oods
A icle
Rapid, E ec i e, and Ve sa ile Ex ac ion o Glu en in Food
wi h Applica ion on Di e en Immunological Me hods
Ve ónica Segu a 1, Jacobo Díaz 2,Ángela Ruiz-Ca nice 1, Alba Muñoz-Suano 3, Ca olina Ca illo-Ca ión3,
Ca olina Sousa 1,Ángel Cebolla 3and Isabel Comino 1,*
Ci a ion: Segu a, V.; Díaz, J.;
Ruiz-Ca nice , Á.; Muñoz-Suano, A.;
Ca illo-Ca ión, C.; Sousa, C.;
Cebolla, Á.; Comino, I. Rapid,
E ec i e, and Ve sa ile Ex ac ion o
Glu en in Food wi h Applica ion on
Di e en Immunological Me hods.
Foods 2021,10, 652.
h ps://doi.o g/10.3390/
oods10030652
Academic Edi o s: Annalisa
De Gi olamo and Vincenzo Lippolis
Recei ed: 5 Feb ua y 2021
Accep ed: 16 Ma ch 2021
Published: 19 Ma ch 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Depa men o Mic obiology and Pa asi ology, Facul y o Pha macy, Uni e si y o Se ille,
41012 Se ille, Spain; [email p o ec ed] (V.S.); [email p o ec ed] (Á.R.-C.); [email p o ec ed] (C.S.)
2Clinical Analysis Se ice, Hospi al Uni e si a io INGESA, 51003 Ceu a, Spain; [email p o ec ed]
3Biomedal S.L., 41900 Se ille, Spain; [email p o ec ed] (A.M.-S.);
[email p o ec ed] (C.C.-C.); [email p o ec ed] (Á.C.)
*Co espondence: [email p o ec ed]; Tel.: +34-954-556-452
Abs ac :
One o he main conce ns in glu en analysis is o achie e e icien ex ac ion o glu en
p o eins. Con en ional e hanol-based ex ac ion solu ions a e ine icien and, because o his, i is
necessa y o use educing agen s o acids o p ope solubiliza ion. The ex ac ion ecommended
by CODEX S anda d 118-1979 ( e ised 2008) u ilizes Cock ail solu ion (pa en WO 02/092633 A1).
Howe e , i is ha m ul wi h a disgus ing odo and is no compa ible wi h some immunological
echniques. He e, he e sa ili y and ex ac ion capaci y o a new Uni e sal Glu en Ex ac ion
Solu ion (UGES) (pa en ES 2 392 412 A1) we e e alua ed using di e en me hodological condi ions,
ood ma ices, and a ious immunological me hods. UGES includes sa e compounds o bo h he
use and he en i onmen , and i displayed simila ex ac ion e iciency o ha o he ex ac ion
me hod ecommended o sandwich enzyme-linked immunoso ben assay (ELISA). The ex ac ion
ime was signi ican ly educed om 100 o 40 min, depending on he ype o he sample. Fu he mo e,
unlike he cu en ly used solu ion, UGES is compa ible wi h compe i i e ELISA.
Keywo ds: celiac disease; glu en p o eins; glu en- ee die ; glu en ex ac ion solu ion
1. In oduc ion
Glu en is a complex mix u e o p olamin and glu elin s o age p o eins, which is p esen
in ce ain ce eals such as whea , ba ley, ye, oa s, and hei de i a es. P olamins mainly
comp ise monome ic p o eins ha a e insoluble in wa e and sal solu ions bu soluble in
aqueous alcohols [
1
]. P olamins con ibu e o he cohesi eness and ex ensibili y o glu en.
Glu elins a e polyme ized by in e chain disul ide bonds and a e insoluble in wa e , sal
solu ions, and aqueous alcohol. Glu elins play a ole in he main enance o he elas ici y
and s eng h o he glu en [
2
–
4
]. These common die a y p o eins ha e unusual biochemical
p ope ies ha include a high abundance o glu amine and p oline esidues, which ende
hem esis an o deg ada ion by gas oin es inal p o eases [5,6].
The consump ion o glu en p o eins d i es ad e se immune eac ions in p edisposed
indi iduals who su e om whea alle gy, non-celiac glu en sensi i i y (NCGS), de ma i is
he pe i o mis, glu en a axia, o celiac disease (CD) [
7
]. CD is one o he mos equen
hype sensi i i ies, wi h a global se op e alence o 1.4% [
8
]. This disease is igge ed by
he p esence o pep ides gene a ed by he agmen a ion o glu en p o eins ha a e no
diges ed by human p o eases. These pep ides c oss he in es inal ba ie and cause an
abe an immune eac ion. This esul s in illous a ophy accompanied by in es inal mani-
es a ions, such as abdominal pain, bloa ing, nausea, omi ing, dia hea, and ex ain es inal
mani es a ions ha include a axia, anemia, and os eopo osis [9–15].
Fo decades, compliance o a s ic glu en- ee die (GFD) has been accep ed as he
only e ec i e ea men o CD [
16
] and, mo e ecen ly, o pa ien s wi h o he glu en-
Foods 2021,10, 652. h ps://doi.o g/10.3390/ oods10030652 h ps://www.mdpi.com/jou nal/ oods
Foods 2021,10, 652 2 o 16
ela ed diso de s [
17
,
18
]. Adhe ence o a GFD can be di icul , due o lack o awa eness,
c oss-con amina ion o oods, and inadequa e labeling and es ing mechanisms [
13
,
19
–
22
].
The lack o o he ea men s o glu en-associa ed diso de s makes sensi i e and low-cos
analy ical me hods necessa y o he quan i ica ion o glu en con en in all kinds o ha m ul
samples, including oods, be e ages, cosme ics, and medicines [23–26].
The Codex Alimen a ius and Food and D ug Adminis a ion ecommend ha he
quan i ica ion o glu en in ood should be based on sensi i e and speci ic me hods wi h a de-
ec ion limi o
≤
20 ppm [
27
,
28
]. Howe e , he GFD Ce i ica ion O ganiza ion and Celiac
Suppo Associa ion equi e lowe limi s o
≤
10 ppm
≤
5 ppm glu en, espec i ely [
26
].
Glu en analy ical me hods should be alida ed and calib a ed agains ce i ied e e ence
ma e ials. In addi ion, he me hods should use an an ibody ha eac s wi h he ce eal
p o ein ac ions oxic o hose wi h CD and ha does no c oss- eac wi h o he ood
componen s, o p e en unde - epo ing o o e - epo ing glu en [18,29,30].
One o he majo conce ns in glu en analysis is o achie e he e icien ex ac ion
o glu en p o eins om simple samples (e.g., aw ma e ials and unp ocessed p oduc s)
and complex samples (e.g., hea -p ocessed p oduc s o samples wi h a complex ma ix
composi ion). Con en ional e hanol-based solu ions ine icien ly ex ac glu en om
p ocessed oods because, du ing he p ocessing o same oods, he gene a ed pep ide
p o ile is e y dependen on nume ous pa ame e s (e.g., ing edien s, ime, empe a u e,
ype o e men ing o ganisms, o enzymes used). As a esul , some gliadins/glu elins
a e no in hei na i e o m. To sol e his p oblem, he use o educing agen s, acids,
o enzymes is necessa y o dis up he disul ide bonds be o e hei solubiliza ion in alcohol
solu ions du ing he ex ac ion p ocedu e [31–34].
The cu en ecommended o icial me hod is an ex ac ion p ocedu e wi h Cock ail solu-
ion. Howe e , his solu ion con ains guanidine hyd ochlo ide (2 M) and 2-me cap oe hanol
(250 mM) which a e ha m ul o use s, while 2-me cap oe hanol has an unpleasan odo .
In addi ion, Cock ail solu ion is no compa ible wi h some immunological assays ha ely
on he di ec con ac o an an ibody wi h he sample ex ac , such as he compe i i e enzyme-
linked immunoso ben assay (ELISA) [
34
–
36
]. The e o e, he sea ch o glu en ex ac ion
solu ions ha a e e icien in ex ac ing p o eins, compa ible wi h di e en immunological
me hods, and sa e o he use is an impo an aim.
Based on he de elopmen o compa ible solu ions, he Uni e sal Glu en Ex ac ion
Solu ion (UGES) is p oposed as a new glu en ex ac ion solu ion. UGES ea u es solubilizing
and an isep ic agen s in an e hanol solu ion, wi h no componen s ha a e oxic o he
use . He e, we e alua ed he e iciency, e sa ili y, and ex ac ion capaci y o UGES using
di e en me hodological condi ions and ood ma ices o di e ing complexi y. Fu he mo e,
we assessed he compa ibili y o his solu ion wi h a ious immunological me hods, such as
ELISA and la e al low immunoassay (LFIA), compa ed wi h adi ional ex ac ion solu ions.
2. Ma e ials and Me hods
2.1. Reagen s
All chemicals we e o analy ical g ade o highe . All aqueous sol en s and solu ions
we e p epa ed wi h double-dis illed wa e . P olamin Wo king G oup (PWG) gliadin
p o ided by he Wo king G oup on P olamin Analysis and Toxici y was used as he
e e ence ma e ial. Gliadin solu ion (2 mg/mL) was p epa ed in 60% ( / ) e hanol.
2.2. Samples
A wide a ie y o comme cial samples, labeled and no labeled as being glu en- ee,
we e selec ed and pu chased in supe ma ke s. Solid samples we e ho oughly g ound o
a ine powde o mix u e using an elec ic blende (John Os e Manu ac u ing Company,
Mexico, Mexico). Liquid samples we e homogenized by igo ous o exing using a
Licuos appa a us (Del alab, Ba celona, Spain). The p oduc s we e g ouped in o wo
ood ca ego ies: hea -p ocessed o complex composi ion samples and non-hea -p ocessed
samples. In o ma ion abou he nu i ional composi ion and ing edien s was di ec ly
Foods 2021,10, 652 3 o 16
collec ed om he ood manu ac u e ’s websi e and he p oduc label. Rice lou was used
as he nega i e con ol, and whea lou was used as he posi i e con ol.
2.2.1. Diges ion o he Gliadin S anda d
To ep oduce gas ic diges ion
in i o
, PWG gliadin was suspended in 0.03 N hy-
d ochlo ic acid and incuba ed a 37
◦
C o 10 min wi h s i ing. Pepsin (Sigma Ald ich,
S Louis, MO, USA) was added and incuba ed a 37
◦
C o 60 min wi h s i ing. The eac-
ion was hen s opped by incuba ing gliadin in a d y ba h a 95
◦
C o 5 min o inac i a e
he enzyme. Gas ic diges s we e adjus ed o pH 6.0 wi h sodium phospha e bu e and
subjec ed o simula ed duodenal diges ion by sequen ial addi ion o bo ine panc ea ic
ypsin (Sigma Ald ich, S Louis, MO, USA) and ype II bo ine panc ea ic-
α
-chymo ypsin
(Sigma Ald ich, S Louis, MO, USA) a 37
◦
C o 30 min. The eac ion was hen s opped by
in oducing PWG gliadin in a d y ba h a 95 ◦C o 5 min [37].
2.2.2. Spiked Samples
Di e en comme cial samples labeled as being glu en- ee we e spiked wi h PWG gliadin
o diges ed PWG gliadin o check o a possible ma ix e ec ha could in e e e wi h he
analysis and o e alua e he eco e y. Fo each ma ix, ou samples we e analyzed (glu en
le el: 0, 10, 20, and 40 ppm). The sample (0.5 g) in a 50 mL ube was spiked wi h gliadin dilu ed
in 60% e hanol, adjus ing he concen a ion o gliadin o each sample. The samples we e
incuba ed a 4
◦
C un il analysis. The pe cen age glu en eco e y (R) in oods was calcula ed
om he a e age measu ed (M) and spiked (S) le el using he equa ion R = (M/S) ×100.
2.3. Glu en Ex ac ion P ocedu es
2.3.1. Aqueous E hanol Ex ac ion
Samples (1 g) we e weighed and ans e ed o indi idual polyp opylene ubes
(Je Bio il, Elgin, IL, USA). Ten millili e s o 60% ( / ) e hanol we e added, and he ubes
we e incuba ed in a o a y shake o 1 h a oom empe a u e. Each suspension was
cen i uged a 2500
×
g o 10 min, and each supe na an was collec ed. In he case o
RIDASCREEN Gliadin compe i i e (R-Biopha m AG, Da ms ad , Ge many), an e hanol
solu ion (60%) con aining 10% liquid ish gela in was equi ed o he ex ac ion o samples
con aining polyphenols. These samples included bee , mal , and hops.
2.3.2. Cock ail Ex ac ion Solu ion
Samples (0.25 g) we e weighed and indi idually ans e ed o p opylene ubes. They we e
ex ac ed wi h an ex ac ion solu ion con aining 2-me cap oe hanol as a educing agen and
guanidine hyd ochlo ide as a chao opic agen (Cock ail solu ion R7006, pa en WO 02/092633
A1, R-Biopha m AG, Da ms ad , Ge many). B ie ly, 2.5 mL o he ex ac ion solu ion was added
o each sample, ollowed by incuba ion a 50
◦
C in a wa e ba h o 40 min. Then, 7.5 mL o
80% ( / ) e hanol was added and incuba ed in a o a y shake o 1 h a oom empe a u e.
Las ly, each suspension was cen i uged a 2500
×
g o 10 min, and he supe na an was collec ed.
2.3.3. UGES Ex ac ion
UGES is based on a hyd oalcoholic solu ion (less han 40%) and a ginine as key
componen s o p omo e glu en solubili y (pa en ES 2 392 412 A1).
Samples (0.5 g) we e weighed and indi idually ans e ed o p opylene ubes.
They we e hen ex ac ed wi h 5 mL o UGES (Hygiena, Se ille, Spain). Fo samples
con aining polyphenols (including annins) and cosme ics con aining an ioxidan s, 0.5 g o
he special polyphenol addi i e (Hygiena, Se ille, Spain) was added. Each suspension was
cen i uged a 2500×g o 10 min, and he supe na an was ans e ed o a clean ube.
Foods 2021,10, 652 4 o 16
2.4. Techniques Employed
2.4.1. Enzyme-Linked Immunoso ben Assays (ELISAs)
Glu en in oods was de e mined using he RIDASCREEN
®
FAST Gliadin (R-Biopha m)
and RIDASCREEN
®
Gliadin compe i i e (R-Biopha m) ELISA ki s. Bo h assays u ilize
he R5 monoclonal an ibody (moAb) [
38
,
39
]. All samples we e analyzed acco ding o he
manu ac u e ’s ins uc ions.
2.4.2. Sodium Dodecyl Sul a e Polyac ylamide Gel Elec opho esis (SDS-PAGE) and
G12/A1 Wes e n Blo ing
SDS-PAGE and immunoblo ing we e pe o med unde s anda d condi ions. Sam-
ples we e dilu ed in SDS-PAGE dena u ing bu e (62.5 mM T is–HCl pH 6.8, 5% 2-
me cap oe hanol, 2% SDS, 0.001% b omophenol blue, and 10% glyce ol) [
40
–
42
]. P o eins
esol ed by 12.5% SDS-PAGE we e ans e ed o a poly inylidene di luo ide (PVDF) mem-
b ane. The memb anes we e incuba ed wi h G12 o A1 moAbs o/n a 4
◦
C. A e washing,
an i-mouse immunoglobulin G (IgG) alkaline phospha ase conjuga ed an ibody (Sigma
Ald ich) was added and incuba ed o 1 h a oom empe a u e.
2.4.3. La e al Flow Immunoch oma og aphic Assays (LFIA)
The LFIA Glu enTox S ick was used o de elop he new analy ical me hod (Hygiena,
Se ille, Spain). These s icks a e based on he G12 and A1 moAbs. The s icks we e calib a ed
agains PWG gliadin. The ood samples we e ex ac ed wi h 10 mL o UGES pe g o ood.
A e ex ac ion, he samples we e dilu ed 1:10 in dilu ion solu ion. The es s icks we e
dipped in o he dilu ion solu ion (100
µ
L) in a well o a mic o i e pla e. The esul s we e
de e mined quan i a i ely a e 30 min. The Glu enTox
®
Reade (Hygiena, Se ille, Spain)
was used o quan i ica ion by con inuous scanning o he s icks using an op ical de ec o .
2.5. S a is ical Analysis
Da a analyses was pe o med using SPSS 25.0 o Windows (IBM SPSS S a is ics,
A monk, NY: IBM Co p). Sca e diag ams we e used as g aphic indica o s o he com-
mu abili y be ween he alues gene a ed by each pai o ex ac ion solu ions. The equa ion
o he line was calcula ed using he nonpa ame ic Passing–Bablok eg ession [
43
–
45
].
This me hod is a s a is ical p ocedu e ha allows aluable es ima ion o analy ical me hod
ag eemen and possible sys ema ic bias be ween hem. The ma e ial was deemed com-
mu able i he a io ob ained o a e e ence ma e ial using he wo me hods was consis en
wi h he p edic ion in e al (ISO, 2009). The Lin conco dance co ela ion coe icien is
he conco dance ( Lin) be ween a new es o measu emen and a gold s anda d es o
measu emen . This coe icien combines a p ecision measu e, ep esen ed by he Pea son’s
co ela ion coe icien ( Pe ason), wi h an accu acy measu e, ep esen ed by he bias co ec-
ion coe icien (Cb). The McB ige scale was used o mo e s ingen ly quali y he s eng h
o he ag eemen . Fo con inuous a iables, he scale was almos pe ec o alues >0.99,
subs an ial o alues o 0.95 o 0.99, mode a e o alues o 0.90 o 0.95, and poo o alues
<0.90. Di e ences be ween g oups we e s a is ically compa ed by he median using he
Wilcoxon es .
3. Resul s and Discussion
3.1. Cha ac e iza ion o UGES P o ocol
Du ing he p ocessing o some oods, he hea ea men o cooked and baked p oduc s
leads o he o ma ion o p o ein agg ega es and glu en-de i ed pep ides in insoluble
ma ices. This esul s in a he e ogeneous mix u e wi h a nonuni o m dis ibu ion o glu en,
which makes analyses e en mo e di icul . I is necessa y o op imize he ex ac ion sys ems
o p oduce he comple e elease and eco e y o bo h p olamins and glu elins [
46
,
47
].
The ime and empe a u e o incuba ion du ing he ex ac ion p ocess a e wo c i ical
ac o s o conside [48].
Foods 2021,10, 652 5 o 16
In his wo k, he e ec o empe a u e on UGES was e alua ed (Figu e 1). Se e al sam-
ples we e ex ac ed a wo condi ions: incuba ion in wheel agi a ion a oom empe a u e
and incuba ion in a wa e ba h a 50
◦
C. In bo h cases, he ime was ixed a 1 h, and he
amoun o ex ac ed glu en was quan i ied by sandwich ELISA (Figu e 1A). Unhea ed
p ocessed ood o simple samples (maize lou , oa lakes) did no need hea o achie e
he comple e ex ac ion o glu en. In con as , hea ea men was needed o comple e
ex ac ion in samples wi h a complex composi ion (mix u e ood addi i es) and o hea -
p ocessed samples (co n b ead).
Foods 2021, 10, x FOR PEER REVIEW 5 o 17
insoluble ma ices. This esul s in a he e ogeneous mix u e wi h a nonuni o m dis ibu-
ion o glu en, which makes analyses e en mo e di icul . I is necessa y o op imize he
ex ac ion sys ems o p oduce he comple e elease and eco e y o bo h p olamins and
glu elins [46,47]. The ime and empe a u e o incuba ion du ing he ex ac ion p ocess
a e wo c i ical ac o s o conside [48].
In his wo k, he e ec o empe a u e on UGES was e alua ed (Figu e 1). Se e al
samples we e ex ac ed a wo condi ions: incuba ion in wheel agi a ion a oom em-
pe a u e and incuba ion in a wa e ba h a 50 °C. In bo h cases, he ime was ixed a 1 h,
and he amoun o ex ac ed glu en was quan i ied by sandwich ELISA (Figu e 1A).
Unhea ed p ocessed ood o simple samples (maize lou , oa lakes) did no need hea o
achie e he comple e ex ac ion o glu en. In con as , hea ea men was needed o
comple e ex ac ion in samples wi h a complex composi ion (mix u e ood addi i es) and
o hea -p ocessed samples (co n b ead).
Figu e 1. Op imiza ion o ime and empe a u e in he Uni e sal Glu en Ex ac ion Solu ion
(UGES) p o ocol. (A) E ec o empe a u e in di e en ypes o samples. (B) E ec o ime in simple
Figu e 1.
Op imiza ion o ime and empe a u e in he Uni e sal Glu en Ex ac ion Solu ion (UGES)
p o ocol. (
A
) E ec o empe a u e in di e en ypes o samples. (
B
) E ec o ime in simple and
complex solid samples. (
C
) E ec o ex ac ion ime in liquid samples wi hou suspended solids.
The expe imen s we e pe o med in duplica e, and he mean ±s anda d de ia ion (SD) is shown.
The in luence o di e en incuba ion pe iods (2 min o 1 h) was s udied. Maize lou
(b1) and a p ecooked mea ood (b2) we e selec ed o op imize he ex ac ion ime. The b1
Foods 2021,10, 652 6 o 16
example was ex ac ed by o a ion a oom empe a u e. The b2 example was hea -ex ac ed.
A minimum o 30 o 40 min was necessa y o achie e he comple e glu en ex ac ion o
bo h samples (Figu e 1B). Acco ding o hese esul s, he ex ac ion ime was ixed a
40 min o solids samples o con aining polyphenol, annins, o an ioxidan s, ega dless o
he use o hea ing in he ex ac ion.
Se e al addi ional assays we e ca ied ou wi h liquid samples wi hou solids in
suspension, such as milk, juices, o ganic d inks, bee s, and so d inks, o e alua e i he
ex ac ion ime o hese samples could be educed. The esul s ob ained by compe i i e
ELISA indica ed a educ ion in he ex ac ion ime o hese samples because hey we e
sa is ac o y wi h only 2 min o agi a ion compa ed o he ex ac ion ca ied ou in solid
samples (40 min) (Figu e 1C).
These indings indica ed ha he op imum ex ac ion p ocedu e depends la gely on
he ype o sample. Howe e , he ad an age o UGES is i s adap abili y o di e en ypes
o samples wi h sligh modi ica ions o he p o ocol, as desc ibed in Table 1.
Table 1.
Scheme o he s eps in he ex ac ion p ocedu e wi h UGES depending on he ype o sample. RT, oom empe a u e.
Ex ac ion P o ocol
Type o Sample Amoun o Sample Addi i es UGES Volume P ocedu e
Liquid Wi hou solids in
suspension 1 mL 9 mL Shake 1–2
min
Solid
Non-hea -p ocessed
and simple
composi ion
1 g 10 mL
Incuba e 40
min a RT
(wheel
agi a o )
Cen i uge 10
min a 2500×g
Solid Hea -p ocessed o
complex composi ion
1 g 10 mL
Incuba e 40
min a 50
◦
C
(wa e ba h)
Cen i uge 10
min a 2500×g
Solid o
liquid
Con aining
polyphenols, annins,
o an ioxidan s
1 g 1.0 g gela in
0.4 g PVP 10 mL
Incuba e 40
min a 50
◦
C
(wa e ba h)
Cen i uge 10
min a 2500×g
PVP, poly inylpy olidone.
3.2. UGES Ex ac ion E ec i eness Assessed by Sandwich ELISA
3.2.1. De e mina ion o Glu en in Comme cial Food Samples
Ha ing cha ac e ized UGES using di e en ma ices, we nex e alua ed he applica-
ion o UGES o he analysis o di e en comme cial oods, which we e labeled and no
labeled as being glu en- ee, by compa ison o s anda d ex ac ion p ocedu e using 60%
e hanol o Cock ail solu ion.
UGES was highly e icien o simple and complex ma ices, e en wi h he mal p o-
cessing. The glu en amoun ex ac ed wi h UGES was always highe (be ween 1.1- and
5.3- old inc ease) han ha ex ac ed wi h 60% e hanol (Table 2). In addi ion, we ound
i e ood samples wi h a glu en con en <20 ppm using 60% e hanol solu ion bu >20 ppm
glu en using UGES. The g ea e inc eases wi h UGES compa ed wi h 60% e hanol co -
esponded o hea -p ocessed samples, such as co n b ead, linseed oas snack ood, and
chips. Glu en p o eins con ain in e molecula bonds, which allow hem o o m a ne wo k
when cooked o baked [
2
]. The esul s ob ained demons a ed ha he use o educing and
disagg ega ing agen s is, hus, necessa y o b eak disul ide bonds du ing he ex ac ion
p ocedu e. E hanol–wa e solu ions used as ex ac an s solubilize monome ic p olamins
in non-p ocessed ood like lou . Howe e , hei e iciency is low when p ocessed ood is
analyzed because p o ein agg ega ion occu s due o disul ide bond o ma ion [34,49,50].
Foods 2021,10, 652 7 o 16
Table 2. Glu en con en in ppm (mg/kg) o di e en samples by sandwich ELISA wi h 60% e hanol and Uni e sal Glu en
Ex ac ion Solu ion (UGES).
Glu en (ppm)
Food Sample 60% E hanol Solu ion UGES *
Non-hea -
p ocessed samples
Maize lou 23 26.0 (×1)
Co n semolina 11.7 15.7 (×1.3)
Oa lakes 60.3 66.8 (×1.1)
Glucose sy up 29.3 33.7 (×1.2)
Wo ces e sauce 4 5.2 (×1.3)
Yogu ce eal ba 11 14 (×1.3)
Ga lic soup 30.2 33 (×1.1)
Hea -p ocessed o complex
composi ion samples
Rice ce eals 7 11 (×1.6)
Glu en- ee-b ead <3 4.6 (×1.5)
Co n b ead 3.2 15.7 (×4.9)
Glu en- ee b eadc umbs 4.3 8.2 (×1.9)
Mix u e ood addi i es <3 5.2 (×1.7)
Spel c acke 77.2 115.2 (×1.5)
Dog d y ood 13.3 28.2 (×2.1)
Linseed oas snack 14.9 78.8 (×5.3)
Honey co n pops 5.5 24.9 (×4.5)
Chips a 5.3 21.1 (×4.0)
Dough lou 7.9 40.2 (×5.1)
Popco n 6 16.1 (×2.7)
* Values in b acke s a e he yield ac o compa ed o ha wi h 60% e hanol solu ion and UGES.
Addi ionally, 93 comme cial ood samples, labeled and no labeled as being glu en-
ee, we e analyzed using UGES and Cock ail solu ion (Table 3). Glu en was unde ec able,
less han he limi o quan i ica ion (<LOQ) in 51 o he samples by bo h ex ac ion me hods
(da a no shown). No signi ican di e ences in he glu en con en o he ood we e e iden
using UGES and Cock ail solu ion; he yield ac o s ound we e 0.5 o 1.7 imes. Howe e ,
he “chips a” sample con ained <20 ppm glu en using Cock ail solu ion and >20 ppm using
UGES. This esul is e y impo an because indi iduals wi h CD can only inges p oduc s
wi h a glu en con en <20 ppm.
A s a is ical e alua ion o he ex ac ion p ocess was pe o med by compa ing UGES
e sus e hanol and UGES e sus Cock ail solu ion. Passing–Bablok linea eg ession
analysis wi h a p edic ion in e al o 95% was calcula ed o desc ibe he commu abili y
o he esul s. Lin’s conco dance co ela ion coe icien (
Lin
) was de e mined using a
p ecision measu e (
Pea son
) and an accu acy measu e (C
b
). Las ly, he di e ences be ween
g oups we e s a is ically compa ed using he Wilcoxon es .
The s a is ical analysis o he glu en concen a ions ob ained by UGES and 60% e hanol
showed ha he da a we e no commu able (Figu e 2B(b1)), since he p ecision and accu acy
we e low (
Pea son
= 0.8252, C
b
= 0.8144). This indica ed a poo ag eemen s eng h, acco ding
o he McB ige scale (<0.90), wi h
Lin
= 0.6720 (Figu e 2A). In addi ion, hese esul s showed
ha UGES had a be e ex ac ion capaci y han 60% e hanol solu ion, since he e was a
signi ican di e ence in he esul s acco ding o he Wilcoxon es (p< 0.0001)
(Figu e 2A).
Howe e , UGES and Cock ail solu ion we e commu able acco ding o Passing–Bablok
eg ession (Figu e 2B(b2)) wi h high p ecision and accu acy coe icien s (
Pea son
= 0.9694,
C
b
= 0.9984) (Figu e 2A). Lin’s co ela ion conco dance coe icien (
Lin
) was 0.9679,
indica ing subs an ial ag eemen s eng h acco ding o he McB ige scale (>0.95).
Fu he mo e, no signi ican di e ence was ound (p= 0.2381) acco ding o he Wilcoxon es ,
indica ing ha Cock ail solu ion could be eplaced by UGES.
Foods 2021,10, 652 8 o 16
Table 3.
Glu en con en in ppm (mg/kg) o di e en samples by sandwich ELISA wi h Cock ail solu ion and Uni e sal
Glu en Ex ac ion Solu ion (UGES).
Glu en (ppm)
Food Sample Cock ail Solu ion UGES *
Non-hea -
p ocessed samples
Maize lou 27 26.0 (×1.0)
Co n semolina 14.8 15.7 (×1.1)
Oa lakes 64 66.8 (×1.0)
Glucose sy up 36.3 33.7 (×0.9)
Wo ces e sauce 5.3 5.2 (×1.0)
Yogu ce eal ba 13 14 (×1.1)
Ga lic soup 30.8 33.0 (×1.1)
Hea -p ocessed o complex composi ion samples
Rice ce eals 14 11 (×0.8)
Glu en- ee-b ead 3.9 4.6 (×1.2)
Co n b ead 16.7 15.7 (×0.9)
Glu en- ee b eadc umbs 5.6 8.2 (×1.5)
Mix u e ood addi i es 4.9 5.2 (×1.1)
Spel c acke 111 115.2 (×1.0)
Dog d y ood 35.4 28.2 (×0.8)
Linseed oas snack 89.4 78.8 (×0.9)
Honey co n pops 38.3 24.9 (×0.7)
Chips a 17.8 21.1 (×1.2)
Dough lou 46.5 40.2 (×0.9)
Popco n 17.4 16.1 (×0.9)
Appe ize s a 74.8 65.2 (×0.9)
Appe ize s b 80.0 70.1 (×0.9)
Chips b 28.5 23.5 (×0.8)
Cookies a 7.9 7.7 (×1.0)
Cookies b 9.0 9.2 (×1.0)
Co n a epa 7.8 4.0 (×0.5)
Fishmeal 19.6 19.5 (×1.0)
In usions a 58.4 80.0 (×1.4)
In usions b 80.0 80.0 (×1.0)
Len il g ains 77.7 40.6 (×0.7)
Mea p epa a ion 7.5 12.5 (×1.7)
Pea lou 7.6 8.3 (×1.1)
Pla e o len ils 15.7 14.1 (x0.9)
Quinoa lou 80.0 80.0 (×1.0)
Rice pape 8.5 10.2 (×1.2)
Rice p epa a ion 80.0 80.0 (×1.0)
Spice a 80.0 80.0 (×1.0)
Spice b 78.9 69.8 (×0.5)
Spice c 39.3 47.1 (×1.2)
Swee a 80.0 80.0 (×1.0)
Swee b 80.0 80.0 (×1.0)
Swee c 80.0 80.0 (×1.0)
Swee d 80.0 80.0 (×1.0)
* Values in b acke s a e he yield ac o compa ed o ha wi h Cock ail solu ions and UGES.
Foods 2021,10, 652 9 o 16
Foods 2021, 10, x FOR PEER REVIEW 9 o 17
A s a is ical e alua ion o he ex ac ion p ocess was pe o med by compa ing UGES
e sus e hanol and UGES e sus Cock ail solu ion. Passing–Bablok linea eg ession
analysis wi h a p edic ion in e al o 95% was calcula ed o desc ibe he commu abili y o
he esul s. Lin’s conco dance co ela ion coe icien ( Lin) was de e mined using a p eci-
sion measu e ( Pea son) and an accu acy measu e (Cb). Las ly, he di e ences be ween
g oups we e s a is ically compa ed using he Wilcoxon es .
The s a is ical analysis o he glu en concen a ions ob ained by UGES and 60%
e hanol showed ha he da a we e no commu able (Figu e 2B(b1)), since he p ecision
and accu acy we e low ( Pea son = 0.8252, Cb = 0.8144). This indica ed a poo ag eemen
s eng h, acco ding o he McB ige scale (<0.90), wi h Lin = 0.6720 (Figu e 2A). In addi ion,
hese esul s showed ha UGES had a be e ex ac ion capaci y han 60% e hanol solu-
ion, since he e was a signi ican di e ence in he esul s acco ding o he Wilcoxon es
(p < 0.0001) (Figu e 2A). Howe e , UGES and Cock ail solu ion we e commu able ac-
co ding o Passing–Bablok eg ession (Figu e 2B(b2)) wi h high p ecision and accu acy
coe icien s ( Pea son = 0.9694, Cb = 0.9984) (Figu e 2A). Lin’s co ela ion conco dance coe -
icien ( Lin) was 0.9679, indica ing subs an ial ag eemen s eng h acco ding o he
McB ige scale (>0.95). Fu he mo e, no signi ican di e ence was ound (p = 0.2381) ac-
co ding o he Wilcoxon es , indica ing ha Cock ail solu ion could be eplaced by
UGES.
Figu e 2. S a is ical e alua ion o he ex ac ion p ocedu e. (A) Compa ison o ag eemen be ween pai s o assays o
esul s o he ood samples by sandwich ELISA using di e en ex ac ion solu ions di e en . S a is ical analysis was
pe o med by he Wilcoxon es . The p- alues a e gi en in he igu e. (B) Sca e diag am wi h eg ession line and con-
co dance bands o eg ession line; (b1) ep esen a ion o he 60% e hanol solu ion e sus Uni e sal Glu en Ex ac ion
Figu e 2.
S a is ical e alua ion o he ex ac ion p ocedu e. (
A
) Compa ison o ag eemen be ween pai s o assays o esul s
o he ood samples by sandwich ELISA using di e en ex ac ion solu ions di e en . S a is ical analysis was pe o med by
he Wilcoxon es . The p- alues a e gi en in he igu e. (
B
) Sca e diag am wi h eg ession line and conco dance bands
o eg ession line; (
b1
) ep esen a ion o he 60% e hanol solu ion e sus Uni e sal Glu en Ex ac ion Solu ion (UGES);
(
b2
) ep esen a ion o he Cock ail solu ion e sus UGES. The solid lines ep esen he eg ession line d awn o he ood
samples, and he dashed lines ep esen he 95% p edic ion in e al.
3.2.2. Reco e y o Gliadins in Spiked Food Samples
An impo an app oach in he analysis o glu en is o use an ex ac ion sys em ha
leads o a eco e y close o 100% o ensu e ha he p oduc s sui able o consump ion by
people wi h glu en- ela ed diso de s a e uly ee o glu en. To check his, comme cial
samples labeled as glu en- ee (b ead, soup, chocola e, and spices) we e spiked wi h
a PWG gliadin ex ac o p oduce glu en concen a ions o 10, 20, o 40 ppm glu en
(Figu e 3A).
Each spiked expe imen was pe o med ou imes. These samples we e
ex ac ed wi h UGES and Cock ail solu ion, and he ex ac ed amoun o glu en was
quan i ied by sandwich ELISA.
Foods 2021,10, 652 16 o 16
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