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Gluten, Celiac Disease, and Gluten Intolerance and the Impact of Gluten Minimization Treatments with Prolylendopeptidase on the Measurement of Gluten in Beer


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There is an increase in the number of people adopting a gluten-free diet, with one-fifth of Australian consumers avoiding certain food or drinks for allergy or intolerance reasons. For most consumers, the belief that a gluten-free diet is healthier than a gluten containing diet is unsupported by a formal diagnosis. However, for a small group of subjects who have gluten sensitive disorder, a lifelong, gluten-free diet is required, including avoidance of beer. Prolylendopeptidase enzymes (PEP) cleave gluten proteins after the abundant proline residues and have the potential to destroy gluten proteins and remove or minimize immunoreactive peptides from food. However, PEP treatment may also confound measurement of gluten concentration in food and beverages by destroying epitopes that are used to enumerate gluten peptides – we ask, “does PEP treatment destroy celiac reactive epitopes or merely disguise them from ELISA enumeration?” There is now sufficient data to show that treatment of gluten peptides with bacterial PEP in combination with other proteases, or the fungal Aspergillus niger PEP alone, reduces the immunoreactivity of gluten peptides, with celiac T-cells to near zero. This is also accompanied by destruction of key epitopes that are used by antibodies to enumerate gluten peptides during ELISA reactions. Thus, both immunoreactivity and ELISA measurements are reduced to near zero by PEP treatment. However, definitive evidence of the safety of treated beer for celiacs ideally requires a double-blind crossover, dietary challenge. Consuming sufficient beer to present a suitable load of hordein peptides is not possible, and presenting hordeins in the same form as encountered in treated beer is difficult. The effect of protease treatments on the safety of treated gluten for the remainder the celiac-like diseases, including gluten ataxia and dermatitis herpetiformis, and the larger spectrum of glutenrelated disorders, including gluten intolerance and the IgE-mediated allergic responses Bakers asthma, gluten allergy, WDEIA, and urticaria, cannot be definitively assessed until the epitopes involved have been defined. The gluten sensitive disorders and the gluten proteins are reviewed, and the effects of proteolysis on several archetypal gluten peptides are examined. Keywords: Beer, celiac disease, gluten, hordeins, prolylendopeptidase.
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Gluten, Celiac Disease, and Gluten Intolerance and
the Impact of Gluten Minimization Treatments with
Prolylendopeptidase on the Measurement of Gluten in Beer
Gregory J. Tanner,1 CSIRO Plant Industry, GPO Box 1600 Canberra, ACT 2601, Australia and CSIRO Food Futures
Flagship, Riverside Corporate Park, 5 Julius Avenue, North Ryde, NSW 2113, Australia; Michelle L. Colgrave, CSIRO
Animal, Food and Health Sciences, 306 Carmody Rd, St Lucia, QLD 4067, Australia and CSIRO Food Futures Flagship,
Riverside Corporate Park, 5 Julius Avenue, North Ryde, NSW 2113, Australia; and Crispin A. Howitt, CSIRO Plant
Industry, GPO Box 1600 Canberra, ACT 2601, Australia and CSIRO Food Futures Flagship, Riverside Corporate Park,
5 Julius Avenue, North Ryde, NSW 2113, Australia
J. Am. Soc. Brew. Chem. 72(1):36-50, 2014
There is an increase in the number of people adopting a gluten-free
diet, with one-fifth of Australian consumers avoiding certain food or
drinks for allergy or intolerance reasons. For most consumers, the belief
that a gluten-free diet is healthier than a gluten containing diet is unsup-
ported by a formal diagnosis. However, for a small group of subjects who
have gluten sensitive disorder, a lifelong, gluten-free diet is required,
including avoidance of beer. Prolylendopeptidase enzymes (PEP) cleave
gluten proteins after the abundant proline residues and have the potential
to destroy gluten proteins and remove or minimize immunoreactive pep-
tides from food. However, PEP treatment may also confound measure-
ment of gluten concentration in food and beverages by destroying
epitopes that are used to enumerate gluten peptides – we ask, “does PEP
treatment destroy celiac reactive epitopes or merely disguise them from
ELISA enumeration?” There is now sufficient data to show that treatment
of gluten peptides with bacterial PEP in combination with other prote-
ases, or the fungal Aspergillus niger PEP alone, reduces the immunoreac-
tivity of gluten peptides, with celiac T-cells to near zero. This is also
accompanied by destruction of key epitopes that are used by antibodies to
enumerate gluten peptides during ELISA reactions. Thus, both immuno-
reactivity and ELISA measurements are reduced to near zero by PEP
treatment. However, definitive evidence of the safety of treated beer for
celiacs ideally requires a double-blind crossover, dietary challenge. Con-
suming sufficient beer to present a suitable load of hordein peptides is not
possible, and presenting hordeins in the same form as encountered in
treated beer is difficult. The effect of protease treatments on the safety of
treated gluten for the remainder the celiac-like diseases, including gluten
ataxia and dermatitis herpetiformis, and the larger spectrum of gluten-
related disorders, including gluten intolerance and the IgE-mediated
allergic responses Bakers asthma, gluten allergy, WDEIA, and urticaria,
cannot be definitively assessed until the epitopes involved have been
defined. The gluten sensitive disorders and the gluten proteins are re-
viewed, and the effects of proteolysis on several archetypal gluten pep-
tides are examined.
Keywords: Beer, celiac disease, gluten, hordeins, prolylendopeptidase.
Está en aumento el número de personas adoptando una dieta libre de
gluten, con la quinta parte de los consumidores en Australia evitando
ciertas comidas o bebidas por razones de alergia o de intolerancia. La
creencia de que una dieta libre de gluten es más saludable que una dieta
con gluten no tiene soporte en un diagnóstico formal. Sin embargo, un
pequeño grupo de personas con un desorden por sensibilidad al gluten
requieren de una dieta libre de gluten para toda la vida; eso incluye no
beber cerveza. Enzimas prolil endopeptidasas (EPE) rompen las proteínas
del gluten de los residuos abundantes de prolina y tienen el potencial de
destruir proteínas de gluten y remover o minimizar la cantidad de pépti-
dos imunoreactivos de la comida. Sin embargo, el tratamiento puede
también confundir la medición de la concentración de gluten en comidas
y bebidas mediante la destrucción de epítopos usados para enumerar
péptidos de gluten, por lo que preguntamos, “¿destruye realmente el
tratamiento EPE epítopos reactivos celíacos o será que únicamente los
disfraza para que no sean detectados por ELISA?” Ahora existen suficien-
tes datos para demostrar que el tratamiento de péptidos de gluten con
EPE bacterial en combinación con otras proteasas (o también el EPE del
hongo Aspergillus niger solo) reduce la imunoreactividad de péptidos de
gluten con células-T celíacos a casi cero. Esto también está acompañado
por la destrucción de epitopos claves que son utilizados para enumerar los
péptidos de gluten en una reacción ELISA, por lo que tanto las medicio-
nes imunoreactivas como las de ELISA son reducidas a prácticamente
cero por un tratamiento EPE. Sin embargo, para tener evidencia suficiente
en cuanto a la seguridad de cerveza tratada para celíacos requiere ideal-
mente de un desafío dietético cruzado doble-ciego. No es posible consu-
mir suficiente cerveza como para presentar una carga adecuada de pépti-
dos de hordeína, y presentar las hordeínas de la misma forma como se
encuentran en cerveza tratada es muy difícil. El efecto de un tratamiento
con proteasa sobre la seguridad de gluten tratado para el restante de enfer-
medades similares (tales como ataxia gluténico y dermatitis herpetifor-
mis) y la gama mayor de trastornos relacionados con el gluten (inclu-
yendo intolerancia al gluten y las respuestas mediadas por IgE, como
asma de panadero, alergia al gluten, WDEIA y urticaria) no puede ser
evaluado definitivamente hasta que sea definido los epitopos involucra-
dos. Los trastornos de sensitividad al gluten y las proteínas del gluten son
revisados en este artículo, así como también se examina los efectos de la
proteólisis sobre varios péptidos del gluten arquetípicos.
Palabras claves: Cerveza, Enfermedad celíaca, Enzimas prolilendopep-
tidasas (EPE), Gluten, Hordeínas
There is considerable increase in the number of people adopt-
ing a gluten-free diet. The size of the global gluten-free food
industry, with a value in excess of $6 billion in 2011, is predicted
to grow by US$1.2 billion over the next five years (11). Eighteen
percent of Australian consumers now avoid certain food or drinks
for allergy or intolerance reasons (11). However, for most con-
sumers, the belief that a gluten-free diet is more healthy than a
gluten containing diet is unsupported by research (92). A gluten-
free diet may actually be more unhealthy, due to a higher fat and
sugar content and a lower content of fiber, minerals, and complex
carbohydrates (156).
For the small group of subjects who have a gluten sensitive dis-
order, a lifelong gluten-free diet is required (9), including avoid-
ance of beer. Seventy percent of Australian celiacs reported they
1 Corresponding author. E-mail:
* The e-Xtra logo stands for “electronic extra” and indicates that Figures 2, 4, and 5
appear in color in the online edition.
2014 American Society of Brewing Chemists, Inc.
Effect of PEP on Gluten Measurement in Beer / 37
used to drink beer before diagnosis and ranked loss of beer as a
significant impost of a gluten-free diet (Tanner, personal commu-
nication). Beer has been consumed in its present hopped form
since medieval times. It predominantly consists of a 1 mg/mL
solution of two dominant, water soluble, heat stable protein fami-
lies: serpin Z4 (protein Z) and lipid transfer proteins (LTP 1 and
2) that are dramatically enriched as the brewing process proceeds
(109,144) and are important determinants of the foaming proper-
ties of the finished beer (96). In addition to these dominant pro-
teins, beer contains trace levels of gluten proteins due to the bar-
ley or wheat seed storage proteins, the hordeins (38) and the
gliadins/glutenins, respectively.
Although gluten concentration is measured in parts per million
(ppm, µg/mL, or µg/g), trace gluten levels produce an adverse
reaction in celiacs, associated with a raft of adverse health out-
comes such as increased rates of intestinal malignancy, a 10-fold
increased risk of intestinal cancer, a 3- to 6-fold increase in the
risk of non-Hodgkin lymphoma, and a 28-fold increased risk of
intestinal T-cell lymphoma (70) as well as increased rates of ane-
mia, osteoporosis, neurologic deficits, and autoimmune disorders
such as diabetes (133).
The measurement of gluten (hordein) in beer is possible using
mass spectrometry (144). Gluten concentration can also be esti-
mated by enzyme-linked immunosorbent assay (ELISA); how-
ever, there is a significant problem in defining and producing an
appropriate standard to enable quantitation (143). Pre-treatment
of the beer with protease preparations can confound ELISA meas-
urements by destroying the antibody binding epitopes and dis-
guising the peptide from ELISA enumeration. Proteolysis may
not necessarily reduce the clinical immunoreactivity of the hydro-
lyzed peptides.
This review specifically addresses developments in the analysis
of hordein/gluten-derived materials in beer, especially in relation
to the effect of gluten minimization treatments such as the use of
prolylendoprotease enzymes (PEP). We conclude that treatment
of gluten peptides with PEP can reduce the immunoreactivity
celiac T-cells toward gluten peptides to near zero.
The nomenclature of the spectrum of gluten-related disorders
has recently been reviewed (118), clarifying the confusing diver-
sity of disorders (Fig. 1). Adverse reactions to gluten may be
firstly divided into T-cell mediated, autoimmune complaints in-
cluding celiac disease, gluten ataxia (75) (an unsteady gait/bal-
ance disorder), and dermatitis herpetiformis (a blistering skin
rash), which are all associated with adverse and inappropriate
reactions to dietary gluten manifested by specific and characteris-
tic damage to the small intestine, the cerebellum, and the skin,
respectively, and mediated by clonal populations of particular T-
cells that are amplified in response to a dietary challenge with one
or more of the gluten proteins.
Celiac Disease
Approximately 1% of most global populations suffer from ce-
liac disease (CD); however, recent estimates using a combined
serology analysis in Australia have revised this upward to 1.3% in
men and 1.9% in women (3). Not all celiacs exhibit symptoms; up
to 50% of adult celiacs and 90% of child celiacs have “silent” CD
and do not exhibit symptoms of intestinal discomfort (33–35,61).
They do, however, exhibit measurable damage to the intestinal
villi. The remaining celiacs can be divided into those with classic
symptoms (Fig. 1, symptomatic; the “tip of the celiac iceberg”
[35]) and those whose biology is somehow committed to develop-
ment of CD, but as yet have not manifested measurable damage
(Fig. 1, potential). Symptoms of CD manifest from weeks to years
after first exposure. Increasing rates of detection of CD are seen
in most jurisdictions, although the reasons are unknown. A transi-
ent epidemic of CD in Swedish infants was due to early introduc-
tion of gluten after breast feeding had ceased (82). Changes in the
gluten content induced by plant breeding have been recently ruled
out as a cause of increased rates of CD (90). Developments in
analysis of celiac disease have been recently reviewed (47) and
are discussed in detail below.
Gluten Allergy
The second group of gluten related disorders involve an IgE
mediated allergic response and includes respiratory allergy (Bak-
ers asthma), gluten allergy, wheat-dependent exercise-induced
anaphylaxis (WDEIA, asthma) and contact skin allergy (urticaria;
Fig. 1). Unlike the first group of complaints, the allergic complaints
involve rapid reactions from minutes to hours after exposure. IgE-
gluten allergy may be life threatening in its rapidity and severity.
Gluten Intolerance
A third group reacting adversely to gluten is those suffering
from gluten intolerance. Gluten intolerance has an unknown etiol-
ogy and is currently the subject of active investigation (18,25),
and may affect 3–4% of the population (81). Symptoms of ab-
dominal discomfort typically develop hours to days after expo-
sure, but are not associated with either IgE- or celiac phenome-
nology (27). The symptoms of gluten intolerance decrease
following withdrawal of dietary gluten and return with re-com-
mencement of gluten consumption (18). An additional double
blind crossover dietary challenge, in which low-fermentable,
poorly-absorbed, short-chain carbohydrates (FODMAPs) were
first reduced, did not reproduce the dose-dependent benefit of
reducing gluten (19).
Different classes of proteins correlate with certain disorders,
and in some cases disorders have been linked to specific amino
acid epitopes (Table I).
Wheat Allergy
All patients with wheat allergy had an IgE-mediated reaction to
gluten, in particular ω5-gliadin (148). The majority had IgE that
reacted with α-, β-, ω-, and γ-gliadins and low molecular weight
glutenin subunits (LMW-GS). A quarter of subjects also reacted to
high molecular weight glutenin subunits (HMW-GS).
Fig. 1. The spectrum of gluten related disorders (redrawn with permission
from Sapone et al [118], published by BioMed Central).
38 / Tanner, G. J., Colgrave, M. L., and Howitt, C. A.
Wheat Dependent Exercise Induced Anaphylaxis (WDEIA)
WDEIA is a complex disorder caused by gluten consumption
that sensitizes airways to subsequent exercise. The period be-
tween consumption and exercise induced asthma may be several
days. All patients with WDEIA had an IgE-mediated reaction to
ω5-gliadin (148).
Bakers Asthma
Occupational asthma (bakers asthma) is an allergy principally
to the albumin/globulin fraction of wheat, in particular α-amyl-
ase/trypsin inhibitor, and the 9 kDa LTP. Subjects also reacted to
several LMW-GS in the gluten fraction (95,108).
CD is now a well understood T-cell mediated disorder that oc-
curs when the extensive intestinal immune system reacts inappro-
priately toward dietary gluten, as if it were an invading microor-
ganism. Specific epitopes involved in provoking a celiac response
have been recently reviewed (136). There is a single principle
causative event: the deamidation of key glutamine residues in
partially digested, proline-rich gluten peptides to glutamate resi-
dues by human intestinal tissue transglutaminase (TG2, also
known as tTG) (4,121). In response to this deamidation, two pro-
cesses are set in train: a rapid response by the innate immune
system leading to the increase in intraepithelial lymphocytes, and
a slower adaptive response initiated by the binding of deamidated
gluten peptides to protein receptors on human leukocyte antigen
presenting cells (HLA-DQ2 or less frequently -DQ8) (67,140).
This stimulates a massive clonal expansion of particular T-cells,
which also carry a homing integrin that targets the intestinal epi-
thelium (134). Both innate and adaptive immune responses lead to
the destruction of intestinal mucosa and villous atrophy. In addi-
tion, the induced T-cells activate B-cells and the production of
characteristic antibodies targeted against gluten proteins and glu-
ten complexes with TG2 (135).
The introduction of a negative charge, due to the conversion of
specific glutamine to glutamate residues in gluten peptides, al-
lows the initiation of the celiac response.
The Special Case of Alpha-Gliadin, the First Cloned Prolamin
Gene, and Celiac Disease
The cloning of the first prolamin gene, α-gliadin, by Don
Kasarda (91) rapidly led to the recognition of the central role
played by deamidated glutamine residues in the recognition by
CD4+ T-cells in HLA DQ2 celiac subjects (4,8). Early epitope
mapping of α-gliadin with T-cell populations isolated from the
peripheral blood of celiacs showed that a central peptide, α-glia-
din57-73 QLQPFPQPQLPYPQPQS, could account for the bulk of
the immunoreactivity of the entire α-gliadin protein, and within
that peptide, one specific glutamine Q65 (underlined) was essen-
tial for reactivity (4). Shan (121) identified a related peptide Glia-
known as the “toxic 33mer,” as the principle immunoreactive
peptide in a closely related gene, α2-gliadin. This peptide con-
tains six overlapping T-cell epitopes that stimulate a reaction in
most celiacs, with the key glutamines underlined above. All 14
cultured, gut-derived T-cell lines isolated from adult Scandinavian
celiacs reacted to this peptide. However, many α-gliadin genes
have a deletion that covers this region of the gene and hence only
fragments of α-gliadin57-89 and α-gliadin57-73 are represented in
most α-gliadin genes (157).
Most, But Not All, Celiacs React to the Same Epitopes
The celiac response is heterogeneous, but archetypal prolamin
peptides can be identified. Camaraca showed that the response
towards prolamins of 14 cloned intestinal T-cell lines, isolated
from adult HLA-DQ2 celiac patients, was heterogeneous and
12/14 lines reacted to different prolamins (31). However, the ma-
jority of T-cell lines reacted to α-gliadins, in particular α-gliadin57-
73, α-gliadin139-153, and α-gliadin102-118. Koning showed that only
half of cultured T-cell lines isolated from children celiacs were
reactive to peptides spanning α-gliadin57-75 (154). There is also a
wide variation in sensitivity; the reactivity of T-cell populations to
the same peptides differ by a factor of 100 (145). Some sensitive
celiacs react to as little as 1 mg of gluten (16,17).
It has been 60 years since dietary gluten was identified by
William Dicke as the cause of celiac disease (53,89). Over the
past 13 years since the initial identification of the key celiac
epitopes in α-gliadin (4,121), enormous progress has been made
using non-biased populations of T-cells induced by a dietary glu-
ten challenge, isolated 6 days after the challenge from circulating
peripheral blood (5). Most importantly, these T-cell libraries
maintain the diversity of the original T-cell population and have
not been skewed by rounds of sub-culture necessary with cultured
intestinal biopsies. Massively parallel processing, investigating
the stimulation of gamma-interferon release induced in T-cells
from celiacs by synthetic prolamin peptides, has allowed identifi-
cation of all celiac immunoreactive epitopes, in all published pro-
lamins, a massive undertaking (13,151).
Epitope mapping with T-cells isolated from celiacs has identi-
fied three key immunoreactive peptides. These highly immuno-
genic peptides were derived from α-gliadin (ELQPFPQPELPYPQ
PQ), ω-gliadin/C-hordein (EQPFPQPEQPFPWQP), and B-hor-
Immunoreactive Sequences That Provoke Response to Gluten
Clinical manifestation Proteins Representative sequence Onset (118) Occurrence
Gluten (food) allergy ω5-gliadins Not known Minutes 0.1%
Bakers asthma α-amylase inhibitor and
LTP (148)
Not known Minutes to hours 0.1%
of underlined residues abolishes IgE binding (148)
Minutes to 1 day 0.1%
Non-celiac gluten
Gluten (18) Not known Hours to days 6-10% (18)
Irritable bowel syndrome Wheat (158,161), beef,
pork, and lamb (158)
Not known Hours to days 10%
Celiac disease HMW and LMW-GS,
hordeins, avenins,
secalins, avenins (in
some subjects) (151)
α-gliadin57-73: QLQPFPQPQLPYPQPQS; α-gliadin: Glia257–89
ELQPFPQPELPYPQPQ; ω-gliadin/C-hordein:
Weeks to years 1%
Effect of PEP on Gluten Measurement in Beer / 39
dein (EPEQPIPEQPQPYPQQ), and account for 90% of the celiac
specific response elicited by the full complement of wheat, barley,
and rye proteins (151).
There are many possible celiac immunoreactive epitopes, but
representative archetypal prolamin peptides can be identified.
In addition to the diversity in gluten related disorders, there is a
complex diversity in the primary structures of the gluten like pro-
teins (Fig. 2), which are collectively known as prolamins, due to
the high frequency of prolines and glutamines in the primary se-
quence. Prolamins are a family of closely homologous, alcohol-
soluble, seed storage proteins consisting of gluten in wheat (Triti-
cum spp. L., composed of a mixture of gliadins and glutenins),
hordeins in barley (Hordeum vulgare L.), secalins in rye (Secale
cereal L.), and avenins in oats (Avena sativa L.).
Unfortunately, there is not a single gluten protein; rather, wheat
gluten is a complex mixture of several hundred related proteins,
containing members of the monomeric α-, γ-, and ω-gliadins and
the high and low molecular weight glutenins, which form poly-
mers in vivo (126). The hordeins consist of four protein families:
the B-, C-, D-, and γ–hordeins. The B- and C- hordeins account
for 70% and 20% of the hordeins, respectively, while the D- and
γ-hordeins are minor components accounting for less than 1% and
5% of total hordeins, respectively (127). The B- and C-hordeins
are both multi-gene families with 2D protein gels showing up-
wards of 10 individual B- and C-hordeins. The D- and γ-hordeins
are coded for by one and three genes respectively, with 2D protein
gels showing approximately five D-hordein isoforms.
The secalins are also multi-gene families (117,123,147) of four
protein families with the prolamins accounting for 65% of seed
protein, and within that, the γ-75k secalins accounting for about
half of the prolamin, followed by γ-40k secalins (24%), the ω-
secalins (17%), and HMW secalins at 7% of prolamin (66).
The oat avenins are multi-gene families of at least 20 proteins
(125), with homology to the α- and γ-gliadins of wheat, the B-
hordeins of barley, and the γ-secalins of rye (114). These genes
are distributed across a single chromosome (151) and do not con-
tain homologous sequences to the gliadin-33-mer or -17-mer
(114); however, they do contain immunoreactive peptides QQPFV
QQQQQPFVQ and QQPFMQQQQPFMQP with the repetitive
epitopes PFVQQQ and PFMQQQ (114).
All of the above prolamins are immunoreactive with celiac T-
cells as they share repeated runs of amino acid sequence with
other celiac immunoreactive prolamins. However, it has now been
clearly established that approximately 10% of celiacs have a gen-
uine T-cell mediated reaction to avenins (80,138,142,151). The
reaction of an individual celiac depends upon the concentration of
immunoreactive prolamins and the degree of immunoreactivity of
the prolamins. The published prolamin content for the full suite of
cereals and pseudocereals discussed here was unclear and was
therefore determined using standard techniques (Table II). The
absolute prolamin concentration decreases from wheat and barley
to rye to oats with about 35% of total protein as prolamin, except
in oats. This in part explains why some cereals are less immuno-
reactive than others, e.g., oats are less immunoreactive with most
celiacs than are wheat and barley. The 10% of celiacs who react
to oats may represent a cohort of celiacs who are very sensitive
and react to the low level of avenins found in oats or they may be
subjects who can mount a T-cell response to avenins for other
reasons. Thus, consumption of uncontaminated oats is suitable for
most, but not all celiacs.
Prolamins are, by definition, alcohol soluble proteins with a
high proportion of glutamine and proline. Such prolamins also
occur in the gluten-free grains such as maize (Zea mays L.), rice
(Oryza sativa L.), and sorghum (Sorghum bicolor L. Moench),
which are distant relatives of wheat (Fig. 2); however, these prola-
mins are distantly related to the gluten proteins of wheat. The
gluten-free cereals maize, sorghum, and rice diverged from the
common line 50 million years before the gluten-containing cere-
Fig 2. Distribution of gluten proteins in the cereal grains (redrawn from Colgrave et al [39] with permission). Celiac patients react to immunoreactive
prolamins (in red) produced by members of the Triticae and Avenae tribes, including Triticum aestivum (wheat), Secale cereale (rye) and
vulgare (barley). Wheat and oats are hexaploid which complicates traditional breeding. A small percentage of celiacs also exhibit a genuine reaction to
avenins from Avena sativa (oats) and maize (Zea mays). Beer is produced by brewing and fermentation of sugars derived from cereal grains, most com-
monly barley and wheat, but rice, corn, millet, sorghum and oats are often used as adjuncts. Beer can also be brewed from the non-cereal grains, such as
40 / Tanner, G. J., Colgrave, M. L., and Howitt, C. A.
als evolved (90). Maize prolamins (zeins) may provoke a celiac
response in some celiacs (30), but are generally considered safe
for most celiacs to consume (105). The prolamins from rice (105)
and sorghum (112) do not contain homologous sequences to the
33-mer gliadins or the 17-mer-gliadin and also lack the extensive
and repetitive PSQQ and QQQP epitopes present in wheat glia-
dins and glutenins (14). Rice and sorghum do not provoke a celiac
response. The amount and proportion of total protein represented
by alcohol soluble protein in maize and sorghum is lower than
wheat and barley and is lowest in rice (Table II).
Beer may be brewed using the gluten-free grains sorghum, mil-
let, and teff (94). Millet (Eleusine coracana) and teff (Egrostis
tef) are members of the Chloridae family and are also distantly
related to wheat (146) (Fig. 2). The alcohol soluble proteins in
these grains are considered to be safe for celiacs (25,105) and the
level and proportion of alcohol soluble protein are similar to the
level in the gluten-free cereals, with the notable exception of teff,
which had the highest alcohol soluble protein level of all tested
grains (Table II).
In rice, there are three families of prolamins (sometimes called
oryzeins): the 10 kDa, 13 kDa, and 16 kDa prolamins encoded by
single, multiple (up to six), and single genes, respectively (128).
In maize, the 22 kDa and 19 kDa zeins are encoded by large
multi-gene families with over 20 members (137). In sorghum,
there are four families of kafirins (46): α-kafirins (the most abun-
dant, 80–85% of total kafirin) at 23 and 25 kDa, β-kafirins (7–
13%) at 19 kDa, and γ-kafirins (10–20%) at 20 kDa (112). A
fourth group of kafirins, related to the δ-zeins of maize, has been
identified from cDNA sequences (14).
In addition to the gluten-free cereals above, chia, quinoa, buck-
wheat, and amaranth are also commonly used as gluten-free
grains (62), but are pseudograins and not true grasses. Buckwheat
(Fagopyrum esculentum) is related to rhubarb. Quinoa (Chenopo-
dium spp.) is related to beets and spinach, whereas chia (Salvia
hispanica) is related to the mint family (152). The Amaranth fam-
ily (Amaranthaceae) consists three main species, Amaranthus
caudatus (best known as the ornamental “Loves-lies-bleeding”),
A. cruentus, and A. hypochondriacus (41), also related to spinach
and sugar-beet.
The prolamins in the gluten-free grains and pseudograins are
less well studied than the gluten proteins; however, most do not
contain the epitopes known to provoke celiac disease and other
gluten related disorders and are generally considered safe for celi-
acs to consume (11,12,105). In addition, the concentration of
alcohol soluble protein and the proportion of total protein in these
grains is substantially lower than in the gluten positive cereals and
resembles that seen in rice (Table II). However, these grains may
not be universally safe—a detailed study of quinoa showed most
cultivars did not possess quantifiable amounts of celiac-toxic
epitopes by ELISA, but two cultivars had celiac immunoreactive
epitopes that could activate immune responses in some patients
with celiac disease (159).
The central element in explaining the celiac pathogenesis of
various peptides lies in their susceptibility to be deamidated by
Fritz Koning’s group recognized that the spacing between
glutamine and proline, the second most abundant amino acid in
gluten, played an essential role in the specificity of deamidation
(153). They identified several algorithms for predicting TG2 de-
amidation of Q by mass spectrometry (MS) analysis of TG2
treated peptides. The epitope numbering scheme for TG2 and
DQ2(8) follow that established for proteases (Fig. 3). In se-
quences containing QP and QXXP, the Q was not a target for
TG2. In contrast, the sequences QXP, QXXZ where Z = Y, W, M,
L, I, or V and QXPZ (Z as above) favor deamidation of the Q.
Two predictive algorithms were identified that could identify de-
amidation of Q4 residues:
First algorithm XXXQ4XP6Q7XZ (Z = YFWIL)
Second algorithm XXXQ4XPQ7XPZ (Z = YFWIL)
Similar peptides with a consensus sequence of QXP, where the
Q was deamidated, were also identified by a separate mass spec-
trometry approach (59,99,153).
The above algorithms commonly reveal matches in the gluten,
hordein, and secalin prolamins, whereas the avenins rarely reveal
matches explaining the relative lack of immunotoxicity of avenins
Total Protein and Prolamin Content of Cereals
and Pseudocereals, Ranked by Prolamin Content
Grain Total proteina Prolaminb ± SE % Prolaminc
Teff 13.81 ± 0.25 4.80 ± 0.14 35%
Barley 14.46 ± 0.18 4.52 ± 0.13 31%
Wheat 14.11 ± 0.09 4.46 ± 0.12 32%
Sorghum 9.01 ± 0.08 3.35 ± 0.03 37%
Rye 8.79 ± 0.04 3.28 ± 0.01 37%
Maize 7.61 ± 0.10 3.10 ± 0.05 41%
Millet 12.13 ± 0.52 3.08 ± 0.06 25%
Chia 24.05 ± 1.65 2.84 ± 0.07 12%
Oats 11.58 ± 0.31 1.84 ± 0.03 16%
Buckwheat_Hitachi 8.81 ± 0.11 1.84 ± 0.09 21%
Amaranth 15.71 ± 1.17 1.82 ± 0.04 12%
Buckwheat_Botan 8.31 ± 0.37 1.77 ± 0.05 21%
Quinoa 12.73 ± 0.95 1.29 ± 0.12 10%
Rice 7.71 ± 0.24 1.20 ± 0.16 16%
a Mean total protein (mg/100 mg flour) was determined in triplicate by
DUMAS and is calculated as 6.63N ± SE.
b Mean prolamin ± SE, was determined as alcohol soluble proteins (mg/100
mg flour) extracted from 20 mg triplicate whole-meal flour samples, in 0.5
mL solution of 50% (v/v) isopropyl alcohol, 1%(w/v) DTT, for 30 s in a
Savant Beadbeater and sedimented. Extraction was repeated twice more and
the supernatants combined and protein measured by Bradford (21) calibrated
against gamma-globulin standard.
c Mean prolamin is expressed as % of mean total protein.
Fig. 3. Protease substrate binding (redrawn with permission from
Simpson [130]). Each amino acid residue in the target peptide is num-
bered consecutively from P4 to P3, and fits into a substrate binding pocke
correspondingly numbered S4 to S3. Cleavage occurs at the scissile bond
(arrowed) between P1 and P1.
Effect of PEP on Gluten Measurement in Beer / 41
The binding mechanism of HLA-DQ2 to a particular proline
(P) in the alpha-gliadin peptides has been deduced by X-ray crys-
tallography of DQ2.5-glia-α1a (DQ2-QLQPFPQPELPY) protein
complex (93). The proline at P6 (P above) is an important anchor
residue as it participates in an extensive hydrogen-bonding inter-
action. The nine-amino acid core sequences of celiac disease rele-
vant gluten CD4+ T-cell epitopes, restricted by HLA-DQ2 and
-DQ8 molecules, has been recently summarized (136). The gluta-
mate introduced by TG2 was usually in position 4 (P4), P6, or P7
in HLA-DQ2.5 restricted epitopes. Proline dominates at P1, P6,
and P8. In addition, there are several databases where T-cell
epitopes are listed, including and www. Various software solutions also exist for predicting
T-cell epitopes in peptide strings, as reviewed (26). In addition, a
scheme that predicts the relative toxicity of proteins, as measured
by the density of T-cell epitopes on particular proteins, has been
developed to identify the epitopes involved in wheat allergy and
celiac disease (85,86).
The epitope specificity of DQ2 and DQ8 T-cell receptors may
be conveniently summarized using web logo software (42) as in
Figure 4, which shows the most common epitopes as well as cap-
turing the epitope diversity of these receptors. The frequency of
each amino acid in a given position is proportional to the height
of each amino acid single letter code in the figure. It can be seen
that the most common DQ2 epitope is PQPEQEQ(F)PQ, where a
Q or F is equally encountered at the seventh residue (Fig. 4 up-
per). Similarly, the most common DQ8 epitope is EGP(S,Y)F(Q,
Y)QPSPE where either a P or S or Y is encountered at the third
residue, and F or Q or Y is encountered at the fourth residue (Fig.
4 lower).
The global gluten-free food industry had a value in excess of $6
billion in 2011, predicted to grow by US$1.2 billion over the next
five years (11). Regulation of the upper limit of gluten in gluten-
free food is managed by a number of jurisdictions worldwide,
recently reviewed (48) and which all tend to follow the lead set by
the Food and Agriculture Organization of the United Nations
(FAO) and laid down in the Codex Alimentarius, the guiding leg-
islation regulating food safety (37). The Codex standard states
that gluten in gluten-free food shall not exceed 20 ppm. This ex-
panding economic market currently depends on two approved
antibodies for validation of the gluten-free status of food prod-
ucts. These two ELISA sandwich kits have been ring-tested and
accepted by the FAO for measuring gluten concentrations in flour
and food (124,131).
First Generation Antibodies
The key first generation ELISA kits are based on one of two
The Mendez R5 antibody (RidaScreen) (74,104) uses the
mouse monoclonal R5 antibody raised against rye secalins
by Mendez (88) and which recognizes QQPFP, QQQFP,
LQPFP, and QLPFP epitopes.
The second FAO accepted sandwich ELISA kit is based on
the Skerritt antibody (131) (ELISA Systems & Tepnel, now
acquired by Neogen) that uses the mouse monoclonal anti-
body MAb41201, which is functionally equivalent to
M12224 raised to wheat ω-gliadins (79) and recognizes the
epitopes PQPQPFPQE and PQQPPFPEES (132).
Many similar antibodies have been raised against defined glu-
ten fractions. Other monoclonal antibodies (lFRN 0061 and
00614), which bind to the S-poor prolamins of barley, wheat, and
rye, have a high affinity for the major repeat motif, PQQPFPQQ
present in the LMW glutenins of wheat (24). Panels of antibodies
have also been developed against HMW glutenin subunits (106).
Five monoclonal antibodies were raised against an enriched C-
hordein fraction and were specific for the members of the sulfur-
rich hordein family including C-, B-, γ-1, and γ-2 hordeins (115).
The detection of gluten levels with ELISA calibrated with a
commercial gliadin preparation can be accurate in some circum-
stances, e.g., the detection of wheat gluten in a series of deliber-
ately contaminated oat samples by various sandwich ELISA kits
was recently examined (2) with only the RidaScreen kit accu-
rately detecting gluten at zero, 20, and 100 ppm contamination
levels. However, confusion is encountered when an ELISA read-
ing, commonly calibrated with a commercial gliadin standard, is
converted to total gluten concentration by doubling the gliadin
concentration (50), since gliadin in wheat is roughly half of the
sum of gluten and gliadin. Such an approximation does not hold
Fig. 4. Amino acid epitope specificity of DQ2 (upper) and DQ8 (lower)
T-cell receptors. The figure summarizes the frequency that the indicated
amino acid occurs in the designated position of the 9 amino acid T-cell
epitope (136). The height of the symbols is proportional to the frequency
that the designated amino acid is found in the indicated position. The
graphical representations were generated with the WebLogo software tool
v2.8.2 (42).
42 / Tanner, G. J., Colgrave, M. L., and Howitt, C. A.
for barley, rye, oats, or even some wheat lines. The effect of anti-
body specificity, extraction procedures, and different reference
materials on the accuracy of gluten quantitation, as opposed to the
reproducibility of the determination, has been recently discussed
However, accurate measurement of different prolamins remains
a thorn in the side of ELISA techniques. The development of
standardized gluten sources represents a step in the right direction
toward accurate determination of gluten in flour. However, this is
confounded because there is not a single gluten protein, but many
hundreds in biological materials and the proportion of each pro-
tein varies with plant growth conditions (1) and genotype. The
definition of a suitable gluten standard for beer is even more diffi-
cult because of the modification of gluten proteins produced by
the brewing process and the enormous relative enrichment of heat
stable and water soluble proteins during brewing (83,109). Detec-
tion of hordeins in malt and beer by sandwich and competitive
ELISA has been reported (54,55,103); however, these determina-
tions are calibrated against commercial wheat gliadins, which are
unrepresentative of hordeins that have been malted and mashed.
Second Generation Antibodies
A second developmental phase of anti-gluten reagents involves
antibodies raised against biologically relevant peptides involved
in anti-gluten biochemistry such as the G12 monoclonal antibody
directed against the toxic 33mer (76) of α-gliadin (40,76), which
encodes six partially overlapping T-cell epitopes and is a potent
stimulator of T-cells (121). This peptide also contains the α-glia-
din56-75 that has been identified as the dominant immunoreactive
peptide for celiacs (4). Measurement of this peptide in beer may
indicate relative levels of immunoreactivity of peptides in beer
Various hordein preparations are detected with dramatically dif-
ferent sensitivity by anti-gluten antibodies (Table III) (143). The
dissociation constant (Kd) for a given ELISA reaction with differ-
ent hordeins can vary by three orders of magnitude when com-
pared to the Kd of commercial gliadin preparations supplied with
the ELISA kits. The Kd of the same hordein determined by
ELISA using different antibodies varied by up to two orders of
magnitude. For example, commercial gliadin preparations were
detected equally by both antibodies, but C-hordeins from RisØ 56
or cv. Sloop were detected with 25- and 28-fold greater sensitiv-
ity, respectively, by the ELISA system kit than the RidaScreen kit.
Furthermore, the reaction of the ELISA system kit towards C-
hordeins from cv. Sloop were detected with 230-fold higher sensi-
tivity than were B-hordeins from cv. Sloop. It is clear that differ-
ent hordein preparations are not detected equally by ELISA.
Accurate determination of hordein requires that the hordein
standard used to calibrate the ELISA reaction be identical in com-
position to the hordeins present in the test substance. In practice,
this is almost impossible to arrange, as the detailed prolamin com-
position of the food being analyzed is almost always unknown.
Modification during processing can also occur (144). The use of a
commercial gliadin preparation is convenient and reproducible,
but can lead to gross under- and over-estimation of gluten levels if
the hordein composition is unknown.
Recent comparison of all seven commercially available sand-
wich ELISA kits suggested that the main limiting factors of
method validation was the lack of reference materials (28,29). An
early suggestion to use the peptide LGQQQPFPPQQPY is a par-
tial solution (36) to the problem of quantitating ELISA reactions,
as are the use of second generation antibodies, which bind to pep-
tides involved in the celiac response.
The use of competitive ELISA has been recommended as more
reproducible, particularly for measuring gluten in beer (72,74).
However, the problems encountered in calibrating sandwich
ELISA assays are also encountered with competitive ELISA as-
says. In this case, the competing protein added to displace the
measured protein from the bound antibody must have the same
composition as the protein being measured; a requirement un-
likely to be addressed by a single commercial standard.
It is important to measure apples with apples: The ELISA
standard must be the same as the peptide being measured (143).
The detection of gluten by MS offers the prospect of avoiding
the pitfalls involved with calibrating and comparing the many and
various ELISA methods in use to measure gluten in foodstuffs. In
addition, MS can detect and differentiate between proteins arising
from wheat, barley, rye, and oats (48,49). An example of how
detection and confirmation of prototypic peptides by multiple
reaction monitoring (MRM) is accomplished is depicted in Figure
5. The m/z of the parent ion, a quadruply charged ion from a γ-1-
hordein tryptic peptide, QGVQIVQQQPQPQQVGQCVLVQGQ
GVAQPQQLAQMEAIR, may be detected as 1074.54. This ion is
selected in the first quadrupole of a triple quadrupole mass spec-
trometer. The parent ion is fragmented in a process known as
collision-induced dissociation (result of peptide collision with
stationary gas molecules) in the second quadrupole of the mass
spectrometer. The collisions result in dissociation, or fragmenta-
tion, of the parent ion that occur at the peptide amide bonds. Frag-
mentation in this type of mass spectrometer occurs in a predicta-
ble pattern producing a ladder of y-ion fragments, where charge
remains on the C-terminal fragment, and b-ion fragments, where
charge remains on the N-terminal fragment. The masses of the
fragments can be measured and selected in the third quadrupole
of the mass spectrometer. In this way, only peptides that have both
the parent ion mass (Q1 mass) and fragment ion mass (Q3 mass)
will pass through to the detector for quantification. This pair of
ions (Q1-Q3) is known as an MRM transition. In practice, it not
necessary to detect all the fragment ions. One MRM transition is
Comparison of Kd of Purified Prolamins for ELISA Systems
and RidaScreen Sandwich Assays (Reproduced
from Tanner et al [144] with Permission)
Hordeina ELISA Systems
Kd ± SE (ppb) RidaScreen
Kd ± SE (ppb)
Intra-antibody variation
ULG 2.0_T 57 ±0.7 Not detected
Risø 1508_T 64 ±9.0 Not detected
Sloop_C 84 ±0.7 15.8 ±0.3 5.3
Risø 56_C 212 ±24 7.6 ±0.3 28
Risø 56_T 261 ±58 10.5 ±0.2 25
Gliadin 343 ±6.0 390 ±15 0.9
Sloop_T 670 ±160 26 ±1.0 26
Risø 56_γ 3,640 ±180 560 ±90 6.5
Sloop_B 19,400 ±3,600 2,100 ±360 9.2
a The suffix _T refers to total hordein; _C refers to FPLC purified C-hordeins;
_γ refers to FPLC purified γ-hordeins; _B refers to FPLC purified B-
hordeins. Commercial gliadin standards used are denoted as Gliadin. Serial
dilutions of purified hordein from the indicated fractions were added in the
range 10 parts per billion (ppb) to 2 ppm, sufficient to produce a hyperbolic
response (when plotted on linear axes) for each ELISA kit. The Kd (dose
required to produce a half maximal response) was determined from the
hyperbolic curve of best fit using GraphPAD Prism. The experiment was
repeated twice and the average Kd ± SE is reported.
Effect of PEP on Gluten Measurement in Beer / 43
used as the quantifier, e.g., the intense y11 ion in this example,
and two additional ions (b9 and b11) are selected as qualifiers,
i.e., to confirm the peptide identity. There are several calibration
strategies for MS. The simplest approach is standard addition, i.e.,
to spike increasing aliquots of purified gluten fractions into the
unknown solution. The concentration of each prototypic peptide
is determined by the method of standard addition. A more elegant
solution is to spike known amounts of synthetic stable isotopi-
cally labeled protetypic peptides into the unknown solution and
determine the unknown concentration by direct comparison of
peak heights (or areas) in the MS. This has the advantage of com-
paring identical peptides that are analyzed simultaneously. The
isotopically labeled peptides coelute, but are distinguishable from
the unlabeled (native) peptides by a predictable mass difference.
Comparison of the peak ratios allows quantification of the native
peptides and hence proteins.
An immunoreactive peptide can be cleaved at one site, allowing
the daughter peptides to remain immunoreactive toward celiacs
(Fig. 6A). This single cleavage may disguise an ELISA epitope
from enumeration, preventing the use of ELISA to estimate resid-
ual immunoreactivity (Fig. 6B). HPLC and MS can be used to
indicate loss of peak height due to cleavage of the parent peptide,
but the resultant daughter peptides may elute elsewhere on the
HPLC chromatogram and not be recognized by the operator (Fig.
6C). A mass spectrometer can be programmed to look for particu-
lar ions (Fig. 5) and in doing so provide both quantification and
confirmation of the sequence of the proteolytic fragments. The
immunoreactivity of the daughter peptides can be estimated by
sequence inspection or by an algorithm, but the most convincing
evidence is provided by the lack of reaction of celiac specific T-
cells in small scale biochemical immunoscreening (Fig. 6D).
Celiac specific T-cells may be isolated from circulating periph-
eral blood 6 days after a 3 day dietary challenge (6). They may
also be isolated from cultured intestinal biopsies (110). In the
presence of an immunoreactive epitope, T-cells react by dividing,
as estimated by the uptake of radioactive thymidine, or by cell
counting. Stimulated T-cells also release gamma-interferon. The
proportion of a population of T-cells that release gamma-inter-
feron after a short culture period (typically overnight and less than
one cell cycle) with the test peptide may be enumerated using
gamma-interferon specific immunoplates (IFN-γ ELISPOT; Fig.
6D) (5). The higher the proportion of a T-cell population reacting
to a given peptide, the higher was the immunoreactivity of that
peptide. Peptides must be first pre-treated with protolytic en-
zymes including trypsin, chymotrypsin, and elastase to resemble
intestinal digestion. Deamidation of digested peptides with puri-
fied TG2 increases the immunoreactivity (93) and the T-cell prep-
aration normally contains sufficient antigen presenting cells to
transfer the restricted peptide to the appropriate T-cells.
On a large scale, the best indication of lack of immunoreactive
peptides is obtained using a double-blind dietary challenge cross-
over experiment where a sufficient number of established celiacs,
who are maintaining a gluten-free diet, are fed the treated or un-
treated food over time. About 20 subjects are often chosen and
divided between two treatments. However, if an infrequent section
of the celiac population is being searched for, such as the propor-
tion of celiacs who react to oats, larger numbers of subjects are
needed to ensure adequate detection of the trait. After the treat-
ment period, the roles are reversed. The length of the treatment
period is difficult to judge. Sufficient gluten must be ingested to
invoke a celiac response within the treatment period. Dose/treat-
ment periods vary from 20 g gluten daily for 3 days (sufficient to
mount a T-cell response in circulating blood); 3 g gluten daily for
14 days (sufficient to elicit mucosal damage in 75% of subjects
[97]); or longer periods (up to 90 days) have been used with
smaller dietary challenge of 50 mg gluten/day, provoking a sig-
Fig. 5. Mass spectrometry detection of prototypic peptides for quantification by multiple reaction monitoring (MRM). The m/z of the precursor ion of a
γ1-hordein peptide, QGVQIVQQQPQPQQVGQCVLVQGQGVAQPQQLAQMEAIR, may be predicted or experimentally determined. In this example,
the m/z is 1074.544+. Fragmentation of the precursor ion by collision-induced dissociation yields a ladder of peptide fragment ions, typically b- and y-
ions. MRM transitions are selected either from the experimental data (MS/MS spectrum) or determined bioinformatically and consist of pairs of precur-
sor ion m/z and fragment ion m/z values. Typically, three MRM transitions are selected per peptide: the most intense transition acts as the quantifier,
whereas the remaining two transitions act as qualifiers, i.e., to confirm the identity of the peptide.
44 / Tanner, G. J., Colgrave, M. L., and Howitt, C. A.
nificant decrease in villous height/crypt depth (32). The gold
standard for lack of immunotoxicity of dietary components is
shown by unchanged small intestinal histology and lack of villous
atrophy, measured by the median villous height/crypt depth in the
biopsies of small-intestinal mucosa. This can be accompanied by
low intraepithelial lymphocyte counts and low and unchanged
serology indicators including anti-TG2, anti-endomysial, and
anti-gluten antibodies without an increase in celiac reactive T-
cells in peripheral blood 6 days after dietary challenge (5). The
above indicators are well understood for subjects with celiac dis-
ease. However, the proportion of consumers avoiding gluten for
other reasons, e.g., gluten intolerance, is approximately 10 times
larger than those who suffer from celiac disease. At present, glu-
ten intolerance is not accompanied by established and well known
clinical indicators (18,19).
Proteases are characterized by the amino acid at the active site
and their specific inhibitors. For example, cysteine proteases (EC
3.4.22) have a cysteine at the active site and may be specifically
inhibited by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane
(E64). Similarly, aspartic proteases (EC 3.4.23) are inhibited by
pepstatin A; serine proteases (EC 3.4.21) are inhibited by 4-(2-
amino-ethyl)benzenesulfonylflouride (AEBSF). Metalloproteases
(EC 3.4.24) are inhibited by metal binding compounds, e.g., 1,10-
phenanthroline and EDTA. Further, endoproteases attack internal
peptide linkages while exoproteases attack only from the N-termi-
nus (aminopeptidases) or C-terminus (carboxypeptidases) (84).
Proteases are often synthesized as an inactive pro-protease, which
is activated by a specific internal cleavage.
During malting, prolamins are exposed to up to 42 endoprote-
ases, including 27 cysteine endoproteases, seven serine endopro-
teases, four metalloproteases, and four aspartate endoproteases in
green (unroasted) barley malt (160). In addition, there are a vari-
ety of human digestive enzymes such as trypsin, chymotrypsin,
and pepsin that may act on prolamins during digestion.
Protease substrate specificity is determined by the order of spe-
cific amino acids (Fig. 3; P4 to P4; with cleavage at the scissile
bond between P1 and P1) that bind to the substrate pockets of the
protease (Fig. 3; S4 to S4).
This experiment has been unintentionally conducted many times:
it is carried out each time a celiac or gluten intolerant inadvertently
consumes a malted barley product and feels poorly. On this basis, it
is unlikely that the native barley proteases or human digestive
enzymes can destroy immunoreactive peptides as consumption of
malted products invariably induces symptoms in celiacs.
This supposition is reinforced by examination of predicted
cleavage patterns for a variety of archetypal celiac immunoreac-
tive epitopes (Table IV):
α-gliadin-257-73, QLQPFPQPQLPYPQPQS (4,91)
α-gliadin57-89 (toxic33mer),
α-gliadin: ELQPFPQPELPYPQPQ (151)
ω-gliadin/C-hordein EQPFPQPEQPFPWQP (151)
The question we have to answer is simply: Does cleavage of
gluten by a protease result in a sequence that can be deamidated
by TG2, i.e., have a QXP core and bind to HLA-DQ2(8)? If so,
the product is likely to remain immunoreactive toward celiacs.
Determination of the treated amino acid sequence by MS would
establish the safety of treated gluten proteins.
Fig. 6. Shortfalls in gluten analysis. Proteolysis (arrows) may destroy amino acid epitopes recognized by ELISA, leaving T-cell epitopes either intact (A)
or hydrolyzed (B). HPLC analysis (C) may not recognize daughter peptides, particularly in a busy chromatogram typically seen in HPLC of proteolysis
products. Toxicity of peptides is best measured with an unselected population isolated from circulating peripheral blood, 6 days after a dietary challenge
(5), using gamma-interferon ELISPOT and estimating the proportion of T-cells that release gamma-interferon when presented with a immunoreactive
peptide (D). Gamma-interferon releasing cells may be enumerated by ELISPOT following a short incubation. T-cell division may be estimated by thymi-
dine uptake or by cell counting after a longer incubation.
Effect of PEP on Gluten Measurement in Beer / 45
Human Digestive Enzymes
The cleavage pattern of prolamins with other proteases can be
reliably predicted by various algorithms, e.g., ExPASy “Peptide
Cutter” ( and the resultant
peptides examined for potential immunoreactivity with celiac T-
cells (Table IV). Examination of the predicted cleavage patterns
of the archetypal gluten peptides shows that, although all peptides
are cut to some degree by the array of proteases, all proteolysis
products remain immunoreactive to celiac T-cells. This is illus-
trated with the α-gliadin2-33mer, which is cleaved by chymotryp-
sin (three cleavages) and pepsin (six cleavages; Table IV). All
proteolytic products contain immunoreactive epitopes and are
likely to provoke a response in celiacs. Similar cleavages are ob-
tained with all the archetypical peptides, resulting in predicted
immunoreactive hydrolysis products.
The Malt Proteases
The cysteine endoproteases and the metalloproteases appear to
account for bulk of hordein degradation during germination/
malting; however, most of the proteases, including the cysteine,
serine, and metalloproteases, are inhibited by endogenous protein
and non-protein factors, confounding estimation of total activity
in crude extracts (84).
Cysteine endoprotease (EP-A and EP-B). EP activity ac-
counts for 90% of the proteolytic activity in germinating barley
and accounts for up to 90% of the total activity in degrading pro-
lamins, with EP-B being one of the major enzymes responsible
for degrading hordeins (130). Hordeins are naturally cleaved by
two endoproteases: EP-A (three isoforms) and the more abundant
EP-B (two isoforms), both present in green barley malt. The
cleavage specificity of purified EP-A and EP-B was determined
by Davey using C-hordein with cleavage occurring at FRQQ,
FQQP, VQQP, LQQP, and LQSP. Specificity was subse-
quently also determined with 2-aminobenzoyl-P2-P1-P1- P2-
tyrosine(NO2)-aspartic amino acid substrates (44,45), which
showed that the cysteine EPs preferred Phe, Leu, or Val at P2. Arg
was preferred over Gln at P1, whereas Pro at P2, P1, or P1
greatly reduced activity. In general, EP-B2 has a similar specific-
ity to TG2 and cleaved gluten peptides at QXP (129) residues.
Separate in vitro experiments with rat intestine showed the EP-B2
cleaved peptides lost the capacity to bind to HLA-DQ2 (and
-DQ8) and in the presence of PEP, gluten was completely de-
stroyed and detoxified as judged by HPLC, T-cell proliferation,
and ELISA (64,129). The EP-B enzymes are synthesized during
germination and are excreted into the starchy endosperm (101); it
is likely that this enzyme plays a significant part in the degrada-
tion of hordeins during germination. Added to celiac diets and
supplemented with PEP enzymes, it could play a useful role in
reducing the immunoreactivity of gluten peptides.
Metalloproteases. Metalloprotoease activity accounted for 9%
of the proteolytic activity in malt and the purified metalloprote-
ases degrade C- and D-hordeins in vitro; however, the presence of
metalloprotease inhibitors in malt has led to conflicting results in
studies of these enzymes (84).
Serine carboxypeptidases (Ser-CP). Ser-CP are the major
exopeptidase in germinating cereals. At least two serine-carboxy-
peptidases (Ser-CPs) are also expressed de novo in the germinat-
ing barley grain and secreted into the endosperm, where they are
involved in cleavage of hordeins (43). The dominant Ser-CP, malt
carboxypeptidase III, cleaves the C-terminal peptide bond be-
tween a Phe, Met, Ile, or Val in the P1 position and Phe, Leu, Met,
or Ala in the P1 position (22). The other Ser-CP, malt carboxypep-
tidase II, exhibits a strong preference for substrates containing
Lys and Arg as C-terminal amino acid residues, but it also hydro-
lyzes substrates with hydrophobic amino acid residues in this
position (23). Cleavage at these residues is unlikely to destroy the
archetypal celiac immunoreactive epitopes. It is unlikely that the
Ser-CP play a role in degradation of hordeins during malting.
Aspartate endoproteases (HvAP). The aspartic protease from
barley, HvAp, appears to consist of four isoforms, but they are not
secreted into the endosperm and do not appear to be involved in
mobilization of hordeins during malting (101).
Serine endoproteases. Serine endoprotease-1 (60), which is
not secreted into the endosperm, and the serine protease hordoly-
sin (149) both appear not to be involved in hordein degradation in
malted barley.
Dietary supplementation with PEP (100,119) and germinating
cereal proteases (64,129,139) and EP-B2 (above) appear to be a
useful means of minimizing immunoreactive peptides in the ce-
liac diet. The reaction of whole gluten in food matrices in the
intestine is complex and not exactly predicted from the results of
in vitro proteolysis of purified protein fractions; however, in vivo
studies are encouraging, as recently reviewed (155).
Using mass spectrometry, HPLC and ELISA with a monoclonal
antibody that recognizes an immunodominant gluten epitope, and
a T-cell proliferation assay, Ehren et al (58) showed that simulated
digests of whole gluten and whole wheat bread were digested and
detoxified effectively by aspergillopepsin from Aspergillus niger
supplemented with dipeptidyl peptidase IV from A. oryzae.
The Special Case of Aspergillus niger Prolylendopeptidase
Prolyl oligopeptidase (87) (POP) and prolylendopeptidase
(sometimes prolylendoprotease, post-proline cleaving enzyme,
PEP) are serine proteases (EC belonging to the S9A
peptidase family, which hydrolyze peptide bonds at the carboxyl
side of internal prolines.
POP enzymes are found in small quantities in mammals, where
they are involved in cleavage and maturation of peptide hormones
and neuropeptides, and have pharmaceutical relevance to depres-
sion, blood pressure, and amnesia. Crystal structures (87,120)
have shown that movement of a seven-bladed propeller gating
Cleavage of α-Gliadin57-89 by Human Digestive Enzymesa
Name of enzyme No. of cleavages Positions of cleavage sites Toxic products
Chymotrypsin-high specificity (C-term to [FYW], not before P) 1 33 Yes
Chymotrypsin-low specificity (C-term to [FYWML], not before P) 3 1,3,33 Yes
Pepsin (pH>2) 6 1, 2, 11, 18, 25, 32 Yes
a Cleavages predicted by Peptide Cutter (http// cutter/) are indicated. These enzymes do not cut: Arg-C proteinase; Asp-N endopeptidase;
Asp-N endopeptidase + N-terminal Glu; Caspase1-10; Chymotrypsin-high specificity (C-term to [FYW], not before P); Glutamyl endopeptidase; LysC; LysN;
Prolyl oligopeptidase (POP); Trypsin.
46 / Tanner, G. J., Colgrave, M. L., and Howitt, C. A.
mechanism in the POP enzymes only allows peptides smaller than
30 residues (about 3 kDa) to enter the catalytic site.
The bacterial PEP enzymes can act on larger peptides and pro-
teins (63,141) and are found widely distributed among microor-
ganisms. They can hydrolyze peptides with a minimum length of
three amino acids, but no defined maximum length (20,63). The
kinetics and specificity of three bacterial PEPs showed that the
enzymes from F. meningosepticum (Fm) and Myxococcus xanthus
(Mx) cleaved the peptides PQPQLPYPQPQLP and LQLQPFPQP
Sphingonionas capsulate (Sc) only cleaved the shorter peptide.
For the 13-mer peptide, the FM PEP enzyme preferentially
cleaved at the PQPQLPYPQPQLP site while the MX PEP en-
zyme preferentially cleaved at PQPQLPYPQPQLP. The Sc PEP
enzyme had comparable preference for both cleavage sites. PEP
from F. meningosepticum has a preference for PXP tripeptides
(20), with Pro in S1 and S3 positions, and Leu and Phe but not
Pro in the S2 position.
The fungal enzymes, especially the A. niger endoprotease (An-
PEP) and PEP enzymes present in sourdough fermentations are
particularly suited for remediation of celiac diets, function well at
intestinal pH, and have been shown to lower immunoreactivity of
gluten in vitro (77,78,99,100,102,107,116,141,150,155) and in
vivo (51,69,77,78,100,107,111,113,116,121,122,141,150,155). An-
PEP cleaves internal peptide linkages on the C-terminal side of
proline residues (63), e.g., cleavage of α-gliadin-2 takes place at
QLQPFPQPQLPYPQPQS (141). There is an abundance of
evidence that these enzymes, in combination with other proteases
or An-PEP alone, can reduce gluten immunoreactivity; three
examples are sufficient to illustrate this approach:
1. In a benchmark experiment, the 33-mer α-gliadin-2 peptide
(7) was digested by PEP from F. meningosepticum, combined
with small intestinal brush-border membrane enzymes in vitro
using perfused rat intestine (121). The brush-border membrane
enzymes have dipeptidyl peptidase activity and break down re-
maining peptides into amino acids, dipeptides, or tripeptides. Ex-
amination of the digestion products by HPLC showed the gliadin-
33mer was cleaved completely. More importantly, the stimulatory
effect of the digested preparation on clonal T-cells was abolished,
indicating that the digestion products were not immunotoxic to
celiac T-cells.
2. The An-PEP, available commercially as Brewers Clarex
(DSM) (57), was initially developed to de-bitter casein extracts
(56) and prevent chilling haze (98), but may hydrolyze gluten
proteins during brewing. Addition of Brewers Clarex at 2.5 g/hL
for 90 h reduced ELISA measured gluten below detectable limits
(73). More convincing evidence was obtained by analyzing the
proteolytic products. The An-PEP was active at intestinal pH and
destroyed T-cell epitopes as measured by mass spectrometry, T-
cell proliferation assays, ELISA, reverse-phase HPLC, SDS-
PAGE, and Western blotting (141).
3. A combination of EP-B2 from barley and bacterial PEP from
S. capsulata, in conjunction with a normal intestinal digestion
protocol with pepsin, trypsin, chymotrypsin, elastase, and carbox-
ypeptidase, reduced the concentration of gluten proteins to near
zero measured by HPLC and a lack of stimulation of cultured T-
cell lines was observed (65,129).
A PEP enzyme has recently been designed by protein engineer-
ing to have optimal activity at stomach pH (pH 2–4); have re-
sistance to common digestive proteases; suited to recombinant
production in a soluble form; and has specificity for the common
proline-glutamine (PQ) motif found in immunogenic α-gliadin
oligopeptides (68). This enzyme has 100-fold greater proteolytic
activity for gluten and 800-fold preference in substrate specificity
toward immunogenic portions of gluten peptides containing PQ
epitopes in P2 and P1 sites of the peptide. It is likely that this
enzyme is capable of digesting and destroying most, if not all,
immunoreactive gluten epitopes. Expression of this activity in a
transgenic yeast or addition of the purified enzyme to the brewing
cycle may assist in reducing immunoreactive malt proteins in-
volved with celiac disease. The role that this protease treatment
may play in reducing gluten intolerance and, for example, wheat
allergy, is limited by the lack of knowledge of the specific
epitopes and the clinical aspects of these other complaints.
It appears that treatment of gluten peptides with fungal An-PEP
alone reduces celiac immunoreactivity measured by cultured T-
cells to near zero. Concomitant mass spectrometry, HPLC,
ELISA, and western blot measurements of PEP-treated gluten
peptides showed the level is reduced to near zero. This biochemi-
cal evidence is supported by feeding trials, recently reviewed
(15,155), which support the efficacy of PEP pretreatment in
reducing the adverse effects of dietary gluten in some (51,52,
113), but not all situations (150). However, there is approximately
a 100-fold variation in the sensitivity toward the same gluten pep-
tide shown by T-cell populations isolated from different celiacs
(145). This may translate to a large range in the sensitivity of the
celiac population toward gluten peptides. This possibility is illus-
trated by the case of the communion wafer (16,17). Consumption
of as little as 1 mg of gluten per day, in the form of a communion
wafer, was sufficient to prevent mucosal recovery in one ex-
tremely sensitive celiac. Avoidance of the wafer resulted in com-
plete mucosal recovery within 6 months. This extreme sensitivity
in a single individual is in contrast to a study that demonstrated
that 10 mg of gluten (the equivalent of 500 g of food containing
20 ppm) daily for 3 months had no effect on the individuals in-
volved (32).
Definitive evidence of the safety of treated beer for celiacs ide-
ally requires a double-blind crossover dietary challenge. In this
experiment, the effect on circulating T-cells (4) and mucosal ap-
pearance (71) of a large number of celiacs, including sensitive
subjects, who have been challenged with either PEP-treated beer
or untreated beer followed by a crossover to the other treatment
regime, would provide convincing evidence for the efficacy of A.
niger PEP on eliminating gluten peptides for the whole celiac
population. In order to achieve this, subjects would have to drink
10 L of an average beer (at 100 ppm) per day to consume suffi-
cient hordein (1 g) for a useful short term challenge. Sourcing
volunteers for such an experiment may not be a problem, but eth-
ics approval would be unlikely. The use of barley flour, diluted
with rice flour to obtain sufficiently low levels, may be more ac-
ceptable; however, it introduces a new problem that the hordein
composition of flour differs from that of beer (144). Thus, while
the in vitro studies regarding digestion of gluten using peptidases
are promising for celiacs, until clinical evidence demonstrating
the safety of this approach is produced, a definitive answer cannot
be given.
The problem of the effects of protease treatments on the safety
of treated gluten for the remainder the celiac like diseases, includ-
ing gluten ataxia and dermatitis herpetiformis, must await defini-
tion of the epitopes responsible. Treatment of food for the larger
spectrum of gluten-related disorders, including gluten intolerance
and the IgE-mediated allergic responses bakers asthma, gluten
allergy, WDEIA, and urticaria, also all await examination of the
effect of added PEP on the epitopes responsible.
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... The regulatory threshold of 20 ppm intact gluten was based on studies examining the immunopathogenicity of intact gluten. Whether the biological activity is the same for gluten peptides that are produced during fermentation is unknown (29,(56)(57)(58)(59). ...
... Several proteases [PEP derived from Aspergillus niger (AN-PEP), Sphingomonas capsulate, EP-B2 (cysteine endoprotease from germinating barley), ALV003 (mixture of cysteine endoprotease and PEP), and Pseudolysin (lasB)] have been recently used to enzymatically hydrolyze gluten proteins in an attempt to prevent proliferative responses in gluten specific T cells (58,(62)(63)(64)(65)(66)(67)(68)(69)(70). The Aspergillus niger derived PEP (AN-PEP) and the ALV003 have been evaluated in clinical trials for their effectiveness in mitigating gluten-induced immune responses in celiac patients (71,72). ...
... Variability in the quantities and proportions of gluten proteins among wheat, rye, and barley cultivars exists and this makes the establishment of a universal standard or reference material problematic (93)(94)(95)(96). Although reference materials comprised of both wheat gliadin and barley hordein have been proposed for gluten analysis, currently there is no certified reference material and moreover no suitable reference material is available for the detection of fermented-hydrolyzed gluten (9,58,(97)(98)(99)(100). Wheat gluten was chosen as a calibrant in order to avoid excluding any gluten protein fraction (gliadins or glutenins) from the analysis. ...
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Celiac disease (CD) affects ~1 in 141 individuals in the United States, requiring adherence to a strict gluten-free diet. The Codex Standard and the European Commission states that gluten level of gluten-free foods must not exceed 20 ppm. The FDA requires food bearing the labeling claim “gluten-free” to contain <20 ppm gluten. Accurate quantitation of gluten in fermented-hydrolyzed foods by antibody-based methods is a challenge due to the lack of appropriate reference materials and variable proteolysis. The recent uses of proteases (e.g., proline endopeptidases or PEP) to hydrolyze immunopathogenic sequences of gluten proteins further complicates the quantitation of immunopathogenic gluten. The commercially available antibody-based methods routinely used to detect and quantitate gluten are not able to distinguish between different hydrolytic patterns arising from differences in fermentation processes. This is a severe limitation that makes accurate quantitation and, ultimately, a detailed evaluation of any potential health risk associated with consuming the food difficult. Utilizing gluten-specific antibodies, a recently developed multiplex-competitive ELISA along with western blot analysis provides a potential path forward in this direction. These complimentary antibody-based technologies provide insight into the extent of proteolysis resulting from various fermentation processes and have the potential to aid in the selection of appropriate hydrolytic calibration standards, leading to accurate gluten quantitation in fermented-hydrolyzed foods.
... AN-PEP is available in the market as Brewers Clarex, for example. A very low amount of this product (about 2.5 g/hL) can reduce gluten content below detectable limits [20,113]. It is generally added at the beginning of fermentation. ...
... The right choice of germination conditions, for example, 8 days at 18 °C with 48% of moisture, improves the peptidase activity of these enzymes. Furthermore, also cysteine proteases (EP-A and the major abundant EP-B) play an important role in the degradation of hordein during germination, unlike the other enzymes present in green barley malt, such as metalloproteases, serine carboxypeptidases, aspartate endoproteases and serine endoproteases [20]. ...
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Celiac disease (CD) is an immune-mediated gluten-sensitive enteropathy. Currently, it affects around 1% of world population, but it is constantly growing. Celiac patients have to follow a strict gluten-free (GF) diet. Beer is one of the most consumed beverages worldwide, but it is not safe for people with CD. It has a gluten content usually above the safe threshold (20 ppm), determined by the official method for hydrolyzed foods (R5-competitive-ELISA). The demand on the market for GF beers is increasingly growing. This review aims to provide a comprehensive overview of different strategies to produce GF beer, highlighting strengths and weaknesses of each approach and taking into account technological and sensory issues. GF cereals or pseudocereals have poor brewing attitudes (if used as main raw material) and give the beer unusual flavour. Instead, enzymatic treatments allow traditional brewing process followed by gluten content reduction. A survey on 185 GF-producing breweries (both industrial and craft) from all over the world have been considered to assess which approach is most used. Beers brewed with GF cereals and pseudocereals (used in well-balanced proportions) are more common than gluten-removed (GR) beers, obtained by enzymatic treatment.
... Research has been conducted to remove these proteins from beer using separation, filtration, or enzymatic methods [5,6]. While these treatments are effective at reducing gluten levels, the safety of these treated beers for gluten-sensitive individuals is not certain [7]. Research from Fiedler et al. [8] and Fiedler et al. [9] showed that gluten peptides, including those with known immunogenic sequences, could still be detected by mass spectrometry in enzymatically-treated beers. ...
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The mashing step underpins the brewing process, during which the endogenous amylolytic enzymes in the malt, chiefly β-amylase, α-amylase, and limit dextrinase, act concurrently to rapidly hydrolyze malt starch to fermentable sugars. With barley malts, the mashing step is relatively straightforward, due in part to malted barley’s high enzyme activity, enzyme thermostabilities, and gelatinization properties. However, barley beers also contain gluten and individuals with celiac disease or other gluten intolerances should avoid consuming these beers. Producing gluten-free beer from gluten-free malts is difficult, generally because gluten-free malts have lower enzyme activities. Strategies to produce gluten-free beers commonly rely on exogenous enzymes to perform the hydrolysis. In this study, it was determined that the pH optima of the enzymes from gluten-free malts correspond to regions already typically targeted for barley mashes, but that a lower mashing temperature was required as the enzymes exhibited low thermostability at common mashing temperatures. The ExGM decoction mashing procedure was developed to retain enzyme activity, but ensure starch gelatinization, and demonstrates a modified brewing procedure using gluten-free malts, or a combination of malts with sub-optimal enzyme profiles, that produces high fermentable sugar concentrations. This study demonstrates that gluten-free malts can produce high fermentable sugar concentrations without requiring enzyme supplementation.
... While "gluten-free" beers are often produced from malted barley worts using enzymatic treatments with Aspergillus niger prolyl-endopeptidase, these treatments do not actually remove all risk of gluten peptides persisting in the beer, nor assess the potential immunogenicity of all the peptide hydrolysates formed by its action, nor the limitations in the most common ELISA-based assays used for gluten detection, and thus in the USA, beers produced in this manner cannot be labelled gluten-free [12][13][14][15][16][17]. Brewing a true gluten-free beer then necessitates the utilization of alternative brewing ingredients that do not contain gluten and alternative processes to better utilize these ingredients. ...
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A successful wort fermentation depends on both the sugar and the free amino nitrogen (FAN) content of a wort. The primary goal of the mashing step is to generate fermentable sugars, as FAN is regarded as being primarily determined by malt quality; however, the role of mashing in modifying FAN has not been extensively studied, especially with respect to non-barley brewing materials. In this study, the FAN content of gluten-free (GF) worts varied greatly from barley (73–490 mg/L vs. 201 mg/L, respectively) and yielded different amino acid profiles, including lower proline and higher γ-aminobutyric acid concentrations. While most of the amino acids were present in the malt or generated in a brief window early in the mashing, significant increases in amino acid concentrations could be generated by mashing at temperatures below 55 °C. Overall, GF malts are promising brewing ingredients that can produce quality worts if appropriate mashing conditions are implemented.
... Nevertheless, the studies indicated that people diagnosed with gluten-related health complications such as celiac disease and gluten sensitivity have gluten intolerance symptoms after exposure to gluten-rich foodstuffs. Such people should always take gluten-free meals to avoid the associated adverse complications with gluten intake (Tanner et al., 2014). Additionally, it is essential to seek medical diagnosis and advice if one experiences irregular reactivity toward gluten-rich nutrients and to avoid unnecessary damage to one's digestive system. ...
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Gluten refers to the protein component evident in cereal grains such as barley, wheat and rye and is responsible for maintaining the food shape. Gluten is mainly contained in bread, cereals, and pasta in a high percentage through which gluten nutrients are consumed. Excessive gluten consumption is associated with celiac disease and gluten sensitivity health conditions, which affect the capability of the human gut to absorb nutrients into the bloodstream.
... These authors came to the conclusion that this exogenous enzyme, added during fermentation or to the finished product, can make the beer gluten free without affecting foam stability [66]. Tanner et al. [69], employing mass spectrometry, succeeded in confirming these results. In addition to microbial enzymes, a number of endogenous proteases contained in cereal seeds have been found to destroy immunotoxic gluten peptides. ...
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This article consists of a study of the literature and an assessment of available data on the production of gluten-free beer and its constituents. The article shows how the FAO/WHO Codex Alimentarius Commission for Nutrition and Foods for Special Dietary Uses defines celiac disease, gluten-free products, and gluten-free beer. It describes diet-dependent diseases, which require a gluten-free diet, and groups of potential consumers of gluten-free beer. This article describes the use of oats as a raw material for the production of brewing malt and its usefulness in the production of beer. It specifies how the technological process of standard beer production needs to be modified so that the product meets the requirements of patients with celiac disease. The article also provides an overview of literature data on the production of gluten-free beer from pseudocereal malts, such as sorghum malt, buckwheat malt, amaranth malt, and quinoa malt.
Some endogeneous and exogeneous enzymes participate in the brewery and winery technologies. Industrial enzymes provide quantitative advantages (increased juice yields) and qualitative advantages (enhanced extraction and flavor) for processing (shorter maceration, settling, and filtration time). This review aims to explain the flow process of brewing and wine-making, discuss different enzymes used in brewery and wine-making industry. Also, this chapter summarizes the key enzymes used at different stages of wine-making and brewing, and the challenges of the exogeneous, commercial and immobilized enzymes. Finally, the use of immobilized enzymes is presented as a significant strategy to improve catalyst during brewing and wine-making.
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ABSTRACT: Barley (Hordeum vulgare) is the fourth most cultivated crop in the world in terms of production volume, and it is also the most important raw material of the malting and brewing industries. Barley belongs to the grass (Poaceae) family and plays an important role in food security and food safety for both humans and livestock. With the global population set to reach 9.7 billion by 2050, but with less available and/or suitable land for agriculture, the use of biotechnology tools in breeding programs are of considerable importance in the quest to meet the growing food gap. Proteomics, as a member of the “omics” technologies, has become popular for the investigation of proteins in cereal crops and particularly barley and its related products such as malt and beer. This technology has been applied to study how proteins in barley respond to adverse environmental conditions including abiotic and/or biotic stresses, how they are impacted during food processing including malting and brewing, and the presence of proteins implicated in celiac disease. Moreover, proteomics can be used in the future to inform breeding programs that aim to enhance the nutritional value and broaden the application of this crop in new food and beverage products. Mass spectrometry analysis is a valuable tool that, along with genomics and transcriptomics, can inform plant breeding strategies that aim to produce superior barley varieties. In this review, recent studies employing both qualitative and quantitative mass spectrometry approaches are explored with a focus on their application in cultivation, manufacturing, processing, quality, and the safety of barley and its related products.
The recently growing demand of gluten-reduced beer is leading to the development of diverse approaches to be applied in brewing. The current work focuses on the development of an innovative and sustainable biocatalytic tool for the continuous production of gluten-reduced beer, based on the application of immobilized prolyl endopeptidase from Aspergillus niger (AN-PEP). This food-grade protease has been immobilized on A. niger chitosan beads and applied, for the first time, for the reduction of gluten in a commercial beer from barley malt. The immobilization procedure was optimized for maximizing the specific activity of the biocatalyst (0.016 I.U./mgBSAeq) and the best performance was reached using an immobilization solution at an initial protein concentration of 0.3 mgBSAeq/mL. The immobilization increased the thermal stability of the protease, which showed similar catalytic properties in synthetic beer (toward the synthetic substrate Z-Gly-Pro-pNA) when it was applied at 20 °C or at 50 °C. The continuous treatment in fluidized bed reactor (FBR), containing 10 g of immobilized AN-PEP (corresponding to 0.0036 gBSAeq), was optimized varying the flow rate (Qv). The suitable conditions to achieve reduction of the intact gluten of authentic beer was Qv of 728 mL/min. The continuous treatment in FBR allowed us to reduce the initial gluten content (65 mg/kg) in the commercial beer from barley malt, reaching the concentration of 19 mg/kg after 9 h and 15 mg/kg after 10 h of treatment.
The cultivated oat, Avena sativa L., is an allohexaploid (2n = 6x = 42) with three genomes (called A, C and D) derived from related wild species. Its first recorded appearance is in central Europe at about 1000 BC, and it is generally regarded as a secondary crop which developed from weeds in cultivated wheat and barley (Thomas, 1995). The world production of oats has been declining steadily over recent years and it currently ranks 6th in world production among the cereals (after wheat, maize, rice, barley and sorghum) with total annual yields of 30–40 million metric tonnes. It is well suited to cool moist climates with major production areas in the former Soviet Union, the USA, Canada, Poland, Germany, Australia and Scandinavia (see Hoffman, 1995).
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Beer is derived from grist materials that contain gluten-type protein; therefore, it has long been assumed that it should not be consumed by those with celiac disease. However, because a significant objective of the malting and brewing process is protein precipitation and modification, beer may be rendered low in gluten from these processes alone. The levels of prolamin have been monitored throughout the brewing process with the RIDASCREEN Gliadin competitive R5 enzyme-linked immunosorbent assay method. The barley malt contained 6,832.3 ± 61 mg/kg, but only 131.1 ± 1 mg/kg of prolamin remained after fermentation. Addition of prolyl endoproteinase lowered the level of prolamin still further, to levels below the reliable limit of detection. A significant difference in foam stability was observed between the control and the prolyl endoproteinase-treated gluten-free beer, but no other beers were significantly affected by the addition of this enzyme.
The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgareL.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P2-P1-P1′-P2′-tyrosine(NO2)-aspartic acid, in which cleavage occurs between P1 and P1′, showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P2. Arginine was preferred to glutamine at P1, whereas proline at P2, P1, or P1′ greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower K m values.
Kernels of the rye cultivars Danko and Halo were milled into white flour and compared with flour of the wheat cultivar Rektor. Flour proteins were extracted stepwise with a salt solution (albumins-globulins), 60% ethanol (prolamins), and 50% 2-propanol under reducing conditions (glutelins). The quantification by reversed-phase HPLC indicated that the extractable proteins of both rye flours consisted of ≈26% albuminsglobulins, 65% prolamins, and 9% glutelins. Compared with wheat flour, rye flours comprised significantly higher proportions of nonstorage proteins (albumins-globulins) and lower proportions of polymerized storage proteins (glutelins). SDS-PAGE revealed that the prolamin fractions of rye contained all four storage protein types (HMW, γ-75k, ω, and γ-40k secalins), whereas the glutelin fractions contained only HMW and γ-75k secalins. The quantification of secalin types by RP-HPLC showed a close relationship between the two cultivars. The γ-75k secalins contributed nearly half (≈46%) of the total storage proteins, followed by γ-40k secalins (24%) and ω secalins (17%); HMW secalins (≈7%) were minor components, and 6% of eluted proteins were not identified. The amino acid composition of γ-40k secalins corresponded to those of γ-gliadins of wheat, whereas γ-75k secalins were characterized by higher contents of glutamine and proline. Matrix-assisted laser desorption/ionization and time of flight mass spectrometry (MALDI-TOF MS) indicated molecular masses of about 52,000 (γ-75k) and 32,000 (γ-40k), respectively. Nterminal amino acid sequences were homologous with those of wheat γgliadins except for position 5 (asparagine in γ-75k and glutamine in γ-40k secalins) and position 12 (cysteine in γ-75k secalins). The N-terminal amino acid sequences of HMW and ω-secalins were homologous with those of the corresponding protein types of wheat. Gel-permeation HPLC of prolamin fractions revealed that rye flours contained a significantly higher proportion of ethanol-soluble oligomeric proteins than wheat flour.
In the course of our work we aimed to develop a product from gluten-free raw materials (millet, sorghum and buckwheat) that is similar to beer made of barley malt but is consumable by coeliacs. Our measurements were started by qualification of cereal/pseudo-cereal grains. Next malts were made of them with different steeping, germination and kilning parameters, and their most important quality characteristics were determined. Qualification of grains were done by grading, determination of thousand-kernel and hectolitre weight, and protein content, while malts were examined with congress mashing, Hartong mashing and lauter test, as well. Gelatinization point of the starch found in grains and malts were determined by Brabender amyloviscograph which helped to set the temperature of beta-amylase rest in future mashings. The gelatinization points were higher in our samples, than in the barley's starch. Optimization of mashing was continued with malts that fulfilled requirements needed for brewing. Mashing programs were written for each raw material with the help of our laboratory mashing equipment, and resulting worts were analysed (for extract content), then carbohydrate content was measured by HPLC, alpha-amylase activity by Phadebas test, and free alpha-amino nitrogen (FAN) content by the ninhydrin method. Those worts were selected for further fermentation tests that had the highest extract and FAN content, best filtration time and appropriate sensory characteristics. Optimal malting temperatures and time periods, aeration and water uptake were determined, and then the duration and temperature of protein and enzyme rests of mashing were set. The malting process that proved to be the most suitable for brewing requirements (high extract content, good lautering characteristics, high FAN content) has the following parameters: steeping with 25 degrees C water for 18 h with aeration in every 5 h; germination at 15 degrees C for 84 h; kilning at 50 degrees C for 48 h.
The application of nitrogen (N) fertilizer in malting barley is necessary to obtain good yield, but it also influences kernel protein content, which affects malting quality. The negative correlation between kernel protein content and malting extract is due to the hordein proteins. The aim of this study was to determine the effect of N application timing on hordein fractions and how this influences malt quality. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to determine the hordein fractions in the two- and six-row barley parents and their doubled-haploid progeny, for two seasons for one location, and two locations in the second season. There were no changes in hordein level in response to timing of N application over years and locations. The two-row parent had the lowest hordein level and the six-row progeny had the highest. Total hordein content of the two-row progeny and six-row parent were similar. The supply of additional N in the form of fertilizer rather than the timing of all N applications influenced the production of all hordein fractions. There were significant correlations between hordein fractions and malting quality that also were reported in other studies. Kernel plumpness showed significant negative correlations with D, C, and B hordeins and total hordein content when half of the fertilizer was applied at planting and the other half at the six-leaf stage. Absorption showed significant positive correlation with C hordeins and a negative correlation with the B-to-C ratio, also when the second half of the N was given at the six-leaf stage.
The changes in malt proteins during the brewing process and the roles of malt proteins in the formation of beer haze were investigated in this study. Coomassie blue stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed that proteins with molecular weights of ∼40, 25–29 and 6.5–17 kDa were the major components of beer haze protein, originating primarily from malt water-soluble protein, partly from hordein. Two-dimensional electrophoresis and mass spectrometry analysis highlighted malt protein modification occurring during the brewing process. It was found that most of the malt hordeins disappeared during the brewing process, except B and γ3 hordein and storage protein (Hordeum vulgare). The results also suggested that the heat- and proteolysis-stable hydrophobic proteins such as barley trypsin inhibitor CMe protein, germin E (H. vulgare) and protein Z might be the important haze-promoting proteins, and that B and γ3 hordein might be the minor haze-active proteins in beer but were a critical factor in the formation of beer haze. Haze stability is of great importance to brewers as it is the first characteristics by which a consumer judges the quality of their beer. It is conventionally accepted that proline-rich hordeins from malt play a major role in the formation of haze. In this study, it was found that B and γ3 hordein might be the minor haze-active proteins, but the heat- and proteolysis-stable hydrophobic proteins such as barley trypsin inhibitor CMe (BTI-CMe) protein, germin E (Hordeum vulgare) and protein Z might be the important haze-promoting proteins. This study made a further confirmation on this issue that nonhordein proteins are regarded as haze-active and laid the theoretical foundation for further investigations to determine the mechanism of the formation of beer haze. An improved understanding of the impact of brewing on malt proteins could potentially provide further scope for optimizing beer quality.