Heat and pressure treatments effects on peanut allergenicity
ABSTRACT Peanut allergy is recognized as one of the most severe food allergies. The aim of this study was to investigate the changes in IgE binding capacity of peanut proteins produced by thermal-processing methods, including autoclaving. Immunoreactivity to raw and thermally processed peanut extracts was evaluated by IgE immunoblot and skin prick test in patients with clinical allergy to peanut. Roasted peanut and autoclaved roasted peanut were selected for IgE ELISA experiments with individual sera, immunoblot experiments with antibodies against peanut allergens (Ara h 1, Ara h 2 and Ara h 3), digestion experiments, and circular dichroism spectroscopy. In vitro and in vivo experiments showed IgE immunoreactivity of roasted peanut proteins decreased significantly at extreme conditions of autoclaving. Circular dichroism experiments showed unfolding of proteins in autoclave treated samples, which makes them more susceptible to digestion. Autoclaving at 2.56 atm, for 30 min, produces a significant decrease of IgE-binding capacity of peanut allergens.
- [Show abstract] [Hide abstract]
ABSTRACT: The in vitro digestion of heated whey protein aggregates having different structure and physicochemical properties was evaluated under simulated gastric conditions. Aggregates were formed by heating whey protein isolates (WPI) at 3-9% w/w initial protein concentration and pH 3.0-7.0. Results showed that high protein concentration led to formation of larger WPI aggregates with fewer remaining monomers. Aggregates formed at high protein concentrations showed slower degradation rate compared to those formed at low protein concentration. The effect of initial protein concentration on peptide release pattern was not apparent. Heating pH was a significant factor affecting digestion pattern. At pH above the isoelectric point, the majority of the proteins involved in the aggregation, and aggregates formed at pH 6.0 were more susceptible to pepsin digestion than at pH 7.0. At acidic conditions, only small amount of proteins was involved in the aggregation and heated aggregates were easily digested by pepsin, while the remaining unaggregated proteins were very resistant to gastric digestion. The potential physiological implication of these results on satiety was discussed.Food Chemistry 02/2014; 145C:473-480. · 3.26 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Food induced allergic manifestations are reported from several parts of the world. Food proteins exert their allergenic potential by absorption through the gastrointestinal tract and can even induce life threatening anaphylaxis reactions. Among all food allergens, legume allergens play an important role in induction of allergy because legumes are a major source of protein for vegetarians. Most of the legumes are cooked either by boiling, roasting or frying before consumption, which can be considered a form of thermal treatment. Thermal processing may also include autoclaving, microwave heating, blanching, pasteurization, canning, or steaming. Thermal processing of legumes may reduce, eliminate or enhance the allergenic potential of a respective legume. In most of the cases, minimization of allergenic potential on thermal treatment has generally been reported. Thus, thermal processing can be considered an important tool by indirectly prevent allergenicity in susceptible individuals, thereby reducing treatment costs and reducing industry/office/school absence in case of working population/school going children. The present review attempts to explore various possibilities of reducing or eliminating allergenicity of leguminous food using different methods of thermal processing. Further, this review summarizes different methods of food processing, major legumes and their predominant allergenic proteins, thermal treatment and its relation with antigenicity, effect of thermal processing on legume allergens; also suggests a path that may be taken for future research to reduce the allergenicity using conventional/nonconventional methods.Plant Foods for Human Nutrition 11/2012; 67(4):430-41. · 2.42 Impact Factor
Heat and pressure treatments effects on peanut allergenicity
Beatriz Cabanillasa,⇑,1, Soheila J. Malekib,1, Julia Rodrígueza, Carmen Burbanoc, Mercedes Muzquizc,
María Aránzazu Jiméneza, Mercedes M. Pedrosac, Carmen Cuadradoc, Jesús F. Crespoa
aServicio de Alergia, Hospital Universitario 12 de Octubre. Instituto de Investigación Hospital 12 de Octubre (i+12), Avenida de Córdoba s/n, 28041 Madrid, Spain
bUS Department of Agriculture, Agriculture Research Service, Southern Regional Research Center, 1100 Robert E. Lee Boulevard, New Orleans, LA, United States
cDepartamento de Tecnología de Alimentos, SGIT-INIA, Ctra. de La Coruña km 7.5, 28040 Madrid, Spain
a r t i c l ei n f o
Received 13 July 2011
Received in revised form 9 September 2011
Accepted 27 October 2011
Available online 7 November 2011
a b s t r a c t
Peanut allergy is recognized as one of the most severe food allergies. The aim of this study was to inves-
tigate the changes in IgE binding capacity of peanut proteins produced by thermal-processing methods,
including autoclaving. Immunoreactivity to raw and thermally processed peanut extracts was evaluated
by IgE immunoblot and skin prick test in patients with clinical allergy to peanut. Roasted peanut and
autoclaved roasted peanut were selected for IgE ELISA experiments with individual sera, immunoblot
experiments with antibodies against peanut allergens (Ara h 1, Ara h 2 and Ara h 3), digestion experi-
ments, and circular dichroism spectroscopy. In vitro and in vivo experiments showed IgE immunoreactiv-
ity of roasted peanut proteins decreased significantly at extreme conditions of autoclaving. Circular
dichroism experiments showed unfolding of proteins in autoclave treated samples, which makes them
more susceptible to digestion. Autoclaving at 2.56 atm, for 30 min, produces a significant decrease of
IgE-binding capacity of peanut allergens.
? 2011 Elsevier Ltd. All rights reserved.
Peanut allergy is one of the most severe food allergies due to its
life-threatening nature and persistency (Sicherer & Sampson,
2007). Peanut allergy prevalence seems to have increased in the
western world during the past decades. An estimate of the preva-
lence of peanut allergy in US children was 1.4% in 2008 compared
with 0.8% in 2002 and 0.4% in 1997 in a self-reported population
survey (Sicherer, Muñoz-Furlong, Godbold, & Sampson, 2010).
Foods, including peanuts, are subjected to different processing
methods to improve their quality, preservation, safety, suitability
for specific product applications, etc. (Sathe & Sharma, 2009). The
types of modifications that food proteins may undergo during pro-
cessing includes protein unfolding and aggregation and chemical
modification (Mills, Sancho, Rigby, Jenkins, & Mackie, 2009). These
modifications could alter food allergenicity, i.e. increasing or
decreasing IgE reactivity (Cabanillas-Martín, Crespo, Burbano, &
Rodríguez, 2010; Cuadrado et al., 2009). Therefore, understanding
food processing seems to be important in food allergenicity.
It has been recognized that roasted peanuts are more allergenic
than raw peanuts (Beyer et al., 2001; Chung & Champagne, 1999,
2001; Kopper et al., 2005; Maleki, Chung, Champagne, & Raufman,
2000; Maleki et al., 2003). Roasted peanut extracts bind IgE from
patients with peanut allergy at approximately 90-fold higher levels
than raw peanuts, and the protein modifications induced by Mail-
lard reaction contribute to the observed effect (Chung & Cham-
pagne, 1999, 2001; Maleki et al., 2000). Previous studies have
demonstrated that autoclaving at 2.56 atmospheres (atm) for
30 min (min) and ‘‘instantaneous controlled pressure drop (DIC)’’
at 6 bar (5.9 atm) during 3 min reduced IgE-binding capacity of lu-
pine allergens, whereas it was only slightly affected by boiling,
microwave and extrusion-cooking, demonstrating the high ther-
mal resistance of the major lupine allergens (Alvarez-Alvarez
et al., 2005; Guillamón et al., 2008).
Autoclaving at 2.56 atm, for 30 min, produced a relevant de-
crease in the IgE-binding capacity of lentil and chickpea allergens.
However, several immunoreactive proteins still remained in these
legumes upon harsh autoclaving (Cuadrado et al., 2009).
We sought to investigate the changes in IgE binding capacity of
peanut proteins produced by thermal-processing methods, includ-
2. Materials and methods
2.1. Patients and sera
Serum samples from 54 patients sensitized to peanut were used
in this study. Nineteen of them (Nos. 1–19) had a clinical history of
peanut allergy, confirmed on the basis of either a convincing his-
tory of severe systemic anaphylaxis after peanut ingestion or a
0308-8146/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
⇑Corresponding author. Tel.: +34 913908660; fax: +34 913908261.
E-mail address: email@example.com (B. Cabanillas).
1These authors contributed equally to this study.
Food Chemistry 132 (2012) 360–366
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/foodchem
positive double-blind, placebo-controlled food challenge (DBPCFC)
with peanut, a positive skin prick test response and a specific IgE
level to peanut, greater than 0.35 kU/l (median = 2.79 kU/L; range,
0.47?100 kU/L), quantified with the CAP-FEIA System (Phadia,
Uppsala, Sweden). The other 35 patients (Nos. 20–54) were sensi-
tized to peanut, having a specific IgE level to peanut, greater than
0.35 kU/l (median = 5.44 kU/l; range, 0.36?100 kU/L). A serum
pool from 4 out of 19 patients with actual peanut allergy (Nos. 5,
8, 9 and 11) and individual sera from all patients were used in
IgE immunodetection assays. A serum pool from three patients
with specific IgE to Anisakis spp. was used as a negative control.
After informed consent, seven additional patients with con-
firmed clinical allergy to peanut underwent skin testing with raw
and thermally processed peanut extracts. Two healthy subjects
were tested with the same protein extracts as a control group.
The study was approved by the Ethics Committee of the Hospital
Universitario12 de Octubre
ofMadrid (Permission No.
2.2. Plant material, heat treatments and protein extracts
Raw, fried and roasted peanuts (Virginia variety) obtained from
Aperitivos Medina SL (Spain) were used in the study. Raw peanut
seeds (1:10 w/v) were boiled in water, at 100 ?C, for 60 min.
Roasted peanuts seeds were autoclaved using a tabletop autoclave
(CertoClav Multicontrol IPX4, Traun, Austria) at 121 ?C (1.18 atm)
for 15 and 30 min and at 138 ?C (2.56 atm) for 15 and 30 min.
Raw and thermally processed peanut seeds were milled to pass
through a 1 mm sieve (Tecator, Cylotec 1093, Höganäs, Sweden)
and the resulting meal was defatted with n-hexane (34 ml/g of
flour) for 4 h, shaken, and air-dried after filtration of the n-hexane.
Defatted flour was extracted twice in a solution of 50 mM Tris–HCl,
pH 8.0, plus 500 mM NaCl at a 1:10 w/v ratio for 1 h, at 4 ?C, by
stirring. After centrifugation (27000g, for 20 min, at 4 ?C) the
supernatants were dialyzed against H2O (cut-off point, 3.5 kDa),
for 48 h at 4 ?C and freeze-dried. The protein content of each sam-
ple was measured according to the Bradford dye-binding assay
(Bio-Rad, Hercules, CA) using BSA (Sigma, St. Louis, MO) as a
2.3. Immunodetection assays
2.3.1. Protein electrophoresis and IgE immunoblot experiments
SDS–PAGE was performed according to Laemmli (1970). Sam-
ples (10 lg per well) were mixed with Laemmli sample buffer
(Bio-Rad) and 2-mercaptoethanol (Bio-Rad) heated at 90 ?C for
10 min, electrophoresed in 4–20% Tris–HCl linear gradient precast
gel (Bio-Rad). Proteins were visualized with Coomassie brilliant
blue R250 staining. Western blotting was performed by electro-
phoretic transfer to polyvinylidene difluoride (PVDF) membranes
at 250 mA for 100 min, at room temperature, essentially according
to the method of Towbin, Staehelin, and Gordon (1979). After
blocking with 5% BSA (w/v) in PBS, membranes were incubated
overnight with the serum pool from patients with clinical allergy
to peanut (Nos. 5, 8, 9 and 11, 1:5 dilution) or individual sera
(1:10 dilution) washed, and then treated with mouse anti-human
IgE mAbHE-2ascitic fluid
(Sanchez-Madrid, Morago, Corbi, & Carreira, 1984). After washing,
a rabbit anti-mouse IgG peroxidase-conjugated antibody (1:5000
dilution for 1 h; DAKO, Glostrup, Denmark) was added. Detection
of IgE-binding components was achieved by means of enhanced
chemiluminescence, according to the manufacturer’s instructions
(Amersham Biosciences, Little Chalfont, United Kingdom). A serum
pool (1:10 dilution) from three patients with specific IgE to Anisakis
spp. was tested as a negative control.
(1:3000 dilution for2 h)
Specific IgE binding was determined by means of indirect ELISA
in 14 individual sera from patients with clinical allergy to peanut
(Nos. 2, 4, 6, 7, 9, 10, 12, 15, 17) and sera from patients sensitized
to peanut (Nos. 21, 22, 24, 32, 33). The selection of these specific
patients was made attending serum availability. Polystyrene 96-
well microtiter plates (Costar 3590, Corning) were coated with
100 ll of extract at 30 lg/ml in PBS and incubated at 4 ?C over-
night. Wells were washed with PBS and 0.5% Tween 20 (v/v) and
blocked with PBS containing 3% nonfat milk (w/v) and 0.1% Tween
20. Plates were incubated with individual sera (1:2 dilution) and
binding of IgE was detected by mouse anti-human IgE mAb HE-2
ascitic fluid (1:5000 dilution for 1 h) (Sanchez-Madrid et al.,
1984) followed by goat anti-mouse IgG peroxidise-conjugated
(1:2500 dilution for 1 h, Pierce Chemical Co, Rockford, Ill). The per-
oxidase reaction was developed with 50 ll of peroxidase substrate
buffer (Dako). After 30 min, the reaction was stopped with 50 ll of
4 N H2SO4, and the optical density (OD) was measured at 492 nm.
All the tests were performed in triplicate.
2.3.3. Anti Ara h 1, Ara h 2 and Ara h 3 immunoblots
For anti Ara h 1, Ara h 2 and Ara h 3 experiments, defatted flours
were solubilized with extraction buffer (50 mM Tris, 500 mM
NaCl), followed by sonication and centrifugation at 5500g, for
15 min. The supernatant was removed and saved (soluble fraction).
The pelleted fractions, after centrifugation (insoluble fraction),
were solubilized by boiling for 5 min in standard electrophoresis
sample buffer, containing 2% sodium dodecyl sulfate (SDS) and
reducing agent (Invitrogen). The samples in SDS-sample buffer
were centrifuged at 5500g for 15 min. The supernatants were re-
moved, aliquoted and stored at ?20 ?C.
The proteins in the soluble and insoluble fractions were
electrophoresed and transferred to PVDF membranes. Blocking
was carried out for 1 h, at room temperature, in 5% blotto. Chicken
anti-raw Ara h 1 (1:10000), chicken anti-raw Ara h 2 (1:8000) and
chicken anti-raw Ara h 3 (1:5000) (custom synthesized by Sigma
Immunosys, The Woodlands, TX) were diluted in 5% blotto and
incubated with the PVDF membrane for 1 h, at room temperature.
These antibodies have been shown to recognize both raw and ther-
mally processed forms of these proteins (Schmitt, Nesbit, Hurlburt,
(1:100,000) (Sigma Immunosys, The Woodlands, TX), was diluted
in 2% blotto and incubated with membranes for 30 min at room
temperature. Detection was achieved as described above.
2.4. Digestion reactions with trypsin
Roasted peanut and autoclaved roasted peanut were selected
for digestion experiments with trypsin. Soluble fractions were
incubated in the presence of 0.0168 lM trypsin in a solution of
50 mM Tris, 1 mM EDTA at pH 8.4, at 37 ?C. Aliquots were taken
for SDS–PAGE analysis at 1, 3, 5, 10, 30 min, 1 h, 2 h, and overnight
(?15 h). The gels were stained for 1 h using GelCode Blue Stain
Reagent according to manufacturer’s instructions (Pierce, Rockford,
2.5. Circular dichroism (CD) spectroscopy
Far UV (185–250 nm) circular dichroism spectra of roasted pea-
nut and autoclaved roasted peanut (1.18 atm and 2.56 atm, 15 and
30 min) were obtained. Samples were desalted through use of dis-
posable gel filtration columns (Bio-Rad, Hercules, CA) into Milli-Q
water and immediately used in CD measurements. Protein concen-
tration was 0.1 mg/ml and spectra were obtained at room temper-
ature with a JASCO 815 spectropolarimeter equipped with a Peltier
temperature control system (Japan Spectroscopic Co. Ltd., Tokyo,
B. Cabanillas et al./Food Chemistry 132 (2012) 360–366
Japan). Average of two measurements was obtained using Jasco CD
Manager Software. Data was analyzed with CDPro.
2.6. Skin prick tests
Skin prick tests were performed through a standard technique
with sterile needles (Alk-Abelló, Hørsholm, Denmark). Protein
extracts of raw, boiled, fried, roasted and autoclaved roasted pea-
nut (2.56 atm, 30 min) (10 mg/ml protein in PBS); a positive con-
trol (histamine dihydrochloride, 10 mg/ml); and a negative
control (PBS) were applied on the volar side of the forearm in
duplicate. The mean diameters of the wheal were measured after
15 min. To be considered positive, the wheal had to be at least
3 mm greater than that elicited by the negative control.
3.1. Electrophoretic characterization
The SDS–PAGE patterns of the protein extracts of raw, boiled,
fried, roasted and autoclaved roasted peanut seeds are presented
in Fig. 1A1. Similar patterns were observed in raw, boiled, fried
and roasted peanut extracts. Roasted peanut after autoclaving
(1.18 and 2.56 atm) showed less stained bands but an increase of
low molecular weight smear. Western blotting was performed
using a serum pool from four patients with actual peanut allergy
(Nos. 5, 8, 9 and 11) (Fig. 1A2). Similar allergenic proteins were
present in raw, fried and roasted peanut extracts with the excep-
tion of a high molecular weight protein (121 kDa) that is signifi-
cantly reduced in fried peanut extract (Fig. 1A2). The IgE-binding
pattern of boiled peanut extract showed differences compared to
raw, roasted and fried peanut extract: the 65 kDa band (Ara h 1)
and Ara h 2 (17–18 kDa) were less visible in this extract. After
autoclaving of roasted peanut, a reduction in the total IgE-reactive
bands was seen (Fig. 1A2). Bands of 121, 65, 37 kDa and two low
molecular weight proteins (17, 14 kDa) were not bound by IgE in
any autoclaved treatment (1.18 or 2.56 atm). Two immunoreactive
bands of 26 and 33 kDa were still detected at high pressure
(2.56 atm) applied for 15 min, however under the same pressure
conditions applied during 30 min, none of them were recognized.
Western blotting using a serum pool from three patients with
specific IgE to Anisakis spp. (negative control; Fig. 1A3) did not
show any reactive bands.
IgE antibody reactivity to roasted peanut extract and autoclaved
roasted peanut extract under extreme conditions (2.56 atm, 138 ?C,
30 min) was further screened using fifty-four individual sera from
patients sensitized to peanut. Western blotting of roasted (B1) and
autoclaved roasted peanut extracts (B2) are shown in Fig. 1B. A
complex pattern of bands from 13 to 121 kDa was detected in
roasted peanut, with proteins of 65, 26, 22, 20 and 17 kDa recog-
nized by 90%, 70%, 70%, 67.1% and 60% respectively. Twelve out
of 54 (22%) sera recognized some proteins in autoclaved roasted
peanut at 2.56 atm for 30 min.
ELISA assay using roasted peanut and autoclaved roasted pea-
nut (2.56 atm, 138 ?C, 30 min) as a solid phase led to determine
the specific IgE levels in the individual sera from fourteen pea-
nut-sensitized patients. The percentage of the decrease in antigenic
activity was calculated with the formula: (1 ? AH/AN) 100 where AH
is the absorbance value obtained from autoclaved roasted peanut
Fig. 1. A. SDS–PAGE (A1) and western blot (A2 and A3) of raw, boiled, fried, roasted peanut and autoclaved roasted peanut. Western blot A2 was carried out using a serum
pool from 4 patients with actual peanut allergy (Nos. 5, 8, 9 and 11). Western blot A3 was carried out using a serum pool from 3 patients with allergy to Anisakis spp. 1B.
Western blot of roasted peanut (B1) and autoclaved roasted peanut at 2.56 atm, 30 min (B2) incubated with sera from patients with actual peanut allergy (lanes 1–19) and
sera from patients sensitized to peanut (20–54). Sera recognizing some proteins in autoclaved roasted peanut at 2.56 atmospheres for 30 min are marked with an asterisk.
B. Cabanillas et al./Food Chemistry 132 (2012) 360–366
samples and ANis the absorbance value of the roasted peanut pro-
tein extract (Cabanillas et al., 2010).
The results are summarized in Fig. 2. Roasted peanut IgE reac-
tivity was reduced by autoclaving at 2.56 atm, 30 min treatment
in all sera tested. The treatment led to a minimum antigenicity
reduction of 9% (in serum from patient 10) and a maximum antige-
nicity reduction of 96% (in serum from patient 4).
3.3. Ara h 1, Ara h 2 and Ara h 3 following autoclave processing
Solubility of proteins from roasted and autoclaved roasted pea-
nut (1.18 and 2.56 atm, 15 and 30 min) was evaluated by SDS–
PAGE extracting proteins into solution by buffer (soluble portion)
and the pelleted portion of each sample, brought down by centri-
fugation following extraction of soluble material with buffer (insol-
uble portion) (Fig. 3A).
Soluble fractions showed a reduction in the overall level of in-
tact proteins following autoclave treatments compared with insol-
uble fractions. Specific anti Ara h 1, anti Ara h 2 and anti Ara h 3
antibodies used to identify Ara h 1, Ara h 2 and Ara h 3 molecules
in soluble and insoluble fractions of roasted and autoclaved
roasted peanut (Fig. 3B) confirmed the results observed in SDS–
PAGE. A decrease in the relative Ara h 1, Ara h 2 and Ara h 3 levels
could be observed in autoclaved roasted peanut samples with
increased pressure and time, in the soluble fraction compared to
the insoluble fraction. Nevertheless, these results are more evident
in Ara h 1 than Ara h 2 and Ara h 3 immunoblots. The relative level
of Ara h 1 increased in the insoluble fraction of autoclaved roasted
peanut compared to soluble fraction, and high-molecular-weight-
aggregates or oligomers could be observed in the insoluble frac-
tion. However, in spite of these facts, there was a marked decrease
in recognition of Ara h 1, Ara h 2 and Ara h 3 in autoclaved roasted
peanut at 2.56 atm, 30 min even in the insoluble fractions.
3.4. Susceptibility of autoclaved roasted peanut extract to trypsin
To evaluate digestibility, roasted peanut and autoclaved roasted
peanut (1.18 atm, 15 and 30 min) were subjected to trypsin treat-
ment (Fig. 4). SDS–PAGE separation followed by immunodetection
with a serum pool from three patients with clinical allergy to pea-
nut (Nos. 7, 12, 15) indicated that autoclaved roasted peanut, com-
pared to roasted peanut, was more extensively digested by trypsin
within 10 min of treatment, losing most of its capacity to bind IgE
from peanut allergic patients (Fig. 4B).
3.5. CD analysis of the structural alterations
The secondary structure of roasted peanut and autoclaved
roasted peanut (1.18 atm and 2.56 atm, 15 and 30 min) was ana-
lyzed by CD spectroscopy and the far-UV spectra are shown in
Fig. 5. The percentages of a-helix, b-sheet and random coil and/
or loops are shown in the table annexed to Fig. 5. The CD spectrum
of roasted peanut proteins rendered 50% a-helix, 10% b-sheet, and
40% random coil and/or loops. As can be seen in the case of auto-
Fig. 2. ELISA of roasted peanut and autoclaved roasted peanut at 2.56 atm, 30 min, incubated with sera from patients with actual peanut allergy (Nos. 2, 4, 6, 7, 9, 10, 12, 15,
17) and sera from patients sensitized to peanut (Nos. 21, 22, 24, 32, 33). Percentages of decrease in antigenic activity are shown in the table annexed to Fig. 2.
Fig. 3. SDS–PAGE (A) and anti-Ara h 1 (B1), anti-Ara h 2 (B2) and anti-Ara h 3 (B3) western blot of soluble and insoluble fractions of roasted and autoclaved roasted peanut.
B. Cabanillas et al./Food Chemistry 132 (2012) 360–366
claved roasted peanut, most of the a-helical structure was lost
(11% remaining in autoclaved roasted peanut at 2.56 atm,
30 min), random coil and/or loops increased (80% in autoclaved
roasted peanut at 2.56 atm, 30 min) and b-sheets remain.
3.6. Skin prick tests
Table 1 shows the results of skin testing with raw and processed
peanut extract samples performed on seven patients with clinical
allergy to peanut. All patients had skin reactivity to raw, fried
and roasted peanut extract, although six out of seven reactions
were positive with roasted peanut. Boiled peanut extract elicited
positive reactions in one patient. None of the patients reacted to
autoclaved roasted peanut extract. Healthy subjects did not have
skin reactivity to any protein extract.
In this study the effect of different food-processing conditions
on peanut allergenicity was investigated. The results showed that
IgE immunoreactivity of roasted peanut protein extract decreased
significantly at extreme conditions of autoclaving (2.56 atm,
30 min), as shown by in vitro experiments of western blot, ELISA
and in vivo experiments of skin prick tests. These results can be
explained by circular dichroism experiments: most of the a-helical
structure was lost after autoclave treatments. It is known that
many of the IgE binding epitopes in the major allergens of peanuts
(i.e. Ara h 1, Ara h 2 and Ara h 3) are located on the a-helical
regions of these proteins (Barre, Jacquet, Sordet, Culerrier, & Rougé,
2007; Mueller et al., 2011; Shin et al., 1998). However, although
the decrease of IgE-binding proteins is notable, in vitro experi-
Fig. 4. A. SDS–PAGE analysis of the digestion of roasted peanut (A1), autoclaved roasted peanut 1.18 atm, 15 min (A2) and autoclaved roasted peanut 1.18 atm, 30 min (A3)
by trypsin at 0, 1, 3, 5, 10, 30, 60, 120 min and overnight (?15 h). B. Western blot of selected digestion times (0, 10 min and overnight) of roasted peanut and autoclaved
roasted peanut using a serum pool from 3 patients with clinical allergy to peanut (Nos. 7, 12 and 15).
Fig. 5. CD-spectrum of roasted peanut (RP) and autoclaved roasted peanut at 1.18 atm and 2.56 atmospheres (15 and 30 min). Percentages of a-helix, b-strand and random
coil and/or loops are shown in the table annexed to Fig. 5.
B. Cabanillas et al./Food Chemistry 132 (2012) 360–366
ments showed that 22% of sera recognized some proteins in auto-
claved roasted peanut at 2.56 atm, for 30 min, in western blot. ELI-
SA results showed that autoclaved roasted peanut (2.56 atm,
30 min) elicited a decrease of IgE reactivity between 9% and 96%.
These findings suggest that autoclaving produces an important
decrease in IgE-binding properties of roasted peanut due to
changes in the structure of the proteins. Similar results have been
previously reported in other legumes: Malley, Baecher, Mackler,
and Perlman (1975) found that autoclaving green pea at 120 ?C
for 15 min reduced its allergenicity. Also, IgE-binding capacity of
lupine allergens was reduced by autoclaving at 2.6 atm, for
30 min, or by DIC (instantaneous controlled pressure drop) at
6 bar (5.9 atm) for 3 min, whereas it was only slightly affected by
boiling, microwave, and extrusion-cooking (Alvarez-Alvarez et al.,
2005; Guillamón et al., 2008). However, it has been demonstrated
that the allergenicity of the almond major protein (amandin) and a
peach protein in the nectar (Pru p 1) is maintained after autoclav-
ing at 121 ?C, up to 30 min (Brenna et al., 2000; Venkatachalam,
Teuber, Roux, & Sathe, 2002).
Autoclaved roasted peanut, compared to roasted peanut, was
more extensively digested by trypsin within 10 min of treatment,
losing most of its capacity to bind IgE from peanut allergic patients.
It has been demonstrated that heat treatments can increase the
susceptibility to enzymatic digestion of some allergens (Morisawa
et al., 2009). In the present study, circular dichroism experiments
showed unfolding of proteins in autoclave treated samples, which
makes them more susceptible to digestion. Morisawa et al. (2009)
found that B cell epitopes of b-lactoglobulin were impaired by heat
treatment, increasing susceptibility to digestion and facilitating the
enzymatic cleavage of protein sequences that disrupts linear B cell
The degree of processing can dramatically affect the results of
digestibility, solubility and other parameters (Maleki, 2004). Dur-
ing processing, proteins can form oligomers, become denatured,
degraded, aggregated, cross-linked, fragmented and re-assembled
and these changes most often cause a reduction in solubility
(Maleki, 2004). In this way, processing can alter the overall IgE
binding profiles of a particular extract (Schmitt et al., 2010). It
can be hypothesized that the soluble portion of autoclaved roasted
peanut samples assessed in this study contains less allergenic pro-
teins or products due to a reduction in the solubility caused by
autoclave treatment. For this reason, protein profiles, including
peanut allergens: Ara h 1, Ara h 2 and Ara h 3, were assessed in sol-
uble and insoluble fraction of roasted and autoclaved roasted pea-
nut. The results showed that the total level of intact protein
decreases with increased pressure and time in both, soluble and
insoluble fractions, although soluble fractions showed a higher
reduction of proteins than insoluble fractions. Ara h 1, Ara h 2
and Ara h 3 allergens were recognized more extensively by specific
antibodies in insoluble fraction than in soluble fraction. Moreover,
Ara h 1 was present in higher molecular weight aggregates or olig-
omers in the insoluble fraction. It has been demonstrated that
heating of purified Ara h 1 leads to a more structured secondary
conformation of the protein, with an increased content of extended
b-sheet structures leading to the formation of large protein com-
plexes or aggregates (Koppelman, Bruijnzeel-Koomen, Hessing, &
de Jongh, 1999). However, in the present study, a marked decrease
of Ara h 1, Ara h 2 and Ara h 3 allergens was detected at harsh con-
ditions of autoclaving (2.56 atm, 30 min) in both, soluble and insol-
In summary, although further studies are needed to assess the
clinical relevance of our findings, we can conclude that autoclaving
at 2.56 atm for 30 min produces a significant decrease of
IgE-binding capacity of peanut allergens due to changes in their
secondary structure. This treatment could be a technique to im-
prove food safety.
This study was supported by Grant No. AGL2004-07971-C02,
Grant AGL2008-03453-C02, Ministerio de Educación y Ciencia
and Grant No. FI07/00286 Instituto Carlos III Ministerio de Ciencia
Alvarez-Alvarez, J., Guillamón, E., Crespo, J. F., Cuadrado, C., Burbano, C., Rodríguez,
J., et al. (2005). Effects of extrusion, boiling, autoclaving and microwave heating
on lupine allergenicity. Journal of Agricultural and Food Chemistry, 53(4),
Barre, A., Jacquet, G., Sordet, C., Culerrier, R., & Rougé, P. (2007). Homology
modelling and conformational analysis of IgE-binding epitopes of Ara h 3 and
other legumin allergens with a cupin fold from tree nuts. Molecular Immunology,
Beyer, K., Morrow, E., Li, X. M., Bardina, L., Bannon, G. A., Burks, A. W., et al. (2001).
Effects of cooking methods on peanut allergenicity. The Journal of Allergy and
Clinical Immunology, 107(6), 1077–1081.
Brenna, O., Pompei, C., Ortolani, C., Pravettoni, V., Farioli, L., & Pastorello, E. A.
(2000). Technological processes to decrease the allergenicity of peach juice and
nectar. Journal of Agricultural and Food Chemistry, 48(2), 493–497.
Cabanillas, B., Pedrosa, M. M., Rodríguez, J., González, A., Muzquiz, M., Cuadrado, C.,
et al. (2010). Effects of enzymatic hydrolysis on lentil allergenicity. Molecular
Nutrition & Food Research, 54(9), 1266–1272.
Cabanillas-Martín, B., Crespo, J. F., Burbano, C., & Rodríguez, J. (2010). Systemic IgE-
mediated reaction to a dietary slimming bar. Annals of Allergy, Asthma &
Immunology, 104(5), 450.
Chung, S. Y., & Champagne, E. T. (1999). Allergenicity of Maillard reaction products
from peanut proteins. Journal of Agricultural and Food Chemistry, 47(12),
Chung, S. Y., & Champagne, E. T. (2001). Association of end-product adducts with
increased IgE binding of roasted peanuts. Journal of Agricultural and Food
Chemistry, 49(8), 3911–3916.
Cuadrado, C., Cabanillas, B., Pedrosa, M. M., Varela, A., Guillamón, E., Muzquiz, M.,
et al. (2009). Influence of thermal processing on IgE reactivity to lentil and
chickpea proteins. Molecular Nutrition & Food Research, 53(11), 1462–1468.
Guillamón, E., Burbano, C., Cuadrado, C., Muzquiz, M., Pedrosa, M. M., Sánchez, M.,
et al. (2008). Effect of an instantaneous controlled pressure drop on in vitro
allergenicity to lupins (Lupinus albus var Multolupa). International Archives of
Allergy and Immunology, 145(1), 9–14.
Koppelman, S. J., Bruijnzeel-Koomen, C. A., Hessing, M., & de Jongh, H. H. (1999).
Heat-induced conformational changes of Ara h 1, a major peanut allergen, do
not affect its allergenic properties. The Journal of Biological Chemistry, 274(8),
Results of skin prick testing with raw and thermal processed peanuts in patients with clinical allergy to peanut.
SPT (mm) peanutImmunological and clinical features
Patient RawBoiled FriedRoastedAutoclaved roasted CAP-FEIA (kU/L)Diagnostic challenge
CAP-FEIA, Capsulated hydrolic carrier polymer-fluoro-enzyme immunoassay; DBPCFC, double-blind, placebo-controlled food challenge; SPT, skin prick testing.
aNot challenged because of a convincing history of severe anaphylaxis with peanut.
B. Cabanillas et al./Food Chemistry 132 (2012) 360–366
Kopper, R. A., Odum, N. J., Sen, M., Helm, R. M., Stanley, J. S., & Burks, A. W. (2005).
Peanut protein allergens: the effect of roasting on solubility and allergenicity.
International Archives of Allergy and Immunology, 136(1), 16–22.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature, 227(5259), 680–685.
Maleki, S. J. (2004). Food processing: effects on allergenicity. Current Opinion in
Allergy and Clinical Immunology, 4(3), 241–245.
Maleki, S. J., Chung, S. Y., Champagne, E. T., & Raufman, J. P. (2000). The effects of
roasting on the allergenic properties of peanut proteins. The Journal of Allergy
and Clinical Immunology, 106(4), 763–768.
Maleki, S. J., Viquez, O., Jacks, T., Dodo, H., Champagne, E. T., Chung, S. Y., et al.
(2003). The major peanut allergen, Ara h 2, functions as a trypsin inhibitor, and
roasting enhances this function. The Journal of Allergy and Clinical Immunology,
Malley, A., Baecher, L., Mackler, B., & Perlman, F. (1975). The isolation of allergens
from the green pea. The Journal of Allergy and Clinical Immunology, 56(4),
Mills, E. N., Sancho, A. I., Rigby, N. M., Jenkins, J. A., & Mackie, A. R. (2009). Impact of
food processing on the structural and allergenic properties of food allergens.
Molecular Nutrition & Food Research, 53(8), 963–969.
Morisawa, Y., Kitamura, A., Ujihara, T., Zushi, N., Kuzume, K., Shimanouchi, Y., et al.
(2009). Effect of heat treatment and enzymatic digestion on the B cell epitopes
of cow’s milk proteins. Clinical & Experimental Allergy, 39(6), 918–925.
Mueller, G. A., Gosavi, R. A., Pomés, A., Wünschmann, S., Moon, A. F., London, R. E.,
et al. (2011). Ara h 2: crystal structure and IgE binding distinguish two
subpopulations of peanut allergic patients by epitope diversity. Allergy, 66(7),
Sanchez-Madrid, F., Morago, G., Corbi, A. L., & Carreira, J. (1984). Monoclonal
antibodies to three distinct epitopes on human IgE: Their use for determination
of allergen-specific IgE. Journal of Immunological Methods, 73(2), 367–378.
Sathe, S. K., & Sharma, G. M. (2009). Effects of food processing on food allergens.
Molecular Nutrition & Food Research, 53(8), 970–978.
Schmitt, D. A., Nesbit, J. B., Hurlburt, B. K., Cheng, H., & Maleki, S. J. (2010).
Processing can alter the properties of peanut extract preparations. Journal of
Agricultural and Food Chemistry, 58(2), 1138–1143.
Shin, D. S., Compadre, C. M., Maleki, S. J., Kopper, R. A., Sampson, H., Huang, S. K.,
et al. (1998). Biochemical and structural analysis of the IgE binding sites on Ara
h 1, an abundant and highly allergenic peanut protein. The Journal of Biological
Chemistry, 273(22), 13753–13759.
Sicherer, S. H., Muñoz-Furlong, A., Godbold, J. H., & Sampson, H. A. (2010). US
prevalence of self-reported peanut, tree nut, and sesame allergy: 11-year
follow-up. The Journal of Allergy and Clinical Immunology, 125(6), 1322–
Sicherer, S. H., & Sampson, H. A. (2007). Peanut allergy: emerging concepts and
approaches for an apparent epidemic. The Journal of Allergy and Clinical
Immunology, 120(3), 491–503.
Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure and some
applications. Proceedings of the National Academy of Sciences of the United
States of America, 76(9), 4350–4354.
Venkatachalam, M., Teuber, S. S., Roux, K. H., & Sathe, S. K. (2002). Effects of roasting,
blanching, autoclaving, and microwave heating on antigenicity of almond
(Prunus dulcis L.) proteins. Journal of Agricultural and Food Chemistry, 50(12),
B. Cabanillas et al./Food Chemistry 132 (2012) 360–366