DOI: 10.57755/hrh4-k251
title: The evaluation of antigenic reactivity of cow&#39;s milk proteins treated with non-commercial enzyme preparations of microbial and plant origin

description: <p class="MsoNormal" style="text-align:justify;line-height:200%"><span style="font-size:10px"><span style="font-family:&#39;times new roman&#39; , &#39;times&#39; , serif">Allergy to cow&#39;s milk
protein (CMPA) is commonly diagnosed in 1–17.5 %
of infants
and children under age of 5 years, among 1–13.5 % of teenagers and also among
about 0.5-2.4 % of adults. Cow milk contains at least 20 potentially allergenic
proteins, among which casein (CN), serum proteins, β-lactoglobulin (BLG), and ɑ-lactalbumin
(ALA) have been identified as the main allergens recognized by human IgE. In most cases, children
with milk allergies are sensitized to more than one allergen. In the processing
of milk and manufacturing of dairy products, various methods are
applied to reduce the allergenicity of CMP, such as thermal (dry heating,
boiling, or cooking), biochemical,
and chemical processes (enzymatic digestion). To reduce allergenicity, proteins
can be broken down into smaller peptides and amino acids by enzyme hydrolysis. </span><span lang="EN-US" style="line-height:107%;font-family:&#39;times new roman&#39; , serif">The aim of the study was to investigate the use
of two different origin serine proteases isolated from </span><em style="font-size:10px"><span lang="EN-GB" style="line-height:107%;font-family:&#39;times new roman&#39; , serif">Cucurbita</span><span lang="EN-US" style="line-height:107%;font-family:&#39;times new roman&#39; , serif"> ficifolia</span></em></span><span lang="EN-US" style="line-height:107%;font-family:&#39;times new roman&#39; , serif;font-size:11pt"><span style="font-size:10px"> (Asian pumpkin) and
secreted from <em>Yarrowia lipolytica</em>
yeast for reduction the milk protein allergenicity. Analyzed enzymes were
introduced in a sequence, in both combinations, and simultaneously.  </span></span><span style="font-size:10px"><span lang="EN-US" style="letter-spacing:0px;font-family:&#39;times new roman&#39; , serif;color:rgb( 31 , 31 , 31 )">These results demonstrate the potential of enzyme combinations to
enhance the efficiency of epitope destruction in allergenic proteins, providing
valuable insights into the development of hypoallergenic </span><span lang="EN-GB" style="letter-spacing:0px;font-family:&#39;times new roman&#39; , serif"><a href="https://www.sciencedirect.com/topics/food-science/dairy-product" rel="nofollow"><span lang="EN-US" style="color:rgb( 31 , 31 , 31 )">dairy products</span></a></span></span><span lang="EN-US" style="letter-spacing:0px;font-family:&#39;times new roman&#39; , serif;color:rgb( 31 , 31 , 31 );font-size:10px">.</span></p><p class="MsoNormal" style="text-align:justify;line-height:150%"><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif"></span><br /></p><p><br /></p>
description: <p class="MsoNormal" style="text-align:justify;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">The
aim of the study was to investigate the use of two different origin serine
proteases isolated from </span><em><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">Cucurbita</span></em><em><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif"> ficifolia</span></em><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif"> (Asian
pumpkin) and secreted from <em>Yarrowia
lipolytica</em> yeast for reduction the milk protein allergenicity. Analyzed
enzymes were introduced in a sequence, in both combinations, and simultaneously.
</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">The enzymes
at doses of: 150 U per mg of substrate protein of<em> C. ficifiolia</em> protease and 10 U per mg of substrate protein of <em>Y. lipolytica</em> protease were added to 1% whey
proteins isolate (WPI). In experiment ten different hydrolysates were obtained:</span></span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">H1-H4</span></strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif"> sequential hydrolysis
using </span><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">serine proteases isolated from <em>C. ficifolia</em> as first enzyme and <em>Y. lipolytica</em> as a second enzyme</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif"></span></span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">H1: directly after
addition <em>C.</em></span><em><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif"> ficifolia</span></em><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif"> serine protease followed by
thermal inactivation at 100 °C for 10 minutes</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; , &#39;times&#39; , serif;font-size:10px">H2: after 3h of the
hydrolysis by Asian pumpkin serine protease, followed by thermal inactivation
at 100 °C for 10 minutes </span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , &#39;times&#39; , serif;font-size:10px">H3: directly after
addition <em>Y. lipolytica</em> serine
protease, followed by thermal inactivation at 100 °C for 10 minutes </span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , &#39;times&#39; , serif;font-size:10px">H4: after 1h of the hydrolysis
by yeast serine protease, followed by thermal inactivation at 100 °C for 10
minutes </span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><strong><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">H5-H8</span></strong><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">sequential hydrolysis using </span><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">serine
proteases secreted from <em>Y. lipolytica</em>
as a first enzyme and <em>C. ficifolia</em> as
a second enzyme </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; , &#39;times&#39; , serif;font-size:10px">H5: directly after
addition <em>Y. lipolytica</em> serine
protease, followed by thermal inactivation at 100 °C for 10 minutes </span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , &#39;times&#39; , serif;font-size:10px">H6: after 3h of the
hydrolysis by yeast serine protease, followed by thermal inactivation at 100 °C
for 10 minutes </span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">H7: directly after
addition </span><em><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">C.
ficifolia</span></em><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">serine
protease,</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">followed
by thermal inactivation at 100 °C for 10 minutes</span><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif"></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; , &#39;times&#39; , serif;font-size:10px">H8: after 1h of the
hydrolysis by Asian pumpkin serine protease, followed by thermal inactivation
at 100 °C for 10 minutes</span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><strong><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">H9-H10</span></strong><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif"> simultaneously hydrolysis </span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">using </span><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">serine
proteases from <em>C. ficifolia</em> and <em>Y. lipolytica</em> at the same time</span></span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">H9:
</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">directly
after addition </span><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">serine proteases from <em>C. ficifolia</em> and <em>Y.
lipolytica</em>, </span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">followed
by thermal inactivation at 100 °C for 10 minutes</span></span></p><p class="MsoNormal" style="text-align:justify;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">H10:</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif"> after 3h of the
hydrolysis by </span><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">serine proteases from <em>C. ficifolia</em> and <em>Y.
lipolytica</em>, </span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">followed
by thermal inactivation at 100 °C for 10 minutes</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; , &#39;times&#39; , serif;font-size:10px">As a substrates the 1%
solution of n-WPI (native WPI) and ht-WPI (heat treated at 100 °C for 10
minutes WPI) 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; , &#39;times&#39; , serif;font-size:10px">In obtained hydrolysates
the free amino groups (FAG) content and the degree of hydrolysis (DH) were
evaluated.</span></p><p class="MsoNormal" style="text-align:justify;line-height:150%"><span lang="EN-US" style="font-family:&#39;times new roman&#39; , &#39;times&#39; , serif;font-size:10px">WPI
and ht-WPI and their hydrolysates were analyzed for their capacity to bind:</span></p><p class="MsoNormal" style="text-align:justify;line-height:150%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif">a.
</span><span lang="EN-GB" style="font-family:&#39;times new roman&#39; , serif">goat anti-cow’s
whey serum</span><span lang="EN-US" style="font-family:&#39;times new roman&#39; , serif"></span></span></p><p class="MsoNormal" style="text-align:justify;line-height:150%"><span lang="EN-US" style="font-family:&#39;times new roman&#39; , &#39;times&#39; , serif;font-size:10px">b.
IgE and IgG antibodies present in pool sera from patients with CMPA</span></p><p class="MsoNormal" style="text-align:justify;line-height:150%"><span lang="EN-US" style="font-family:&#39;times new roman&#39; , &#39;times&#39; , serif;font-size:10px">c.
IgE and IgG antibodies present in pool sera from healthy children (without
CMPA) </span></p><p class="MsoNormal" style="text-align:justify;line-height:150%"><span lang="EN-US" style="font-family:&#39;times new roman&#39; , &#39;times&#39; , serif;font-size:10px">using
a competitive ELISA and immunoblotting.</span></p><p style="margin-top:12pt;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;margin-bottom:0.0001pt;text-align:justify;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><span lang="EN-GB" style="line-height:200%">The
reactivity of antibodieswith substrates (n-WPI and ht-WPI) and
hydrolysates (H1-H10) was determined using a competitive ELISA with
modification as described by <span style="color:black">Babij
et al. 2015 [</span></span><span style="line-height:200%;color:black">doi: </span><span style="line-height:200%"><a href="https://doi.org/10.1007/s00726-015-2013-2" target="_blank" rel="nofollow noopener noreferrer"><span style="color:black">10.1007/s00726-015-2013-2</span></a><span style="color:black">]</span></span><span lang="EN-GB" style="line-height:200%;color:black">. In the </span><span lang="EN-GB" style="line-height:200%">study four different groups of
pooled children sera and anti-whey serum were used. The final (after mixing
with serum) protein concentrations were 5000, 1666.6, 555.6, 185.2, 61.7, 20.6,
6.8, 2.3, 0.76 </span><span style="line-height:200%">μ</span><span lang="EN-GB" style="line-height:200%">g/mL.
During SDS-PAGE and immunoblotting analysisall samples were separated in gradient polyacrylamide gels (4 - 15
%). Proteins were transfer to 0.2 µm nitrocelulose membrane. After blocking,
the membranes were incubated overnight at 4°C with goat anti-whey IgG serum
(diluted 1:1000); serum pool of the CMPA children or serum pool of the healthy
children (for identification of IgG antibodies dilution 1:2000; for IgE
dilution 1:25). The reaction was developed using Clarity Western ECL as the
substrate. Visualization of the membranes was performed using ChemiDoc Touch
Instruments. The molecular weights of the proteins were estimated using Image
Lab<sup>TM</sup> software by comparison with a standard protein marker
(Precision Plus Protein Dual Xtra Standards). The selected </span><span lang="EN-GB" style="line-height:200%">gels were stained using Coomassie Brilliant
Blue R-250.</span><span lang="EN-GB" style="line-height:200%"></span></span></p><p style="margin-top:12pt;margin-right:0cm;margin-bottom:0cm;margin-left:0cm;margin-bottom:0.0001pt;text-align:justify;line-height:200%"><span lang="EN-GB" style="line-height:200%;font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif">The
pools of sera were obtained from:</span></p><p style="margin-top:12pt;margin-right:0cm;margin-bottom:0cm;margin-left:36pt;margin-bottom:0.0001pt;text-align:justify;text-indent:-18pt;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><span lang="EN-GB" style="line-height:200%">a.<span style="font-stretch:normal;line-height:normal;font-family:&#39;times new roman&#39;">      
</span></span><span lang="EN-GB" style="line-height:200%">19 children (3–5 years old) with
persistent CMPA, defined by a positive result for specific serum IgE and
clinical symptoms of CMPA. Sera were selected from 43 analyzed samples and were
characterized by the high absorbance levels in the reaction with n-WPI in IgE
(pool no. I) and IgG (pool no. II) class of antibodies (absorbance 450nm ≥0.4).</span></span></p><p style="margin-top:12pt;margin-right:0cm;margin-bottom:0cm;margin-left:36pt;margin-bottom:0.0001pt;text-align:justify;text-indent:-18pt;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><span lang="EN-GB" style="line-height:200%">b.<span style="font-stretch:normal;line-height:normal;font-family:&#39;times new roman&#39;">      
</span></span><span lang="EN-GB" style="line-height:200%">26 children (3–5 years old) with no
clinical symptoms of CMPA. Sera were selected from 63 analyzed samples and
characterized by the high absorbance levels in the reaction with n-WPI in IgE
class of antibodies (absorbance 450nm ≥0.4; pool sera no. III).</span></span></p><p style="margin-top:12pt;margin-right:0cm;margin-bottom:0cm;margin-left:36pt;margin-bottom:0.0001pt;text-align:justify;text-indent:-18pt;line-height:200%"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><span lang="EN-GB" style="line-height:200%">c.<span style="font-stretch:normal;line-height:normal;font-family:&#39;times new roman&#39;">      
</span></span><span lang="EN-GB" style="line-height:200%">10 children (3–5 years old)
exhibiting no clinical symptoms of CMPA. Sera were selected out of 63 analyzed
samples and characterized by the high absorbance levels in the reaction with
n-WPI in the IgG class of antibodies (absorbance 450nm ≥0.4; pool sera no. IV).</span></span></p><p style="margin-top:12pt;margin-right:0cm;margin-bottom:0cm;margin-left:36pt;margin-bottom:0.0001pt;text-align:justify;line-height:200%"><span lang="EN-GB" style="line-height:200%;font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif">In
case of competitive ELISA the percentage of antigen-binding inhibition was
calculated using the following equation:</span></p><p>

























































<span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif"><br /></span></p><p style="margin-top:12pt;margin-right:0cm;margin-bottom:0cm;margin-left:36pt;margin-bottom:0.0001pt;text-align:justify;line-height:200%"><span lang="EN-GB" style="line-height:200%;font-size:11pt"><span style="font-size:10px;font-family:&#39;times new roman&#39; , &#39;times&#39; , serif">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></span></p>