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Original article
Discarded cross-allergy between miracle berry (Synsepalum
dulcificum) and peanut
Adri
an Men
endez-Rey,
1,2
*F
atima Jerez-Arroyo,
1
Nicol
as Alegr
ıa-Aravena,
1,3
Josefa Quiroz-Troncoso,
1,2
Raquel Gonz
alez-Martos,
1,4
Marta S
anchez-D
ıez,
1
Carlos Blanco,
5
Ceferino Maest
u Unturbe,
1
Loan Bensadon-Naeder
2
& Carmen Ramirez-Castillejo
1,6,7
*
1 CTB (CTB-UPM) Centro de Tecnolog
ıa Biom
edica, Universidad Polit
ecnica de Madrid, 28223, Pozuelo de Alarc
on, Spain
2Ba
€
ıa Food, Medicinal Gardens S.L, 28008, Madrid, Spain
3 Grupo de Biolog
ıa y Producci
on de C
evidos, Instituto de Desarrollo Regional. Universidad de Castilla-La Mancha, Albacete, Spain
4 GENUD Toledo Research Group, Universidad Castilla-La Mancha, Toledo, Spain
5 Departamento de Alergia, Hospital Universitario de La Princesa, Instituto de Investigaci
on Sanitaria Princesa, RETIC ARADYAL RD16/
0006/0015, Madrid, Spain
6 Oncology Group, Instituto de Investigaci
on Sanitaria San Carlos, Madrid, Spain
7 ETSIAAB, Departamento Biotecnolog
ıa-Biolog
ıa Vegetal, Universidad Polit
ecnica de Madrid, Madrid, Spain
(Received 20 May 2022; Accepted in revised form 23 September 2022)
Summary Peanut allergies are one of the food allergies with the highest prevalence and mortality due to anaphy-
laxis, drastically reducing the quality of life of people who suffer from it. In the case of Novel Foods,
such as the miracle berry from Synsepalum dulcificum, there is insufficient evidence regarding their aller-
genic potential. An exhaustive analysis was carried out to study potential cross-allergy effects between
miracle berries and peanuts. First, both an in silico allergenicity prediction model based on sequence
homology and an in silico structure homology test were designed to evaluate miraculin cross-reactivity
against Ara h allergens. Second, an in vitro ELISA assay using blood sera from peanut-allergic patients
was executed to discard the possibility of cross-reaction with total protein content from the berry. The
obtained results ruled out the presence of cross-allergy, leading to the conclusion that consumption of this
berry presents no risk for patients with this allergy.
Keywords Ara h, ELISA, food safety, in silico analysis, miraculin, Novel Food.
Highlights
•In silico analysis revealed the absence of cross-allergy
with miraculin.
•ELISA assay discarded the cross-reactivity of the
total proteins from miracle berry.
•Miracle berry can be safely consumed by those aller-
gic to peanuts.
Introduction
Food allergies are a health problem with a negative
effect on the quality of life of up to 10% of the world
population (Chafen et al., 2010), and this number is
expected to increase within the next few years (Sicherer
& Sampson, 2014; Renz et al., 2018). People who suf-
fer from this type of pathology exhibit various
symptoms that range from mild discomfort to severe
anaphylactic reactions (Reyes et al., 2020). One of the
most common food allergies is to peanuts, affecting up
to 3% of the world population (Sicherer & Samp-
son, 2018) and 1.6% in Europe specifically (Nwaru
et al., 2014). In addition, it stands out for being the
food allergy with the highest number of deaths due to
anaphylaxis (Turner et al., 2017; Frith & Kate-
laris, 2019). To date, a total of 17 peanut allergens
have been identified, with only 9 of them having clini-
cal significance. These include storage proteins such as
vicilins (Ara h 1 and 3) and albumins (Ara h 2), which
correspond to the major allergens by triggering 90%
of peanut allergy cases (Restani et al., 2005); and aller-
gens belonging to the family of profilins (Ara h 5), Bet
v 1 homologous (Ara h 8) and lipid transfer proteins
(LTP) (Ara h 9), which are minor allergens (Matri-
cardi et al., 2016; Frith & Katelaris, 2019).
The allergy profile that is generated will differ
depending on which allergen is recognised by the
*Correspondendent: E-mail: carmen.ramirez@ctb.upm.es
menendez.rey.adrian@gmail.com
International Journal of Food Science and Technology 2022, 57, 7681–7690
doi:10.1111/ijfs.16114
Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd
on behalf of Institute of Food, Science and Technology (IFSTTF).
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
7681
individual’s immune system and can be distinguished
by the severity of the symptoms. The first profile
responds to the major allergens Ara h 1, 2 and 3 and
is characterised by causing anaphylactic reactions. The
second, caused by the minor allergens Ara h 5, 8 and
9, presents milder symptoms, mainly an oral allergic
syndrome (OAS) that affects throat, mouth and face
causing itching, redness and mild inflammation
(Krause et al., 2009; Klemans et al., 2015; Frith &
Katelaris, 2019; Abrams et al., 2020).
Peanuts can cause cross-allergy with other foods,
mainly with other legumes, such as lupine (Lup an 1),
soy (Gly m 1, 3, 5, 6 y 8) and lentils (Len c 1 y 3); as well
as with other nuts, such as hazelnut (Cor a 2, 9, 11 y 14),
pistachio (Pis v 1, 2 y 3), walnut (Jug r 1, 2 y 4), cashew
(Ana o 3) and almond (Pru du 4 y 6). It also presents
cross-reactivitywithbirchpollen(Betv1y2)andpeach
(Pru p 3) (Chan et al., 2019; Krogulska & Wood, 2020).
The only way to prevent this food allergy is to avoid
the consumption of peanut-based products. However,
the possible presence of traces or contamination from
other foods and of cross-allergy phenomena make this
disease difficult to manage (Taylor & Baumert, 2010).
Furthermore, this fact is hampered by the incorpora-
tion of Novel Foods: foods that have traditionally had
their consumption limited to their native region, and
for which the nutritional content and potential cross-
allergens are unknown. To avoid risk, the European
Commission establishes the need to guarantee the food
safety of any Novel Food through Regulation (EU)
2015/2283 along with the European Food Safety
Authority (EFSA), the agency in charge of studying
the allergenic potential of foods before authorisation
of their commercialisation in Europe.
Miracle berry corresponds to the fruit from Synsepa-
lum dulcificum, a plant located in Eastern Africa which is
characterised by the presence of a glycoprotein, called
miraculin, in its pulp. Miraculin is capable of transform-
ing acidic flavours into sweet flavours (Bartoshuk
et al., 1974; Rodrigues et al., 2016) and this property is
of particular interest for applications such as, the man-
agement of dysgeusia (Soares et al., 2010;Wilken&
Satiroff, 2012), the improvement of the dietary profile of
people with diabetes and obesity (Chen et al., 2006;Oba-
femi et al., 2019), and as a natural alternative to sugar
and other artificial sweeteners. Last June, the EFSA
decreed that miracle berry consumption is not harmful to
human health (Turck et al., 2021). However, in that
same document, the experts of the evaluation panel state
that they consider the public results to date as prelimi-
nary, and that they do not allow definitive conclusions to
be drawn on cross-reactivity (page 14 of the report in the
Allergenicity section (Turck et al., 2021)). To date, sev-
eral studies have been carried out to characterise the
composition of the fruit, including the analysis of its phe-
nolic compounds with antioxidant (Inglett &
Chen, 2011;Duet al., 2014;Heet al., 2016;Haddad
et al., 2020), antidiabetic (Obafemi et al., 2019) and anti-
cancer (G
omezdeCedr
on et al., 2020) properties, lectins
(Men
endez-Rey et al., 2021), vitamins and amino acids
(Njoku et al., 2015), among others. The next required
step is the detailed analysis of this berry’s potential aller-
genic role. Previous commercial ELISA assays suggested
the possible presence of peanut allergens or structural
homologues in miracle berry (data not shown). For this
reason, the objective of the present study was the evalua-
tion of potential cross-allergy effects between miracle
berry and the main peanut allergens, through a combina-
tion of in silico and in vitro assays.
Materials and methods
ROC predictive model design to determine cross-
allergenicity between miraculin and peanut allergens
The predictive model was based on the development of
a Receiver Operating Characteristic (ROC) curve from
two databases (DB). The first DB consisted of allergens
whose cross-reaction was described in the literature
(positive database, n=100) (Table S1) while the second
DB collected allergens that did not present cross-
allergenicity (negative database, n=118) (Table S2).
The protein sequences of the allergens were obtained
from the UniprotKB database (https://www.uniprot.
org) and the presence/absence of cross-allergenicity was
verified through the AllerBase allergen database (http://
bioinfo.unipune.ac.in) (Kadam et al., 2017).
These DBs contained the results of allergen pairwise
sequence alignment (allergen 1 vs. allergen 2) using the
NCBI BLASTp bioinformatics tool (https://blast.ncbi.
nlm.nih.gov), including identity (PI) and query cover
(PC) percentages for each comparison. For all align-
ments, the product between PI and PC was calculated
as an indicator of sequence homology. This quantita-
tive variable was used to obtain the ROC curve using
GraphPad Prism 6 software and to determine the opti-
mal cut-off point of this product that allows distin-
guishing proteins that present cross-allergenicity.
Finally, sequence alignment between miraculin
(P13087) and peanut allergens was carried out. Again,
the product between PI and PC values was calculated
and compared with the cut-off point provided by ROC
curve to identify potential cross-allergens.
Analysis of cross-allergenicity by structural homology
between miraculin and peanut allergens
In this trial, miraculin protein (C5NU63) was struc-
turally compared with peanut allergens (Ara h 1, 2, 3,
5, 8 and 9). In parallel, each peanut allergen structure
was aligned with bovine serum albumin (BSA) (6RJV)
as a negative control since they do not present cross-
Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd
on behalf of Institute of Food, Science and Technology (IFSTTF).
International Journal of Food Science and Technolo gy 2022
Cross-allergy between miracle berry and peanut A. Men
endez-Rey et al.7682
allergy, and with an allergen whose cross-reactivity is
described as a positive control, whose structures are
indicated in the following table (Table 1).
All protein structures were obtained from the Protein
Data Bank (PDB) database. As some allergens pos-
sessed multimeric structures, the monomer unit was
used to carry out the structural alignments. Structures
were coupled using the UCSF Chimera software, a pro-
gram that provided an RMSD (root-mean-square devi-
ation) value, used as an indicator of homology. The
other three parameters, scores that measure the statisti-
cal significance of structural similarity, were calculated
and also served as indicators of sequence homology.
These were P-value, Z-score, and TM-score, which were
obtained using the FATCAT software, the DALI net-
work server, and the TM-align pairwise structural align-
ment algorithm, respectively. These four indicators have
a cut-off threshold from which to consider whether two
structures are homologous or not. Two proteins present
significant structural homology when: RMSD <2, Z-
score >2, P-value <0.001 and TM-score between 0.5
and 1. The calculation of the percentage of identity
(Pident) and the percentage of coverage analysed (Qco-
ver) of the protein sequences was carried out using the
NCBI BLASTp tool, after introducing the protein
sequences from the UniprotKB database.
Analysis of the presence of Ara h inmunodominant
epitopes in miraculin
Immunodominant epitope protein sequences of peanut
allergens were obtained from the AllerBase allergen
database. To check the presence of these in miraculin
protein, a sequence alignment between the different
epitopes and miraculin (P13087) using the NCBI
BLASTp bioinformatics tool was carried out.
Measurement of specific IgE binding against miracle
berry extracts by indirect ELISA with peanut allergy sera
Starting material and protein extraction
The starting material used, lyophilised miracle berry
powder, was provided by the company Ba€
ıa Food
(Medicinal Gardens S.L.), and unprocessed natural
peanuts were provided by Professor Santiago Moreno
from the ETSIAAB of the Polytechnic University of
Madrid. A solid–liquid protein extraction was per-
formed using an aqueous 0.5 M NaCl pH 6.8 solution
that has already been published by He et al.(2015).
The resulting extracts were filtered using a 0.45 lm
syringe filter (Clear Line, Brumath, France) and the
total protein content was quantified using the BCA kit
(Merck Millipore, Massachusetts, USA). Finally, sam-
ples were diluted using phosphate-buffered saline
(PBS) (Corning, New York, USA) until reaching the
desired concentration.
Indirect ELISA with sera from peanut-allergic patients
To analyse the possible cross-allergenicity of the mira-
cle fruit with peanuts, an indirect ELISA test was car-
ried out using serum samples from four patients
allergic to peanuts and from three non-allergic individ-
uals (controls). Allergy to peanut was defined as imme-
diate adverse reactions suggestive of IgE-mediated
allergy, together with demonstration of a peanut-
specific IgE level higher than 3.5 kU L
1
, as deter-
mined by ImmunoCAP
TM
(Thermo Fisher, Mas-
sachusetts, USA). Samples were provided by the
Allergy Dept. of Hospital Universitario de La Princesa
(Madrid, Spain). A 96-well plate was coated with mir-
acle berry extract (test sample), peanut extract (posi-
tive control) and BSA (negative control), diluted in
PBS at a concentration of 0.01 lglL
1
and incubated
overnight at 4 °C. After this, wells were washed with
PBS and blocked with PBS-2% BSA for 1 h at room
temperature (RT). Once blocked, a series of washes
were carried out with PBS +0.1% Tween-20 (Panreac,
Barcelona, Spain). The plate was then incubated with
different blood serum samples at a 1:4 dilution in PBS
for 3 h at RT. After incubation, with the IgE antibod-
ies present in the serum, an exhaustive washing process
was carried out using PBS +0.1% Tween-20. Then,
incubation with HRP-conjugated anti-IgE antibody
(Thermo Fisher, Massachusetts, USA) was carried out
at a 1:2000 dilution in PBS for 45 min at RT. To
remove nonspecific bonds, the washing process was
repeated. Finally, the plate was colorimetrically devel-
oped with TMB reagent (3,30, 5,50-
tetramethylbenzidine) (ACROS Organic, Mas-
sachusetts, USA). After 20 min, the reaction was
stopped by adding 2 M sulphuric acid solution (Pan-
reac, Barcelona, Spain) and the absorbance was mea-
sured at 450 nm using a BIOBASE EL-10A ELISA
microplate reader spectrophotometer (BIOBASE,
Shandong, China).
Statistical analysis
A total of five independent experiments were carried
out and statistical analysis was performed using the 2-
Table 1 Peanut allergens and their respective cross-allergens
used in structural alignment. The PDB codes for each structure
are shown in parentheses
Peanut allergen
Cross-reactive allergen
Ara h 1(3SMH) Len c 1 (2EA7) Lens culinaris
Ara h 2 (3OB4) Lup an Gcong (4PPH) Lupinus angustifolius
Ara h 3 (3C3V) Gly m 6 (1FXZ) Glycine max
Ara h 5 (4ESP) Bet v 2 (1CQA) Betula verrucosa
Ara h 8 (4MA6) Gly m 4 (2K7H) Glycine max
Ara h 9 (2 N81) Pru p 3 (2ALG) Prunus persica
Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd
on behalf of Institute of Food, Science and Technology (IFSTTF).
International Journal of Food Science and Technology 2022
Cross-allergy between miracle berry and peanut A. Men
endez-Rey et al. 7683
way ANOVA test by Tukey’s multiple comparison
test. The mean absorbances of the negative control
(BSA), positive control (peanut) and miracle berry
(sample to be evaluated) were compared in each
patient, representing the level of significance on the
error bars (*if P<0.05; ** if P<0, 01; *** if
P<0.001; **** if P<0.0001; n.s. if P>0.05).
Results
Cross-allergenicity evaluation between miraculin and pea-
nut allergens using a prediction model based on sequence
homology
To study the possible cross-allergenicity of miraculin
with the main food allergens, a prediction model of
allergenicity was developed based on an ROC curve
made with the products between PI and PC (homology
indicator) from the allergen sequence alignments
(Fig. 1).
The robustness of the cross-allergenicity prediction
model was adequate as the area under the curve
(AUC) was 0.96 0.01. The 0.239 value of the
homology indicator was chosen as the cut-off point as
it denoted the highest specificity (95.76%) and sensitiv-
ity (91.00%).
Once the cut-off point to distinguish non-allergenic
and allergenic proteins was established, sequence align-
ment between miraculin (P13087) and peanut allergens
was carried out comparing the product obtained
between PI and PC with the cut-off point (0.239).
After the comparison, it was possible to observe that
the resulting product from all the alignments was
below the cut-off point value indicating no apparent
cross-reactivity (Table 2).
Analysis of cross-allergenicity by structural homology
between miraculin and peanut allergens
Specific analysis of the cross-reactivity between miraculin
and the main peanut allergens (Ara h 1, Ara h 2, Ara h 3,
Ara h 5, Ara h 8 and Ara h 9) was carried out based on
the study of their structural homology. The structural
alignment of each peanut allergen with a previously deter-
mined cross-allergenic allergen (positive control) and with
a structurally different not cross-allergenic protein (BSA,
negative control) was performed in parallel. Sequence
identity (Pident) and sequence coverage (Qcover) percent-
ages were included in all alignments. Moreover, to anal-
yse each alignment’s degree of structural similarity, four
indicators capable of discriminating between homologous
and different proteins were included: RMSD, Z-score, P-
value and TM-score (Fig. 2).
As can be seen, when comparing each peanut allergen
with its respective cross-allergen, RMSD values and
scores of clearly structurally homologous proteins were
obtained, except for Ara h 2 and Lup an GCong, showing
RMSD values <2 (taking into account most of the pro-
tein structure) and Z-score >2, P-value <0.001 and TM-
score >0.5. However, when comparing the allergens Ara
h 1, 2, 3, 5, 8 and 9 with the miraculin protein, the homol-
ogy indicators provided values very similar to those
obtained in the alignment with the negative control
(BSA), typical of non-homologous proteins (RMSD>2,
Z-score <2, P-value>0.001 and TM-score <0.5).
Evaluation of the presence in miraculin of
immunodominant epitopes from Ara h allergens
Despite the lack of sequence and structure homology,
the possible presence of epitopes involved in peanut
Figure 1 ROC curve. Product data between
identity and query cover percentages from
both databases (positive and negative cross-
allergenicity databases) were plotted using
GraphPad Prism 6 statistical analysis soft-
ware.
Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd
on behalf of Institute of Food, Science and Technology (IFSTTF).
International Journal of Food Science and Technolo gy 2022
Cross-allergy between miracle berry and peanut A. Men
endez-Rey et al.7684
allergies in the miraculin protein sequence was also
evaluated. For this, a sequence alignment between
miraculin and the different allergenic epitopes was per-
formed. The immunodominant epitopes from the main
peanut allergens are detailed in Fig. 3. This assay
revealed that none of the peptides were found intact in
miraculin, instead only conserved between 1–4 identi-
cal non-contiguous amino acids, and having, in some
cases, 2 contiguous amino acids at the most.
Measurement of specific IgE binding against miracle
berry extracts by indirect ELISA with sera from peanut-
allergic patients
To analyse possible cross-allergy with this nut in
greater detail, an ELISA test of reactivity of anti-Ara
h IgE antibodies against the miracle berry protein
extract was performed with sera from four peanut-
allergic patients and three control subjects (Fig. 4).
Discussion
Previous studies affirm that miraculin protein is unable
to generate an allergic or toxicological response (Tafa-
zoli et al., 2019). However, the rest of the compounds
present in the fruit have not yet been evaluated omit-
ting the presence of cross-allergy phenomena with
foods such as peanuts.
First, a prediction model based on an ROC curve
elucidated the absence of cross-reactivity between
miraculin and peanut allergens (Table 2) because the
products obtained in the alignment did not exceed the
cut-off point. The low sequence homology may be due
to the fact that none of the peanut allergens belong to
the miraculin protein family, the protease inhibitors I3
(leguminous Kunitz-type inhibitor) family (Takai
et al., 2013).
As this analysis did not exclude the possibility that
they had a similar structure or conserved some
immunodominant epitope, a structural homology anal-
ysis between miraculin and peanut allergens was car-
ried out along with an analysis to evaluate the
presence of the main immunoreactive epitopes of pea-
nut allergies in the miraculin sequence. The structure
alignments between miraculin and the main peanut
allergens (Ara h 1, 2, 3, 5, 8 and 9) provided different
homology indicator values typical of proteins that do
not have structural similarity, as occurred when com-
paring each allergen with our negative control, BSA
(Fig. 2). Moreover, the miraculin sequence did not
contain any complete epitope of the peanut allergens
with complete identity of 6–8 contiguous amino acids
being necessary to be considered cross-allergens as
established by the WHO (FAO & World Health Orga-
nization, 2001). These tests thus allowed us to rule out
Table 2 Results of the cross-allergenicity prediction model
after sequence alignment between miraculin (P13087) and
peanut allergens. All alignments whose products (% identity
and % query cover) were below the cut-off point obtained in
the ROC curve (0.239) were not considered as possible cross-
allergens. Alignment results whose similarity was not signifi-
cant (n.s.) were also included
Peanut (Arachis hypogaea) cross-allergenicity evaluation
Allergens and
isoallergens
Query
cover
(%)
Identity
(%) Product Test result
Ara h 1 (P43238) 44.00% 38.10% 0.168 No cross-
allergy
Ara h 2 (Q6PSU2) 1.00% 66.67% 0.007 No cross-
allergy
Ara h 3.0101 (O82580) 59.00% 33.33% 0.197 No cross-
allergy
Ara h 3.0201
(Q9SQH7)
28.00% 40.00% 0.112 No cross-
allergy
Ara h 5 (Q9SQI9) 47.00% 24.74% 0.116 No cross-
allergy
Ara h 7.0101
(Q9SQH1)
20.00% 27.59% 0.055 No cross-
allergy
Ara h 7.0201 (B4XID4) 5.00% 25.00% 0.013 No cross-
allergy
Ara h 8.0101 (Q6VT83) 33.00% 44.44% 0.147 No cross-
allergy
Ara h 8.0201 (B0YIU5) 33.00% 32.69% 0.108 No cross-
allergy
Ara h 9.0101 (B6CEX8) 13.00% 31.58% 0.041 No cross-
allergy
Ara h 9.0201 (B6CG41) 20.00% 54.55% 0.109 No cross-
allergy
Ara h 10.0101
(Q647G5)
7.00% 80.00% 0.056 No cross-
allergy
Ara h 10.0102
(Q647G4)
7.00% 80.00% 0.056 No cross-
allergy
Ara h 11.0101
(Q45W87)
n.s. n.s. 0.000 No cross-
allergy
Ara h 11.0101
(Q45W86)
n.s. n.s. 0.000 No cross-
allergy
Ara h 12 (B3EWP3) n.s. n.s. 0.000 No cross-
allergy
Ara h 13.0101
(B3EWP4)
26.00% 26.67% 0.069 No cross-
allergy
Ara h 13.0102
(C0HJZ1)
8.00% 31.25% 0.025 No cross-
allergy
Ara h 14.0101
(Q9AXI1)
15.00% 42.11% 0.063 No cross-
allergy
Ara h 14.0102
(Q9AXI0)
12.00% 47.37% 0.057 No cross-
allergy
Ara h 14.0103
(Q6J1J8)
15.00% 42.11% 0.063 No cross-
allergy
Ara h 15 (Q647G3) 9.00% 22.22% 0.020 No cross-
allergy
Ara h 18
(A0A444XS96)
75.00% 21.18% 0.159 No cross-
allergy
Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd
on behalf of Institute of Food, Science and Technology (IFSTTF).
International Journal of Food Science and Technology 2022
Cross-allergy between miracle berry and peanut A. Men
endez-Rey et al. 7685
Figure 2 Structural alignment of peanut allergens Ara h 1, 2, 3, 5, 8 and 9 (camel colour) with miraculin (pink colour). In yellow, the immun-
odominant Ara h epitopes recognised by the specific IgE of the individuals with peanut allergy are indicated. The comparison of its structure
with the respective cross-allergens (coloured in the blue range) and with the BSA protein (grey colour) is included as a positive and negative
control, respectively. Sequence identity (Pident) and coverage (Qcover) percentages are represented in addition to four structural similarity indi-
cators (RMSD, Z-score, P-value and TM-score). n.s. (not significant).
Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd
on behalf of Institute of Food, Science and Technology (IFSTTF).
International Journal of Food Science and Technolo gy 2022
Cross-allergy between miracle berry and peanut A. Men
endez-Rey et al.7686
Figure 4 Graphic representation of the absorbance values obtained in the ELISA assay with the sera of allergic (HUP01-04) and non-allergic
(control 1–3) versus BSA (negative control), peanut extract (positive control) and miracle berry extract. The mean absorbance from five inde-
pendent experiments (n=5) is plotted. A 2-way ANOVA test was performed, comparing the absorbances of the negative control (BSA), posi-
tive control (peanut) and miracle berry (sample to be evaluated) and the level of significance was represented on the error bars (*if P<0.05;
** if P<0.01; *** if P<0.001; **** if P<0.0001). The lack of significance between groups was represented as n.s. (not significant). As can
be seen, the signal obtained in the miracle berry protein extract (dark grey boxes) was similar to that obtained in the negative control (BSA)
(white boxes) in all the analysed serums, providing low reactivity values (not significant). However, in the wells covered with peanut protein
extract (light grey boxes), statistically significant signal (P<0.0001) was observed in the allergic individuals HUP01, HUP02 and HUP03, while
not being observed in HUP04 and in non-allergic individuals.
Figure 3 Protein surfaces of Ara h 1, 2, 3, 5, 8 and 9 peanut allergens. Each allergen appears coloured with a camel tone and the respective
immunodominant epitopes with varied colours. The PDB code used to represent each allergen is included as well as the sequence and position
(number of residues) of each epitope. The sequences of the immunodominant epitopes of each allergen were extracted from the Allerbase aller-
gen database. Graphic representation was made using PyMOL bioinformatics software.
Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd
on behalf of Institute of Food, Science and Technology (IFSTTF).
International Journal of Food Science and Technology 2022
Cross-allergy between miracle berry and peanut A. Men
endez-Rey et al. 7687
cross-reactivity between miraculin and peanut aller-
gens.
The following stage was to discard the presence of
any component of miracle berry that can trigger an
allergic reaction in individuals allergic to peanuts. To
this end an ELISA test was carried out (Fig. 4),
observing that specific IgEs from different individuals
with allergies to peanuts are not capable of binding to
miracle berry protein extract, a result that points to
the absence of components in the fruit that can trigger
an allergic reaction in these patients. Patient HUP04,
the only patient with an LTP profile (Table S3), did
not show signal in the wells coated with peanut extract
possibly because this individual had relatively low
levels of specific IgE, the peanut extract did not have
enough LTP concentration due to low presence in the
nut (Walczyk et al., 2013; Johnson et al., 2016), or
because the extraction method used did not allow for
optimal LTP extraction (Aalberse et al., 2013).
Ultimately, these results rule out cross-allergy
between the miracle berry and peanuts. This fact is a
great relief because peanut shows cross-reactions with
a large number of foods, as occurs with the peach
(specifically between Pru p 3 and Ara h 9 allergens,
both from the LTP family; Romano et al., 2009),
being this food capable of generating anaphylactic epi-
sodes in patients. For this reason, it is vitally impor-
tant to continue the research and provide evidence on
food safety to improve the quality of life of patients
suffering from this type of pathology.
Conclusions
In conclusion, the miracle berry from S.dulcificum
does not contain any allergens from the Ara h family
or structural homologues that can generate a cross-
allergenicity reaction. Therefore, there seems to be no
risk of symptoms appearing in individuals allergic to
peanuts after miracle berry consumption. In addition,
this article provides a methodological basis and evalu-
ate aspects for other researchers about the possible
cross-allergy of another Novel Food and to provide a
new evidence of its food safety.
Acknowledgments
The authors wish to acknowledge that this work was
part of the Project “Actividad biol
ogica e inmu-
nog
enica de Synsepalum dulcificum para el desarrollo
de alimentos funcionales (Biologic and immunogenic
activity of Synsepalum dulcificum for the development
of functional food)” coordinated by the Community of
Madrid and the Polytechnic University of Madrid.
We thank C.E. Gavira O’Neill for English correction
and Dr. Santiago Moreno for providing the peanut
samples.
Author contributions
Adrian Men
endez-Rey: Conceptualization (equal); data
curation (equal); formal analysis (equal); investigation
(equal); methodology (equal); resources (equal); valida-
tion (equal); writing –original draft (equal); writing –
review and editing (equal). F
atima Jerez-Arroyo: Data
curation (equal); investigation (equal); methodology
(equal); writing –original draft (equal). Nicolas
Alegria-Aravena: Data curation (equal); formal analy-
sis (equal); methodology (equal); validation (equal);
writing –review and editing (equal). Josefa Quiroz-
Troncoso: Data curation (equal); formal analysis
(equal); methodology (equal); writing –review and
editing (equal). Raquel Gonz
alez-Martos: Data cura-
tion (equal); formal analysis (equal); methodology
(equal); writing –review and editing (equal). Marta
Sanchez-D
ıez: Data curation (equal); formal analysis
(equal); methodology (equal); writing –review and
editing (equal). Carlos Blanco: Resources (equal);
supervision (equal); writing –review and editing
(equal). Ceferino Maestu Unturbe: Project administra-
tion (equal); resources (equal). Loan Bensadon-Naeder:
Funding acquisition (equal); project administration
(equal); resources (equal).
Funding information
This work was supported by the Community of
Madrid [IND2017/BIO-7827].
Conflict of interest
The authors declare that they have no known compet-
ing financial interests or personal relationships that
could have appeared to influence the work reported in
this paper.
Ethics statement
This study involves the use of blood samples from
human patients. Before starting the study, all the vol-
unteers signed an informed consent as indicated in the
study protocol, approved in Madrid on December 22,
2020, by the Ethics Committee for Drug Research of
the Hospital Universitario de la Princesa (certificate
CEIm 25/20).
Peer review
The peer review history for this article is available at
https://publons.com/publon/10.1111/ijfs.16114.
Data availability statement
Data available on request from the authors.
Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd
on behalf of Institute of Food, Science and Technology (IFSTTF).
International Journal of Food Science and Technolo gy 2022
Cross-allergy between miracle berry and peanut A. Men
endez-Rey et al.7688
References
Aalberse, J.A., Meijer, Y., Derksen, N., an der Palen-Merkus, T.v.,
Knol, E. & Aalberse, R.C. (2013). Moving from peanut extract to
peanut components: towards validation of component-resolved IgE
tests. Allergy,68, 748–756.
Abrams, E., Chan, E. & Sicherer, S. (2020). Peanut allergy: new
advances and ongoing controversies. Pediatrics,145, e20192102.
Bartoshuk, L.M., Gentile, R.L., Moskowitz, H.R. & Meiselman,
H.L. (1974). Sweet taste induced by miracle fruit (Synsepalum dul-
cificum). Physiology & Behavior,12, 449–456.
Chafen, J.J.S., Newberry, S.J., Riedl, M.A. et al. (2010). Diagnosing
and Managing Common Food Allergies: A Systematic Review.
JAMA - Journal of the American Medical Association,303, 1848–
1856.
Chan, E.S., Greenhawt, M.J., Fleischer, D.M. & Caubet, J.C.
(2019). Managing cross-reactivity in those with Peanut allergy.
Journal of Allergy and Clinical Immunology: In Practice,7, 381–
386.
This reference indicates the large number of foods that present
cross-allergy with peanuts. Therefore, in the case of a Novel Food
such as the miracle berry, it is necessary to provide evidence to
ensure that its consumption does not imply any risk for these
patients.
Chen, C.-C., Liu, I.-M. & Cheng, J.-T. (2006). Improvement of insu-
lin resistance by miracle fruit (Synsepalum dulcificum) in fructose-
rich chow-fed rats. Phytotherapy Research,20, 987–992.
de G
omez Cedr
on, M., Wagner, S., Reguero, M., Men
endez-Rey, A.
& de Molina, A.R. (2020). Miracle berry as a potential supplement
in the control of metabolic risk factors in cancer. Antioxidants,9,
1282.
Du, L., Shen, Y., Zhang, X., Prinyawiwatkul, W. & Xu, Z. (2014).
Antioxidant-rich phytochemicals in miracle berry (Synsepalum dul-
cificum) and antioxidant activity of its extracts. Food Chemistry,
153, 279–284.
FAO & World Health Organization. (2001). Evaluation of allergenic-
ity of genetically modified foods:report of a Joint FAO/WHO
Expert Consultation on Allergenicity of Foods Derived from Biotech-
nology.
Frith, K. & Katelaris, C. (2019). Current perspectives on peanut
allergy. Internal Medicine Journal,49, 1480–1487.
Haddad, S., Mohammad, M., Raafat, K. & Saleh, F. (2020). Antihy-
perglycemic and hepatoprotective properties of miracle fruit
(Synsepalum dulcificum) compared to aspartame in alloxan-
induced diabetic mice. Journal of Integrative Medicine,18, 514–521.
He, Z., Tan, J.S., Abbasiliasi, S., Lai, O.M., Tam, Y.J. & Ariff, A.B.
(2016). Phytochemicals, nutritionals and antioxidant properties of
miracle fruit Synsepalum dulcificum. Industrial Crops and Products,
86,87–94.
He, Z., Tan, J.S., Lai, O.M. & Ariff, A.B. (2015). Optimization of
conditions for the single step IMAC purification of miraculin from
Synsepalum dulcificum. Food Chemistry,181,19–24.
Inglett, G.E. & Chen, D. (2011). Contents of phenolics and flavo-
noids and antioxidant activities in skin, pulp, and seeds of miracle
fruit. Journal of Food Science,76, C479–C482.
Johnson, P.E., Sayers, R.L., Gethings, L.A. et al. (2016). Quantita-
tive Proteomic Profiling of Peanut Allergens in Food Ingredients
Used for Oral Food Challenges. Analytical chemistry,88, 5689–
5695.
Kadam, K., Karbhal, R., Jayaraman, V.K., Sawant, S. & Kulkarni-
Kale, U. (2017). AllerBase: a comprehensive allergen knowledge-
base. Database,2017,1–12.
Klemans, R., van Os-Medendorp, H., Blankestijn, M., Bruijnzeel-
Koomen, C., Knol, E. & Knulst, A. (2015). Diagnostic accuracy of
specific IgE to components in diagnosing peanut allergy: a system-
atic review. Clinical and Experimental Allergy,45, 720–730.
Krause, S., Reese, G., Randow, S. et al. (2009). Lipid transfer pro-
tein (Ara h 9) as a new peanut allergen relevant for a
Mediterranean allergic population. The Journal of Allergy and Clin-
ical Immunology,124, 771–778.e5.
Krogulska, A. & Wood, R. (2020). Peanut allergy diagnosis: moving
from basic to more elegant testing. Pediatric Allergy and Immunol-
ogy,31, 346–357.
Matricardi, P.M., Kleine-Tebbe, J., Hoffmann, H.J. et al. (2016).
EAACI molecular allergology User’s guide. Pediatric Allergy and
Immunology,27,1–250.
Men
endez-Rey, A., Gonz
alez-Martos, R., Ye, P. et al. (2021). Quan-
tification of lectins in Synsepalum dulcificum and comparison with
reference foods. Food Chemistry,352, 129341.
Njoku, N.E., Ubbaonu, C.N., Alagbaoso, S.O., Eluchie, C.N. &
Umelo, M.C. (2015). Amino acid profile and oxidizable vitamin
content of Synsepalum dulcificum berry (miracle fruit) pulp. Food
Science & Nutrition,3, 252–256.
Nwaru, B., Hickstein, L., Panesar, S., Roberts, G., Muraro, A. &
Sheikh, A. (2014). Prevalence of common food allergies in Europe:
a systematic review and meta-analysis. Allergy,69, 992–1007.
Obafemi, T.O., Olaleye, M.T. & Akinmoladun, A.C. (2019). Antidia-
betic property of miracle fruit plant (Synsepalum dulcificum Shu-
mach. & Thonn. Daniell) leaf extracts in fructose-fed
streptozotocin-injected rats via anti-inflammatory activity and inhi-
bition of carbohydrate metabolizing enzymes. Journal of
Ethnopharmacology,244, 112124.
Renz, H., Allen, K.J., Sicherer, S.H. et al. (2018). Food allergy. Nat-
ure Reviews Disease Primers,4,1–20.
Restani, P., Ballabio, C., Corsini, E. et al. (2005). Identification of
the basic subunit of Ara h 3 as the major allergen in a group of
children allergic to peanuts. Annals of Allergy, Asthma & Immunol-
ogy,94, 262–266.
Reyes, A.J., Hosein, A.S., Ramcharan, K. & Perot, S. (2020). Ana-
phylaxis and other allergic reactions to food: a global challenge.
BMJ Case Reports,13, e231425.
Rodrigues, J.F., RDS, A., Bastos, S.C., Coelho, S.B. & Pinheiro,
A.C.M. (2016). Miracle fruit: an alternative sugar substitute in
sour beverages. Appetite,107, 645–653.
Romano, A., Fernandez-Rivas, M., Caringi, M., Amato, S., Mis-
trello, G. & Asero, R. (2009). Allergy to peanut lipid transfer pro-
tein (LTP): frequency and cross-reactivity between peanut and
peach LTP. European Annals of Allergy and Clinical Immunology,
41, 106–111.
Sicherer, S. & Sampson, H. (2018). Food allergy: a review and
update on epidemiology, pathogenesis, diagnosis, prevention, and
management. The Journal of Allergy and Clinical Immunology,141,
41–58.
Sicherer, S.H. & Sampson, H.A. (2014). Food allergy: epidemiology,
pathogenesis, diagnosis, and treatment. Journal of Allergy and Clin-
ical Immunology.,133, 291–307.e5.
These authors point out the high prevalence of peanut allergy world-
wide, which supports the importance of studying its cross-allergy
with other foods in order to improve the quality of life of people
who suffer from it.
Soares, H.P., Cusnir, M., Schwartz, M.A. et al. (2010). Treatment of
taste alterations in chemotherapy patients using the “miracle fruit”:
preliminary analysis of a pilot study. Journal of Clinical Oncology,
28, e19523.
Tafazoli, S., Vo, T., Roberts, A. et al. (2019). Safety assessment of
miraculin using in silico and in vitro digestibility analyses. Food
and Chemical Toxicology,133, 110762.
Takai, A., Satoh, M., Matsuyama, T. et al. (2013). Secretion of
miraculin through the function of a signal peptide conserved in the
Kunitz-type soybean trypsin inhibitor family. FEBS Letters,587,
1767–1772.
This reference shows that miraculin belongs to a protein family that
does not correspond to that of any peanut allergen, which could
show the absence of cross-reactivity. However, it is necessary to
carry out the relevant tests (in silico and in vitro) to dismiss this phe-
nomenon.
Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd
on behalf of Institute of Food, Science and Technology (IFSTTF).
International Journal of Food Science and Technology 2022
Cross-allergy between miracle berry and peanut A. Men
endez-Rey et al. 7689
Taylor, S.L. & Baumert, J.L. (2010). Cross-contamination of foods
and implications for food allergic patients. Current Allergy and
Asthma Reports,10, 265–270.
Turck, D., Castenmiller, J., de Henauw, S. et al. (2021). Safety of
dried fruits of Synsepalum dulcificum as a novel food pursuant to
regulation (EU) 2015/2283. EFSA Journal,19, 6600.
This reference justifies the importance of analysing the cross-allergy
of the miracle berry with the peanut, since the experts of the evalua-
tion panel state that the published results are so far preliminary and
do not completely rule out a possible cross-reactivity.
Turner,P.,Jerschow,E.,Umasunthar,T.,Lin,R.,Campbell,D.&
Boyle, R. (2017). Fatal anaphylaxis: mortality rate and risk factors. The
Journal of Allergy and Clinical Immunology. In Practice,5,1169–1178.
Walczyk, N.E., Smith, P.M.C., Tovey, E., Wright, G.C., Fleis-
chfresser, D.B. & Roberts, T.H. (2013). Analysis of crude protein
and allergen abundance in peanuts (Arachis hypogaea cv. Walter)
from three growing regions in Australia. Journal of Agricultural
and Food Chemistry,61, 3714–3725.
Wilken, M.K. & Satiroff, B.A. (2012). Pilot study of "miracle fruit"
to improve food palatability for patients receiving chemotherapy.
Clinical Journal of Oncology Nursing,16, E173–E177.
Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Table S1 Positive cross-allergenicity database. It col-
lects a total of 100 comparisons between pairs of
allergens whose cross-reactivity is described in the lit-
erature (AllerBase). A protein sequence alignment was
carried out using BLASTp software and the sequence
coverage (%), the percent identity (%) and the pro-
duct of both values (homology indicator) were noted.
In addition, the name of each allergen (along with its
UniprotKB code) and the species from which it came
are displayed.
Table S2 Negative cross allergenicity database. It
collects a total of 118 comparisons between pairs of
allergens in which no cross-reactivity has been
described (AllerBase). A protein sequence alignment
was carried out using BLASTp software and the
sequence coverage (%), the percent identity (%) and
the product of both values (homology indicator) were
noted. In addition, the name of each allergen (along
with its UniprotKB code) and the species from which
it came are displayed.
Table S3 Additional information on the four peanut
allergic individuals. The results of the serological
determination of IgE against the different peanut aller-
gens (Ara h 1, 2, 3, 8 and 9), the allergy profile and
the observed symptoms are included. (
1
Positive val-
ues >0.35 kU/L are considered;
2
Lipid Transfer Pro-
tein (LTP);
3
Oral Allergy Syndrome (OAS)).
Ó2022 The Authors. International Journal of Food Science & Technology published by John Wiley & Sons Ltd
on behalf of Institute of Food, Science and Technology (IFSTTF).
International Journal of Food Science and Technolo gy 2022
Cross-allergy between miracle berry and peanut A. Men
endez-Rey et al.7690
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