Removal of Allergens in Some
Food Products Using
, Zhenxing Li
, Ishfaq Ahmed
, Hong Lin
University of Maine, Orono, ME, United States;
Ocean University of China, Qingdao, P.R. China
Food allergy refers to an abnormal or exaggerated immune response triggered
by eating speciﬁc foods or food additives. It was deﬁned by an expert panel of
the National Institute of Allergy and Infectious Diseases as “an adverse health
effect [that] occurs reproducibly on exposure to proteins, or antigens, which
are components of food matrices, thereby causing a speciﬁc immune
response.” Allergic reactions to food may be caused by cells in the immune
system or antibodies in the blood (Boyce et al., 2010). Food allergy is among
the most common disorders and has tended to increase in prevalence in the
past decades thus becoming a signiﬁcant public health concern. Novel proteins
and innovations in food formulations could lead to new cases of allergies. It is
very important to understand the basic terms used, the mechanism of food
allergy in humans, and the detection of allergens before applying any inter-
vention technology for the reduction of food allergens.
About 170 foods or food components have been documented as being
potentially allergenic. According to the Food Allergen Labeling and Consumer
Protection Act of 2004, only eight major foods or food groups, namely eggs,
milk, ﬁsh, shellﬁsh, tree nuts, peanuts, soybeans, and wheat, account for
approximately 90% of all the allergic reactions. Milk and eggs are ubiqui-
tously prominent worldwide, whereas the adverse reactions caused by other
common allergenic foods vary based on geographic region. Radauer et al.
(2008) and Sathe et al. (2016) attempted to classify selected major food
allergens based on their sequence analogy and domain architecture. Most of
the allergens are water-soluble glycoproteins with molecular masses of
10e70 kDa that are illustrated by three main attributes: (1) ability to sensitize
a genetically inﬂuenced individual by activating the creation of IgE antibodies,
Ultrasound: Advances in Food Processing and Preservation. http://dx.doi.org/10.1016/B978-0-12-804581-7.00011-7
Copyright ©2017 Elsevier Inc. All rights reserved. 267
(2) ability to bind those speciﬁc IgE antibodies, and (3) ability to cause an
adverse immunological reaction following IgE binding.
These adverse reactions can be toxic and nontoxic (Fig. 11.1). Among the
nontoxic reactions, those that are not immune-mediated are termed food
intolerance. Nonimmune adverse reactions mostly occur in people having
enzyme defects (i.e., vasoactive amines or lactose intolerance caused by a
deﬁciency of lactase, the enzyme responsible for digesting milk lactose). Such
adverse reactions are more prevalent than immune-mediated reactions. Phar-
macological reactions, another type of food intolerance, are caused by
chemical components of foods, such as tyramine in aged cheese or theobro-
mine in chocolate. Certain food additives such as potassium and sodium sul-
ﬁtes, metabisulﬁtes, gaseous sulfur dioxides, and monosodium glutamate are
also examples of molecules that could cause adverse pharmacological
reactions in susceptible people. On the other hand, the immunological
response encompasses all forms of immune-mediated reactions, including
those caused by the innate and the adaptive immune system (Fig. 11.1).
Nonetheless, immune-mediated reactions are responsible for signiﬁcant
morbidity and health care costs and can even lead to severe life-threatening
reactions (Sicherer and Sampson, 2014; De Silva et al., 2014; Longo et al.,
2013). The symptoms of food allergies include gastrointestinal, respiratory,
cardiovascular, and cutaneous symptoms and in the worst case can lead to
anaphylactic shock. Symptoms that become visible soon, within 1 h or less of
Adverse reactions to food
Innate Immune Responses:
Immune cells, Toll-like
Immune mediated Non-immune mediated
(e.g., lactose intolerance)
(e.g., vasoactive amines,
capsaicin, ethanol, methyl
FIGURE 11.1 Classiﬁcation of immune-response adverse reactions to food.
268 Ultrasound: Advances in Food Processing and Preservation
exposure, are termed immediate-type reactions; those occurring between 1 and
6 h are termed accelerated reactions, and a delayed reaction may occur even
some days after ingestion of the food.
11.1.1 Types of Food Allergy
Food allergy is classiﬁed into four basic types, type I, type II, type III, and type
IV (Coombs and Gell, 1975). The most common and prevalent type of
immune-mediated adverse reaction to food protein is type 1 reactions, which
are characterized by the creation of immunoglobulin E (IgE) antibodies
following sensitization of basophils or mast cells, which is the main process
involved in triggering allergic immune response. In general, these key events
are called sensitization and effecter phases (Fig. 11.2). Sensitization entails
antigen-presenting cells; T cells; Th2 cytokines such as interleukin (IL)-4,
IL-13, and IL-5; IgE production; and cross-linking of allergens with B-cell
FIGURE 11.2 Sensitization and effector phases involved in the development of IgE-mediated
food allergy. Ig, immunoglobulin; IL, interleukin; MHC, major histocompatibility complex.
Removal of Allergens in Some Food Products Using Ultrasound Chapter j11 269
receptors. Sensitization takes place when antigen-presenting cells (i.e.,
dendritic and B cells) ﬁnd allergenic segments or epitopes in the protein
fraction of food or food ingredients. After that, allergen antigens are trans-
ferred to MHC class II molecules and antigen interactions with T cells may
take place. This arrangement triggers allergen-speciﬁc T cells, which subse-
quently generate Th2 cytokines, and encourages the creation of IgE antibodies
by allergen-speciﬁc B cells. The IgE antibodies may attach to the IgE receptor
FcεRI on mast cell and basophil membranes, thereby creating sensitized cells
(He et al., 2013).
On the other hand, proteins that undergo complete digestion do not have
the potential to cause sensitization and may encourage tolerance (Vila et al.,
2001; Barone et al., 2000). However, indigestible proteins can lead to the
production of IgE and sensitization (Untersmayr and Jensen-Jarolim, 2006).
The movement of proteins all the way through the intestinal epithelium may
take place via either the transcellular route or the paracellular route (Reitsma
et al., 2014). The effector phase starts after the cross-linking of the same
allergen with two adjacent IgE molecules on sensitized basophils and mast
cells. Afterward, the triggered cells release proinﬂammatory mediators or
cytokines, which subsequently lead to allergic reactions (He et al., 2013).
IgE-associated food allergies affect 1%e3% of adults and 3%e8% of
children in developed countries. The IgE antibodies lead to instant allergic
reactions (within 2 h) after eating the food, and classic symptoms such as
swelling, itchy rash, and in some cases diarrhea and vomiting could occur. The
severity of the symptoms can vary among patients and in some situations could
cause a life-threatening health condition, such as difﬁculty in breathing and
collapse (anaphylaxis). The detection of food allergenespeciﬁc IgE in body
ﬂuids and serum, as well as the assessment of IgE-mediated cellular and
in vivo responses, is used to identify patients with IgE-associated food allergy.
Eggs, milk, nuts, peanuts, wheat, ﬁsh, sesame, fruits, and vegetables are the
main causes of IgE-associated food allergy. Patients acquire tolerance to a few
foods among these, i.e., eggs, milk, and wheat, whereas allergies to tree nuts,
peanuts, and ﬁsh mostly endure for a lifetime.
The immunoglobulins produced by the body in response to an antigen must
bind initially with the antigen to induce an immune response. The binding sites
of the allergens are called epitopes. The epitopes can be part of a continuous
amino acid chain, which are called linear epitopes, or be a portion of the three-
dimensional folding of a protein, called conformational epitopes. The
disruption of epitopes is often required to alter the reactivity of binding sites of
allergens. Linear epitopes can be altered by fragmentation or genetic modiﬁ-
cation of the amino acid sequence, whereas the conformational epitopes can be
obliterated by altering the structure of the allergen via denaturation, cross-
linking, aggregation, or chemical modiﬁcation. The aforementioned methods
are used to inhibit allergic reactions, as the IgE antibodies may no longer be
able to recognize the allergen.
270 Ultrasound: Advances in Food Processing and Preservation
Similarly, food antigenespeciﬁc IgG is considered responsible for initi-
ating adverse reactions via type II or type III hypersensitivity. Type II is a
cytotoxic reaction between a cell- or tissue-bound antigen and an IgG or IgM
antibody. The clinical cases in which this arises include immune cytopenias
such as immune thrombocytopenia and autoimmune hemolytic anemia. In
contrast, type III is an immune-complex reaction between IgG antibody and
circulating antigen, and takes place in the walls of blood vessels, kidneys,
joints, and skin, exhibiting as serum sickness or in a few cases extrinsic
allergic alveolitis, although no concrete experimental proof is available to
support food allergies relevant to these reactions in patients. Therefore, several
researchers robustly discourage the testing for food antigenespeciﬁc IgG for
diagnosing food allergy (Bock, 2010; Stapel et al., 2008).
On the other hand, type IV hypersensitivity entails food antigenespeciﬁc
T-cell responses that can affect the gut mucosa and subsequently result in
disorders such as celiac disease. Such reactions are mostly gastrointestinal
(constipation. diarrhea, vomiting) and/or skin problems (atopic eczema). The
T-cell immune response is likely to be a delayed reaction, in which symptoms
are ﬁrst visible in 4e28 h after ingesting the food. Celiac disease is charac-
terized by an allergic reaction against the components of wheat gluten,
comprising alkali- or acid-soluble glutenins and alcohol-soluble gliadins, in
conjunction with an autoimmune fraction. Type IV hypersensitivity reactions
could also occur because of food protein-induced enterocolitis causing
inﬂammation of the small and large intestine by directly activating the innate
immune system. Certain milk oligosaccharides and wheat amylase trypsin
inhibitors can induce inﬂammation of the intestines through activation of the
11.1.2 Prevalence of Food Allergy
The diagnosis of food allergy should be done by a food challenge test (the gold
standard), which is conducted as a double-blind placebo-controlled food
challenge test, except for cases in which severe food allergic reaction can be
recognized clearly. However, this practice is not followed nowadays, as it is
time-consuming and laborious. Alternatively, the food allergy prevalence
among the population can be estimated on the basis of interviews/question-
naires (self-reported food allergy) and can also be extended to the skin prick
test or allergen-speciﬁc IgE serology tests. The exact incidence of food
allergies has not been fully known, as discrepancies are found in studies in
which food allergies were self-reported versus those diagnosed by various
assays (e.g., skin test, provocation, or serologic tests).
A metaanalysis was carried out to evaluate the prevalence of food allergy in
Europe that included 30 articles published in the period from January 2000 to
September 30, 2012 (Nwaru et al., 2014). The prevalence of perceived food
allergy to any food was observed to be 5.0% in adults and 6.8% in children.
Removal of Allergens in Some Food Products Using Ultrasound Chapter j11 271
The overall prevalence of challenge-proven food allergy was 0.89% in adults
and 0.99% in children. With regard to sensitization, its prevalence was 2.7%
and 10.1% on the basis of skin prick or allergen-speciﬁc IgE tests, respectively.
The prevalence of food allergy among the US population is more than 2% but
less than 10% (Chafen et al., 2010). A 2013 research study conducted by the
National Health and Nutrition Examination Survey reported that the preva-
lence of self-reported food allergy in the United States was 9.7% in adults and
6.5% in children. In this study, the main allergens reported were milk, shell-
ﬁsh, and peanuts (McGowan and Keet, 2013). In Asia, the prevalence of
challenge-proven food and self-reported food allergies ranged from 1.1% to
3.8% and from 4.8% to 16.7%, respectively. In the Asian population, ﬁsh
seems to be the most reported allergen in comparison with other geographic
locations, which has been attributed to the large consumption of seafood in this
region. In Latin America, fruits and vegetables, such as beans, onions, oranges,
tomatoes, and lettuce, and meats and seafood were the most reported allergens
(Hu et al., 2010; Ho et al., 2012; Chen et al., 2011; Lao-araya and Trakulti-
vakorn, 2012; Wu et al., 2012; Lee et al., 2013).
The severity and prevalence of food allergies seem to be greater than ever.
The severity, frequency, and type of allergic symptoms in patients are inﬂu-
enced by various factors such as genetics; dietary exposures; environmental,
behavioral, and cultural factors; and study designs or methodologies. The
hygiene hypothesis states that the reduction in family size and enhancements
in personal hygiene have led to the increased prevalence of IgE-mediated
allergies in people. Conversely, a diet of organic foods containing lactoba-
cilli and restrictive use of vaccines, antipyretics, and antibiotics (anthro-
posophical lifestyle) have a contributing role in minimizing the incidence of
allergies. The exclusion of the culprit food is the best remedy for both food
intolerance and allergy, although absolute exclusion of the food from the diet
is hard in modern days as allergenic foods are used as ingredients in a variety
of food products.
11.1.3 Detection of Food Allergens
Food allergen detection has been attaining signiﬁcant attention from 2000 to
till date from both regulatory agencies and food industries. Food allergen
diagnosis is based on history, dietary analysis, challenge tests, skin tests, and
measuring speciﬁc IgE in the blood serum. The various methods employed for
assessing food allergenicity to qualify or quantify allergens are imperative in
establishing the efﬁciency of various processing techniques for altering the
reactivity of food allergens. Most food allergies are IgE based, and allergen
reactivity is frequently depicted by its potential to bind IgE antibodies
(Hengel Arjon, 2007).
The most common method used for detecting allergenicity of an antigen
includes in vitro, in vivo, and ex vivo tests. These tests are used to detect the
272 Ultrasound: Advances in Food Processing and Preservation
presence of food-speciﬁc IgE (sensitization). In vitro tests have many bene-
ﬁcial aspects as they are cheap, rapid, and harmless when human subjects are
employed. On the other hand, in vivo assays provide more accurate results in
comparison with in vitro assays. But, it can be costly and time-consuming to
utilize human or animal subjects and the assays may pose threats to human
subjects (Besler, 2001).
11.2 EFFECTS OF ULTRASOUND IN FOOD ALLERGEN
Ultrasound technology is an emerging technology that is used extensively for
food processing and preservation. It is used successfully to deactivate
enzymes, aid extraction, homogenize emulsions, and accelerate dehydration,
ripening, and aging processes (Villamiel and Jong, 2000; Dolatowski et al.,
2007). However, the application of ultrasound in the reduction of food aller-
genicity is yet in its infancy. High-intensity ultrasound is operated by me-
chanical waves within a frequency range of 20e100 kHz (Feng et al., 2011).
The high energy causes physical and chemical modiﬁcations by promoting
formation of sonication bubbles in foods and leads to intermittent compression
and rarefaction until collapse at critical bubble sizes. The increased temper-
ature and pressure (up to 5000 K and 1000 atm, respectively) in the vicinity of
the collapsed cavities is the basis for altering the conformation of allergens and
their reactivity. Moreover, regions of high-velocity gradients and high shear
stress can create microstreams that stimulate chemical and mechanical effects,
resulting in the alteration of the native protein structure into a molten globule
state, formation of new intra-/intermolecular interactions, and even degrada-
tion (Soria and Villamiel, 2010; Lee et al., 2009).
The structures of allergenic food proteins are modiﬁed differently by
unfolding, aggregation, cross-linking, and sometimes oxidation and glyco-
sylation by different processing methods. Conformational changes through
disruption of the linear or conformational epitopes in the proteins by pro-
cessing methods can directly inﬂuence the allergenicity of food products. The
linear epitopes are affected by acidic or enzymatic hydrolysis, whereas the
conformational epitopes can be exposed or hidden by unfolding or aggregation
of allergenic proteins (Rahaman et al., 2015). Allergenicity and antigenic
integrity are sometimes used by many investigators without proper clariﬁca-
tion. The integrity of epitopes is recognized by IgG or IgE antibodies, whereas
allergenicity is the ability of food proteins to induce allergenic sensitization
(Verhoeckx et al., 2015). Thermal and nonthermal processing will modify the
allergenic proteins and inﬂuence the ability of antibodies to bind the modiﬁed
proteins; especially the IgE antibodies ﬁnd it difﬁcult to elicit an allergic
reaction or stimulate the production of an IgE-mediated food allergy. Modi-
ﬁcation of proteins by ultrasound is primarily attributed to the cavitation
phenomenon. The cavitation effects generated by bubble collapse may be
Removal of Allergens in Some Food Products Using Ultrasound Chapter j11 273
responsible for modifying proteins structurally and altering their functionality
(Mawson et al., 2011). Brieﬂy, acoustic waves produced by ultrasound travel
through a medium resulting in a series of compression and rarefaction events.
Attractive forces between molecules in a liquid phase exceed during rarefac-
tion, at high power levels, and form bubbles owing to cavitation. Interference
of each bubble with its neighbor causes a collapse, and the release of energy
increases temperature and pressure in the medium. The cavitation collapse in
aqueous media also generates shear forces that can produce mechanical and
chemical effects. With low intensities (or high frequencies), acoustic stream-
ing is the main mechanism (Leighton, 2007). Acoustic streaming is the motion
and mixing within the ﬂuid without formation of bubbles (Alzamora et al.,
2011). Higher intensities (low frequencies) induce acoustic cavitation (Povey
and Mason, 1998) due to the generation, growth, and collapse of large bubbles,
which cause the liberation of higher energies. In addition, the extreme agita-
tion created by microstreaming could disrupt Van der Waals interactions and
hydrogen bonds in polypeptides, causing protein denaturation (Tian et al.,
2004). Limited studies have been reported on the inﬂuence of ultrasound on
Amponsah and Nayak (2016) investigated the effects of ultrasound-assisted
extraction (UAE) on the recovery and detection of allergenic proteins from
various soy matrices (e.g., soy ﬂour, soy protein isolate, and soy milk). The
investigators also used several buffers [phosphate-buffered saline (PBS),
Laemmli buffer, and urea] to determine the recovery of allergenic soy proteins
after the UAE. The recovery of total proteins from soy ﬂour and soy protein
isolate was higher at 23C than at 4C using UAE, and urea provided
maximum recovery of proteins compared to Laemmli and PBS at both tem-
peratures (Amponsah and Nayak, 2016). However, recovery of total proteins
from soy milk was higher using UAE with Laemmli buffer. The same in-
vestigators reported that protein solubility was increased by the application of
ultrasonic energy and caused protein to undergo physical disruption and
chemical modiﬁcation (Fig. 11.3). Tan et al. (2011) also reported that recov-
ery/yield and structural/functional properties of the protein were inﬂuenced by
extraction process parameters such as pH, salt concentration, and ionic
strength of the medium of extraction. Additionally, Jambrak et al. (2009)
reported an increase in the solubility of soy proteins after ultrasound treatment
(20, 40, and 500 kHz) for 15 min in soy protein isolates (SPI) and soy protein
concentrate (SPC). Karki et al. (2010) also investigated the use of high-power
ultrasound prior to soy protein extraction to simultaneously enhance protein
and sugar release in the extract and concluded that sonication at high ampli-
tude for 120 s gave the highest increase in protein yield of 46% compared with
nonsonicated samples (control). In another study, Albillos et al. (2011)
274 Ultrasound: Advances in Food Processing and Preservation
sonicated protein extracts from roasted almonds in a temperature-controlled
water bath and reported that ultrasonic treatment improved protein extrac-
tion from almonds roasted at 260C and 400C. Choudhary et al. (2013)
reported a reduction of 24% in soy protein allergenicity by the application of
high-intensity ultrasound treatment by a 37-kHz ultrasonic processor for
10 min. Ultrasound treatment at 37 kHz resulted in a reduction of allergenicity
of soy proteins by disrupting the secondary structure of the proteins
(Choudhary et al., 2013).
FIGURE 11.3 Protein proﬁles of extracts obtained from conventional extraction (lanes A/A0eC/C0),
MAE at 60Ce70C(lanes D/D0eF/F0), and UAE at 23C(lanes G/G0eI/I0) with PBS, Laemmli,
and urea buffers, respectively. Lanes AeI, reduced conditions; A0eI0, nonreduced conditions;
J, molecular weight marker. (A) Soy ﬂour; (B) soy protein isolate (SPI), and (C) soy milk. Bands
around 75 and 50 kDa correspond to b-conglycinin and bands around 37 kDa correspond to
glycinin (33 kDa) and the 34-kDa P34 allergenic soy protein. Adapted from Amponsah, A., Nayak,
B., 2016. Effects of microwave and ultrasound assisted extraction on the recovery of soy proteins for
soy allergen detection. Journal of Food Science 81 (11), T2876eT2885.
Removal of Allergens in Some Food Products Using Ultrasound Chapter j11 275
The application of ultrasound (at 20 kHz and 125 mm amplitude for 60 min)
had a great positive impact on dairy processing as it maintained the nutritional
value of milk and did not increase the allergenic potential of b-lactoglobulin
(Stanic-Vucinic et al., 2012). The investigators observed an increase in the
polymerization of b-lactoglobulin with treatment time. Sixty minutes of
treatment time provided a signiﬁcant number of high-mass polymers due to
bonds formed because of disulﬁde exchange, resulting in the formation of new
intermolecular bonds. The use of ultrasound also formed localized high tem-
perature that induced aggregation of up to 50% of the protein at 60 min.
However, ultrasound application under controlled temperature conditions
resulted in only dimer formation, unlike uncontrolled conditions. Most
importantly, circular dichroism measurements conﬁrmed that ultrasound
generated a structure with a higher percentage of a-helical forms and random
coils, whereas untreated samples had exhibited predominantly b-strand
behavior. The calculation of secondary structure fractions conﬁrmed that
different times of ultrasound exposure resulted in different compositions of
secondary structure after refolding. However, the conformational changes due
to ultrasound affected IgE binding of b-lactoglobulin very slightly as observed
by immunoblot and immunoprint. In a separate study, Chandrapala et al.
(2011) observed a 5%e9% decrease in b-strands, but an increase of 10% in the
a-helical form of whey protein concentrate when treated with ultrasound for
Modiﬁcation of proteins structurally and their allergenicity also depend on
the intensity of tandem use of ultrasonic treatment (e.g., ultrasonic treatment
combined with heat). It was reported that denaturation of a-lactalbumin and
b-lactoglobulin in milk was higher when treated with high-intensity ultrasound
in combination with heat (Villamiel and Jong, 2000). This type of synergism
between heat and ultrasound was attributed the reduction in viscosity of the
heated milk, resulting in a better penetration of the ultrasound into the liquid.
However, the high-intensity ultrasound application at 20 kHz and 500 W
power was not efﬁcient at minimizing the allergenicity of milk proteins
(Choudhary et al., 2013).
IgE-binding afﬁnity of Ara h1 and Ara h2, two major peanut allergens, was
moderately reduced (w10%) by ultrasound treatments (at 50 Hz for 5 h)
compared to ultrasonication and protease digestion (trypsin/a-chymotrypsin),
which signiﬁcantly decreased IgE reactivity and increased the solubility of
proteins (Li et al., 2013). The investigators reported that ultrasonic treatment
loosened the structure of peanut protein, cleaved peptide bonds by shear force,
and also enhanced the efﬁciency of enzymatic hydrolysis treatment.
276 Ultrasound: Advances in Food Processing and Preservation
11.2.4 Shrimp and Crustaceans
Shellﬁsh are responsible for inducing allergic reactions in most parts of the
world. Generally, shellﬁsh allergy is severe, lifelong, and potentially fatal
¨thrich and Ballmer-Weber, 2001). Shellﬁsh proteins are more heat stable,
which can lead to shellﬁsh hypersensitivity. The main heat-stable shrimp
allergen is a 34- to 38-kDa protein called tropomyosin, also named Pen a 1 or
Sa-II, and is the main cause of food-related complications, i.e., hypersensi-
tivity (Reese et al., 1999;Nagpal et al., 1989). Tropomyosin has an important
role in muscle contraction, regulation of cellular structure, and motility.
Myosin light chain (20-kDa) and arginine kinase (40-kDa) are the other two
allergens that have been recognized, yet most shrimp allergenicity is attributed
to tropomyosin (Shiomi et al., 2008; Ayuso et al., 2008). Shrimp allergy,
in general, is a type I hypersensitivity. The ﬁrst encounter with a shrimp
allergen will sensitize the individual to future exposures (Leungi and Chu,
1998). The most common symptoms of shrimp allergies include hives, itching,
swelling of the tongue and lips, gastrointestinal symptoms, pulmonary
symptoms, and anaphylactic shock (Jeong et al., 2006). The best way to
prevent shrimp-induced allergies is complete avoidance. However, complete
avoidance is usually difﬁcult because of the addition of food allergens as
ingredients in common foods or unintentional cross-contamination. Therefore,
it is important to develop methods to inactivate or remove food allergens from
In general, processing is considered a potentially efﬁcient way to decrease
the allergenicity of shrimp. Relevant literature on the inﬂuence of ultrasound
on shrimp and crustacean allergens is very scanty. Li et al. (2006) performed a
study on the effects of high-intensity ultrasound on isolated shrimp protein
and shrimp extract. High-intensity ultrasound treatment led to a substantial
reduction in the allergenicity by reducing IgE binding to both crude
shrimp protein extracts and isolated tropomyosin. These ﬁndings were also
corroborated by immunoblot and ELISA analysis. The isolated proteins and
shrimp extract were treated with a frequency of 30 Hz for 130e180 min.
The IgE binding capacity of isolated shrimp protein was decreased by
81.3%e88.5% after ELISA, whereas only a 68.9% reduction in the IgE
binding potential was found in shrimp extracts. Furthermore, during allergen
isolate treatment, new protein fractions with low molecular weights were
formed as time elapsed. Therefore, disintegration of shrimp protein may
take place during high-intensity ultrasound treatment (Li et al., 2006). Yang
et al. (2006) investigated whether the combined application of papain and
ultrasound aids in tenderization of shrimp by minimizing the shear force.
Li et al. (2011) examined the inﬂuence of power ultrasound on the allerge-
nicity and textural attributes of raw and boiled shrimp. Ultrasound treatment
signiﬁcantly reduced the allergenicity of the boiled shrimp compared to raw
Removal of Allergens in Some Food Products Using Ultrasound Chapter j11 277
Crustaceans are also considered a main cause of allergic reactions
because of their high consumption, especially in coastal areas. Crustaceans
are favored by many people because of their nutritive value and delicacy
(Wild and Lehrer, 2005). The major allergen found in crabs is tropomyosin,
which is a myoﬁbrillar protein having two identical subunits of 35e38 kDa
molecular mass and an isoelectric point of 4.5. Tropomyosin has high
stability compared to other proteins and can tolerate grinding, heat, and other
processing methods (Motoyama et al., 2006, 2007). Lee and Park (2004)
reported that common whelk allergens were digested by SGF, but remained
unchanged after thermal processing. This indicates that the key crustacean
allergen is heat stable. Yu et al. (2011) evaluated three different processing
methods (boiling, high-pressure steaming, and combined ultrasound and
boiling) for the degradation of tropomyosin and reduction of its IgE-binding
capacity in crabs, so that it could be easily decomposed during gastrointes-
tinal digestion. SDSePAGE showed very little impact on the digestive
stability of tropomyosin extracted from processed crab in the case of boiling
treatment. On the other hand, high-pressure steaming and combined
ultrasound and boiling enhanced the digestion of tropomyosin. Similarly,
inhibition ELISA and Western blotting also indicated that the reactivity of
IgE/IgG binding of tropomyosin was partially reduced after treatment with
high-pressure steaming and combined ultrasound and boiling. These ﬁndings
suggest that ultrasound treatment could be used to reduce the reactivity of
IgG/IgE binding of tropomyosin, promote tropomyosin degradation in
simulated gastrointestinal digestion, and reduce the occurrence of allergic
The application of ultrasound is a new interventional method targeted toward
reducing the allergenicity of certain food products. Unlike other thermal
treatments such as baking, boiling, roasting, and pasteurization and
nonthermal treatments such as high-pressure processing, very few allergenic
food products have been processed using ultrasound. Therefore, limited
information is available on the application of ultrasound for processing food
products to reduce food allergenicity. As a common practice, ultrasound is
used as a pretreatment method in most cases, prior to food processing oper-
ations. It is also used to extract phytochemicals or other compounds of interest
from food materials. A few investigators have used ultrasound to modify
allergenic proteins structurally and then detect them using a number of in vitro
and in vivo methods. Most of the investigators observed that ultrasound helped
in breaking up the structural integrity of the food products as well as protein
structures depending on the intensity of application. Recovery of total protein
as in the case of soy products may increase after ultrasound treatment, but the
allergenicity is not necessarily reduced in the processed soy products.
278 Ultrasound: Advances in Food Processing and Preservation
However, in some other cases, ultrasound reduced the allergenicity of food
products, such as roasted peanut extracts. It is recommended that more
allergenic foods in solid as well as liquid form should be tested for modiﬁ-
cation of proteins and assessed for any reduction in allergenicity to help the
industry and human beings.
BOX 11.1 In Vitro Tests for Food Allergen Detection
The discovery of IgE allowed various immune assays to enable direct and objective
measurements of the speciﬁcity and extent of the immune response. The
in vitro assays utilize enzyme-linked immunosorbent assay (ELISA), sodium dodecyl
sulfateepolyacrylamide gel electrophoresis (SDSePAGE), radio-allergosorbent tests
(RAST), enzyme-allergosorbent tests (EAST), ImmunoCAP assays (Phadia, Uppsala,
Sweden), and immunoblotting to determine the presence and reactivity of allergens.
RAST and EAST are similar and the enzyme activity evaluated correlates to the
antibodies afﬁxed to enzymes (Sampson, 1999). Among all the in vitro techniques,
immunoassays are used most frequently. Particularly, SDSePAGE, ELISA, and immu-
noblots are corroborated by federal agencies to detect major food allergens such as
milk, peanut, hazelnut, egg white, soybean, and wheat proteins (Merget et al., 1993).
Histamine Release Tests
In the histamine release assay basophils sensitized with an allergen are challenged,
which can result in cross-linking of surface-bound speciﬁc IgE antibodies, thereby
leading to release of histamine from the cells. Histamine can be determined immu-
nochemically by the Immunotech radioimmunoassay, ﬂuorimetrically by coupling to
a ﬂuorophore (o-phthaldialdehyde), or by using an automated ﬂuorimetrichistamine
assay. The histamine release method uses glass-ﬁber-coated plates for histamine
separation from other components of the assay. The histamine concentration is
measured and a doseeresponse curve can be prepared and analyzed by comparing
with an appropriate standard. The liberated histamine content can also be articulated
as a percentage of the total level of histamines of unchallenged cells. On the other
hand, a passive sensitization technique can be applied instead of using blood from
sensitized patients. The basophils of a nonsensitized individual are used and the
receptor-bound IgE that is already present is taken from the donor basophils’ surface,
and subsequently the cells are passively sensitized with human serum havingrelevant
and speciﬁc IgE antibodies (Skov et al., 1997;Poulsen, 2001).
Basophils make up less than 0.5% of the leukocytes in the blood and are
extremely difﬁcult to purify (Gibbs, 2008). Moreover, fresh blood cells from
nonallergic donors or allergic donors are required in case of passive sensitization
for each experiment, and after collection, the blood samples must be processed
immediately, which may present logistical hindrances. Another disadvantage is
that about 10% of basophil donors are nonresponsive, which leads to less reliable
results in terms of detecting individual responsiveness (Palmer et al., 2005). A
stable cell line that could be passively sensitized with serum IgE from allergic
patients could be used to overcome these problems.
Simulated Gastric Fluid (SGF) Assay
The resistance offered by some proteins, particularly transgenic proteins, to pro-
teolytic digestion in SGF can induce allergic reactions. It is assumed that
Removal of Allergens in Some Food Products Using Ultrasound Chapter j11 279
Box 11.1 In Vitro Tests for Food Allergen Detectiondcont’d
nutritionally desirable proteins, when consumed, should be digested rapidly, so
that they can exert fewer adverse health effects. Under acidic conditions (pH 1.2 to
2.0), the stability offered by transgenic proteins to pepsin digestion is usually
presumed to be an effective test to assess the allergenic risk associated with
transgenic proteins (Astwood et al., 1996). However, the relationship between the
digestive stability of a protein in SGF and allergenicity is not always absolute, as
there are reports indicating no correlation between allergenicity and digestive
stability (Fu et al., 2002).
SGF is designed in such a way as to mimic the mammalian gastrointestinal
system. Several factors, such as mechanical breakdown of food tissues, buffering
effect of food ingredients, range of stomach pH, gastric lipase in physiological
amounts, addition of surfactants (phospholipids), peristalsis, etc., can play a role.
Many researchers have reported their ﬁndings in support of the utilization of the
SGF assay. The pepsin activity recommended by many researchers ranges from
5000 to 20,000 units/mg of the test protein. The ratio of pepsin to protein (3:1),
pepsin concentration (3.2 mg/mL), pH (1.2 and 2.0), and different times of
incubation at 37C provide similar results (Flow Chart 11.1). Sometimes, the
nature of the substrate affects the pepsin activity, with a fairly broad range of pH
between 1.2 and 3.5. However, a small shift in these factors can hamper the
pepsin digestion of several allergens.
Although the SGF assay does not exactly mimic in vivo digestion, it still presents
results similar to those of in vivo digestion by the mammalian system (Verma and
Singh, 2013). The lack of consideration of the allergen prevalence in food, food
matrix interactions, or food processing effects is the main reason behind the vague
predictive potential of the SGF assay. The interaction of food matrix might play a
major role, as food components may sequester some of the proteins away from the
pepsin and acid in the gastric ﬂuid. The puriﬁed kiwi allergen Act c 2 was digested
instantly in SGF, but digestion was stopped by fruit pectin both in vivo and in vitro.
Similarly, the association of protein with starch granules in transgenic corn
protected it against digestion in SGF. Therefore, the evaluation of puriﬁed protein
allergenicity in the SGF assay may not always present the desired results.
Simulated Intestinal Fluid (SIF) Assay
It is believed that allergenic proteins are not generally digested completely in the
proteolytic system of the human gastrointestinal system and can be absorbed
through the mucosa of the intestine. Thus, a novel protein is subjected to digestion
in SIF and SGF. Pancreatin is used to prepare SIF for the development of the SIF
assay. For the preparation of the assay, 1 g/100 mL pancreatin is dissolved in
0.05 M KH
at a pH of 7.5. The SIF aliquot is placed in a microcentrifuge tube
and incubated for 10 min at 37C in a water bath. The test protein at a level of
5 mg/mL should be put into the microcentrifuge tubes to initiate the reaction.
Laemmli buffer should be added to each tube at different time intervals (i.e., 0, 0.5,
5, 15, 60, and 120 min). SDSePAGE analysis in conjunction with densitometry
is carried out to compare the degradation at the different time intervals (Flow
The assay conditions can inﬂuence the relative protein digestibility in SIF. The
relative amounts of test protein and enzyme employed in an SIF assay inﬂuence
280 Ultrasound: Advances in Food Processing and Preservation
Box 11.1 In Vitro Tests for Food Allergen Detectiondcont’d
the outcome for a particular protein. Changes in pH and the ratio of enzyme to
protein affects protein digestibility. Various guidelines have been considered for
SIF or SGF digestibility as a predictive tool for estimating the allergenic capability
of proteins. However, it is necessary to develop standardized assay conditions that
can be accepted globally. Some of the major allergens, like cow’s milk, lacto-
peroxidase, conalbumin, soybean, and peanut, are stable in SIF for 120 min.
Similarly, papain and bromelain exhibit higher stability in SIF. Ovomucoid and
lysozyme, well-known allergens in egg, showed higher stability (up to 60 and
120 min, respectively) in SIF (Fu et al., 2002). Hence, the in vitro digestion assay
can be employed to establish the allergenicity of a novel protein. Occasionally,
small portions of degraded protein in the gastrointestinal tract are enough to
stimulate allergenic reactions.
RAST and EAST
The radio-allergosorbent tests employs antibodies that are bound to radioisotopes
to measure serum IgE. The allergen is adsorbed to a solid phase and a serum
sample is used to incubate it. The IgE antibodies in the serum are speciﬁc to the
selected allergen and are cross-linked with the immobilized allergen. A secondary
antibody, which is conjugated to a radioisotope, reacts with the IgE antibodies, and
subsequently radioactivity is assessed. Furthermore, the ﬁndings are quantiﬁed via
a standard curve (Falagiani et al., 1994).
The enzyme-allergosorbent tests employs the same principle as the RAST,
although the antibodies used in EAST detection are conjugated to enzymes (i.e.,
alkaline phosphatase) and as a result enzyme activity is evaluated (Merget et al.,
1993). RAST and EAST are advantageous, as multiple samples can be tested at the
same time and there is no need for the patient’s presence during the test (Poulsen,
2001). Yet, qualitative discrepancies during the solid-phase and sample prepara-
tion among analysts could impose quantiﬁcation issues. Moreover, IgG antibodies
may intrude during the analysis, as they vie with IgE antibodies for analogous
allergenic determinants (Falagiani et al., 1994).
The concept of the ImmunoCAP test is similar to that of EAST and RAST. However,
it shows higher sensitivity and improved results and is designed in such a way as to
minimize the hurdles seen in EAST and RAST (Johansson, 2004). The best part of
an ImmunoCAP test is the three-dimensional solid phase that reduces nonspeciﬁc
binding by means of non-IgE-binding antibodies. ImmunoCAP tests can be
conducted in less than 20 min and reagent preparation is designed in such a way
as to minimize conformational epitope losses (Hamilton and Williams, 2010;
Diaz-Vazquez et al., 2009).
Sodium dodecyl sulfateepolyacrylamide gel electrophoresis (SDSePAGE) is used
to evaluate the existence or absence of allergens or to measure changes in the
electrophoretic pattern of a protein (Shapiro et al., 1967). Fluctuations in the
molecular weight, like dimerization, can also be observed in SDSePAGE. Proteins
or allergens often get aggregated owing to treatment conditions and may enlarge in
Removal of Allergens in Some Food Products Using Ultrasound Chapter j11 281
Box 11.1 In Vitro Tests for Food Allergen Detectiondcont’d
size and so get stuck in the pores of the polyacrylamide gel or washed away. In
contrast, if treatment conditions lead to fragmentation of proteins, then the frag-
ments that are too small for the gel resolution will pass through the gel swiftly and
eventually result in buffer loss. In addition, the bands of proteins with stimulated
intramolecular cross-linking may have a smudged appearance in polyacrylamide
gels. The intramolecular cross-linking can hamper the protein from being wholly
linearized during the denaturing conditions. This analytical method is advanta-
geous, as it is cheap and results can be acquired in a short period of time.
However, SDSePAGE could not measure the IgE-binding reactivity of the allergens
(Taheri-Kafrani et al., 2009).
The analysis of allergens by immunoblotting includes Western blot and dot-blot
analysis. Western blot analysis involves the separation of proteins by molecular
weight via PAGE. Afterward, the proteins are transferred to a membrane of
nitrocellulose or polyvinylidene ﬂuoride (PVDF) and subjected to antibody
assessment assays (Towbin et al., 1979). Western blotting has many beneﬁts, as
protein bands can be analyzed individually for the determination of variations in a
speciﬁc allergen. This method of detection is relatively easy, fast, and inexpensive.
A shortcoming of Western blotting is that proteins are usually tested at their
primary or linear level and conformational epitopes might not be represented
(Aalberse, 2000). Additionally, the new binding epitopes, which were once
invisible within the protein, may be exposed. It is important to point out that as
molecular weight is used to separate the proteins, so those that are too small or too
large for the gel or blotting membrane resolution may not be precisely evaluated.
In the case of dot-blot analysis, the samples are directly adsorbed onto a
membrane such as nitrocellulose and evaluated by using antibody detection
(Singh and Knox, 1985). In this analysis, conformational epitopes are not affected,
because no denaturing conditions are involved, although membrane adsorption
may lead to unfolding of proteins. The immunogenicity of the whole sample is
analyzed in this method, as proteins are not separated by molecular weight.
However, single proteins may also be determined, in case the sample contains
separated proteins. Likewise, the blotting membrane resolution is the limiting
factor for extremely low or high molecular weight proteins.
ELISA includes indirect ELISA and competitive inhibition ELISA (Ci-ELISA), which
is used to determine the IgE-binding capacity of the allergens. The method is used
to detect either single protein or entire sample reactivity, based on the antibodies
used. In this method proteins are adsorbed to the surface of the ELISA plate wells
and detected via suitable antibodies (Wachholz et al., 2005; Kemeny and Chantler,
1988). Sometimes, hydrophobic interactions could lead to the adherence of pro-
tein to the plate and can hinder or mask the conformational epitopes. Moreover,
strong hydrophobic interactions between protein and the polystyrene material may
cause denaturation and unfolding of the protein. Therefore, it is essential to choose
appropriate materials for performing ELISA (Butler et al., 1997).
282 Ultrasound: Advances in Food Processing and Preservation
BOX 11.2 In Vivo Tests for Detection of Food Allergens
In vivo assays include the skin prick test (SPT) and oral food challenge (OFC) to
detect food allergens.
Skin Prick Test
Skin Prick tests can be used to determine speciﬁc IgE sensitization. The skin is
marked for testing with a panel of appropriate allergens for the patient, selected on
the basis of the clinical history and knowledge of the allergens commonly found in
the locality. Positive and negative comparator tests using histamine and saline also
should be performed to prove that the skin is capable of demonstrating a positive
reaction and to prevent the interpretation of false-positive results occurring as a
result of dermatographism. It is necessary for the patient not to take any medi-
cations such as antihistamines prior to testing. The SPT is performed by injecting a
minute amount of allergen under the skin using a device, i.e., a lancet. This allows
the test protein to act together with IgE antibodies on the surface of skin mast cells.
Mast cells degranulate in the presence of food-speciﬁc IgE antibodies and release
mediators that result in localized wheal and ﬂare. After 10e15 min the results are
interpreted by reference to the control tests. Saline and histamine are employed as
negative and positive controls, respectively. The development of a small red circle
or wheal greater than 3 mm on the skin indicates the sensitiveness of the patient
toward the allergen. However, wheal size can vary depending on allergen and
subject (Sampson, 1999; Merget et al., 1993). SPT cannot quantify the allergens
and is employed only to ﬁnd the presence or absence of an allergy. This method is
not a perfect technique to determine allergy, however, as it can cause false-positive
wheals in patients with atopic dermatitis (Byrne et al., 2010). The subject must not
be given any drugs like antihistamines, which can inﬂuence the ﬁndings of the test.
Moreover, SPT is based on the quality of the allergen extract employed for testing,
so subjective results could vary among evaluators (Poulsen, 2001). The advantages
of SPT include good sensitivity, quick results, and the potential to test any antigen.
The disadvantages include possible danger of anaphylaxis, discomfort inherent in
the procedure, and the contraindicating inﬂuence of medications such as
decongestants, antihistamines, bronchodilators, beta blockers, and theophylline
(Sampson and Metcalfe, 1992).
Oral Food Challenge
Oral food challenge (OFC) test is considered to be the “gold standard” and pro-
vides more accurate results with regard to food allergy. In this study, it is necessary
for the subject to consume test foods. However, this practice could be hazardous
for the patient, because the patient is directly subjected to allergens. It can induce
severe reactions in the case of acute IgE-mediated reactions, enterocolitis syn-
drome, and severe atopic dermatitis. Especially those who are vulnerable to
anaphylaxis must not participate in this type of study (Byrne et al., 2010; Merget
et al., 1993). The safety of OFC studies was tested by Perry et al. (2004), who found
severe allergic reaction in 28% of the participants. The participants consuming
10 g of lyophilized protein without any visible symptoms are considered not
allergic, yet need to be validated further (Sampson, 1999). Such tests are often not
Removal of Allergens in Some Food Products Using Ultrasound Chapter j11 283
Box 11.2 In Vivo Tests for Detection of Food Allergensdcont’d
performed because of their high cost, complexity, and time requirements. None-
theless, animal models could be used to avoid such problems, but still these
models are not always comparable to the human body.
Animal models are used to develop a standardized allergen exposure protocol in
an animal rather than humans, as ethical concerns are related around performing
food allergen challenge studies in humans. Owing to the challenges in performing
experiments with humans in controlled studies, animal models are studied to
predict whether a novel protein has the potential to induce the production of IgE in
the animal. Additionally, their relevance to the human condition must also be
considered. Different allergen exposure models have been attempted in several
species, with each species having advantages over others. Rodents present many
advantages, as they are easily available, easy to handle, and genetically stable. The
rodent models can be evaluated for their response to a range of exposure sites.
Several strains can be evaluated for the relevance to the sensitivity of humans to a
given allergen (Madsen and Pilegaard, 2003). Additionally, rodents are helpful in
understanding allergen mechanisms owing to the availability of an enormous array
of reagents for the researchers. Mostly IgE binding is assessed to specify sensiti-
zation in animals; however, biomarkers of sensitization can also include cell
receptors and cytokines for rodent models. Similarly, other species, such as swine
and dog models, proffer closer approximations of clinical symptoms of humans.
The swine model is particularly useful for illustrating peanut allergen sensitivity
with greater correlations to human peanut allergy (Helm et al., 2002).
An authenticated allergen model that kindles the process of sensitization in
humans is not easy to perform because of the lack of well-deﬁned allergic re-
sponses in animals that would remain consistent among allergens and could be
considered parallel with the human allergic response. From a mechanistic
perspective on optimizing novel allergy vaccines, the models need to reﬂect
especially the aspects related to human disease but not the natural process of
sensitization, while a very high correlation to the sensitization process occurring in
humans is needed for a predictive model. Furthermore, allergen preparation,
adjuvant selection, and the duration between sensitization and allergen challenge
are not easy to determine for all but only a small number of well-characterized
proteins. Moreover, the consistency across the study sites of an animal model’s
response to a well-studied allergen (i.e., ovalbumin) remains obscure. Studies on
animal models have led to improvement in animal models, such as including
appropriate positive and negative study controls, allergen preparation standardi-
zation, animal selection, and the proper genetic strain (Helm et al., 2003; Knippels
et al., 2004). The commonly used animal models permit the exploration of
mechanisms at the molecular and cellular levels to study therapeutic strategies
(Adel-Patient et al., 2005). Several model allergens over a wide range of sensi-
tivities can be accommodated by an animal model, yet in animals, the develop-
ment of a standardized protocol for food allergens still remains a challenge for
their use as a predictive tool.
284 Ultrasound: Advances in Food Processing and Preservation
Box 11.2 In Vivo Tests for Detection of Food Allergensdcont’d
Mediator Release Assay
The mediator release assay imitates the type I allergic reaction mechanism
utilizing rat basophilic leukemia (RBL) cells, which express the human FcεR1
receptor in a recombinant manner (Vogel et al., 2005; Kaul et al., 2007). These
cells help in the binding of human IgE antibodies and possess all the functional
properties of basophils and mast cells. b-Hexosaminidase, which is found in the
granules and released along with histamine, has been selected as a surrogate
marker for histamine release. This enzyme hydrolyzes the added substrate,
thereby leading to coloration that can be analyzed via a spectrophotometer.
The mediator release assay allows the measurement of allergens in different
samples and the biological activity or potency of the allergens and the investi-
gation of cross-reactivities among the allergens. The potency of allergens can be
indicated by determining the requirement of doses for initiating efﬁcient cell
degranulation and values corresponding to the allergen dose that induces up to
50% of the maximum release (EC
values). Generally, sera having a higher
percentage of allergen-speciﬁc versus total IgE antibodies can be used in this test.
In the case of sera obtained from peanut-allergic patients, the most effective sera
must have at least 15 kU/L of peanut-speciﬁc IgE and 50 kU/L of total IgE, and
have more than 10% peanut-speciﬁc IgE (Dibbern et al., 2003). In addition, the
concentration of FcεRI on the transfected RBL cell lines may be a limiting factor,
for example, RBL SX-38 cells have about 100,000 receptors/cell versus 500,000
receptors/cell on the basophils from allergic individuals (Wiegand et al., 1996;
MacGlashan, 2007). The low percentage of serum-speciﬁc IgE in conjunction
with low expression level of FcεRI on the RBL cells could lead to the loading of
most of the IgE receptors with nonspeciﬁc IgE antibodies. Many serum factors and
also the extent of the allergic subject’s clinical response severity have been
regarded as imperative factors to induce mediator release with these cell lines.
T-Cell Polarization Assay
T-cell polarization assays are employed to examine the reactivity mediated by
T cells toward allergens and allergenic proteins. Abnormal T-cell responses to
allergens predominated by extended Th2 cells lead to allergic diseases (Akdis
et al., 2004). Allergen-speciﬁc CD4
Th2 cells exude large quantities of IL-4 and
IL-13, which stimulate the creation of allergen-speciﬁc IgE antibodies, which
results in immediate allergic symptoms (Christensen et al., 2008; Akdis, 2009).
Allergen-speciﬁc Th2 cells can also take part directly in clinical late-phase
reactions in target organs such as skin and lung, in addition to this indirect
participation in immediate reactions (Bohle et al., 2006).
Removal of Allergens in Some Food Products Using Ultrasound Chapter j11 285
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SGF (0.32% pepsin, pH 1.2) at 37oC.
Incubation of test proteins in SGF from 30 sec-1
hr time periods
Stopping of pepsinolysis using NaHCO3 or NaOH
Observation of protein profile on SDS-PAGE
FLOW CHART 11.1 Simulated gastric ﬂuid (SGF) assay protocol.
SIF (1g/100 mL of pancrean in
0.05 M KH2PO4, pH 7.5) at 37°C
Incubaon of test proteins in SIF for diﬀerent
incubaon me periods
Stopping of reacons at diﬀerent me points
Observaon of protein proﬁle on SDS-PAGE
FLOW CHART 11.2 Simulated intestinal ﬂuid (SIF) assay protocol.
286 Ultrasound: Advances in Food Processing and Preservation
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