DOI: 10.57755/v3cg-qs53
title: The evaluation of antigenic reactivity of peanut proteins treated with non-commercial enzyme preparations of plant origin (Cucurbita ficifolia)

description: <p class="MsoNormal" style="text-align:justify;text-indent:35.4pt;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">Peanut allergies (PA) are
among the most reported and severe kinds of food allergies seen in individuals.
In the past 10 years, the prevalence of peanut allergy has more than doubled in
Western countries. PAs are immunoglobulin E (IgE)-mediated type I hypersensitivity
reactions that can lead to fatal (anaphylactic) reactions.</span></p><p class="MsoNormal" style="text-align:justify;text-indent:35.4pt;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">Peanut allergens are
classified as cupin superfamily (vicillin: Ara h 1 and legumin: Ara h 3),
conglutin (2S albumins; Ara h2, Ara h6, Ara h7), profilin (Ara h5),
pathogenesis related class-10 protein (Ara h8), nonspecific lipid-transfer
protein (Ara h9), oleosins (Ara h10 and Ara h11) and defensins (Ara h 12 and
Ara h 13). The “major allergens” identified in peanuts are defined as antigens
that bind large amounts of IgE from allergic patients and have biological
activity. They include thermostable proteins such as Ara h1, Ara h2, and Ara
h3.</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">           
Currently, the only effective treatment for peanut allergy is the avoidance of
this nut in any form. Avoiding peanuts in the daily diet is difficult owing to
their widespread use in products as an economical protein source. Therefore, it
is necessary to explore strategies to decrease the ability of peanuts to elicit
dangerous allergic responses. For several years, new methods for effectively
reducing the allergenic properties of proteins have been investigated.
Enzymatic hydrolysis is widely used in the food industry and is an efficient
process for disrupting sequential and conformational epitopes.</span></p><p class="MsoNormal" style="text-align:justify;text-indent:35.4pt;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">Enzymes used for the
hydrolysis of plant and animal proteins are mainly commercial preparations of
digestive enzymes (trypsin, chymotrypsin, pepsin), of plant origin (papain), or
of microbiological origin (Neutrase, Alcalase, Corolase) preparation. However,
new enzymes obtained from readily available sources that can efficiently
hydrolyse food proteins and effectively reduce their allergenicity are
constantly being sought. Non-commercial extracellular serine proteases obtained
from Asian pumpkin (<em>Cucurbita ficifolia</em>) are an attractive group of
enzymes that can be used for such purposes.</span></p><p>







<br /></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">This study aimed</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif"> to evaluate the optimal
conditions for hydrolysing peanut protein fraction isolates using Asian pumpkin
protease. The effects of hydrolysis on the immunoreactivity of the resulting
preparations were also investigated.</span></p>
description: <p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">This study aimed</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif"> to evaluate the optimal
conditions for hydrolysing peanut protein fraction isolates using Asian pumpkin
protease. The effects of hydrolysis on the immunoreactivity of the resulting
preparations were also investigated.</span></p><p class="MsoNormal" style="line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">Enzymatic hydrolysis assays of 1% peanut protein isolate (PPI) solution
were carried out in 0.1 M Tris/HCl buffer at pH 8.0 at 37°C and 42°C for 0
(H1.n), 1 (H2.n), 5 (H3.n), and 24 (H4.n) h using serine proteinase from Asian
pumpkin at doses of 150, 300, or 600 U/mg of protein. In experiment twenty four
different hydrolysates were obtained:</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">H1.1-H4.1</span></strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">: PPI hydrolysates obtained by
hydrolysis with Asian pumpkin serine protease at a dose of 150U/mg of protein
at 37 °C (H1.1- directly after addition; H2.1- one hour after addition; H3.1-
five hours after addition; H4.1- twenty-four hours after addition);</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">H1.2-H4.2</span></strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">: PPI hydrolysates obtained by
hydrolysis with Asian pumpkin serine protease at a dose of 150U/mg of protein
at 42 °C (H1.2- directly after addition; H2.2- one hour after addition; H3.2-
five hours after addition; H4.2- twenty four hours after addition);</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">H1.3-H4.3</span></strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">: PPI hydrolysates obtained by
hydrolysis with Asian pumpkin serine protease at a dose of 300U/mg of protein
at 37 °C (H1.3- directly after addition; H2.3- one hour after addition; H3.3-
five hours after addition; H4.3- twenty-four hours after addition);</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">H1.4-H4.4</span></strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">: PPI hydrolysates obtained by
hydrolysis with Asian pumpkin serine protease at a dose of 300U/mg of protein
at 42 °C (H1.4- directly after addition; H2.4- one hour after addition; H3.4-
five hours after addition; H4.4- twenty-four hours after addition);</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">H1.5-H4.5</span></strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">: PPI hydrolysates obtained by
hydrolysis with Asian pumpkin serine protease at a dose of 600U/mg of protein
at 37 °C (H1.5- directly after addition; H2.5- one hour after addition; H3.5-
five hours after addition; H4.5- twenty four hours after addition);</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">H1.6-H4.6</span></strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">: PPI hydrolysates obtained by
hydrolysis with Asian pumpkin serine protease at a dose of 600U/mg of protein
at 42 °C (H1.6- directly after addition; H2.6- one hour after addition; H3.6-
five hours after addition; H4.6- twenty-four hours after addition);</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">Hydrolysis was terminated by thermal inactivation (100°C, 10 min). As
substrates, 1% solutions of PPI (native PPI) and ht-PPI (heat-treated at 100 °C
for 10 minutes PPI) were used.</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">The free amino group (FAG) content and degree of hydrolysis (% DH) were
evaluated in the obtained hydrolysates.</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">The concentration of FAG (μmol Gly/g) was
determined using trinitrobenzene sulfonic acid. The progress of hydrolysis in
obtained samples was monitored by the degree of hydrolysis (% DH) assessment in
H1.n-H4.n, according to the formula: %DH &#61; (N1/N2) x 100, where: N1- free amino
group’s content (FAG) in peanut protein hydrolysates (PPH) [μmol Gly/ml]; N2-
free amino group’s content (FAG) in peanut protein hydrolysates (PPH) after
total hydrolysis [μmol Gly/ml]. </span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">PPI, ht-PPI, and PPI hydrolysates were analysed for their capacity to bind:</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">a. commercially available chicken anti-peanut proteins antibodies (all
hydrolysates; H1.n-H4.n)</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">b. IgE and IgG antibodies present in pool sera from patients with allergy
to peanut proteins (PA) (selected hydrolysates: H1.2; H.3.2; H4.2)</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">c. IgG antibodies present in pool sera from healthy children (without PA)
(selected hydrolysates H1.2; H.3.2; H4.2)</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">using competitive ELISA and immunoblotting. </span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">Serum samples (age 0-18 years) were obtained from 47 patients. Twenty
patients had a history of severe PA, and the specific IgE level of allergic
serum and clinical manifestations confirmed the allergic response. Twenty-seven
patients were classified as healthy children without allergic symptoms. 
Children’s sera were pooled based on their reactivity with PPI, and equal
volumes of serum were mixed. In this study, three different groups of pooled
children sera were used.</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">1.       A pool of sera from eight patients
(0-18 years old) with PA, defined by a positive result for specific serum IgE
and clinical symptoms of PA. Sera were selected out of 20 analyzed samples and
were characterized by the high absorbance levels in the reaction with PPI in
IgE class of antibodies</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">2.       A pool of sera was obtained from
nine patients (0-18 years old) with clinical symptoms of PA. Sera were selected
out of 20 analyzed samples and characterized by the high absorbance levels in
the reaction with PPI in IgG class of antibodies</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">3.       A pool of sera from seven
individuals (0-18 years old) exhibiting no clinical symptoms of PA. Sera were
selected out of 27 analyzed samples and characterized by the high absorbance
levels in the reaction with PPI in the IgG class of antibodies</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif;color:black">The concentrations of immunoglobulins IgE and IgG in
the serum of </span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">patients with and without PA<span style="color:black"> were determined
using commercially available sandwich ELISA kits. </span><span style="color:#222222">The reactivity of IgE and IgG anti-PPI antibodies in sera
obtained from </span>patients with and without PA<span style="color:black"> was
determined using indirect ELISA.</span></span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif;color:black">In the case of competitive ELISA, the percentage of
antigen-binding inhibition was calculated using the following equation:</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif"></span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif;color:black">Inhibition rate (%) &#61; [(Abso−Absx)<em>/</em>Abso] × 100,
where Abso is the mean absorbance value obtained after no-antigen incubation
(serum was incubated with PBST at a ratio of 1:1), and Absx is the mean
absorbance value obtained after incubation with different antigen
concentrations.</span></p><p>











































<br /></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">The reactivity of antibodieswith substrates (PPI and ht-PPI) and
hydrolysates (H1.n-H4.n) was determined using a competitive ELISA with
modifications, as described by <span style="color:black">Babij et al. 2015 [</span>doi: </span><a href="https://doi.org/10.1007/s00726-015-2013-2" target="_blank" rel="nofollow noopener noreferrer"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif;color:black">10.1007/s00726-015-2013-2</span></a><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif;color:black">]. In the </span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">study three
different groups of pooled children sera and chicken anti-peanut proteins
antibodies were used. The final (after mixing with serum/antibodies) protein
concentrations were 5000, 1666.6, 555.6, 185.2, 61.7, 20.6, 6.8 </span><span style="font-family:&#39;times new roman&#39; , serif">μ</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">g/mL (for chicken anti-peanut proteins antibodies analysis) and 5000,
1666.6, 555.6, 185.2, 61.7, 20.6, 6.8, 2.3, 0.8 </span><span style="font-family:&#39;times new roman&#39; , serif">μ</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">g/mL (for children pooled sera analysis). During SDS-PAGE and
immunoblotting analysisPPI, ht-PPI and hydrolysates obtained at higher
temperature (42°C) were separated in 20 % polyacrylamide gels. Proteins were
transferred to a 0.2 µm nitrocellulose membrane. After blocking, the membranes
were incubated overnight at 4°C with chicken anti-peanut proteins antibodies
(diluted 1:5000); serum pool of PA patients or serum pool of healthy
individuals (for identification of IgG antibodies final dilution 1:500; for IgE
dilution 1:20). The reaction was developed using Clarity Western ECL substrate.
<span style="color:black">Membrane visualisation was performed using ChemiDoc
Touch Instruments. The molecular weights of the proteins were estimated using</span>
the Image Lab<sup>TM</sup> software by comparison with a standard protein
marker (Precision Plus Protein Dual Xtra Standards).</span></p>