CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2011;9:24 –29
ORIGINAL ARTICLES—ALIMENTARY TRACT
Safety for Patients With Celiac Disease of Baked Goods Made of Wheat
Flour Hydrolyzed During Food Processing
LUIGI GRECO,* MARCO GOBBETTI,‡ RENATA AURICCHIO,* RAFFAELLA DI MASE,* FRANCESCA LANDOLFO,*
FRANCESCO PAPARO,* RAFFAELLA DI CAGNO,‡ MARIA DE ANGELIS,‡ CARLO GIUSEPPE RIZZELLO,‡
ANGELA CASSONE,‡ GAETANO TERRONE,* LAURA TIMPONE,* MARTINA D’ANIELLO,* MARIA MAGLIO,*
RICCARDO TRONCONE,* and SALVATORE AURICCHIO*
*Department of Pediatrics and European Laboratory for the Study of Food Induced Diseases, University of Naples, Federico II, Naples; and ‡Department of Plant
Protection and Applied Microbiology, University of Bari, Bari, Italy
BACKGROUND & AIMS: Celiac disease (CD) is character-
ized by an inflammatory response to wheat gluten, rye, and
barley proteins. Fermentation of wheat flour with sourdough
lactobacilli and fungal proteases decreases the concentration of
gluten. We evaluated the safety of daily administration of baked
goods made from this hydrolyzed form of wheat flour to pa-
tients with CD. METHODS: Patients were randomly assigned
to consumption of 200 g per day of natural flour baked goods
(NFBG) (80,127 ppm gluten; n ⫽ 6), extensively hydrolyzed
flour baked goods (S1BG) (2480 ppm residual gluten; n ⫽ 2),
or fully hydrolyzed baked goods (S2BG) (8 ppm residual
gluten; n ⫽ 5) for 60 days. RESULTS: Two of the 6 patients
who consumed NFBG discontinued the challenge because of
symptoms; all had increased levels of anti–tissue transglutami-
nase (tTG) antibodies and small bowel deterioration. The 2
patients who ate the S1BG goods had no clinical complaints
but developed subtotal atrophy. The 5 patients who ate the
S2BG had no clinical complaints; their levels of anti-tTG anti-
bodies did not increase, and their Marsh grades of small intes-
tinal mucosa did not change. CONCLUSIONS: A 60-day
diet of baked goods made from hydrolyzed wheat flour,
manufactured with sourdough lactobacilli and fungal pro-
teases, was not toxic to patients with CD. A combined
analysis of serologic, morphometric, and immunohisto-
chemical parameters is the most accurate method to assess
new therapies for this disorder.
Keywords: Celiac Disease; Wheat Flour; Sourdough; Gluten
Challenge.
C eliac disease is characterized by an inflammatory response
to wheat gluten. Gluten contains approximately 15% pro-
line and 35% glutamine residues,1 which limit proteolysis by
gastrointestinal enzymes, thus generating toxic peptides.2
Oral supplementation with microbial peptidases was pro-
posed as an alternative to the gluten-free diet.3,4 However, these
enzymes are easily inactivated in the stomach by pepsin and
acidic pH, thus failing to degrade gluten before exposure to
small intestine. Hence a combination of glutamine-specific en-
doprotease and prolyl-endopeptidase, which rapidly detoxifies
oligopeptides after primary proteolysis, was proposed.5 A new
endoprotease from Aspergillus niger accelerated the degrada-
tion of gluten into the stomach to such an extent that only
traces reached the duodenal compartment.6,7 Unfortunately,
to date, the safety of the oral administration of proteases
with gluten is yet to be demonstrated in patients with celiac
disease (CD).
Currently, cereal baked goods are manufactured by fast pro-
cesses in which traditional long fermentation by sourdough, a
cocktail of acidifying and proteolytic lactic acid bacteria, was
replaced by chemical and/or baker’s yeast leavening agents.
Under these conditions, cereal components are not degraded
during manufacture. We showed that the manufacture of wheat
and rye breads or pasta with durum flours by using selected
sourdough lactobacilli markedly decreased the toxicity of glu-
ten.8 A combination of glutamine-specific endopeptidase,
which extensively hydrolyzed gluten, and prolyl-endopepti-
dases, which rapidly detoxified gluten, was recently pro-
posed.9 Proteins from the pepsin-trypsin digest of the wheat
sourdough did not activate peripheral blood mononuclear
cells (PBMCs) or induce interferon-␥ synthesis by PBMCs.
None of the intestinal T-cell lines from 12 patients with CD
showed immunoreactivity toward pepsin-trypsin digest.
Bread making was optimized to show the processing suit-
ability of the detoxified wheat flour.
This study evaluated the safety of daily administration for 60
days to CD patients of goods made of wheat flour hydrolyzed
during food processing by a mixture of selected sourdough
lactobacilli and fungal proteases.
Materials and Methods
Microorganisms and Enzymes
Lactobacillus alimentarius, 15M, L brevis 14G, L sanfranci-
scensis 7A, and L hilgardii (defined as pool S1) were shown to
hydrolyze gliadins efficiently.8 Six strains of L sanfranciscensis
Abbreviations used in this paper: CD, celiac disease; ELISA, enzyme-
linked immunosorbent assay; EMA, endomysial antibody; Ig, immuno-
globulin; NFBG, natural flour baked goods; PBMC, peripheral blood
mononuclear cell; S1BG, sourdough 1 baked goods; tTG, tissue trans-
glutaminase.
© 2011 by the AGA Institute
1542-3565/$36.00
doi:10.1016/j.cgh.2010.09.025
January 2011
(LS3, LS10, LS19, LS23, LS38, and LS47) (named pool S2) were
selected for their peptidase systems, specifically toward proline-
rich peptides. Lactobacilli strains were isolated from natural
wheat sourdoughs traditionally used for typical Italian bread
making. Strains were propagated for 24 hours at 30°C in MRS
broth (Oxoid, Basingstoke, Hampshire, UK) with fresh yeast
extract (5% vol/vol) and 28 mmol/L maltose at pH of 5.6 and
cultivated to the late exponential phase of growth (⬃12
hours). Fungal proteases from Aspergillus oryzae (500,000
hemoglobin units) and A. niger (3.00 acid protease units/g),
used as improvers in bakery industry, were supplied by BIO-
CAT Inc (Troy, VA).
Hydrolysis of Gluten During Wheat
Sourdough Fermentation
Wheat (Triticum aestivum cv. Appulo) flour had moisture
12.8%, protein, and 10.3% of dry matter. Fermentation formulas
were sourdough 1 (S1), 80 g of wheat flour and 320 g of water
containing ⬃5 ⫻ 108 colony-forming units/g (final cell density
in the dough) of pool 1 and sourdough 2 (S2), the same as S1
formula with the addition of ⬃5 ⫻ 108 colony-forming units/g
of pool 2 and 200 ppm of both fungal proteases. Sourdoughs
S1 and S2 were fermented for 48 hours at 37°C. Spray drying
then removed water, and milled flours were used to produce
baked goods.
Making Sweet Baked Goods
The formulas to make biscuits and cakes were sour-
dough 1 (S1) baked goods (S1BG), 100 g of S1 hydrolyzed
wheat flour, 50 g of butter, 50 g of sucrose, and water as
required; sourdough 2 (S2) baked goods (S2BG), the same as
S1BG formula except for the use of S2 instead of S1; and
nonfermented baked goods (natural flour baked goods
[NFBG]), the same as S1BG formula except for the use of
nonprocessed wheat (Triticum aestivum cv. Appulo) flour. Sweet
goods were of excellent taste for all 3 flours used.
Immunologic and Chemical Analyses
R5 monoclonal antibody and the horseradish peroxi-
dase-conjugated R5 antibody were used for gluten analysis. The
R5-based sandwich enzyme-linked immunosorbent assay
(ELISA) was carried out with the Transia plate detection kit
(Diffchamb; Västra, Frölunda, Sweden) at Centro National de
Biotecnologia (Madrid, Spain). For R5-based Western blot anal-
ysis, after one-dimensional sodium dodecylsulfate–polyacryla-
mide gel electrophoresis, proteins were electrotransferred onto
polyvinylidene difluoride membranes and incubated with R5-
horseradish peroxidase, and the blots were developed by immu-
nodetection with enhanced chemiluminescence system (Amer-
sham Pharmacia, Piscataway, NJ).
Patients
Sixteen CD patients (median age, 19 years; range, 12–23
years) accepted to enter the study. All patients were in good
health on a gluten-free diet for at least 5 years.
Challenge Protocol
Gluten challenge to confirm the initial diagnosis was
part of the diagnostic work up to few years ago, but we limited
the gluten challenge to children with very young age or uncer-
tain initial diagnosis. Gluten challenge was frequently re-
SAFETY OF BAKED GOODS FOR PATIENTS WITH CD 25
quested by teenagers who wished to confirm the gluten depen-
dency after many years of gluten-free diet. When the request
was denied, most teenagers did the challenge themselves, with-
out controls. This 25-year strategy led to one of the best levels
of compliance to the gluten-free diet.
The study protocol, approved by the Ethical Committee of
the University “Federico II,” Italy, was proposed to teenagers on
a waiting list for a diagnostic gluten challenge. The protocol
was thoughtfully explained, and their direct informed consent
was obtained in all cases.
Patients were on strict gluten-free diet during challenge.
Anti–tissue transglutaminase (tTG) and anti-endomysial (EMA)
antibodies were evaluated, and a small intestinal biopsy was
carried out before the challenge. Four patients were excluded
for the presence of anti-tTG antibodies or damaged duodenal
mucosa. Twelve patients were randomized to treatments by
random number. The first 6 patients consumed NFBG, the
following 2 patients consumed S1BG, and the last 5 patients
consumed S2BG. All consumed daily about 200 g of baked
goods, corresponding to 8 g of native gluten. The challenge
lasted 60 days. After 30 days, patients underwent a dietary
interview, and anti-TG2 and anti-EMA antibodies were evalu-
ated. At 60 days, biopsy and serology were repeated.
Serology, Morphometric, and
Immunohistochemistry Analyses
Serum IgA EMA was detected by indirect immunoflu-
orescence.10 Serum immunoglobulin (Ig) A anti-TG2 was iden-
tified by ELISA technique (Eurospital, Trieste, Italy).10
One fragment from each biopsy was fixed in 10% formalin,
paraffin embedded, sectioned, and stained with hematoxylin-
eosin. A fragment was embedded in OCT compound (Bio
Optica, Milano, Italy) for immunohistochemistry. Villous to
crypt height ratio was computed, and the number of intra-
epithelial lymphocytes, CD3, and TCR␥␦⫹ was computed
per millimeter of epithelium. Marsh–Oberhuber grading was
applied. Immunohistochemistry was done by standard pro-
cedures.11
Duodenal biopsies from 12 of 13 patients were investigated
for the presence of intestinal deposits of IgA anti-TG2 antibod-
ies, as previously reported.12
To evaluate crypt proliferation, we detected Ki-67 in 11 of 13
biopsies. After preincubation of 10 minutes with rabbit normal
serum (1:100; Dako, Glostrup, Denmark), the primary mouse
monoclonal antibody Ki-67 (1:200; Dako) was applied on 4-␮m
cryostat sections for 1 hour. Rabbit anti-mouse (1:25; Dako)
was then applied for 30 minutes, followed by a 30-minute step
with alkaline phosphatase and monoclonal mouse anti–alkaline
phosphatase immunocomplexes (mouse APAAP 1:40; Dako).
Incubation with fuchsin was the ending step. Crypt prolifera-
tion index was obtained by counting cells positive for anti–
Ki-67 antibody as a percentage of the total enterocytes within
vertical crypts. Five sections with well-oriented crypts were an-
alyzed per patient.
Statistical Analysis
Wilcoxon signed-rank test for paired (before/after) data
was adopted (Tables 1 and 2).
26 GRECO ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 9, No. 1

Table 1. Parameters of CD Patients Before/After Challenge With NFBG

Time of challenge Marsh CD3 (cells/mm TCR␥␦ (cells/mm CD25 (cells/mm2
(d) EMA TG2 (U/mL) grade Vh/Cd epithelium)a epithelium)b lamina propria)c

A.An 0 Negative 5.6 T0 2.6 21.6 6 1

30 Light positivity 12.4 T1 3.5 59.6 15.3 20
A.Am 0 Negative 1.7 T0 2.2 35 10 5
30 Positive 18.8 T2 1.8 80.8 18.5 24
S.R. 0 Negative 1.6 T0 2.5 24 5.5 7
60 Positive ⬎200 T3b 0.5 80 35 102
F.I. 0 Light positivity 1.9 T0 2.3 38 8.6 5
60 Positive 111.4 T3b 0.3 114 33.5 144
G.S. 0 Negative 0.3 T0 3 36 11.7 8
60 Positive ⬎200 T3b 0.7 80 15.4 98
L.R. 0 Negative 0.5 T1 2 43 9.7 58
60 Positive 176.2 T3b 0.6 79 14 88
P value, Wilcoxon test, 0 vs 60 days .028 .023 .074 .028 .028 .025

NOTE. Wilcoxon signed-rank test for paired data (before/after challenge) was performed. P value statistically significant if ⬍.05.
T0, normal duodenal mucosa; T1, architecturally normal duodenal mucosa with increased intraepithelial lymphocyte infiltration; T2, presence of
crypt hyperplasia; T3b, subtotal atrophy; Vh/Cd, villous to crypt height ratio.
aNormal value, ⬍34.
bNormal value, ⬍3.4.
cNormal value, ⬍4.

Results symptoms such as malaise, abdominal pain, and diarrhea. Four

Protein Content of the Wheat Flours Used for
Making Baked Goods
The gluten concentration of native nonfermented
wheat (Triticum aestivum cv. Appulo) flour used for making
NFBG was ⬃80,127 ⫾ 123 ppm (Table 3). After fermentation of
wheat flour by selected sourdough lactobacilli of pool 1 (sour-
dough S1), the residual concentration of gluten markedly de-
creased (⬃97%) to 2480 ⫾ 86 ppm. The residual gluten was
below 10 ppm when hydrolysis was carried out by combining
sourdough S1 with fungal enzymes routinely used in bakery.
The absence of gluten was confirmed by R5 antibody– based
Western blotting (data not shown). The spray-dried flour used
for making S2BG contained a mixture of water/salt soluble
low-molecular mass peptides and free amino acids (1.80% ⫾
0.06%). Indeed, no organic nitrogen was detectable in the glia-
din fraction, and the level of glutenins decreased to traces
(0.05% ⫾ 0.01%). The concentration of free amino acids con-
firmed the differences found for hydrolysis of gluten; it varied
from 1032 ⫾ 32 (NF) to 5320 ⫾ 64 (S1) and 15,320 ⫾ 102
mg/kg (S2).
Clinical Challenge With Nonfermented Baked
Goods
Six CD patients consumed daily 200 g of NFBG that
contained ⬃80,127 ⫾ 123 ppm of gluten. Two patients (A.An
and A.Am) interrupted the challenge after 4 weeks because of
Table 2. Anti-TG2 IgA Deposits in Intestinal Mucosa
TG2 deposits TG2 deposits
at time 0 at time 60 ␹2
Natural gluten 1/5 5/5 P ⫽ .009
Extensively hydrolyzed 0/2 2/2 P ⫽ .045
Fully hydrolyzed 2/5 1/5 P ⫽ .49
patients reached the end point of 60 days with no complaints.
All 6 showed a marked increase in anti-EMA and anti-TG2
antibodies (Table 1). Biopsies showed significant deterioration
with lymphocyte infiltration, increased CD3 and gamma-delta
lymphocytes. Subtotal mucosal atrophy developed in all cases
(Table 1). Data after challenge were significantly different by
prechallenge values (Table 1).
Challenge With Sourdough 1 Baked Goods
Two CD patients consumed 200 g of S1BG that con-
tained ⬃2480 ⫾ 86 ppm of residual gluten. They had no
clinical complaints during the 60 days. One showed increased
antibodies (Table 4), and both showed increased CD3 and
gamma-delta intraepithelial lymphocytes with subtotal atrophy
after challenge.
Challenge With Sourdough 2 Baked Goods
Five patients consumed 200 g of S2BG made of wheat
flour fermented with sourdough pools 1 and 2 and fungal
proteases. None of the 5 patients had clinical complaints dur-
ing the 60 days. None produced anti-TG2 antibodies; none had
any modification of the small intestinal mucosa (Table 4). No
increase of CD3 and gamma-delta cells was observed. Marsh
grade was unchanged after the challenge. No significant
changes before/after the challenge in any of the variables were
observed (Table 4) (Figure 1).
Anti-tissue Transglutaminase Immunoglobulin
A Deposits in the Intestinal Mucosa and
Crypt Proliferation
Table 2 shows that all CD patients challenged with
native gluten developed deposits of TG2 IgA in the mucosa;
similarly, both patients who ate extensively hydrolyzed gluten
showed clear IgA deposits. On the contrary, CD patients who
ate fully hydrolyzed flour showed, after 60 days, fewer deposits
than those observed at time 0.
January 2011 SAFETY OF BAKED GOODS FOR PATIENTS WITH CD 27

Table 3. Protein Nitrogen of the Flours

Albumins/globulins (%) Gliadins (%) Glutenins (%) Free amino acids (mg/kg) Gluten (ppm)

NF 0.3 ⫾ 0.02 0.72 ⫾ 0.08 0.85 ⫾ 0.08 1032 ⫾ 32 80,127 ⫾ 123

Extensively hydrolyzed S1 1.61 ⫾ 0.04 0.07 ⫾ 0.01 0.18 ⫾ 0.03 5320 ⫾ 64 2480 ⫾ 86
Fully hydrolyzed S2 1.80 ⫾ 0.06 ND 0.05 ⫾ 0.01 15,320 ⫾ 102 8⫾2

NOTE. Data are means from 4 independent samples analyzed twice.

ND, not detected.

Ki-67, a specific marker of crypt proliferation, was evaluated
in 11 of 13 biopsies. Figure 2 shows that both the first 2 groups
(native and extensively hydrolyzed gluten) had a clear increase
in crypt proliferation after 60 days, whereas no significant
increase in crypt proliferation was observed in the third group
(fully hydrolyzed gluten).
Discussion
Most studies on the development of therapeutic strat-
egies alternative to the gluten-free diet are based on indirect
outcome such as fecal output, permeability, nutritional and
functional markers. Unfortunately, it is impossible to evaluate
toxicity if the product is not tested in vivo for an acceptable
length of time.
This study tests the toxicity of a new wheat-based food for
CD patients in vivo with the gold standard method. It might be
argued that the first group of patients underwent a challenge
with native flour whose toxicity was expected; the patients,
before random allocation, requested an appropriate gluten
challenge to confirm the diagnosis for life.
The use of food-grade sourdough lactobacilli for extended
fermentation of wheat flour is an example of biotechnology
derived by traditional processes that, unfortunately, were re-
cently replaced by fast-acting chemical and/or baker’s yeast
leavening agents.8 Fermentation with selected lactobacilli added
with fungal proteases, routinely used as an improver in bakery
industries, decreased the concentration of gluten to below 10
ppm. Despite the markedly reduced concentration of gluten,
the resulting spray-dried flour was still adequately workable. As
shown in this and other studies,9,13 the hydrolyzed flour is
suitable for making sweet baked goods and also bread and
pasta if supplemented with gluten-free structuring agents.
As expected, 60 days of consumption of native gluten con-
taining wheat flour (NFBG) were enough to elicit clinical symp-
toms and intestinal damage in CD patients. The ingestion of
baked goods (S1BG) made of wheat flour with markedly de-
creased concentration of gluten (3% of the native content,
⬃2480 ppm) did not provoke symptoms, but it induced TG2
antibodies to increase in 1 patient and was sufficient to increase
inflammation in the small intestinal mucosa. Five patients who
completed the 60-day challenge with baked goods made of
wheat flour extensively digested by lactobacilli and fungal pro-
teases showed no clinical symptoms, neither an increase of
anti-TG2 antibodies nor a modification of the architecture or
the grade of inflammation of the intestinal mucosa.
Crypt proliferation as well as anti-TG2 IgA deposits in the
mucosa did not increase after challenge with fully hydrolyzed
gluten, whereas it clearly increased in the other 2 groups.
This study showed that it is possible to evaluate completely
the clinical, serologic, and histologic features in CD patients
challenged with test meals. D-xylose urine secretion and 72-
hour quantitative fecal fat were proved to be inadequate to
detect intestinal damage in CD patients undergoing new treat-
ments such as oral administration of prolyl-endopeptidases.4

Table 4. Parameters of CD Patients Before/After Challenge With S2BG

Time of challenge TG2 CD3 (cells/mm TCR␥␦ (cells/mm CD25 (cells/mm2
(d) (U/mL) Marsh grade Vh/Cd epithelium)a epithelium)b lamina propria)c

F.I. 0 1.6 T0 2.3 39 5.6 6

60 1.0 T0 2.1 38 8.6 5
I.C. 0 1.9 T0 3 3.7 0.9 11
60 1.1 T0 2.5 11 3.8 9
R.R. 0 0.3 T1 3 53 11.5 3
60 0.3 T1 3.5 56 17.8 4
I.I. 0 0.5 T0 3.5 31 8.4 21
60 0.3 T0 3 36 12.8 21
C.C. 0 0.4 T0 3.5 32 21 3
60 0.7 T0 3.5 47 18 7
P value, Wilcoxon test, 0 vs .273 1 .45 .08 .176 .854
30 days

NOTE. Wilcoxon signed-rank test for paired data (before/after challenge) was performed. P value statistically significant if ⬍.05.
T0, normal duodenal mucosa; T1, architecturally normal duodenal mucosa with increased intraepithelial lymphocyte infiltration; Vh/Cd, villous
to crypt height ratio.
aNormal value, ⬍34.
bNormal value, ⬍3.4.
cNormal value, ⬍4.
28 GRECO ET AL
Figure 1. (A, B) Density of ␥␦⫹ intraepithelial lymphocytes in jejunal biopsy from patient F.I. at the beginning (A) and after 60 days of challenge (B)
with S2BG. (C, D) Density of ␥␦⫹ intraepithelial lymphocytes in jejunal biopsy from patient S.A. at the beginning (A) and after 60 days of challenge
(B) with S1BG. Arrows refer to gd⫹ intraepithelial lymphocytes.
Our group showed that increased intestinal permeability to
sugars and even increase of EMAs were good predictors of
histologic relapse in gluten challenge of 15 CD patients.13
Gliadin antibodies increased early (already 7 days after the
reintroduction of gluten into the diet), but in many cases they
returned to normal thereafter. Most importantly, all patients
relapsed after 2–3 months of gluten-containing diet. The same
results occurred in our cohort of patients who underwent the
challenge with native gluten (NFBG). It is very important to
realize that indirect methods of ascertainment of gluten-in-
duced damage are not adequate to evaluate the long-term
tolerance to gluten. Any short-term trial for few weeks is more
Figure 2. Ki-67 detection as an index of crypt proliferation. Both the
first 2 groups (NFBG and S1BG) had a statistically significant increase (*)
in crypt proliferation after 60 days, whereas no increase in crypt prolif-
eration was observed in the third group (S2BG).
CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 9, No. 1
likely to originate frustrated hopes in the patients than real
solutions.
We showed that markedly reduced levels of gluten (3% of the
native gluten, eg, 2480 ppm) are able to induce changes in the
intestinal mucosa without clinical symptoms. Two patients
developed villous atrophy after 60 days of challenge with the
above concentration of gluten. However, serologic tests (anti-
tTG and anti-EMA) were less sensitive, as already noted in
children with slight dietary transgression to the gluten-free diet.
Recently, Catassi et al14 showed that a daily dose of 0.05 g of
gluten for 90 days induced intraepithelial infiltration in CD
patients with no antibody response.
The recent discovery of immunodominant gluten peptides
(eg, 33-mer epitope) resistant to intestinal digestion led to the
identification of a novel opportunity for reducing gluten tox-
icity, such as bacterial-derived endopeptidases. However, there
are still problems with the delivery of phosphoenolpyruvate
enzymes and with their stability under acidic gastric environ-
ment and efficient mixing with gluten. We showed that the
small amount of residual gluten after extensive fermentation
(preparation S1BG) was sufficient to start the deleterious im-
mune response to gluten. This is the reason why the adminis-
tration of oral enzymes with a gluten meal is theoretically not
likely to suppress the immune response to gluten. Under opti-
mal conditions, healthy humans are able to digest up to 90.3%
of the wheat protein from bread.15 When 8000 mg of gluten
(150 g of bread) are given to healthy subjects, 800 mg (10%) of
January 2011
this gluten will resist duodenal and jejunal degradation and will
be presented to intestinal mucosa. This undigested fraction is
likely to contain the digestion-resistant immunodominant
epitopes of gluten. The oral administration of endopeptidases
has to be 100 times more efficient than the physiological di-
gestive machinery, during a small time lag, to be effective. This
is the obvious reason why the administration of massive
amounts of purified recombinant prolyl-endopeptidase to-
gether with 4 g of gluten (10% of the daily dose) to gluten-
sensitive rhesus macaques did not prevent a marked increase of
anti-gliadin and anti-tTG antibodies.16 Bethune et al16 showed
that the administration of peptidases with the meal did cur-
rently enhance the immunoresponse by exposing the small
intestinal mucosa more rapidly to immunopeptides from frag-
mented gluten proteins.
In conclusion, the findings of this study and the above
considerations provide the rationale for exploring therapies
that could reduce the toxicity of gluten for CD patients beyond
the standard gluten-free diet. A wheat flour– derived product is
shown to be not toxic after administration for 60 days to CD
patients. Hydrolysis of gluten to below 10 ppm was achieved
through the activity of complementary peptidases located in
the cytoplasm of lactobacilli routinely used in sourdough fer-
mentation. The addition of fungal proteases, used in bakery to
modify the elasticity and resistance of gluten, was required to
reach the complete gluten degradation. The primary proteolysis
of wheat proteins was required; after this stage, medium-sized
polypeptides, including the 33-mer and similar peptides, are
efficiently transported into lactobacillus cytoplasm and sub-
jected to peptidase activities.9 A period of 60 days, although
repeatedly shown to be sufficient to evaluate gluten toxicity in
the majority of patients, might not be long enough to evaluate
toxicity in all CD subjects who might show different sensitivity
to gluten. Prolonged trials have to be planned to state the safety
of the baked goods manufactured by applying this rediscovered
and adapted biotechnology. An extended fermentation with
lactic acid bacteria and fungal enzymes naturally present or
used in sourdough biotechnology could have well lowered the
exposure to massive amount of gluten in our past. There is no
doubt that the phenotypic disclosure of CD, which appears
today as a global epidemic, might be related to a threshold of
early exposure.11 In the future, the use of cereals through such
biotechnology could also improve the nutritional and sensory
properties of baked goods containing hydrolyzed gluten as
compared with products made of naturally gluten-free ingredi-
ents.
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Reprint requests
Address requests for reprints to: Prof. Luigi Greco, MD, PhD, Depart-
ment of Pediatrics, Via S. Pansini 5, Edificio 11, 80131 Naples, Italy.
e-mail: ydongre@unina.it; fax: 390-81-546-9811.
Acknowledgments
The authors acknowledge Giuliano, Inc, Italy for participation in the
project.
Conflicts of interest
The authors disclose no conflicts.