4) chestnut chestnut samples. samples. Molecular Molecular weight weight marker marker was was Precision Precision Plus Plus (P+). (P+). 3. Materials and Methods

3.1.In Plant this Material study, andtheProcessing influence of moist thermal treatments on the imunoreactivity of cashew, pistachio, peanut, hazelnut, almond, and chestnut has been analyzed. The results in Figures 1–6 showedCashew that heat (Anacardium and pressure occidentale treatment, type at the 320) harshest and peanut conditions (Arachys considered hypogaea (138, Virginia) °C, 2.56 obtained atm, 30 min)from produced Productos a higher Manzanares decrease S.L. of (Spain)the IgG- andbinding pistachio capacity (Pistachia of the six vera treeKerman), nuts proteins hazelnut than (Corylisboiling withoutavellana, pressure Negreta), or andsoft almondconditions (Prunus of he dulcis,at and Marcona)pressure. Interestingly, from the Germoplasm although Bankthe treatments of Institut of de heatRecerca and pressure i Tecnologia seemed Agroalimentaries to affect cashew (IRTA-Mas to a greater de exten Bover,t than Tarragona, pistachio Spain) allergens were (Figures used for 1 and this 2)study. both Anbesides additional peanut commercial proteins (Figure variety 3) of seemed chestnut to (Castaneabe more sativaresistant), Miquelenca, to technological was purchased processing to thanCooperativa others (hazelnut, Secallona (Viladrau, almond and Gerona, chestnut Spain).) (Figures Whole nuts 4–6). seeds In the were specific immersed case in of distilled cashew water and pistachio,(1:5 w/v) andthe boiledeffects (100of thermal◦C, 30 andprocessing 60 min, on (B30 their and allergenic B60)) or autoclaved properties usinghave anbeen autoclave addressed Compact by a limited40 Benchtop number (Priorclave, of studies London,that usedUK) assays at 121that◦ evaluatedC, 120 kPa IgE for-b 15inding and 30in W minestern (AU1 blot and or AU2)ELISA and [42] at. In138 our◦C, study, 256 kPa, we have for 15 applied and 30 thermal min (AU3 treatments and AU4) to(Figure cashew7 a).and Untreated, pistachio that boiled included and autoclaved not only boilingnut seeds with wereout pressure ground andand defattedheat/pressure with n-hexanetreatments (34 at mLsoft/g conditions, of flour) for but 4 h,also shaken, harsher and conditions air-dried ofafter heat filtration and pressure of the (138 n-hexane. °C, 2.56 Defatted atm, during flour from15 and untreated 30 min), sampleswhich turned was the to controlbe the most for boiled efficient and treatmentautoclaved to samples.decrease the Hazelnuts allergenic and properties almonds of were both ground tree nuts and considered defatted within our n-hexane study. The (34 harshest mL/g of conditions of heat and pressure applied in our study produced a degradation of all the tree nuts studied (cashew, pistachio, peaenut, hazelnut, almond, and chestnut), with an increased protein fragmentation seen as an intense smear in the low molecular weight area in the SDS-PAGE. Such alteration in the electrophoretic and IgG-binding patterns after heat and pressure treatments cannot be explained by a potential loss of solubility of proteins due to the thermal treatments, since the experiments carried out with strong conditions of protein solubilization (flours directly solubilized in SDS-PAGE sample buffer) showed the same pattern of protein degradation for heat and pressure treated samples. The degradation of proteins after harsh heat/pressure treatments obtained in our study reminds to the degradation produced by some enzymatic treatments [33,43,44].

Molecules 2020, 25, x FOR PEER REVIEW 8 of 11

3. Materials and Methods

3.1. Plant Material and Processing Cashew (Anacardium occidentale, type 320) and peanut (Arachys hypogaea, Virginia) obtained from Productos Manzanares S.L. (Spain) and pistachio (Pistachia vera Kerman), hazelnut (Corylis avellana, Negreta), and almond (Prunus dulcis, Marcona) from the Germoplasm Bank of Institut de Recerca i Tecnologia Agroalimentaries (IRTA-Mas de Bover, Tarragona, Spain) were used for this study. An additional commercial variety of chestnut (Castanea sativa), Miquelenca, was purchased to Cooperativa Secallona (Viladrau, Gerona, Spain). Whole nuts seeds were immersed in distilled water (1:5 w/v) and boiled (100 °C, 30 and 60 min, (B30 and B60)) or autoclaved using an autoclave Compact 40 Benchtop (Priorclave, London, UK) at 121 °C , 120 kPa for 15 and 30 min (AU1 and AU2) and at 138 °C, 256 kPa, for 15 and 30 min (AU3 and AU4) (Figure 7a). Untreated, boiled and autoclaved nut seeds were ground and defatted with n-hexane (34 mL/g of flour) for 4 h, shaken, and air-dried after Molecules 2020, 25, 954 8 of 11 filtration of the n-hexane. Defatted flour from untreated samples was the control for boiled and autoclaved samples. Hazelnuts and almonds were ground and defatted with n-hexane (34 mL/g of flour)flour) forfor 44h h and and air-dried air-dried after after filtration filtration of of the the n-hexane. n-hexane. High-pressure High-pressure experimentalexperimentalconditions conditions werewere carriedcarried out according to to Omi Omi et et al. al. [45 [45] and] and Kato Kato et al. et [ al.46] [.46 Hazelnut]. Hazelnut and almond and almond defatted defatted flours flourswere dissolved were dissolved in distilled in distilled water water(1:4 w/v) (1:4 20 w/ v)h before 20 h before HHP HHPtreatment treatment (Figure (Figure 7b). 7Theb). Theflours flours were weresubjected subjected to HHP, to HHP, using using pressures pressures of 300, of 400, 300, 500 400,, and 500, 600 and MPa 600 MPafor 15 for min 15 in min a multivessel in a multivessel high- high-pressurepressure equipment equipment (HHP, (HHP, ACB, ACB, France) France) at at20 20°C◦ C(Figure (Figure 66).The).The nitrogen nitrogen contentscontents ofof thethe samples samples werewere determined determined byby LECOLECO analysisanalysis accordingaccording toto standardstandard proceduresprocedures basedbased onon DumasDumas methodmethod [[4747].]. TheThe totaltotal proteinprotein content content was was calculated calculated as as N N x x 5.3 5.3 [ 47[47].]. The The analyses analyses were were carried carried out out in in duplicate. duplicate.

FigureFigure 7. 7. SchemeScheme of of (aa)) boiling boiling and and autoclave autoclave treatments treatments and and (b)) high high hydrostatic hydrostatic pressurepressure (HHP) (HHP) treatmenttreatment of of tree tree nut nut samples. samples.

3.2.3.2. ProteinProtein ElectrophoresisElectrophoresis andandImmunoblot ImmunoblotExperiments Experiments Protein electrophoresis of the defatted flours was carried out as previously described [33]. Defatted flours from untreated and treated samples were dissolved in standard electrophoresis sample bu ffer. The same amount of protein (20 µg) calculated from LECO analysis from each sample was electrophoresed in 4–20%. Proteins were visualized with Coomassie Brilliant Blue (Bio-Rad, Hercules, CA, USA). For Western blot, proteins were transferred to polyvinylidene difluoride (PVDF) membranes ((Millipore Corp., Bedford, MA, USA). Blocking was carried out for 1 h at room temperature in PBS plus 0.5% Tween-20 (PBST) containing 3% milk (blocking solution). IgG mouse anti-11S (dilution 1:10,000), anti-2S (1:25,000), anti-chitinase (1:200), anti TLP (1:5000), and anti-LTP (1:500) were diluted in blocking solution and incubated with the PVDF membranes for 1 h. Membranes were washed and then treated with alkaline phosphatase (AP) conjugated goat anti mouse antibody (1:5000) (Sigma, Saint Louis, MO, USA) diluted in blocking solution. Detection was achieved by means of BCIP/NBT substrate (Sigma, Saint Louis, MO, USA). The signal was measured using ChemiDoc (Bio-Rad, Hercules, CA, USA). For IgE Western blot, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and the membranes were incubated overnight at 4 ◦C with pool sera from 6 patients with tree nuts, washed and then treated with Horseradish peroxidase (HRP) conjugated mouse anti-human IgE (1:10,000 Molecules 2020, 25, 954 9 of 11 dilution for 30 min at RT) (Sigma, Saint Louis, MO, USA). Detection of IgE-binding proteins was achieved by means of enhanced chemiluminescence, according to the manufacturer’s instructions (Thermo Scientific, Waltham, MA, USA). The signal was measured using CCD camera system of ChemiDoc (Bio-Rad, Hercules, CA, USA).

4. Conclusions Thermal and pressure treatment (autoclaving) was able to decrease IgE binding properties of pistachio, cashew, peanut, hazelnut, almond, and chestnut. Autoclave 138 ◦C (256Kpa) for 30 min showed the most relevant reduction in IgG reactivity in all the tree nuts. After autoclave at 138 ◦C for 30 min, IgG immunodetection of Pis v1, Pis v 2, Pis v 5, Ana o 2, Ana o 3, Ara h 9, Cor a 9, Cor a 14, Cor a 8, Pru du 6, Pru du 2S, Pru du 3, Cas s 5, Cas s 8, and Cas s TLP is strongly diminished. More studies are necessary in order to obtain more conclusive results, such as basophil activation assay with human cells.

Supplementary Materials: The following are available online. Figure S1: Immunodetection in cashew, Figure S2. Immunodetection in PISTACHIO, Figure S3. Western blot in hazelnut (3 patients), Figure S4. Western blot in almond (pool 4 patients), Figure S5. Immunodetection in Chesnut. Author Contributions: Formal analysis, C.C., A.S., F.V., I.B. and R.L.; funding acquisition, C.C.; investigation, C.C., A.S., I.B. and R.L.; supervision, C.C.; writing— original draft, C.C.; writing—review and editing, C.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministerio de Ciencia, Innovación y Universidades, grant number AGL2017-83082-R. Acknowledgments: The authors want to acknowledge Araceli Diaz-Perales (CBGP_UPM/INIA) for sharing the IgG antibodies with us. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Sample Availability: Samples of the compounds (cashew, pistachio, peanut, hazelnut, almond, and chestnut processed) are available from the authors.

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