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Review
Effect of processing on conformational changes of food proteins
related to allergenicity
Toheder Rahaman, Todor Vasiljevic, Lata Ramchandran
*
Advanced Food Systems Research Unit, College of Health and Biomedicine, Victoria University, Melbourne, VIC, Australia
article info
Article history:
Received 11 August 2015
Received in revised form
5 November 2015
Accepted 3 January 2016
Available online 6 January 2016
Keywords:
Food allergens
Epitopes
Processing
Conformation
Digestibility
Allergenicity
abstract
Background: Food allergy is one of the major health concerns worldwide that has been increasing at an
alarming rate in recent times. Foods undergo various processing steps before consumption that could
affect conformation of food proteins, their digestion and thereby allergenicity.
Scope and approach: This review summarizes the effect of various processing methods on structural
changes of major food allergens and how these changes affect their digestibility as well as allergenicity.
This information could be a base line for selecting suitable food processing parameters for management
of food allergies.
Key findings and conclusions: Most physical processes (heat, pressure, radiation, and ultrasound) affect
conformational epitopes (destroy, mask or expose) of food proteins by altering their secondary and
tertiary structures whereas the linear/sequential epitopes are affected mainly through bio-chemical
(fermentation and enzymatic hydrolysis) treatments. Processing may also influence the interaction of
food proteins with other ingredients via Maillard reaction and give rise to formation of new allergenic
compound (neo-allergens). Processing induced changes to food proteins can largely affect their sus-
ceptibility to gastrointestinal digestion, absorption kinetics and consequently their allergenic response to
immune system. Therefore, allergenic potential of food proteins may be minimized by selecting
appropriate parameters during processing. Allergenicity of certain food proteins can also be modulated
through optimized formulation with other food matrices. However, depending on the method of pro-
cessing, intensity of treatment and molecular characteristics of allergen food proteins, allergenicity can
be increased, decreased or remain unaltered.
Crown Copyright ©2016 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Food allergy is one of the major health concerns worldwide
affecting 1e3% of adults and 4e6% of children, and in the last two
decades the rate has increased considerably (Lack &Du Toit, 2014).
It can seriously affect the quality of life of both, patients and their
family, even more than chronic childhood diseases (Arasi et al.,
2014). Food allergies are the adverse immune reactions to specific
foods that result in either instant severe life threatening symptoms
such as acute urticaria, angioedema, bronchospasm and anaphy-
laxis or delayed symptoms including atopic dermatitis and allergic
gastrointestinal disorders. Although many foods are reported for
their allergic reactions, more than 90% of food allergies are caused
by cow's milk, egg, fish, crustaceans, peanuts, tree nuts, wheat and
soybeans, which are referred as “The Big Eight”(T€
or€
ok et al., 2014).
In the early history of food allergy, a report of sensitivity to
cooked but not raw fish (Mills &Mackie, 2008) created an interest
to know whether processing affected food allergy or not. It is now
understood that processing may either reduce or enhance the
allergic potential of food proteins or sometimes have no effect at all.
For example, Chinese traditional water boiling and frying of egg
showed higher allergenic potential than steamed egg and tea-
boiled egg (Liu et al., 2013). In addition, some treatments can
induce formation of new allergenic compounds (neo-allergens) by
prompting interactions between different ingredients (Verma,
Kumar, Das, &Dwivedi, 2012).
Foods are processed in diverse ways before consumption in
order to improve functional, nutritional and sensory attributes, as
well as for preservation and detoxification. Commonly applied
processing techniques include thermal, high pressure, radiation,
high intensity ultrasound, and bio-chemical approaches. Different
processing methods alter the structure of food proteins in different
*Corresponding author.
E-mail address: lata.ramchandran@vu.edu.au (L. Ramchandran).
Contents lists available at ScienceDirect
Trends in Food Science &Technology
journal homepage: http://www.journals.elsevier.com/trends-in-food-science-
and-technology
http://dx.doi.org/10.1016/j.tifs.2016.01.001
0924-2244/Crown Copyright ©2016 Published by Elsevier Ltd. All rights reserved.
Trends in Food Science &Technology 49 (2016) 24e34
ways and possible structural modifications include unfolding, ag-
gregation, cross-linking between the ingredients and chemical
modifications such as oxidation and glycosylation (Lepski &
Brockmeyer, 2013). Such processing induced conformational
changes can directly influence the allergenicity by disrupting
conformational or linear epitopes. Conformational epitopes can be
exposed or hidden by unfolding or aggregation of proteins
(Rahaman, Vasiljevic, &Ramchandran, 2015), respectively, whereas
sequential epitopes can be affected by acidic or enzymatic hydro-
lysis (Kasera, Singh, Lavasa, Prasad, &Arora, 2015) and extreme
Maillard reactions (Toda, Heilmann, Ilchmann, &Vieths, 2014).
Processing induced physico-chemical changes of food proteins may
further affect gastrointestinal digestibility, absorbance kinetics
through mucosa as well as their presentation to the immune sys-
tem and thereby influence their allergenicity (Table 1). However,
the degree of structural alteration and allergenicity depends on the
processing method used, extent and exposure time, and presence
of other ingredients for example salt, sugar etc. (Verma et al., 2012).
Avoidance of allergic foods is the most common management
strategy for sensitive individuals which may consequently lead to a
number of nutritional deficiency syndromes. Although oral and
subcutaneous desensitization therapies have also been practiced
for a long time, their efficacies are not always satisfactory (Patriarca
et al., 2007). Thus, there is a need to seek alternate strategies such
as selective processing for minimizing the allergenic severity of
foods. To select appropriate processing methods, it is very impor-
tant to understand how these procedures alter the structure of food
proteins both at a microscopic and macroscopic level and their
subsequent gastrointestinal digestibility, all of which can influence
their allergenicity. Several reviews (Lepski &Brockmeyer, 2013;
Mills, Sancho, Rigby, Jenkins, &Mackie, 2009; Paschke, 2009;
Shriver &Yang, 2011; Verhoeckx et al., 2015) have compiled the
effect of processing on allergenicity of various foods but it is not
well explained how the processing induced conformational
changes affect digestibility. Resistance to gastrointestinal digestion
is one of the main characteristics that allow food proteins retaining
intact epitopes to invoke allergic reaction. Therefore the present
review focuses on processing induced conformational changes of
major food proteins and its relation to their digestibility and
allergenicity. Such information is critical in the selection of
appropriate parameters during food processing as an effective
alternate in the management strategies of food allergies.
2. Various processing and their effect on food protein
structure, digestibility and allergenicity
2.1. Effect of thermal treatments
Thermal treatment is the conventional and most commonly
used processing technique for many foods in order to reduce their
Table 1
Summary of effect of different processing methods on conformation, digestibility and allergenicity of food allergens.
Allergen Processing
methods
Conformational change Digestibility and allergenic consequence References
Ara h1 and Ara
h2 from
peanut
Roasting Compact globular covalent aggregates and Maillard
products (neo-allergen)
Less susceptible to protease and enhanced
allergenicity
(Blanc et al., 2011; Maleki &
Hurlburt, 2004)
Boiling Loss of
b
barrel with adopting random coil and
formation of branched rod-shaped aggregates
More susceptible to hydrolysis and
decreased allergenicity
Wheat protein
allergen
Baking Formation of aggregates through Maillard reaction
and inter-peptide linkage
Decreased digestibility and enhanced
allergenicity
(Pasini et al., 2001)
b
-lg in cow milk Sterilization Unfolding followed by covalent aggregation and
Maillard reaction
Increased susceptibility to peptic hydrolysis
and reduced allergenicity
(Bu et al., 2009; Peram et al.,
2013)
Pasteurization Exposure of conformational epitopes Enhanced uptake through epithelium with
increased allergenicity
(Bu et al., 2009)
Heating with
wheat matrix
Complex structure formation between wheat and
b
-
lg
Reduced digestibility and bio-availability to
immune system
(Bloom et al., 2014)
High pressure Unfolding of protein molecule with exposure of
cleavage site
Enhanced digestibility and reduced
allergenicity
(L
opez-Exp
osito et al., 2012)
Radiation Protein agglomeration Unaltered (Lee et al., 2001)
Ultrasound Formation of oligomers and
b
sheet to
a
helix
transition
Increased digestibility but allergenicity is
unaltered
(Stanic-Vucinic et al., 2012)
Casein in cow
milk
Pasteurization,
Sterilization
Rheomorphic, no conformational change Unaffected (Morisawa et al., 2009)
Egg ovalbumin Moist heat Denaturation and aggregation Lower permeability through enterocyte
resulting in reduced allergenic potential
(Shin et al., 2013; Watanabe
et al., 2014)
High pressure Loss of conformational and sequential epitopes Enhanced digestibility and reduced
allergenicity
L
opez-Exp
osito et al., 2008
Egg ovomucoid Moist heat Heat stable Unaltered (Juli
a et al., 2007; Shin et al.,
2013)
Heating with
wheat flour in
pasta
Formation of insoluble aggregates Reduced allergenicity (Kato et al., 2001)
Tropomyosin
from shrimp
Moist heat Formation of new allergic compound through
Maillard reaction
Digestibility remain unaltered and
allergenicity increased
(Kamath et al., 2013)
High Pressure Unfolding of protein with loss of
a
helix Improved digestibility and reduced
allergenicity
(Jin et al., 2015)
Ultrasound Denaturation and fragmentation Increased digestibility but allergenicity
remain unaltered
(Li, Lin, Cao, &Jameel, 2006)
Walnut Moist heat Fragmentation of protein molecules Enhanced susceptibility to digestion and
reduced allergenicity
(Cabanillas et al., 2014)
Soy allergen
(glycicnin)
Moist heat Formation of soluble aggregates Slight decrease of peptic digestibility but no
change of allergenicity
(van Boxtel, van den Broek,
Koppelman, &Gruppen, 2008)
High pressure Increased hydrophobicity, SH and
a
helix content Increased digestibility and reduced
allergenicity
(Penas et al., 2006)
T. Rahaman et al. / Trends in Food Science &Technology 49 (2016) 24e34 25
pathogen load, increase shelf life and improve quality. It includes
boiling, cooking, baking, roasting, frying, grilling, pasteurization
and sterilization. Heat induced structural changes of food proteins
have been extensively investigated (Blanc et al., 2011; Rahaman
et al., 2015; Verma et al., 2012) and the suggested mechanisms
include initial unfolding of a protein molecule, loss of secondary
and tertiary structure, formation of intra and/or inter-molecular
covalent and non-covalent interactions. Such structural alter-
ations can express, mask or destroy conformational epitopes in
food proteins and thereby influence allergenicity, while the
sequential epitopes remain unaffected (Fig. 1). Thermal treatment
not only affects the allergenicity of food proteins through confor-
mational changes, but also by influencing their interactions with
other food ingredients. One such interaction is Maillard reaction. It
is a non-enzymatic condensation of Nε-group of amino acid resi-
dues (lysine) in protein with the carbonyl group of reducing sugars
to form glycosamine (Renzone, Arena, &Scaloni, 2015). These gly-
cosamines undergo further rearrangement to form advanced gly-
cation end products (AGEs). In addition, heating can also alter the
susceptibility of proteins to gastrointestinal digestion and their
absorption through mucosa and thereby modulate their allergenic
potential (Fig. 2).
2.1.1. Heat labile food allergens and their allergenic consequence
Heat induced antigenic changes of beta lactoglobulin (
b
-lg), a
major cow milk allergen, has been summarized in Fig. 3. Heating up
to 90
C causes unfolding of the
b
-lg molecule, exposure of
conformational epitopes and enhanced susceptibility to proteolysis
resulting in increased allergenicity (Bu, Luo, Zheng, &Zheng, 2009;
Kleber, Krause, Illgner, &Hinrichs, 2004) as determined by in vitro
ELISA inhibition test with sera from allergic patients. An in vivo
study (Roth-Walter et al., 2008) detailed the mechanism of higher
IgE reactivity as a consequence of pasteurization. Aggregated
b
-lg
could be taken up more extensively by Peyer's patches in the in-
testinal mucosa, which leads to greater production of IgE although
anaphylactic symptoms were elicited only by absorbed soluble
b
-lg.
Thus measuring IgE levels by ELISA may not always reflect accu-
rately the anaphylactic score. This makes it necessary to compare
serum level of IgE with anaphylactic degree after oral challenging
with processed milk allergens. Raising the temperature further to
100 and 120
C can lead to irreversible aggregation with covalent
and hydrophobic interactions, consequently masking and/or
destroying conformational epitopes and reduction of allergenicity.
Such heat induced changes showed higher susceptibility to peptic
hydrolysis (Peram, Loveday, Ye, &Singh, 2013), which resulted in
cleavage of protein sequences with disruption of linear epitopes
(Morisawa et al., 2009) consequently weakening their allergenicity.
Moreover, heat induced glycation also influenced the allergenicity
of
b
-lg. Most of the
b
-lg epitopes contain one or more lysine resi-
dues and condensation of these lysine residues with reducing
sugars (Maillard reaction) during heating results in modification of
linear epitopes (Taheri-Kafrani et al., 2009) with significant
reduction of IgE reactivity. However, high level of glycation reduced
the susceptibility of
b
-lg to proteolysis resulting in increased IgE
reactivity of hydrolysate (Corzo-Martínez, Soria, Belloque,
Villamiel, &Moreno, 2010) regardless of the type of reducing
sugar. Similar reduction in allergenicity of egg white proteins
subjected to heating has also been reported. Heating of egg white at
100 and 120
C resulted in denaturation and aggregation of oval-
bumin (OVA). Although the aggregation showed higher stability to
Fig. 1. Schematic diagram of heat induced changes of antigenic epitopes.
T. Rahaman et al. / Trends in Food Science &Technology 49 (2016) 24e3426
digestion, its lower absorption and poor/delayed sensitivity to
immune system resulted in reduced IgE production in mice model
(Watanabe et al., 2014).
Conditions such as pH and ionic strength are important factors
for heat mediated denaturation of some proteins such as
b
-lg
(Schmitt et al., 2009) and thus can influence related changes in
allergenicity. For example, compared to neutral pH, heating of
b
-lg
at acidic pH (pH 3) leads to unfolding of protein molecule, exposure
of
b
strands and partial acid hydrolysis, which could contribute to
the appearance of some new epitopes resulting in enhanced anti-
genicity (Rahaman et al., 2015). Decreased immune reactivity of egg
ovomucoid (OM) has been reported to be higher at an alkaline pH
(9.5) than at pH 7.5 (Lee et al., 2002) which was attributed to
irreversible denaturation of OM with alteration of allergenic epi-
topes at basic pH.
2.1.2. Heat stable food allergens
Although many food allergens are sensitive to heat and their
allergenicity is changed accordingly, some are stable to heat
denaturation and digestion (Mills et al., 2009), consequently their
allergenicity remains unaltered or can be affected only when
exposed to extreme temperatures. A characteristic feature of such
proteins is presence of intra or inter molecular cystine that de-
termines their structural integrity as well as antigenic stability
(Maeno, Matsuo, &Akasaka, 2013). One such major heat stable
allergen is egg OM that is stable to digestion even after boiling for
1 h. Immune reactivity of OM did not decrease after heating
regardless of temperature and heating time (Shin, Lee, Ahn, Lee, &
Han, 2013). This is attributed to its clearly defined three tandem
domains interlinked by strong disulphide bonds (Juli
a et al., 2007)
which allows for maintained solubility after heating rather than
aggregating thereby retaining its reactivity to patients' IgE (Kato,
Oozawa, &Matsuda, 2001). Tropomyosin, a major allergen in
crustacean food (Lopata, O'hehir, &Lehrer, 2010) also exhibits heat
resistance, which is attributed to its
a
-helical coiled-coil dimeric
form (Ozawa, Watabe, &Ochiai, 2011). Although
a
-helical sec-
ondary structure of tropomyosin collapsed upon heating above
80
C, the native structure could be regained upon cooling and
consequently its antigenicity measured as binding to specific IgG by
ELISA remains unaltered (Usui et al., 2013). Contrary to this,
increased binding affinity of tropomyosin to patients' sera IgE after
boiling has also been observed (Kamath, Rahman, Komoda, &
Lopata, 2013) that has been ascribed to its high content of lysine
residue which readily reacts with reducing sugars to form some
non-native structures (neo-allergens). Such discrepancies between
the studies could be due to use of different antibody, IgG vs IgE.
Variation of primary structure, binding pattern and heat response
of the epitopes for two different antibodies could also have resulted
in these contrary findings.
IgE reactivity of the milk protein casein is also unaffected when
milk is heated at 90
C(Bloom et al., 2014). The four components of
casein
a
s1
-,
a
s2
-,
b
- and
k
-casein possess ill defined, disordered
mobile structure (rheomorphic) and they lack a co-operative
transition of unfolding or partial folding during heating. Also due
Fig. 2. In general heat induced conformational changes and their consequences in digestion, absorption and immune reactivity of food allergens.
T. Rahaman et al. / Trends in Food Science &Technology 49 (2016) 24e34 27
to their dynamic structure, casein components contain many linear
epitopes rather than conformational that could explain why their
immune reactivity remains unaltered upon heating (Mills et al.,
2009).
2.1.3. Dry vs moist heat
Stability and allergenic potential of allergens can also be affected
by type of heat i.e. dry or moist heat. For example, allergenicity of
seed storage globular protein Ara h1 and Ara h2 found in peanuts is
enhanced by dry roasting but diminished after boiling in water
(Blanc et al., 2011). Upon roasting, Ara h1 undergoes covalent cross
linking and hydrophobic interactions and forms compact polymers.
Such aggregation makes the protein inaccessible to gastrointestinal
digestion to some extent, allowing absorbance of large fragments
through intestinal mucosa containing more IgE binding sites
resulting in enhanced allergenicity (Maleki &Hurlburt, 2004). In
contrast, boiling of Ara h1 formed branched rod-shaped aggregates
with loss of some secondary structure and consequently lowers IgE
binding ability (Blanc et al., 2011). Likewise significantly higher IgE
reactivity of OVA in fried egg compared to boiled egg was also re-
ported (Shin, Han, &Ahn, 2013). However, none of these studies
explained the cause of such structural and allergenic variation
between roasted and boiled peanut allergens. Variation of tem-
perature arising from the two different methods of heating, mode
of heat treatment, as well as variations in moisture content in dry vs
moist heat could govern differently the rate of certain interactions
(hydrophobic, covalent etc.) in protein molecules that could have
influenced their allergenicity. Samadi and Yu (2011) have also re-
ported that dry and moist heating (autoclaving) of soybean to the
same temperature (120
C) resulted in significantly different vari-
ations in protein structure, digestibility and consequently allerge-
nicity. Further, it has been shown that (Kroghsbo et al., 2014)
roasted peanuts had lower rat basophilic leukemia (RBL) degran-
ulation capacity than blanched peanut whereas extracted Ara h1
from roasted peanut had higher RBL elicitation. Thus, although
many studies referred to the allergenic potential of purified
allergen rather than that of whole food, the values always do not
represent each other possibly due to the effect of other ingredients
in the allergic foods. Therefore, it is essential to determine effect of
processing on allergenicity of both purified allergens and whole
food to obtain a more holistic evaluation for its application in food
manufacturing.
Baking, another form of dry heat application can alter confor-
mation, digestibility as well as allergenicity of potential wheat flour
Fig. 3. Schematic model for alteration of epitopes in
b
-lg to bind with antibody at different level of heat treatment.
T. Rahaman et al. / Trends in Food Science &Technology 49 (2016) 24e3428
allergens (Petitot, Abecassis, &Micard, 2009). Immunoblotting of
in vitro digested unheated bread dough, bread crumb and crust
with sera from wheat allergic patients resulted in almost complete
disappearance of IgE binding protein in dough but showed persis-
tence of such proteins in bread crumb and crust (Pasini, Simonato,
Giannattasio, Peruffo, &Curioni, 2001; Simonato et al., 2001). Heat
induced covalent protein aggregation of bread crumb (<100
C) and
Maillard reaction, in addition, for bread crust (>180
C), make the
proteins less susceptible to proteolysis thereby allowing passage of
large IgE-reactive fragments through gastrointestinal tract where
they could elicit more allergic reaction. Interestingly,
a
-amylase
inhibitor protein (responsible for wheat flour inhalation allergy
called “baker's asthma”) disappeared in digested bread crumb and
crust although this protein remained in bread dough even after
pancreatic digestion. This could explain why patients suffering
from baker's asthma do not show allergic reaction upon ingestion
of baked products like bread.
2.1.4. Heat mediated interaction of food allergens with other food
proteins and allergenicity
Several studies have established that allergenicity of certain
food proteins could be modulated through optimized formulation.
Cooking of food proteins with other food matrix can affect gastro-
intestinal susceptibility and thereby potentially influence their
allergenicity (Nowak-Wegrzyn &Fiocchi, 2009). About 50e70% of
cow milk allergic children tolerated baked milk products (muffin
and waffle) (Nowak-Wegrzyn et al., 2008) and adding baked milk
products into the daily diet accelerated development of their
tolerance to unheated milk compared to strict avoidance diet (Kim
et al., 2011). Whey proteins (
b
-lg and
a
-la) of baked milk in muffin
and waffle showed significantly lower immune activity with pa-
tients' sera by Western blotting than milk heated alone but reac-
tivity of casein remained unaltered (Bloom et al., 2014). Formation
of disulphide bonded protein complexes through the interaction of
wheat and milk proteins could possibly result in decreased
bioavailability of allergic proteins to immune system consequently
reducing their allergenicity. Similarly, more than 50% of egg allergic
patients tolerated baked egg products with wheat flour in the form
of muffins (baked at 176
C for 30 min) and waffles (baked at 260
C
for 30 min) whereas 19% patients tolerated only heated egg
(Lieberman, Huang, Sampson, &Nowak-We˛grzyn, 2012). Antige-
nicity of egg OM reduced significantly when these were baked with
wheat flour at 180
C for 10 and 30 min and rate of reduction was
positively correlated with increase of heating time (Shin, Lee, et al.,
2013). Irreversible denaturation of egg proteins due to disulphide
exchange reaction with gluten proteins during baking could have
resulted in reduced immune reactivity. Thermal treatment of
hazelnut proteins alone did not affect the stimulatory activity of
basophil of patients with systemic allergy while presence of wheat
proteins decreased such activities. Presence of protein rich food
matrix such as hazelnut and peanut extract with cow milk and
apple allergens can also significantly reduce gastrointestinal di-
gestibility, epithelial transport and thereby reduce their allerge-
nicity (Schulten, Lauer, Scheurer, Thalhammer, &Bohle, 2011).
Incorporation of almond flour in chocolate mousse and Victorian
sponge cake decreased the enzymatic degradation of almond pro-
teins and their allergenicity (Mandalari et al., 2014).
Thus, it is apparent that matrix proteins create a competitive
environment with allergen for enzymatic cleavage as well as active
epithelial transport which resulted in insufficient/delayed presen-
tation of allergic protein to immune system that could help in
formulating another effective means of managing allergies.
2.2. Non thermal applications
Although thermal application can be used to alter allergenic
potential of many foods, sometimes it can have a negative impact
on product qualities by changing organoleptic properties, colour
and nutrient content (Shriver &Yang, 2011). With non-thermal
procedures, food preserves its original characteristics, appears
fresher and sometimes is more nutritious than heat treated foods.
Thus, alternative non thermal approaches have been experimented
and their effects on food allergenicity have been investigated. These
procedures include high pressure, ultrasound, gamma radiation,
microbial fermentation and enzymatic hydrolysis.
2.2.1. High pressure treatment
High pressure (HP) is an emerging non-thermal technique in
food industries to inhibit the growth of microorganism as well as
increase the shelf life of food without affecting its organoleptic
properties. High pressure techniques mostly affect the non-
covalent interactions (hydrogen, ionic and hydrophobic bonds) in
protein molecules thereby affecting secondary and tertiary struc-
tures and consequently modulating their digestibility and allerge-
nicity. The possible mechanisms for modulation of allergenicity of
food proteins through high pressure treatment are summarized in
Fig. 4.
2.2.1.1. Susceptible allergens and their response to HP.
Combined high hydrostatic pressure (600 MPa) and enzymatic
hydrolysis of
b
-lg significantly reduced its in vitro reactivity with
IgE in allergic individuals when tested by indirect ELISA (Bonomi
et al., 2003). In vivo studies (L
opez-Exp
osito, Chic
on, Belloque,
L
opez-Fandi~
no, &Berin, 2012) in sensitized mice also proved that
hydrolysate under high hydrostatic pressure (400 MPa) are
immunologically inert. HP induces unfolding of
b
-lg and loss of
b
sheet and
a
helix at the expense of unordered structure. Such high
pressure induced unfolding facilitated enzymatic digestion result-
ing in shorter peptide fragments (7e10 residues long,
MW <1.5 KDa) which might not have enough epitopes to react
with antibodies leading to reduced allergenicity (Zeece, Huppertz,
&Kelly, 2008). In contrast, Chic
on, L
opez-Fandi~
no, Alonso, and
Belloque (2008) reported that although HP treatment (up to
400 MPa) enhanced susceptibility to proteolysis, its in vitro IgE
reactivity did not decrease. Such contradictory findings could be
the result of differences in methods of combined HP and enzymatic
treatment vis-a-vis enzymatic hydrolysis followed by HP. Moreover,
pressure level, holding time and temperature could also be influ-
encing factors that modulate allergenicity of HP treated
b
-lg.
Similarly, egg OVA treated at 400 MPa (L
opez-Exp
osito et al.,
2008) and Gly 1 allergen in soybean whey at 200e300 MPa
(Penas, Pr
estamo, Polo, &Gomez, 2006) showed much more
enhanced peptic digestion and significantly lower in vitro reactivity
of resulting hydrolysate with specific antibody compared to that
treated at atmospheric pressure. However, allergenicity was not
totally abolished due to residual antigenic effect of some peptide
fragments. Application of high pressure (300 MPa, 15 min) to soy
seed during sprouting could be a novel approach to produce hy-
poallergenic soybean sprouts (Pe~
nas, Gomez, Frias, Baeza, &Vidal-
Valverde, 2011). Pressure treatment of squid tropomyosin (Tod p1
allergen) up to 400 MPa (Jin et al., 2015) caused unfolding of protein
molecule with conversion of
a
helix to
b
sheet, increased free SH
and surface hydrophobicity. Such unfolding resulted in exposure of
target residues for enzymatic hydrolysis consequently increasing
proteolysis and reducing allergenicity. However, further increase of
pressure up to 600 MPa caused slight decrease of surface hydro-
phobicity without remarkable change in proteolysis and hence in
allergenicity.
T. Rahaman et al. / Trends in Food Science &Technology 49 (2016) 24e34 29
High pressure treatment up to 200 MPa did not affect the im-
mune reactivity of crushed peanut whereas at 400, 600 and
800 MPa immune reactivity was reduced significantly (Huang,
Yang, &Wang, 2014). Dhakal et al. (2014) investigated the effect
of high pressure treatment (450e600 MPa, for up to 600 s) on
immune reactivity of both conformational and linear epitopes of
almond milk using monoclonal antibody against both types of
epitopes. High pressure treated almond milk showed total inhibi-
tion in binding of conformational epitopes with specific antibody
and 50% inhibition for linear epitopes. Pressure induced denatur-
ation, formation of aggregation and loss of solubility caused inac-
cessibility of antibody to the epitopes.
Dynamic high pressure microfluidization (DHPM) is a new high
pressure technique that differs from hydrostatic pressure and in-
volves integrated action of strong shear, high speed-bumping,
prompt pressure release and cavitation in the foods being pro-
cessed. Treatment of
b
-lg with DHPM below 80 MPa caused in-
crease of antigenicity (Zhong et al., 2011) whereas above 80 MPa
allergenicity reduced significantly. DMPH up to 180 MPa caused
unfolding of purified Ara h2 from peanut that exposed hydrophobic
residues, decreased
a
helix at the expense of
b
sheet and caused
reduction of SeS bonds resulting in reduced immune reactivity (Hu
et al., 2011). However, both these studies used polyclonal antibody
against specific proteins to assess the allergenicity of DHPM treated
proteins which does not always mimic the original allergenic
response. Therefore, there is a need to further investigate the effi-
cacy of DHPM on allergenicity of food allergens using patients' sera
in vitro as well as by challenging the treated allergens in vivo.
2.2.1.2. Some allergens resistant to HP. Allergenicity of apple aller-
gens Mald 1 and Mald 3 under the superfamily of thaumatin-like
proteins (TLPs) are least affected by HP (Husband et al., 2011).
Eight intra-molecular disulphide bonds contribute to the folding of
protein that could also offer resistance to pressure and proteolysis
(Marzban et al., 2009). Similarly, cod allergen (Gad m1) (Somkuti,
Bublin, Breiteneder, &Smeller, 2012) and largemouth bass
allergen (Liu, Tao, Liu, Chen, &Xue, 2012) did not show any change
in immunogenic potential after treatment with high pressure.Thus,
pressure induced changes in allergenicity largely depend on vari-
ation of microstructure in different food proteins.
2.2.2. Radiation
Radiation has been a successful technique for food preservation
with minimal alteration in nutritive and sensory characteristics of
the food. Radiation brings about conformational changes such as
fragmentation, aggregation, cross linking and amino acid modifi-
cation in food proteins that can modulate their immune reactivity
(Luo et al., 2013). Such changes are triggered by free oxygen radicals
generated through radiolysis of water during radiation of proteins
in solution.
Irradiation (10 and 50 kGy, applied at 10
C at the rate of 10 kG/
h) brought about modification in conformational epitopes found in
peanuts that resulted in significantly lowered allergenic cytokine
production (IL-4) by splenocytes of sensitized mice (Oh et al.,
2009). In another study (Luo et al., 2013), irradiation (1, 3, 5 and
10 kGy at 10
C) of isolated peanut allergen Ara h 6 and whole
peanut extract also caused remarkable decrease in IgG binding
activity in ELISA with the increase of irradiation dose. However, up
to 5 kGy, IgG reactivity of whole peanut extract was higher than Ara
h6 that could be attributed to the effect of other components which
could help in protecting the epitopes on Ara h6. A similar trend was
also observed (Zhenxing, Hong, Limin, &Jamil, 2007) for irradiated
(3e15 kGy, at 10
C at 1 kGy/h) shrimp. Shrimp protein extract
showed decrease of IgE reactivity with increasing dose whereas
whole shrimp muscle presented a higher reactivity only up to 5 kGy
after which it decreased.
Irradiation of wheat germ agglutinin (WGA) caused initial
fragmentation of polypeptide chain followed by formation of large
Fig. 4. Mechanisms for reducing food protein allergenicity using high pressure processing.
T. Rahaman et al. / Trends in Food Science &Technology 49 (2016) 24e3430
insoluble amorphous aggregates resulting in lowered allergenicity
(Vaz et al., 2012). Dose dependent effect of irradiation on cow milk
allergy was investigated by Lee et al. (2001) where they found that
up to 5 kGy, the IgE reactivity of isolated
b
-lg increased and above
this dose, the protein underwent agglomeration with masking of
some epitopes resulting in reduced allergenicity. In contrast, irra-
diation (3e10 kGy applied at 13 Gy/min) of liquid and lyophilized
cow milk and whey showed increased recognition of anti
b
-lg IgG
(Kaddouri et al., 2008). Such differences could be due to the forms
of sample viz. isolated
b
-lg and
b
-lg in milk matrix and also type of
antibody used. Thus it is apparent that allergenic response of whole
food extract and purified allergen after radiation are different
which can be of significance to the food industries. Secondly,
different studies used different dose rates and hence the effect of
various dose rates for a given dosage of radiation on the allerge-
nicity food proteins is not yet clear.
However, irradiation treatment of whole almonds, cashew nuts
and walnuts did not alter conformation of allergens as well as their
allergenicity (Su, Venkatachalam, Sathe, Teuber, &Roux, 2004). In
contrast, negative impact of radiation on gliadin and wheat flour
with increased immune reactivity has been reported (Leszczynska,
Ła˛cka, Szemraj, Lukamowicz, &Zegota, 2003). Such opposing
observation could be due to the variation of transmission media i.e.
solid state vs solution which can impact the effectivity of the ra-
diation treatment. In general, radiation of allergens in liquid me-
dium could be the most effective mode of treatment to reduce
allergenicity.
2.2.3. Ultrasound
High intensity ultrasound is an emerging technique in food in-
dustries, frequently used for homogenization (mayonnaise), filtra-
tion (dairy whey solutions and fruit juice), tenderization (meat),
and dehydration (fruits and vegetables) processes. High intensity
ultrasound uses high energy mechanical waves (20e100 kHz)
which induces cyclic generation and collapse of cavities (sonication
bubbles) followed by formation of localized region of high pressure
and temperature surrounding these collapsed cavities which can
bring about conformational changes to food proteins and thereby
influence their allergic reactivity (Shriver &Yang, 2011).
There was no effect of high intensity ultrasound (30 Hz, 800 W)
at 0
Conin vitro (ELISA) IgE reactivity of a shrimp protein extract
whereas at 50
C reactivity reduced by 2.5 fold (Zhenxing, Caolimin,
&Jamil, 2006). Such differences was possibly due to the heat
induced decrease in viscosity of sample matrix which allowed
better penetration of ultrasound into the food system resulting in
conformational changes as well as reduction of allergenicity
compared to ultrasound treatment alone. However, so far there are
no reports on the digestibility of ultrasound treated shrimp aller-
gens and allergenic consequence of resulting hydrolysates. Also, all
the studies are limited to in vitro, and hence, in vivo studies with
oral food challenge are required for proper assessment. Exposure of
b
-lg to high intensity ultrasound can lead to formation of non-
native oligomers and dimers,
b
-sheet to
a
helix conversion, and
exposure of tryptophan (Trp) residues providing a less compact and
more dynamic structure compared to untreated
b
-lg. Although
these conformational changes enhanced the susceptibility of
b
-lg to
peptic hydrolysis, no remarkable change was observed in patients'
sera IgE reactivity, basophil activation test and skin prick test
(Stanic-Vucinic et al., 2012). Ultrasound treatment alone reduced
IgE-binding affinity of major peanut allergens Ara h1 and Ara h2
moderately (by about 10%) whereas ultra-sonication followed by
protease digestion (trypsinealpha chymotrypsin) significantly
increased the solubility of proteins and decreased IgE reactivity (Li,
Yu, Ahmedna, &Goktepe, 2013).
2.2.4. Fermentation
Fermentation is one of the traditional techniques for processing
and preservation of food. Microbial enzymatic hydrolysis of food
proteins during fermentation produces some bioactive peptides
with potential health benefits such as anticancer and anti-
hypertensive properties (Sah, Vasiljevic, McKechnie, &Donkor,
2014) while destroying some antigenic epitopes resulting in
decreased allergenicity (Fotschki, Szyc, &Wr
oblewska, 2015).
Moreover, consumption of probiotics in fermented foods can in-
fluence secretion of certain type of cytokines that could also affect
allergenicity through immune-modulatory mechanism (Bu, Luo,
Zhang, &Chen, 2010).
Lactic acid bacteria (LAB) (Lactobacillus acidophilus 5622, 20243,
20242 and Lactobacillus kefiri 20587) can potentially reduce
(measured by indirect ELISA using polyclonal antibody) the anti-
genic response of
b
-lg by 70% in sweet whey and more than 90% in
skim milk (Kleber, Weyrich, &Hinrichs, 2006) compared to unfer-
mented milk. Usage of co-culture (Streptococcus thermophilus
subsp. salivarius) synergistically accelerated the hydrolysis of
b
-lg
resulting in further splitting the linear epitopes and reducing their
antigenic potential. In vitro IgE-binding affinity of
a
-la,
b
-lg,
a
- and
b
-casein decreased by 55.12%, 49.66%, 2.62% and 2.02% respectively,
when reconstituted skim milk was fermented with Lactobacillus
casei (Shi et al., 2014). Combining strains of Lactobacillus bulgaricus
with Lactobacillus helveticus caused much higher reduction in
allergenicity of
a
-la and
b
-lg in skim milk (Bu et al., 2010) compared
to the individual strains alone. The extent of changes in antigenicity
also depends on the fermentation time. Antigenicity gradually
decreased from 0 to 12 h of fermentation and slightly increased
thereafter (Bu et al., 2010) which was attributed to breaking down
of oligopeptides by bacterial peptidase into smaller fragments with
exposure of some hidden epitopes. Fermentation with L. helveticus
(Ehn, Allmere, Telemo, Bengtsson, &Ekstrand, 2005) did not affect
the reactivity of
b
-lg with IgE although it caused 80% hydrolysis,
indicating strain specificity in the allergenic potential of LAB. Thus,
pre-treatment such as heating, selection of bacterial strain and
fermentation time are the important factors affecting changes of
milk protein allergenicity by fermentation. All these studies
assessed changes in antigenicity of milk allergens as a result of their
partial hydrolysis during fermentation, however, further changes
during gastrointestinal digestion, absorption through mucosa and
final allergenic consequences have not been studied. Thus, oral
challenges with fermented products and resulting anaphylaxis
needs to be further investigated.
Lactobacillus fermentation of sourdough caused acidification
and reduction of disulphide bonds of gluten resulting in increased
activity of cereal proteases, which improved the digestibility of
gluten by the consumers (G€
anzle, Loponen, &Gobbetti, 2008).
In vitro digestion of fermented sourdough bread showed almost
complete disappearance of potential peptides to induce gut asso-
ciated T cell mediated immune response (celiac disease) due to pre-
proteolytic activity by selected LAB which resulted in tolerance of
gluten by 80% patients (Di Cagno et al., 2004).
2.2.5. Enzymatic hydrolysis
One of the common characteristics of food allergen is their
resistance to gastrointestinal digestion; therefore pre-hydrolysis
with enzymes is one of the most effective methods of modifying
immune reactivity of food proteins. As a result of hydrolysis
conformational epitopes rapidly collapsed whereas linear epitopes
are cleaved and their further existence depends on the degree of
hydrolysis and type of enzyme used (Sabadin, Villas-Boas, de Lima
Zollner, &Netto, 2012).
Many studies have been performed to assess the allergenicity of
hydrolysed milk or whey protein concentrate and all showed
T. Rahaman et al. / Trends in Food Science &Technology 49 (2016) 24e34 31
significantly lower reactivity of resulting hydrolysate with serum
from allergic patients (Duan, Yang, Li, Zhao, &Huo, 2014; Sabadin
et al., 2012). Although extensively hydrolysed whey and casein
formulae and amino acid preparations were tolerated by most of
the allergic patients without any reactions, allergic reactions such
as atopic dermatitis have been reported for several individuals
(Meulenbroek et al., 2014). Such reactions of cow milk allergy pa-
tient to hydrolysed formula are attributed to the residual antige-
nicity of the small peptides (Bu, Luo, Chen, Liu, &Zhu, 2013). There
is no straight relationship between molecular mass of peptides in
hydrolysate and their residual allergenicity. Peptides with molec-
ular weight of 3000 Da (Bu et al., 2013) or even smaller peptides
(Puerta, Diez-Masa, &de Frutos, 2006) can provoke allergic reac-
tion. Such discrepancies could be ascribed to the type of enzyme
and hydrolysis model used degree of hydrolysis and sensitivity of
the patients. Whey protein hydrolysate prepared with alcalase
enzyme significantly lowered immune reactivity of
a
-la and
b
-lg
(Wr
oblewska et al., 2004) and the effectivity can be maximized by
controlling pH, temperature and enzyme-substrate ratio (Zheng,
Shen, Bu, &Luo, 2008). Peptic and tryptic hydrolysis of heated
whey protein had significantly lower allergenicity than the un-
heated hydrolysate (Kim et al., 2007). Heat induced unfolding of
protein molecule with exposure of cleavage sites for enzymes
resulting in enhanced proteolysis, destruction of epitopes and
thereby reduction of allergenicity could be the most plausible
explanation.
Peptic hydrolysis of soybean 2S protein only slightly reduced the
allergenicity while chymotrypsin hydrolysis following peptic hy-
drolysis increased the allergenicity (Sung, Ahn, Lim, &Oh, 2014).
Thus, allergenic epitopes of soybean 2S protein may not be fully
destroyed after peptic digestion and chymotrypsin treatment
possibly due to exposure of some hidden epitopes.
3. Conclusion
Different processing approaches affect physico-chemical prop-
erties of food proteins in different ways which in turn influence
their gastrointestinal digestion, bioavailability and allergenicity.
Inherent molecular characteristics of allergen, type of method used
in processing, intensity of treatment, environment condition (pH,
ionic strength etc.) and food matrix structure largely influence the
conformational changes related to digestibility and allergenicity of
food proteins. In thermal processing, moist heat usually reduces the
allergen reactivity of food proteins by changing protein structure,
alteration of IgE binding conformational epitopes and increased
digestibility. In contrast, dry heat such as baking of wheat flour and
roasting of nuts most often leads to formation of new epitopes
(neo-allergens) via Maillard reaction and reduction of digestibility
resulting in increased allergenicity. Reduction of allergenicity by
non-thermal techniques is attributed to conformational change of
protein with enhanced susceptibility to proteolysis. High pressure
followed by enzymatic hydrolysis appears to be one of the effective
approaches in minimizing allergenicity of many foods. Microbial
fermentation and selective enzymatic hydrolysis can break the
sequence of linear epitopes and extensively reduce the allergenic
potential of dairy proteins.
Although total elimination of allergenic potential by processing
is unlikely, minimization of elicitation threshold could be achieved
through selection of proper conditions. Moreover, development of
tolerance to allergic individuals by challenging hypoallergenic
processed foods may also be an alternative therapeutic strategy.
Although many studies have linked processing induced change of
IgE reactivity of food proteins to their allergenic potentials, this
change always does not translate to clinical symptoms. Thus, other
assessments such as release of allergic mediator (histamine and
cytokines), oral challenge and skin prick test also need to be per-
formed before concluding the effect of any processing method on
allergenic potential of any food protein. Increased understanding of
the impact of various processings on structure, digestibility and
allergenic consequence of food allergens could be applied at in-
dustrial level to develop novel processing strategies aimed at
reducing the prevalence of food allergies.
Acknowledgements
The authors are grateful to Australian Government Award
“Endeavour Postgraduate Scholarships”for granting the fund to the
PhD scholar.
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