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A potential practical approach to reduce Ara h 6 allergenicity by gamma irradiation
Food Chemistry 136 (2013) 1141–1147
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A potential practical approach to reduce Ara h 6 allergenicity by gamma irradiation Chunping Luo a,b, Chunqiu Hu a,d, Jinyan Gao a,c, Xin Li a,c, Zhihua Wu a,e, Anshu Yang a,e, Hongbing Chen a,e,⇑ a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China Taizhou Vocational College of Science & Technology, Taizhou 318020, China c Department of Food Science, Nanchang University, Nanchang 330047, China d Department of Nutrition and Food Hygiene, Anhui Medical University, Hefei 230032, China e Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China b
a r t i c l e
i n f o
Article history: Received 3 February 2012 Received in revised form 1 September 2012 Accepted 25 September 2012 Available online 2 October 2012 Keywords: Gamma irradiation Peanut allergy Ara h 6 Protein structure Allergenicity
a b s t r a c t Peanut allergen Ara h 6 was isolated and irradiated at 1, 3, 5, or 10 kGy, and a whole peanut protein extract (WPPE) was also treated by irradiation. Alteration in structure of Ara h 6 was characterised by circular dichroism (CD) spectroscopy, ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy and SDS–PAGE, and antigenicity was evaluated by immunoblotting and indirect ELISA with anti-Ara h 6 polyclonal antibody. Irradiation induced significant changes in the secondary and tertiary structures of Ara h 6, and the antigenicity of both purified Ara h 6 and WPPE were reduced upon increasing the irradiation doses. Moreover, a good correlation between the loss in a-helix and IgG binding to Ara h 6 was observed. This indicated that irradiation might be an efficient approach to reduce or eliminate peanut allergenicity. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Peanut allergy is common, frequently severe and typically permanent (Bock, Munoz-Furlong, & Sampson, 2007; Pumphrey & Gowland, 2007; Rona, Keil, Summers, & Gislason, 2007). The prevalence of peanut allergy has been estimated to be between 0.6% and 1% of the US and EU populations (Sicherer, 2002; Tariq et al., 1996). David et al. (1997) have shown that peanut was the third most frequent allergenic food in young Australian and Asian children. Additionally, the incidence of peanut allergy seems to be increasing during recent decades (Ben-Shoshan et al., 2009; Grundy, Matthews, Bateman, Dean, & Arshad, 2002; Sicherer, MunozFurlong, & Sampson, 2003). An explanation is that the popularity and consumption of peanut products have increased (Du Toit et al., 2008). Obviously, the investigations on how to prevent it are increasing. Since peanut allergy is mainly triggered by the immunoglobulin E (IgE) recognition of peanut allergens, reducing the allergenic potency or levels of peanut allergens may be an effective approach to lower the allergenic risk. However, previously described methods to reduce the allergenicity of peanut allergens were found to be inefficient, and it was reported that
⇑ Corresponding author at: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China. Tel.: +86 791 88333529; fax: +86 791 88333708. E-mail address: [email protected] (H. Chen). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.09.091
many peanut allergens were resistant to cooking and digesting (Koppelman, Hefle, Taylor, & de Jong, 2010; Mondoulet et al., 2005). Irradiation has recently become one of the successful techniques to preserve food with minimum interruption of its nutritional and sensory properties (Farkas, 2006; Osterholm & Norgan, 2004). Hundreds of animal feeding investigations of irradiated food have been carried out since 1950, and the studies include subchronic and chronic changes in metabolism, histopathology, function of most systems, reproductive effects, growth, teratogenicity, and mutagenicity. Some of the studies have indicated adverse effects, while no consistent pattern has emerged (Shea, 2000). Consequently, the WHO joint committee concluded that the irradiation of any food commodity up to an overall average dose of 10 kGy presents no toxicological hazard, and toxicological testing of foods so treated is no longer required (WHO, 1988). WHO and FDA have concluded that irradiated food is safe under specific conditions (WHO, 1994, 1999). Moreover, any foods irradiated at levels up to 10 kGy are safe for human consumption without any microbiological hazard and any special nutritional problems (Shea, 2000). Oh et al. (2009) have evaluated the allergenicity of irradiated peanut extract using splenocytes from peanut-sensitised mice and draw a conclusion that the allergenicity of peanut extracts could be reduced by irradiation treatment, and irradiated peanuts might provide a novel immunogen for an immunotherapy of peanut allergy. It is the first and only report on the gamma irradiation
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treatment of peanut allergens, and the structural alteration of irradiation-treated peanut allergens was not defined. However, proteins exposed to irradiation would present structural alterations caused by fragmentation, cross-linking, aggregation, and amino acid modification, affecting their secondary and tertiary structures and their immunoreactivity (Poms & Anklam, 2004). Accordingly, Oh et al. (2009) thought that alteration of epitopes by denaturation of the peanut, after irradiation, might have induced a lower response by T cells. In addition, studies have shown that modification of allergens, by irradiation at levels up to 10 kGy, is a feasible approach to reduce or abolish the allergenicity of egg, milk, wheat and shrimp (Antônio et al., 2012; Lee et al., 2001; Seo et al., 2007; Sinanoglou, Batrinou, Konteles, & Sflomos, 2007). Therefore, before the irradiation process can be generally recommended for reducing allergenicity of peanut allergens, much more information about the process remains to be investigated. Moreover, Ara h 6 was identified as one of the major peanut allergens, sharing 59% sequence identity and a high degree of structural conservation with Ara h 2, another major peanut allergen (Kleber-Janke, Crameri, Appenzeller, Schlaak, & Becker, 1999; Marsh et al., 2008). Studies in children have demonstrated that Ara h 6 and Ara h 2 were the most commonly recognised peanut allergens, and IgE reactivity to them was a risk factor for the most serious reactions (Flinterman et al., 2007; Pastorello et al., 2001). Currently, there is only one natural counterpart of Ara h 6 and purified Ara h 6 was described by another two independent groups with a molecular weight of approximately 15 and 14.981 kDa, identified by SDS–PAGE and mass spectroscopy, respectively (Koppelman et al., 2005; Suhr, Wicklein, Lepp, & Becker, 2004), and the prepared Ara h 6 needs to be subjected to biochemical and immunological studies. Consequently, our work aims to define the irradiation-induced alterations in structure and antigenicity of a model peanut allergen, Ara h 6. Additionally, studies have shown that the ingredients of the food matrix can have a great impact on the conformation and antigenic properties of proteins during processing (Grimshaw et al., 2003; Lee, Lee, & Song, 2003; Maleki & Hurlburt, 2004). Herein, the antigenic alteration of whole peanut protein extract was investigated to discover whether irradiation processing, under various conditions, could have unpredictable effects on the Ara h 6 allergenicity in the complex food matrices. 2. Materials and methods 2.1. Preparation of whole peanut protein extract (WPPE) Raw peanuts (Arachis Hypogaea, Xianghua, China) were purchased from a local supplier (Nanchang, China) and were stored at 20 °C until used. After shelling, the skins were removed, and the kernel was ground in liquid nitrogen, using a mortar and pestle. The meal was then defatted by stirring with 5 volumes of precooled acetone for 1 h at 4 °C, and it was recovered by centrifuging at 6000g for 20 min at 4 °C. After being defatted three times, the meal was allowed to dry in the air and suspended in 5 vol of 50 mM Tris–HCl (pH 7.2), followed by stirring for 12 h at 4 °C. Then the suspension was centrifuged at 6000g for 20 min at 4 °C, and the supernatant was collected as WPPE. 2.2. Preparation of Ara h 6 WPPE (10 mg/ml) was loaded onto a DEAE-Sepharose Fast Flow column (bed dimension, 26 300 mm2, GE Healthcare, USA) previously equilibrated with 50 mM Tris–HCl (pH 7.2, loading buffer) at room temperature. The column was washed with loading buffer until the A280 of the effluent was less than 0.2. Proteins were eluted with a linear gradient in a loading buffer (0–0.2 M NaCl in 500 ml
of loading buffer) at a flow rate of 1.5 ml/min. Fractions were collected and analysed by SDS–PAGE, and Ara h 6 appeared to be a band of 15 kDa and essentially pure (>95%), calculated by a densitometer scanning of an SDS–PAGE gel stained with Coomassie Brilliant Blue. Further identity confirmation of Ara h 6 was performed with MALDI-TOF-MS fragmentation analysis of selected tryptic peptides with protein-specific sequences (Applied Biosystems, Framingham, MA, USA). Ara h 6 -containing fractions were pooled and concentrated by ultrafiltration with an Amicon Ultra-15 filter with a MWCO of 3.0 kDa (Millipore, USA), and the protein concentration was measured according to the Bradford method. 2.3. Protein content Protein concentration was determined by the Bradford method, measuring absorbance at 595 nm. Bovine serum albumin was used as a standard. 2.4. Production of polyclonal antibody Polyclonal antibody against Ara h 6 was produced from New Zealand male rabbits. Before immunising, 1 ml of blood from a non-injected rabbit was collected by the ear-bleeding method as a negative serum blank. Two rabbits were immunised by subcutaneously injecting into their shaved backs 0.2 mg of purified Ara h 6 in Freund’s complete adjuvant (Sigma, USA). Subsequent injections of Ara h 6 in incomplete Freund’s adjuvant (Sigma, USA) were performed three times, at 2-week intervals, and the immune responses of rabbits were monitored by indirect ELISA. The rabbits were finally bled and each antiserum was separated by centrifuging at 4000g for 10 min at 4 °C. All sera were stored at 80 °C until used. 2.5. Gamma irradiation The purified Ara h 6 (0.2 mg/ml, 30 ml) and WPPE (2 mg/ml, 10 ml) were put into a plastic tube with a cap, and irradiated in a cobalt-60 gamma-irradiator (Jiangxi Academy of Agricultural Sciences, Nanchang, China) in the presence of air. The applied dose levels were 1, 3, 5 and 10 kGy, and the temperature during irradiation treatment was set at 4 °C. Non-irradiated Ara h 6 was used as a control. 2.6. Ultraviolet (UV) absorption spectroscopy UV absorption spectra analysis was performed by a UV spectrophotometer (UV-VS 2501PC, Shimadzu Corporation, Japan) at room temperature (25 °C). All the samples were homogeneously mixed (vortexed) and scanned from 200 to 400 nm. 2.7. Surface hydrophobicity Surface hydrophobicity was determined by the 1-anilinonaphthalene-8-sulfonate (ANS) (Sigma, USA). A volume of 20 ll of ANS solution (5 mM in 0.01 M PBS, pH 7.4) was added to 4 ml of Ara h 6 solutions (0.1 mg/ml) and homogeneously mixed by vortex. The relative fluorescence was measured with a spectrophotofluorometer (F-4500, Hitachi, Japan). The excitation wavelength was 390 nm, and the emission wavelength was from 400 to 650 nm at a scanning speed of 1500 nm /min. 2.8. Circular dichroism (CD) Conformational changes in the secondary structure of irradiated protein samples were analysed using a CD spectropolarimeter (J810, JASCO, Japan). The homogeneously mixed proteins, at a con-
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centration of 0.5 mg/ml were used for measurements. CD spectra were scanned at the far UV range from 250 to 190 nm, at a rate of 100 nm/min, with a bandwidth of 1.0 nm at room temperature. The spectra represented an average of three consecutive scans. Results of each sample were averaged, and the mean residue ellipticity (h) was expressed as deg cm2 dmol1, followed by calculating the secondary structure contents by JASCO secondary structure software (JASCO, Japan). 2.9. Reducing and non-reducing SDS–PAGE SDS–PAGE was performed essentially according to Laemmli (1970), using the Mini Protean system (BioRad, USA). A 5% stacking gels and 15% separating gels were used in SDS–PAGE analysis. For reducing SDS–PAGE, 3% (v/v) b-mercaptoethanol was added to 2 non-reducing sample buffer [20 mM Tris, pH 6.7, 2 mM EDTA, 2% (w/v) SDS, 25% (v/v) glycerin, 0.02% (w/v) bromphenol blue)]. Samples were mixed (1:1, v/v) with the reducing/non-reducing sample buffer, boiled for 5 min, followed by centrifuging at 4000g for 5 min, and the supernatants were loaded onto the gel. Gels were stained with Coomassie brilliant blue R-250 and destained in a de-staining solution (7.5% HAc (v/v), 5% methanol (v/v) in water). After de-staining, gels were scanned, followed by calculating the band intensity with Quantity One software (Bio-Rad).
IgG binding abilities were defined by the following equation:
IgG binding abilitiesð%Þ ¼ B=B0 100 2.12. Statistical analysis Appropriate data were analysed for statistical significance using ANOVA procedures (SPSS for Windows 2003, Microsoft Corp., version 13.0, Chicago, IL, USA) and Fisher’s least significant difference (LSD, p < 0.05) test. 3. Results 3.1. UV absorption spectra of irradiated Ara h 6 The UV absorbance of irradiated Ara h 6 was assayed at a wavelength of 200–400 nm. Fig. 1 shows the UV spectra of the purified Ara h 6 irradiated at various doses. The non-irradiated Ara h 6 had a weak absorbance at 280 nm, while a rise in UV absorbance of irradiated Ara h 6 was observed. The maximum absorbance value increased from 0.12 (control) to 0.16, 0.77, 1.42, and 1.64 after being irradiated at 0, 1, 3, 5, 10 kGy, respectively. In addition, compared with the control samples, the location of absorption peak in irradiation-treated samples underwent a blue shift of 1–3 nm.
2.10. Immunoblotting 3.2. Surface hydrophobicity of irradiated Ara h 6
2.11. IgG binding by indirect ELISA The binding abilities of the rabbit IgG to the irradiated protein samples were tested by indirect ELISA. The 96-well microtitre plate was coated with 100 ll of purified Ara h 6 (2 lg/ml) or WPPE (6.25 lg/ml) in coating buffer (0.015 M Na2CO3, 0.035 M NaHCO3, pH 9.6) overnight at 4 °C. The wells were washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and subsequently blocked with 5% skimmed milk in PBS, incubating for 1 h at 37 °C. The wells were again washed and 100 ll of serially diluted sera (1:10000 dilutions in PBS) were added, followed by incubation for 1 h at 37 °C. After further washing, 100 ll of goat anti-rabbit IgG conjugated with horseradish peroxidase (1:5000 dilutions in PBS) were then added to each well, followed by incubation for 1 h at 37 °C, for detecting the bound immunogen. The plates were further washed, and the colour was developed by adding a substrate solution (100 ll) of o-phenylene diamine (0.5 mg/ml, Sigma, USA), 0.03% hydrogen peroxide in 0.1 M citrate buffer (pH 5.5) to each well for 15 min at 37 °C. The reaction was terminated with 50 ll of 2 M sulphuric acid, and the absorbance of irradiated samples (B) and non-irradiated samples (B0) was read at 490 nm with a Bio-Rad Microplate Reader (Model 1860, BioRad, USA).
The surface hydrophobicity of irradiated Ara h 6 was determined by ANS. Fig. 2 shows the ANS induced fluorescence spectra of the irradiated Ara h 6 at various doses. It indicates that the ANSfluorescence peak of non-irradiated Ara h 6 was very weak at 450 nm, and the 1 kGy-treated sample had a profile similar to the control sample. However, upon increasing the irradiation dose to 3, 5 and 10 kGy, the ANS-fluorescence intensities of samples were increased 4.37-, 8.58-, and 11.2-fold, compared with the control samples, respectively. Moreover, there were no significant differences of the location of the ANS-fluorescence peak between the irradiation-treated samples and the non-irradiated ones. 3.3. Circular dichroism (CD) spectra of irradiated Ara h 6 Fig. 3 indicates the far-UV CD spectra of the purified Ara h 6 irradiated at various doses. The spectrum of non-irradiated Ara h 6 had a strong positive peak at 196 nm with pronounced double negative peaks at 208 and 222 nm, and it crossed zero ellipticity at 199 nm. Irradiation treatment affected CD spectra of Ara h 6 significantly. Both the intensity of positive peak at 196 nm and the two negative peaks at 208 and 222 nm decreased with increased
Optical density
SDS–PAGE gels were prepared, under reducing conditions as described above, and the separated proteins were transferred to polyvinyldifluoride membranes (PVDF, Millipore, USA) by applying a constant current of 50 mA for 2 h at room temperature. The membranes were blocked with TBS buffer (50 mM Tris–HCl, 150 mM NaCl, pH 7.5) containing 1%Tween-20 (TBST) for 1 h at room temperature, followed by overnight incubation with the rabbit antiAra h 6 serum (1:5000 dilutions in TBST) at 4 °C. Then the membrane was washed three times in TBS and incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:5000 dilutions in TBST, Sigma, USA) for 2 h at room temperature. After washing for a further three times, the peroxidase-positive images were developed by incubating the membrane in 20 ml of TBS containing 12 mg 4-chloro-1-naphthol, 4 ml of ethanol and 12 ll of 30% H2O2 at room temperature for 10 min.
Wavelength(nm) Fig. 1. UV absorption spectra of irradiated Ara h 6 at various doses.
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Relative intensity
diated at 1 or 3 kGy, and a trace spread from the starting point of separating gel at the dose of 5 kGy. Interestingly, no bands were observed in SDS–PAGE when the dose reached 10 kGy because Ara h 6 formed solely insoluble aggregates. Additionally, a similar result was found in the samples of WPPE (Fig. 4b), except that images of over 72 kDa emerged at 3 kGy and above. The immunoblotting images in Fig. 4c and d demonstrate that the irradiation affected the image definition more than did SDS– PAGE. Intensities of the intact Ara h 6 and WPPE declined, and the degrees of the aggregation rose with increase of irradiation doses. Remarkably, heavier images (above 15 kDa) existed in WPPE than in the purified Ara h 6. 3.5. IgG binding abilities of irradiated Ara h 6
Wavelength(nm)
IgG binding abilities of the purified Ara h 6 and WPPE treated at various doses, using anti-Ara h 6 polyclonal antibody for ELISA checking, are shown in Fig. 5. There was a clear decrease in IgG binding to Ara h 6 with the increase of irradiation doses. However, the IgG binding abilities of WPPE were retained better than were those of the purified Ara h 6 at the same irradiation dose. However, when they were irradiated at 10 kGy, only 5% IgG binding abilities were found in both WPPE samples and the purified Ara h 6 samples.
Fig. 2. Surface hydrophobicity of irradiated Ara h 6 at various doses.
irradiation dose. Interestingly, with a 10 kGy irradiation dose, the positive peak disappeared and showed a negative peak at 200 nm. This CD spectral alteration indicated the changes of the secondary structure content of Ara h 6 after irradiation (as shown in the graph insets). A loss of a-helix and b-turn were observed from 1 to 10 kGy, and the content of random coils showed a decline at 1–3 kGy and a rise at 5–10 kGy. Moreover, the b-strand showed an opposite alteration, and particularly, a transition from b-strand to b-turn occurred as the doses increased.
4. Discussion Significant alterations in conformational and antigenic properties of peanut allergen Ara h 6 were induced during irradiation treatment, the extent of which depended on the irradiation doses. 1 kGy treatment had little influence on the tertiary structure of Ara h 6, while it had an important impact on the secondary structure and antigenicity of Ara h 6. Moreover, the higher the dose applied, the greater were the changes in conformation and antigiencity of Ara h 6. Similar findings were reported in that the structure and
3.4. SDS–PAGE and immunoblotting profile of irradiated Ara h 6 SDS–PAGE patterns of purified Ara h 6 and WPPE, at various irradiation doses, are shown in Fig. 4. Fig. 4a indicates that irradiation could cause a decrease of the intensity of the purified Ara h 6 image (15 kDa). A new image (20 kDa) appeared when it was irra-
Percentage(%)
50 40
Helix Strand
30
Turn 20
Random
10
Molar ellipticity(deg·cm2/dmol)
0 Control
1
3
5
10
Irradiation doses(kGy)
Wavelength(nm) Fig. 3. Circular dichroism spectra of irradiated Ara h 6 at various doses. Graph insets represent the corresponding secondary structural content of irradiated Ara h 6 at various doses.
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IgG binding abilities [B/B0 ×100(%)]
Fig. 4. SDS–PAGE patterns of purified Ara h 6 (0.2 mg/ml) (a) and WPPE (2 mg/ml) (b) irradiated at various doses and the corresponding immunoblotting using anti-Ara h 6 polyclonal antibody (c and d). M, molecular weight marker; Con, Control samples.
120 100
WPPE Purified Ara h 6
80 60 40 20 0 Control
1
3
5
10
Doses(kGy) Fig. 5. IgG binding abilities of purified Ara h 6 and WPPE treated at various doses, using anti-Ara h 6 polyclonal antibody for ELISA checking. B and B0 represent ELISA data for defining the binding of irradiated samples to anti-Ara h 6 polyclonal antibody and that of non-irradiated samples, respectively.
antigenicity of shrimp allergens and milk allergens could be altered by irradiation (Byun et al., 2000; Lee et al., 2001). A loss of secondary and tertiary structures of irradiated Ara h 6 was defined by (CD) spectroscopy, UV absorption spectroscopy, and fluorescence spectroscopy. The changes in the UV spectra of Ara h 6 suggested that the native protein structure unfolded, which implied that the hydrophobic amino acids of the proteins were partially exposed to the solvent. Typically, the ANS fluorescence anal-
ysis fully proved the alteration of hydrophobic amino acids, which was in conformity with the UV absorption spectra analysis. This phenomenon has also been observed in other proteins, such as bovine serum albumin and myoglobin (Gaber, 2005; Lee & Song, 2002). Moreover, the large difference between irradiation-treated Ara h 6 and the non-irradiated one (control) was manifested in the loss of the secondary structure of the proteins and the change in the local environment of ordered structure of a polypeptide chain defined by far-UV CD spectra. It clearly supported the idea that oxygen radicals, generated during irradiation of proteins in solution, disrupt the ordered structure of proteins, resulting in structural unravelling of proteins in solution. Changes in the second and tertiary structures caused by gamma irradiation led to protein misfolding and, subsequently, to aggregation. Irradiated proteins can be converted to higher molecular weight aggregates due to the generation of inter-protein crosslinking reactions, hydrophobic and electrostatic interactions, as well as the formation of disulfide bonds (Oliveira, de la Hoz, Silva, Torriani, & Netto, 2007; Xu & Chance, 2005). In this work, Ara h 6 and WPPE formed more soluble aggregates upon increased irradiation dosing, while the aggregates formed insoluble precipitates under 10 kGy irradiation. However, this was not unique, as similar phenomena exist in other irradiated food proteins, such as b-lactoglobulin (Lee et al., 2001), bovine and porcine serum plasma (Lee et al., 2003). Moreover, since reducing and non-reducing SDS– PAGE can define the role of disulfide bonds in the formation of protein aggregation, caused by irradiation treatment, and no difference was observed between the migration patterns of the bands
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in reduced and non-reduced SDS gels (data not shown), this indicated that the disulfide bond was not responsible for the production of protein aggregation in the irradiated Ara h 6. Irradiation-induced alteration of protein structure could affect its antigenicity. In the present study, a clear decrease in a-helix content (Fig. 3) upon elevated irradiation doses was observed, and it correlated well with the decline in IgG binding to Ara h 6 (Fig. 5). Besides, the aggregation gradually became greater upon increasing irradiation doses from 1 to 5 kGy, and it implied that conformational epitopes on Ara h 6 were easily lost by irradiation treatment. Actually, previous works have demonstrated that irradiation treatment could destroy conformational epitopes by denaturation of the proteins and alter linear epitopes by aggregation, fragmentation, and amino acid modification (Kim et al., 2002; Li, Lin, Cao, & Khalid, 2007; Mengna, Mahesh, Suzanne, Keneth, & Shridhar, 2004). In addition, Oh et al. (2009) have evaluated the changes of allergenicity and cytokine production profiles after exposure of irradiated peanut extract in a peanut-allergy mouse model. A higher level of production of IFN-c and IL-10 cytokines was observed, when the cells were stimulated with 10 kGy of irradiated peanut extract, which indicated the rise of the Th1/Th2 ratio in response to treatment with gamma-irradiated peanut extract and showed that the allergenicity of peanut extract could be reduced by gamma irradiation, which caused down-regulation of Th2 lymphocyte activity in the peanut-sensitised mice. Combining the finding of Oh et al. (2009) with this work, a positive conclusion was suggested, that gamma irradiation could reduce the antigenicity and allergenicity of peanut allergens, and the allergenicity reduction primarily relies on irradiation-induced alteration of protein structure. Components of a peanut can prevent the antigenicity of Ara h 6 from decreasing at low irradiation doses, but they lose this function at high doses. Indirect ELISA revealed that the IgG binding to WPPE was stronger than that of the purified Ara h 6 from 1 to 5 kGy (Fig. 5), and the immunoblotting images above 15 kDa, found in WPPE, were much thicker than those in the purified Ara h 6 at low doses (1–5 kGy). This might be due to the fact that other components in the WPPE, such as other proteins and lipids, could help to protect the epitopes on Ara h 6 from damage by the free radicals at low doses. However, as the irradiation doses were increased to 10 kGy, all the proteins formed insoluble aggregates, thus losing their protective effects on Ara h 6. This finding is also in agreement with the previous studies on shrimp allergens, in which it was shown that other components in shrimp muscle, such as lipids, could help to protect the allergens from damaging by the free radicals at low irradiation dose, but lost their function at 15 kGy (Li et al., 2007). In conclusion, the results provided important insights into how Ara h 6 responded to irradiation and the changes of protein structure and antigenicity. Since the structural alteration and antigenic decrease co-existed in irradiated Ara h 6, we could infer that irradiation may reduce the allergenicity of Ara h 6 (at a permitted dose of 10 kGy for food irradiation). Reasonably, we think that the changes of structural and antigenic properties of irradiated Ara h 6 may also emerge in other peanut allergens, and irradiation is an alternative approach to reduce peanut allergenicity. Acknowledgements The work was supported by ‘‘National Natural Science Foundation of China’’ (No. 21162019), National Science and Technology Support, Project, China (No. 2012BAK17B02, 2011BAK10B03) and Research Program of State Key Laboratory of Food Science and Technology (SKLF-MB-201002, SKLF-TS-201109).
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
A potential practical approach to reduce Ara h 6 allergenicity by gamma irradiation Chunping Luo a,b, Chunqiu Hu a,d, Jinyan Gao a,c, Xin Li a,c, Zhihua Wu a,e, Anshu Yang a,e, Hongbing Chen a,e,⇑ a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China Taizhou Vocational College of Science & Technology, Taizhou 318020, China c Department of Food Science, Nanchang University, Nanchang 330047, China d Department of Nutrition and Food Hygiene, Anhui Medical University, Hefei 230032, China e Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China b
a r t i c l e
i n f o
Article history: Received 3 February 2012 Received in revised form 1 September 2012 Accepted 25 September 2012 Available online 2 October 2012 Keywords: Gamma irradiation Peanut allergy Ara h 6 Protein structure Allergenicity
a b s t r a c t Peanut allergen Ara h 6 was isolated and irradiated at 1, 3, 5, or 10 kGy, and a whole peanut protein extract (WPPE) was also treated by irradiation. Alteration in structure of Ara h 6 was characterised by circular dichroism (CD) spectroscopy, ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy and SDS–PAGE, and antigenicity was evaluated by immunoblotting and indirect ELISA with anti-Ara h 6 polyclonal antibody. Irradiation induced significant changes in the secondary and tertiary structures of Ara h 6, and the antigenicity of both purified Ara h 6 and WPPE were reduced upon increasing the irradiation doses. Moreover, a good correlation between the loss in a-helix and IgG binding to Ara h 6 was observed. This indicated that irradiation might be an efficient approach to reduce or eliminate peanut allergenicity. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Peanut allergy is common, frequently severe and typically permanent (Bock, Munoz-Furlong, & Sampson, 2007; Pumphrey & Gowland, 2007; Rona, Keil, Summers, & Gislason, 2007). The prevalence of peanut allergy has been estimated to be between 0.6% and 1% of the US and EU populations (Sicherer, 2002; Tariq et al., 1996). David et al. (1997) have shown that peanut was the third most frequent allergenic food in young Australian and Asian children. Additionally, the incidence of peanut allergy seems to be increasing during recent decades (Ben-Shoshan et al., 2009; Grundy, Matthews, Bateman, Dean, & Arshad, 2002; Sicherer, MunozFurlong, & Sampson, 2003). An explanation is that the popularity and consumption of peanut products have increased (Du Toit et al., 2008). Obviously, the investigations on how to prevent it are increasing. Since peanut allergy is mainly triggered by the immunoglobulin E (IgE) recognition of peanut allergens, reducing the allergenic potency or levels of peanut allergens may be an effective approach to lower the allergenic risk. However, previously described methods to reduce the allergenicity of peanut allergens were found to be inefficient, and it was reported that
⇑ Corresponding author at: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China. Tel.: +86 791 88333529; fax: +86 791 88333708. E-mail address: [email protected] (H. Chen). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.09.091
many peanut allergens were resistant to cooking and digesting (Koppelman, Hefle, Taylor, & de Jong, 2010; Mondoulet et al., 2005). Irradiation has recently become one of the successful techniques to preserve food with minimum interruption of its nutritional and sensory properties (Farkas, 2006; Osterholm & Norgan, 2004). Hundreds of animal feeding investigations of irradiated food have been carried out since 1950, and the studies include subchronic and chronic changes in metabolism, histopathology, function of most systems, reproductive effects, growth, teratogenicity, and mutagenicity. Some of the studies have indicated adverse effects, while no consistent pattern has emerged (Shea, 2000). Consequently, the WHO joint committee concluded that the irradiation of any food commodity up to an overall average dose of 10 kGy presents no toxicological hazard, and toxicological testing of foods so treated is no longer required (WHO, 1988). WHO and FDA have concluded that irradiated food is safe under specific conditions (WHO, 1994, 1999). Moreover, any foods irradiated at levels up to 10 kGy are safe for human consumption without any microbiological hazard and any special nutritional problems (Shea, 2000). Oh et al. (2009) have evaluated the allergenicity of irradiated peanut extract using splenocytes from peanut-sensitised mice and draw a conclusion that the allergenicity of peanut extracts could be reduced by irradiation treatment, and irradiated peanuts might provide a novel immunogen for an immunotherapy of peanut allergy. It is the first and only report on the gamma irradiation
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treatment of peanut allergens, and the structural alteration of irradiation-treated peanut allergens was not defined. However, proteins exposed to irradiation would present structural alterations caused by fragmentation, cross-linking, aggregation, and amino acid modification, affecting their secondary and tertiary structures and their immunoreactivity (Poms & Anklam, 2004). Accordingly, Oh et al. (2009) thought that alteration of epitopes by denaturation of the peanut, after irradiation, might have induced a lower response by T cells. In addition, studies have shown that modification of allergens, by irradiation at levels up to 10 kGy, is a feasible approach to reduce or abolish the allergenicity of egg, milk, wheat and shrimp (Antônio et al., 2012; Lee et al., 2001; Seo et al., 2007; Sinanoglou, Batrinou, Konteles, & Sflomos, 2007). Therefore, before the irradiation process can be generally recommended for reducing allergenicity of peanut allergens, much more information about the process remains to be investigated. Moreover, Ara h 6 was identified as one of the major peanut allergens, sharing 59% sequence identity and a high degree of structural conservation with Ara h 2, another major peanut allergen (Kleber-Janke, Crameri, Appenzeller, Schlaak, & Becker, 1999; Marsh et al., 2008). Studies in children have demonstrated that Ara h 6 and Ara h 2 were the most commonly recognised peanut allergens, and IgE reactivity to them was a risk factor for the most serious reactions (Flinterman et al., 2007; Pastorello et al., 2001). Currently, there is only one natural counterpart of Ara h 6 and purified Ara h 6 was described by another two independent groups with a molecular weight of approximately 15 and 14.981 kDa, identified by SDS–PAGE and mass spectroscopy, respectively (Koppelman et al., 2005; Suhr, Wicklein, Lepp, & Becker, 2004), and the prepared Ara h 6 needs to be subjected to biochemical and immunological studies. Consequently, our work aims to define the irradiation-induced alterations in structure and antigenicity of a model peanut allergen, Ara h 6. Additionally, studies have shown that the ingredients of the food matrix can have a great impact on the conformation and antigenic properties of proteins during processing (Grimshaw et al., 2003; Lee, Lee, & Song, 2003; Maleki & Hurlburt, 2004). Herein, the antigenic alteration of whole peanut protein extract was investigated to discover whether irradiation processing, under various conditions, could have unpredictable effects on the Ara h 6 allergenicity in the complex food matrices. 2. Materials and methods 2.1. Preparation of whole peanut protein extract (WPPE) Raw peanuts (Arachis Hypogaea, Xianghua, China) were purchased from a local supplier (Nanchang, China) and were stored at 20 °C until used. After shelling, the skins were removed, and the kernel was ground in liquid nitrogen, using a mortar and pestle. The meal was then defatted by stirring with 5 volumes of precooled acetone for 1 h at 4 °C, and it was recovered by centrifuging at 6000g for 20 min at 4 °C. After being defatted three times, the meal was allowed to dry in the air and suspended in 5 vol of 50 mM Tris–HCl (pH 7.2), followed by stirring for 12 h at 4 °C. Then the suspension was centrifuged at 6000g for 20 min at 4 °C, and the supernatant was collected as WPPE. 2.2. Preparation of Ara h 6 WPPE (10 mg/ml) was loaded onto a DEAE-Sepharose Fast Flow column (bed dimension, 26 300 mm2, GE Healthcare, USA) previously equilibrated with 50 mM Tris–HCl (pH 7.2, loading buffer) at room temperature. The column was washed with loading buffer until the A280 of the effluent was less than 0.2. Proteins were eluted with a linear gradient in a loading buffer (0–0.2 M NaCl in 500 ml
of loading buffer) at a flow rate of 1.5 ml/min. Fractions were collected and analysed by SDS–PAGE, and Ara h 6 appeared to be a band of 15 kDa and essentially pure (>95%), calculated by a densitometer scanning of an SDS–PAGE gel stained with Coomassie Brilliant Blue. Further identity confirmation of Ara h 6 was performed with MALDI-TOF-MS fragmentation analysis of selected tryptic peptides with protein-specific sequences (Applied Biosystems, Framingham, MA, USA). Ara h 6 -containing fractions were pooled and concentrated by ultrafiltration with an Amicon Ultra-15 filter with a MWCO of 3.0 kDa (Millipore, USA), and the protein concentration was measured according to the Bradford method. 2.3. Protein content Protein concentration was determined by the Bradford method, measuring absorbance at 595 nm. Bovine serum albumin was used as a standard. 2.4. Production of polyclonal antibody Polyclonal antibody against Ara h 6 was produced from New Zealand male rabbits. Before immunising, 1 ml of blood from a non-injected rabbit was collected by the ear-bleeding method as a negative serum blank. Two rabbits were immunised by subcutaneously injecting into their shaved backs 0.2 mg of purified Ara h 6 in Freund’s complete adjuvant (Sigma, USA). Subsequent injections of Ara h 6 in incomplete Freund’s adjuvant (Sigma, USA) were performed three times, at 2-week intervals, and the immune responses of rabbits were monitored by indirect ELISA. The rabbits were finally bled and each antiserum was separated by centrifuging at 4000g for 10 min at 4 °C. All sera were stored at 80 °C until used. 2.5. Gamma irradiation The purified Ara h 6 (0.2 mg/ml, 30 ml) and WPPE (2 mg/ml, 10 ml) were put into a plastic tube with a cap, and irradiated in a cobalt-60 gamma-irradiator (Jiangxi Academy of Agricultural Sciences, Nanchang, China) in the presence of air. The applied dose levels were 1, 3, 5 and 10 kGy, and the temperature during irradiation treatment was set at 4 °C. Non-irradiated Ara h 6 was used as a control. 2.6. Ultraviolet (UV) absorption spectroscopy UV absorption spectra analysis was performed by a UV spectrophotometer (UV-VS 2501PC, Shimadzu Corporation, Japan) at room temperature (25 °C). All the samples were homogeneously mixed (vortexed) and scanned from 200 to 400 nm. 2.7. Surface hydrophobicity Surface hydrophobicity was determined by the 1-anilinonaphthalene-8-sulfonate (ANS) (Sigma, USA). A volume of 20 ll of ANS solution (5 mM in 0.01 M PBS, pH 7.4) was added to 4 ml of Ara h 6 solutions (0.1 mg/ml) and homogeneously mixed by vortex. The relative fluorescence was measured with a spectrophotofluorometer (F-4500, Hitachi, Japan). The excitation wavelength was 390 nm, and the emission wavelength was from 400 to 650 nm at a scanning speed of 1500 nm /min. 2.8. Circular dichroism (CD) Conformational changes in the secondary structure of irradiated protein samples were analysed using a CD spectropolarimeter (J810, JASCO, Japan). The homogeneously mixed proteins, at a con-
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centration of 0.5 mg/ml were used for measurements. CD spectra were scanned at the far UV range from 250 to 190 nm, at a rate of 100 nm/min, with a bandwidth of 1.0 nm at room temperature. The spectra represented an average of three consecutive scans. Results of each sample were averaged, and the mean residue ellipticity (h) was expressed as deg cm2 dmol1, followed by calculating the secondary structure contents by JASCO secondary structure software (JASCO, Japan). 2.9. Reducing and non-reducing SDS–PAGE SDS–PAGE was performed essentially according to Laemmli (1970), using the Mini Protean system (BioRad, USA). A 5% stacking gels and 15% separating gels were used in SDS–PAGE analysis. For reducing SDS–PAGE, 3% (v/v) b-mercaptoethanol was added to 2 non-reducing sample buffer [20 mM Tris, pH 6.7, 2 mM EDTA, 2% (w/v) SDS, 25% (v/v) glycerin, 0.02% (w/v) bromphenol blue)]. Samples were mixed (1:1, v/v) with the reducing/non-reducing sample buffer, boiled for 5 min, followed by centrifuging at 4000g for 5 min, and the supernatants were loaded onto the gel. Gels were stained with Coomassie brilliant blue R-250 and destained in a de-staining solution (7.5% HAc (v/v), 5% methanol (v/v) in water). After de-staining, gels were scanned, followed by calculating the band intensity with Quantity One software (Bio-Rad).
IgG binding abilities were defined by the following equation:
IgG binding abilitiesð%Þ ¼ B=B0 100 2.12. Statistical analysis Appropriate data were analysed for statistical significance using ANOVA procedures (SPSS for Windows 2003, Microsoft Corp., version 13.0, Chicago, IL, USA) and Fisher’s least significant difference (LSD, p < 0.05) test. 3. Results 3.1. UV absorption spectra of irradiated Ara h 6 The UV absorbance of irradiated Ara h 6 was assayed at a wavelength of 200–400 nm. Fig. 1 shows the UV spectra of the purified Ara h 6 irradiated at various doses. The non-irradiated Ara h 6 had a weak absorbance at 280 nm, while a rise in UV absorbance of irradiated Ara h 6 was observed. The maximum absorbance value increased from 0.12 (control) to 0.16, 0.77, 1.42, and 1.64 after being irradiated at 0, 1, 3, 5, 10 kGy, respectively. In addition, compared with the control samples, the location of absorption peak in irradiation-treated samples underwent a blue shift of 1–3 nm.
2.10. Immunoblotting 3.2. Surface hydrophobicity of irradiated Ara h 6
2.11. IgG binding by indirect ELISA The binding abilities of the rabbit IgG to the irradiated protein samples were tested by indirect ELISA. The 96-well microtitre plate was coated with 100 ll of purified Ara h 6 (2 lg/ml) or WPPE (6.25 lg/ml) in coating buffer (0.015 M Na2CO3, 0.035 M NaHCO3, pH 9.6) overnight at 4 °C. The wells were washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and subsequently blocked with 5% skimmed milk in PBS, incubating for 1 h at 37 °C. The wells were again washed and 100 ll of serially diluted sera (1:10000 dilutions in PBS) were added, followed by incubation for 1 h at 37 °C. After further washing, 100 ll of goat anti-rabbit IgG conjugated with horseradish peroxidase (1:5000 dilutions in PBS) were then added to each well, followed by incubation for 1 h at 37 °C, for detecting the bound immunogen. The plates were further washed, and the colour was developed by adding a substrate solution (100 ll) of o-phenylene diamine (0.5 mg/ml, Sigma, USA), 0.03% hydrogen peroxide in 0.1 M citrate buffer (pH 5.5) to each well for 15 min at 37 °C. The reaction was terminated with 50 ll of 2 M sulphuric acid, and the absorbance of irradiated samples (B) and non-irradiated samples (B0) was read at 490 nm with a Bio-Rad Microplate Reader (Model 1860, BioRad, USA).
The surface hydrophobicity of irradiated Ara h 6 was determined by ANS. Fig. 2 shows the ANS induced fluorescence spectra of the irradiated Ara h 6 at various doses. It indicates that the ANSfluorescence peak of non-irradiated Ara h 6 was very weak at 450 nm, and the 1 kGy-treated sample had a profile similar to the control sample. However, upon increasing the irradiation dose to 3, 5 and 10 kGy, the ANS-fluorescence intensities of samples were increased 4.37-, 8.58-, and 11.2-fold, compared with the control samples, respectively. Moreover, there were no significant differences of the location of the ANS-fluorescence peak between the irradiation-treated samples and the non-irradiated ones. 3.3. Circular dichroism (CD) spectra of irradiated Ara h 6 Fig. 3 indicates the far-UV CD spectra of the purified Ara h 6 irradiated at various doses. The spectrum of non-irradiated Ara h 6 had a strong positive peak at 196 nm with pronounced double negative peaks at 208 and 222 nm, and it crossed zero ellipticity at 199 nm. Irradiation treatment affected CD spectra of Ara h 6 significantly. Both the intensity of positive peak at 196 nm and the two negative peaks at 208 and 222 nm decreased with increased
Optical density
SDS–PAGE gels were prepared, under reducing conditions as described above, and the separated proteins were transferred to polyvinyldifluoride membranes (PVDF, Millipore, USA) by applying a constant current of 50 mA for 2 h at room temperature. The membranes were blocked with TBS buffer (50 mM Tris–HCl, 150 mM NaCl, pH 7.5) containing 1%Tween-20 (TBST) for 1 h at room temperature, followed by overnight incubation with the rabbit antiAra h 6 serum (1:5000 dilutions in TBST) at 4 °C. Then the membrane was washed three times in TBS and incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:5000 dilutions in TBST, Sigma, USA) for 2 h at room temperature. After washing for a further three times, the peroxidase-positive images were developed by incubating the membrane in 20 ml of TBS containing 12 mg 4-chloro-1-naphthol, 4 ml of ethanol and 12 ll of 30% H2O2 at room temperature for 10 min.
Wavelength(nm) Fig. 1. UV absorption spectra of irradiated Ara h 6 at various doses.
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Relative intensity
diated at 1 or 3 kGy, and a trace spread from the starting point of separating gel at the dose of 5 kGy. Interestingly, no bands were observed in SDS–PAGE when the dose reached 10 kGy because Ara h 6 formed solely insoluble aggregates. Additionally, a similar result was found in the samples of WPPE (Fig. 4b), except that images of over 72 kDa emerged at 3 kGy and above. The immunoblotting images in Fig. 4c and d demonstrate that the irradiation affected the image definition more than did SDS– PAGE. Intensities of the intact Ara h 6 and WPPE declined, and the degrees of the aggregation rose with increase of irradiation doses. Remarkably, heavier images (above 15 kDa) existed in WPPE than in the purified Ara h 6. 3.5. IgG binding abilities of irradiated Ara h 6
Wavelength(nm)
IgG binding abilities of the purified Ara h 6 and WPPE treated at various doses, using anti-Ara h 6 polyclonal antibody for ELISA checking, are shown in Fig. 5. There was a clear decrease in IgG binding to Ara h 6 with the increase of irradiation doses. However, the IgG binding abilities of WPPE were retained better than were those of the purified Ara h 6 at the same irradiation dose. However, when they were irradiated at 10 kGy, only 5% IgG binding abilities were found in both WPPE samples and the purified Ara h 6 samples.
Fig. 2. Surface hydrophobicity of irradiated Ara h 6 at various doses.
irradiation dose. Interestingly, with a 10 kGy irradiation dose, the positive peak disappeared and showed a negative peak at 200 nm. This CD spectral alteration indicated the changes of the secondary structure content of Ara h 6 after irradiation (as shown in the graph insets). A loss of a-helix and b-turn were observed from 1 to 10 kGy, and the content of random coils showed a decline at 1–3 kGy and a rise at 5–10 kGy. Moreover, the b-strand showed an opposite alteration, and particularly, a transition from b-strand to b-turn occurred as the doses increased.
4. Discussion Significant alterations in conformational and antigenic properties of peanut allergen Ara h 6 were induced during irradiation treatment, the extent of which depended on the irradiation doses. 1 kGy treatment had little influence on the tertiary structure of Ara h 6, while it had an important impact on the secondary structure and antigenicity of Ara h 6. Moreover, the higher the dose applied, the greater were the changes in conformation and antigiencity of Ara h 6. Similar findings were reported in that the structure and
3.4. SDS–PAGE and immunoblotting profile of irradiated Ara h 6 SDS–PAGE patterns of purified Ara h 6 and WPPE, at various irradiation doses, are shown in Fig. 4. Fig. 4a indicates that irradiation could cause a decrease of the intensity of the purified Ara h 6 image (15 kDa). A new image (20 kDa) appeared when it was irra-
Percentage(%)
50 40
Helix Strand
30
Turn 20
Random
10
Molar ellipticity(deg·cm2/dmol)
0 Control
1
3
5
10
Irradiation doses(kGy)
Wavelength(nm) Fig. 3. Circular dichroism spectra of irradiated Ara h 6 at various doses. Graph insets represent the corresponding secondary structural content of irradiated Ara h 6 at various doses.
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IgG binding abilities [B/B0 ×100(%)]
Fig. 4. SDS–PAGE patterns of purified Ara h 6 (0.2 mg/ml) (a) and WPPE (2 mg/ml) (b) irradiated at various doses and the corresponding immunoblotting using anti-Ara h 6 polyclonal antibody (c and d). M, molecular weight marker; Con, Control samples.
120 100
WPPE Purified Ara h 6
80 60 40 20 0 Control
1
3
5
10
Doses(kGy) Fig. 5. IgG binding abilities of purified Ara h 6 and WPPE treated at various doses, using anti-Ara h 6 polyclonal antibody for ELISA checking. B and B0 represent ELISA data for defining the binding of irradiated samples to anti-Ara h 6 polyclonal antibody and that of non-irradiated samples, respectively.
antigenicity of shrimp allergens and milk allergens could be altered by irradiation (Byun et al., 2000; Lee et al., 2001). A loss of secondary and tertiary structures of irradiated Ara h 6 was defined by (CD) spectroscopy, UV absorption spectroscopy, and fluorescence spectroscopy. The changes in the UV spectra of Ara h 6 suggested that the native protein structure unfolded, which implied that the hydrophobic amino acids of the proteins were partially exposed to the solvent. Typically, the ANS fluorescence anal-
ysis fully proved the alteration of hydrophobic amino acids, which was in conformity with the UV absorption spectra analysis. This phenomenon has also been observed in other proteins, such as bovine serum albumin and myoglobin (Gaber, 2005; Lee & Song, 2002). Moreover, the large difference between irradiation-treated Ara h 6 and the non-irradiated one (control) was manifested in the loss of the secondary structure of the proteins and the change in the local environment of ordered structure of a polypeptide chain defined by far-UV CD spectra. It clearly supported the idea that oxygen radicals, generated during irradiation of proteins in solution, disrupt the ordered structure of proteins, resulting in structural unravelling of proteins in solution. Changes in the second and tertiary structures caused by gamma irradiation led to protein misfolding and, subsequently, to aggregation. Irradiated proteins can be converted to higher molecular weight aggregates due to the generation of inter-protein crosslinking reactions, hydrophobic and electrostatic interactions, as well as the formation of disulfide bonds (Oliveira, de la Hoz, Silva, Torriani, & Netto, 2007; Xu & Chance, 2005). In this work, Ara h 6 and WPPE formed more soluble aggregates upon increased irradiation dosing, while the aggregates formed insoluble precipitates under 10 kGy irradiation. However, this was not unique, as similar phenomena exist in other irradiated food proteins, such as b-lactoglobulin (Lee et al., 2001), bovine and porcine serum plasma (Lee et al., 2003). Moreover, since reducing and non-reducing SDS– PAGE can define the role of disulfide bonds in the formation of protein aggregation, caused by irradiation treatment, and no difference was observed between the migration patterns of the bands
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in reduced and non-reduced SDS gels (data not shown), this indicated that the disulfide bond was not responsible for the production of protein aggregation in the irradiated Ara h 6. Irradiation-induced alteration of protein structure could affect its antigenicity. In the present study, a clear decrease in a-helix content (Fig. 3) upon elevated irradiation doses was observed, and it correlated well with the decline in IgG binding to Ara h 6 (Fig. 5). Besides, the aggregation gradually became greater upon increasing irradiation doses from 1 to 5 kGy, and it implied that conformational epitopes on Ara h 6 were easily lost by irradiation treatment. Actually, previous works have demonstrated that irradiation treatment could destroy conformational epitopes by denaturation of the proteins and alter linear epitopes by aggregation, fragmentation, and amino acid modification (Kim et al., 2002; Li, Lin, Cao, & Khalid, 2007; Mengna, Mahesh, Suzanne, Keneth, & Shridhar, 2004). In addition, Oh et al. (2009) have evaluated the changes of allergenicity and cytokine production profiles after exposure of irradiated peanut extract in a peanut-allergy mouse model. A higher level of production of IFN-c and IL-10 cytokines was observed, when the cells were stimulated with 10 kGy of irradiated peanut extract, which indicated the rise of the Th1/Th2 ratio in response to treatment with gamma-irradiated peanut extract and showed that the allergenicity of peanut extract could be reduced by gamma irradiation, which caused down-regulation of Th2 lymphocyte activity in the peanut-sensitised mice. Combining the finding of Oh et al. (2009) with this work, a positive conclusion was suggested, that gamma irradiation could reduce the antigenicity and allergenicity of peanut allergens, and the allergenicity reduction primarily relies on irradiation-induced alteration of protein structure. Components of a peanut can prevent the antigenicity of Ara h 6 from decreasing at low irradiation doses, but they lose this function at high doses. Indirect ELISA revealed that the IgG binding to WPPE was stronger than that of the purified Ara h 6 from 1 to 5 kGy (Fig. 5), and the immunoblotting images above 15 kDa, found in WPPE, were much thicker than those in the purified Ara h 6 at low doses (1–5 kGy). This might be due to the fact that other components in the WPPE, such as other proteins and lipids, could help to protect the epitopes on Ara h 6 from damage by the free radicals at low doses. However, as the irradiation doses were increased to 10 kGy, all the proteins formed insoluble aggregates, thus losing their protective effects on Ara h 6. This finding is also in agreement with the previous studies on shrimp allergens, in which it was shown that other components in shrimp muscle, such as lipids, could help to protect the allergens from damaging by the free radicals at low irradiation dose, but lost their function at 15 kGy (Li et al., 2007). In conclusion, the results provided important insights into how Ara h 6 responded to irradiation and the changes of protein structure and antigenicity. Since the structural alteration and antigenic decrease co-existed in irradiated Ara h 6, we could infer that irradiation may reduce the allergenicity of Ara h 6 (at a permitted dose of 10 kGy for food irradiation). Reasonably, we think that the changes of structural and antigenic properties of irradiated Ara h 6 may also emerge in other peanut allergens, and irradiation is an alternative approach to reduce peanut allergenicity. Acknowledgements The work was supported by ‘‘National Natural Science Foundation of China’’ (No. 21162019), National Science and Technology Support, Project, China (No. 2012BAK17B02, 2011BAK10B03) and Research Program of State Key Laboratory of Food Science and Technology (SKLF-MB-201002, SKLF-TS-201109).
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