APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2007, p. 4499–4507
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 14
Highly Efficient Gluten Degradation by Lactobacilli and
Fungal Proteases during Food Processing: New
Perspectives for Celiac Disease?
Carlo G. Rizzello,1Maria De Angelis,1Raffaella Di Cagno,1Alessandra Camarca,2Marco Silano,3
Ilario Losito,4Massimo De Vincenzi,3Maria D. De Bari,4Francesco Palmisano,4
Francesco Maurano,2Carmen Gianfrani,2and Marco Gobbetti1*
Department of Plant Protection and Applied Microbiology, University of Bari, 70126 Bari, Italy1; Istituto di
Scienze dell’Alimentazione—CNR, 83100 Avellino, Italy2; Division of Food Science, Human Nutrition and
Health, Istituto Superiore di Sanita, I-00161 Roma, Italy3; and Dipartimento di Chimica,
Centro Interdipartimentale di Ricerca SMART, University of Bari, 70126 Bari, Italy4
Received 1 February 2007/Accepted 15 May 2007
Presently, the only effective treatment for celiac disease is a life-long gluten-free diet. In this work, we
used a new mixture of selected sourdough lactobacilli and fungal proteases to eliminate the toxicity of
wheat flour during long-time fermentation. Immunological (R5 antibody-based sandwich and competitive
enzyme-linked immunosorbent assay [ELISA] and R5 antibody-based Western blot), two-dimensional
electrophoresis, and mass spectrometry (matrix-assisted laser desorption ionization–time of flight,
strong-cation-exchange-liquid chromatography/capillary liquid chromatography-electrospray ionization-
quadrupole-time of flight [SCX-LC/CapLC-ESI-Q-TOF], and high-pressure liquid chromatography-elec-
trospray ionization-ion trap mass spectrometry) analyses were used to determine the gluten concentration.
Assays based on the proliferation of peripheral blood mononuclear cells (PBMCs) and gamma interferon
production by PBMCs and intestinal T-cell lines (iTCLs) from 12 celiac disease patients were used to
determine the protein toxicity of the pepsin-trypsin digests from fermented wheat dough (sourdough). As
determined by R5-based sandwich and competitive ELISAs, the residual concentration of gluten in
sourdough was 12 ppm. Albumins, globulins, and gliadins were completely hydrolyzed, while ca. 20% of
glutenins persisted. Low-molecular-weight epitopes were not detectable by SCX-LC/CapLC-ESI-Q-TOF
mass spectrometry and R5-based Western blot analyses. The kinetics of the hydrolysis of the 33-mer by
lactobacilli were highly efficient. All proteins extracted from sourdough activated PBMCs and induced
gamma interferon production at levels comparable to the negative control. None of the iTCLs demon-
strated immunoreactivity towards pepsin-trypsin digests. Bread making was standardized to show the
suitability of the detoxified wheat flour. Food processing by selected sourdough lactobacilli and fungal
proteases may be considered an efficient approach to eliminate gluten toxicity.
Celiac disease (CD) is an inflammatory disorder of the small
intestine that affects genetically predisposed individuals when
they ingest gluten from any Triticum species and similar pro-
teins of barley and rye and their crossbred varieties.
Genes encoding HLA-DQ2 and HLA-DQ8 predispose to
CD by causing the preferential presentation of Pro-rich gluten
peptides that have undergone deamidation by tissue transglu-
taminase (tTG) to mucosal CD4?T cells (37). Gliadin-reactive
CD4?T cells have a key role in the damage of the intestinal
mucosa that culminates with villus atrophy and crypt hyperpla-
sia (12). Several studies have demonstrated the presence of
gliadin-reactive T cells within the celiac mucosa, and T-cell
clones have been established and used to identify specific gli-
adin-derived immunogenic peptides (3, 23). Overall, the lam-
ina propriae of CD patients contain significantly increased
levels of gamma interferon (IFN-?) after challenge with gliadin
(22). The high level of secretion of the Th1 cytokine IFN-? by
peripheral blood mononuclear cells (PBMCs) in response to
incubation with toxic peptides demonstrates a transient, dis-
ease-specific, HLA-DQ2-restricted, CD4?-T-cell response to a
single dominant epitope (1). Importantly, gliadin-induced
IFN-? production contributes to the onset and maintenance of
the chronic inflammatory response at the epithelial layer and
the lamina propria (27), presumably by attracting ?? CD8?
and ?? CD4?T cells and activating the Janus kinase-signal
transducer and activator of transcription 1 pathway.
The prevalence of CD worldwide is increasing; it is esti-
mated to be 0.5 to 2.0% in most of the European countries and
the United States (31). Presently, the only treatment for CD
consists of a life-long gluten-free diet (GFD). The Codex Ali-
mentarius Commission of the World Health Organization and
the FAO distinguishes gluten-free foods as those consisting of
ingredients with a gluten level of ?20 ppm or those which have
been rendered gluten free with a gluten level of ?200 ppm
Several attractive targets for new CD treatments are under
investigation. Complementary strategies aiming to interfere
with the activation of gluten-reactive CD4?T cells include the
inhibition of intestinal tTG activity to prevent the selective
* Corresponding author. Mailing address: Dipartimento di Protezi-
one delle Piante e Microbiologia Applicata, Via G. Amendola 165/a,
70126 Bari, Italy. Phone: 39 080 5442949. Fax: 39 080 5442911. E-mail:
?Published ahead of print on 18 May 2007.
deamidation of gluten immunogenic peptides (25) and the
blockage of the binding of gluten epitopes to the HLA-DQ2
and HLA-DQ8 molecules (21). Other treatments involve cy-
tokine therapy (32), selective adhesion molecule inhibitors
(37), and peptide degradation by prolyl endopeptidases (PEPs)
of microbial origin (24, 29, 34, 35). Beyond therapeutic treat-
ments, there have been recent efforts to use the knowledge on
the toxicity of gluten sequences for developing wheat that is
free of such sequences (18). In the last decades, cereal food
technology has changed dramatically by influencing the dietary
habitudes of entire populations previously naı ¨ve to gluten ex-
posure. Cereal baked goods are presently manufactured by fast
processes in which long-time fermentations by sourdough, a
cocktail of acidifying and proteolytic lactic acid bacteria with or
without Saccharomyces cerevisiae, have been almost totally re-
placed by the use of chemical and/or baker’s yeast leavening
agents. In this technology, cereal components (e.g., proteins)
are not degraded during manufacture (17). Recent studies (6,
7, 10, 11) showed that the manufacture of wheat and rye breads
or pasta with flours made tolerable to CD patients by using
selected sourdough lactobacilli may markedly decrease the tox-
icity of prolamin epitopes but that the concentration of gluten
in these foods remains above 6,000 ppm.
This paper describes the use of a more complex formula,
consisting of sourdough lactobacilli selected for their peptidase
systems and fungal proteases active specifically towards gluten,
than the above-cited studies for the manufacture of wheat
bread having a concentration of gluten of ?20 ppm. Wheat
protein hydrolysis was determined by complementary techniques
such as two-dimensional electrophoresis (2DE), matrix-assisted
laser desorption ionization–time of flight (MALDI-TOF) mass
spectrometry (MS), strong-cation-exchange (SCX)-liquid chro-
matography (LC)/capillary LC (CapLC)-electrospray ionization
enzyme-linked immunosorbent assay (ELISA), and R5-based
Western blot analyses. In vitro assays of PBMC-lymphocyte pro-
liferation and IFN-? production from PBMCs and intestinal T-
cell lines (iTCLs) confirmed the absence of toxicity of hydrolyzed
MATERIALS AND METHODS
Microorganisms and enzymes. Lactobacillus alimentarius 15 M, L. brevis 14G,
L. sanfranciscensis 7A, and L. hilgardii 51B (defined as pool 1) were previously
selected based on their capacity to hydrolyze gliadins (10). L. sanfranciscensis
LS3, LS10, LS19, LS23, LS38, and LS47 (defined as pool 2) were selected based
on their peptidase systems, with particular reference to activities towards Pro-
rich peptides (8). Strains were propagated for 24 h at 30°C in MRS broth (Oxoid,
Basingstoke, Hampshire, United Kingdom) with the addition of fresh yeast
extract (5%, vol/vol) and 28 mM maltose at a final pH of 5.6. When used for
wheat sourdough fermentations, Lactobacillus cells were cultivated until the late
exponential phase of growth was reached (ca. 12 h).
Proteases of Aspergillus oryzae (500,000 hemoglobin units on the tyrosine
basis/g; enzyme 1 [E1]) and A. niger (3,000 spectrophotometric acid protease
units/g; enzyme 2 [E2]), routinely used for bakery applications, were supplied by
BIO-CAT Inc. (Troy, VA).
Wheat sourdough fermentation. The characteristics of the wheat (Triticum
aestivum cv. Appulo) flour used in this study were as follows: moisture content,
12.8%; protein content (approximately equal to the organic nitrogen content
multiplied by 5.70), 10.3% (dry weight); fat content, 1.8% (dry weight); ash
content, 0.6% (dry weight); and total soluble carbohydrate content, 1.5% (dry
weight). The following formulas were used for sourdough fermentation: sour-
dough 1 (S1), 80 g of wheat flour and 320 g of tap water containing 5 ? 108
CFU/g of pool 1 (final density in the dough); S2, the S1 formula with the addition
of 5 ? 108CFU/g of pool 2; S2E1, the S2 formula with the addition of 200 ppm
of E1; S2E2, the S2 formula with the addition of 200 ppm of E2; and S2E12, the
S2 formula with the addition of 200 ppm of both E1 and E2. Doughs were
incubated for 48 h at 37°C with stirring (ca. 200 rpm). A chemically acidified
dough (CAD) without a bacterial and enzyme inoculum was acidified to pH 3.5
by a mixture of lactic and acetic acids (molar ratio, 4:1) and used as the control.
For each condition, four independent fermentations were carried out.
Extraction of wheat flour proteins and electrophoresis. Wheat flour proteins
were selectively extracted by following the method of Weiss et al. (46). For
Tricine-sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
(PAGE), immunological, and MS analyses, protein extraction was carried out
directly with 60% ethanol to include both peptides and proteins that were
hydroalcoholically soluble. The protein concentration was determined by the
Bradford method (5). The organic nitrogen concentration was determined using
the Kjeldahl method. Free amino acids were analyzed by a series 30 amino acid
analyzer (Biochrom Ltd., Cambridge Science Park, United Kingdom) (10).
2DE of aliquots of ca. 30 ?g of protein from extracted fractions was performed
with the Immobiline-polyacrylamide system (6, 10). Isoelectric focusing on im-
mobilized pH gradient strips (Immobiline strips; Amersham Pharmacia Biotech,
Uppsala, Sweden) was carried out to provide a linear gradient of pH 6.0 to 11.0
for gliadin fractions or a nonlinear gradient of pH 3.0 to 10.0 for albumin-
globulin and glutenin fractions by using an IPGphor system. Gels were silver
stained and spot intensities were normalized as reported by Bini et al. (4). Four
gels from independent fermentations were analyzed.
Aliquots of 10 to 20 ?l (ca. 10 ?g of protein) of extracted fractions were also
analyzed by Tricine-SDS-PAGE according to the method of Scha ¨gger and von
Immunological and MALDI-TOF MS analysis. Immunological analyses were
carried out by using R5 antibody-based sandwich and competitive ELISAs and
R5 antibody-based Western blotting. The R5 monoclonal antibody and the
horseradish peroxidase-conjugated R5 antibody were used for gluten analysis.
The R5-based sandwich ELISA (43) was performed with the Transia plate
detection kit by following the instructions of the manufacturer (Diffchamb,
Va ¨stra Fro ¨lunda, Sweden). The R5-based competitive ELISA (15) was per-
formed at the gluten unit of the Centro National de Biotecnologia (Madrid,
Spain). For R5-based Western blot analysis, after one-dimensional SDS-PAGE
or Tricine-SDS-PAGE, proteins were electrotransferred onto polyvinylidene
difluoride membranes, the membranes were incubated directly with R5-
horseradish peroxidase, and the blots were developed by immunodetection
with the ECL Western blotting analysis system (Amersham Pharmacia) (43).
MALDI-TOF MS analysis was carried out with a Voyager De Pro workstation
(PerSeptive Biosystems, United Kingdom). Eight microliters of 50 mM octyl-D-
glucopyranoside detergent and 25 ?l of saturated sinapic acid in 30% (vol/vol)
acetonitrile solution, containing 0.1% (vol/vol) trifluoroacetic acid (TFA), were
added to 100 ?l of gliadin ethanol extracts. The matrix-sample mixture and the
experimental conditions used were described previously (19). A standard of
European gliadins was also included in the analyses.
Off-line bidimensional SCX-LC/CapLC followed by ESI-Q-TOF MS analysis
of gliadin peptides. The SCX-LC apparatus consisted of a binary LC pump
(model no. 1525?; Waters, Milford, MA), an SCX BIO BASIC column (Thermo-
Electron, San Jose, CA), and a Rheodyne six-port injector. SCX-LC separations
were performed at a 0.2-ml/min flow rate by using a binary gradient with (i) a
60% water–40% acetonitrile (vol/vol) mixture (mixture A) and (ii) mixture A
containing 300 mM ammonium acetate (mixture B; elution program, 0 to 40%
mixture B in 50 min, return to initial conditions in 10 min, and column recon-
ditioning for 20 min). UV detection was performed at 240 nm by using a UV-VIS
detector (model no. 2487; Waters). The UV trace, processed by the MassLynx
4.0 software, was employed as a guide for the collection of fractions to be further
separated and analyzed by CapLC-ESI-Q-TOF MS. This stage of analysis was
performed under the conditions described by De Angelis et al. (6).
Kinetics of hydrolysis and high-pressure liquid chromatography (HPLC)-ESI
MS analysis of the 33-mer. A mixture containing the microbial pools 1 and 2, 2
mM 33-mer peptide, and 0.05% (wt/vol) NaN3in 1 ml of 200 mM phosphate
buffer, pH 7.5, was incubated for 48 h at 37°C with stirring (150 rpm). Aliquots
were taken at intervals, and TFA (0.1% [vol/vol] final concentration) was added.
After centrifugation, supernatants corresponding to extracellular solutions were
collected. Harvested cells were resuspended in 100 ?l of 200 mM phosphate
buffer, pH 7.5, and sonicated to obtain the cytoplasmic extracts. Extracellular
solutions and cytoplasmic extracts were stored at ?80°C until MS analysis.
Reverse-phase HPLC coupled with ESI-ion trap MS was adopted for the
analysis of the 33-mer either in the extracellular solutions or in the cytoplasmic
extracts. The HPLC apparatus consisted of a Waters 600 MS chromatographic
pump and a Supelcosil LC-18-DB column (250 by 2.1 mm in diameter; Supelco,
4500 RIZZELLO ET AL.APPL. ENVIRON. MICROBIOL.
Bellefonte, PA) connected to an LCQ ion trap mass spectrometer (Thermo-
Electron) through its ESI interface; the divert/inject six-port valve embedded in
the spectrometer was used for loop injection (injection volume, 40 ?l) of sam-
HPLC separations were performed at a flow rate of 0.16 ml/min by using
gradient elution with (i) water and (ii) acetonitrile, both containing 0.1% (vol/
vol) TFA, according to the following program: 0 to 70% (vol/vol) acetonitrile in
35 min, isocratic elution with 70% acetonitrile for 5 min, return to 0% aceto-
nitrile in 5 min, and column reconditioning for 20 min. The LCQ spectrometer,
completely controlled by the Xcalibur software (ThermoElectron), was operated
in the positive ion mode; MS chromatograms in the total ion current (m/z range,
50 to 2,000) and selected ion monitoring (for an m/z of 1,955.5, corresponding to
the doubly charged ion [M ? 2H]2?of the 33-mer) modes were recorded for
Pepsin-trypsin (PT) digests and patients. Protein fractions selectively ex-
tracted from CAD and sourdoughs were subjected to sequential PT hydrolysis
steps to simulate in vivo digestion (6). After digestion, the PT digests were
heated at 100°C for 30 min to inactivate enzymes and then freeze dried.
Six newly diagnosed CD patients (age range, 18 to 54 years) were included in
the study. CD was diagnosed according to European Society for Paediatric
Gastroenterology, Hepatology and Nutrition criteria (14). All six patients were
on gluten-containing diets at the time of enrollment. A sample of peripheral
blood was taken from each patient on the day of diagnosis by gastrointestinal
For the generation of T-cell lines, biopsy specimens from the small intestines
of six additional patients (age range, 18 to 49 years) undergoing treatment for
CD were obtained. All patients expressed the HLA-DQ2 phenotype.
Culture of PBMCs, MTT assay, and IFN-? evaluation. PBMCs were isolated
from 6 ml of heparinized blood by Lympholyte-H (Cederlane, Hornby, Ontario,
Canada) gradient centrifugation and cultured at a density of 1.0 ? 106cells per
ml in 96-well culture plates in RPMI 1640 culture medium supplemented with
10% fetal bovine serum, streptomycin (50 ng/ml), and penicillin (100 g/ml;
GIBCO-Invitrogen Ltd., Paisley, United Kingdom). After 24 h, PBMCs were
treated in triplicate (independent experiments) with the PT digest of glutenins or
gliadins (0.5 mg/ml). After 24 h of stimulation, PBMCs were harvested for the
MTT [3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide] assay, and
the free supernatant was collected and stored at ?80°C for IFN-? evaluation.
To assess the effect of treatments on the PBMC-lymphocyte proliferation, the
MTT assay (MTT-based cell growth determination kit; Sigma, St. Louis, MO)
was carried out. After 24 h of incubation, measurements were carried out by an
ELISA reader (Bio-Rad, Hercules, CA) at 570 nm by using a 630-nm-pore-size
filter as a reference. The proliferative response in the presence of the culture
medium alone (negative control) was taken as 100%, and the percentage of
inhibition in response to each PT digest was calculated.
The concentrations of IFN-? in PBMC supernatant samples were determined
by commercial ELISA kits according to the instructions of the manufacturer
(Biosource, Camarillo, CA). Standards were run on each plate. Samples from the
different patients were run at the same time. The level of IFN-? production in the
presence of the culture medium alone was taken as 100%, and the responses of
PBMCs treated with PT digests were compared to this standard.
Generation of gliadin-specific iTCLs and T-cell specificity assays. Jejunal
explants were digested with collagenase A as described previously (42). T-cell
lines were established by repeated stimulation (every 7 days) of intestinal cells
with 50 ?g of tTG-deamidated gliadin/ml and autologous irradiated PBMCs in
complete culture medium containing 10% human serum. Gliadin-specific iTCLs
were kept as long-term cell cultures by cyclic restimulation (every 14 days) with
phytohemagglutinin and feeder cells and interleukin-2.
iTCLs were tested for the recognition of gluten-derived fractions from both
CAD and sourdoughs by the detection of IFN-? production by ELISAs. tTG-
treated deamidated PT digests of albumin-globulin, gliadin, and glutenin frac-
tions were used as antigens. Intestinal cells (0.3 ? 105) were plated in 200 ?l of
10% human serum in the presence of irradiated (11,000 rad) autologous or
HLA-matched B lymphoblastoid cell lines transformed with Epstein-Barr virus
and antigens (50 ?g/ml). A tTG-PT-gliadin from T. aestivum cv. Sagittario was
used as a positive control. After 48 h, cell supernatants (50 ?l) were collected for
the assessment of IFN-? production; thereafter, cells were labeled with 0.5 ?Ci
of [3H]thymidine (Amersham Pharmacia)/well and harvested after 16 h. IFN-?
production was analyzed using a sandwich ELISA manufactured in-house with a
pair of anti-IFN-? monoclonal antibodies (purified and biotinylated; Pharmingen-
BD). All experiments were performed in duplicate. T-cell lines were considered
antigen specific when IFN-? production increased more than twofold with re-
spect to that in the medium alone.
Bread making. Baker’s yeast bread was made by short fermentation: a
mixture of 125 g of wheat flour and 64 ml of tap water (containing 1.5%
baker’s yeast) was allowed to ferment for 2 h at 37°C and baked at 250°C for
15 min. Sourdough bread was made using the same formula and conditions
described for S2E12. After fermentation for 48 h at 37°C, water was removed
by spray drying, the flour was milled, and the following formula was used for
bread making: 125 g of fermented wheat flour (moisture content, ca. 12%),
100 ml of tap water (containing 1.5% baker’s yeast), 6% (wt/wt) cornstarch
(Clearam CH 20; product no. E1442; CHIMAB Spa, Pordenone, Italy), and
3% (wt/wt) xanthan gum (product no. E415; CHIMAB Spa). Compared to
that in the baker’s yeast bread formula, the amount of water was increased to
permit the optimal performance of the structuring agents. The dough was
mixed continuously with a high-speed mixer (60 ? g; dough mixing time, 5
min; Chopin and Co., Boulogne-sur-Seine, France), allowed to ferment for
2 h at 37°C, and baked at 250°C for 15 min.
Four independent baking tests were carried out, and loaf volumes (as deter-
mined by rapeseed displacement) and weights were measured.
Immunological analyses. Cell numbers of lactic acid bacteria
in sourdoughs at the end of fermentation ranged from 3 ? 109
to 5 ? 109CFU/g of dough, and the pH was ca. 3.5. The total
bacterial count in the CAD did not exceed 103CFU/g of
After fermentation, samples were analyzed by an R5-based
sandwich ELISA (Table 1). Compared to that in CAD, the
concentration of gluten in the sourdough progressively de-
creased in the order of S1, S2, S2E1, S2E2, and S2E12. The
concentration of residual gluten in S2E12 was 12 ppm. The
same level of gluten was found by an R5-based competitive
ELISA. This sourdough was fermented with microbial pool 1,
consisting of L. alimentarius 15 M, L. brevis 14G, L. sanfranci-
scensis 7A, and L. hilgardii 51B (10); microbial pool 2, consist-
ing of L. sanfranciscensis LS3, LS10, LS19, LS23, LS38, and
LS47 (8); and the addition of proteases from A. oryzae (E1)
and A. niger (E2). None of the other possible combinations not
included in Table 1 gave a concentration of gluten of less than
20 ppm. Relative to the European gliadin standard and the
CAD, no traces of gliadins in S2E12 were detectable by R5-
based Western blotting (Fig. 1). The same was found regarding
hydrolysis end products.
2DE. 2DE analysis of the CAD resolved 218 albumin-glob-
ulin polypeptides with pIs of 4.15 to 9.7 and molecular masses
of 6.0 to 74.5 kDa. No albumin-globulin polypeptides in S2E12
were detectable. Seventy-six gliadin polypeptides in the CAD
were detected by 2DE analysis. Polypeptides had pIs of 6.5 to
TABLE 1. Concentrations of gluten in CAD and sourdoughs
started with different mixtures of lactobacilli and enzymes
as estimated by R5-based sandwich ELISAa
CAD......................................................................................74,592 ? 320
S1 ..........................................................................................20,315 ? 112
S2 ..........................................................................................12,362 ? 86
S2E1...................................................................................... 4,895 ? 92
S2E2...................................................................................... 1,055 ? 45
S2E12....................................................................................12 ? 2
aCAD and sourdoughs were fermented for 48 h at 37°C. Details about sour-
dough compositions, initial cell densities, and enzyme concentrations are given in
Materials and Methods. Data are the means from four independent fermenta-
tions each analyzed twice.
VOL. 73, 2007LACTOBACILLI AND ENZYMES: NEW PERSPECTIVES FOR CD4501
9.9 and molecular masses of 9.2 to 54.1 kDa. No gliadin
polypeptides in S2E12 were detectable. 2DE analysis of the
CAD resolved 193 glutenin polypeptides with pIs of 4.10 to
9.45 and molecular masses of 11.05 to 81.8 kDa (Fig. 2A).
Although marked hydrolysis was found, some glutenin
polypeptides persisted in S2E12 (Fig. 2B). Of the 193 spots
identified for the CAD, 160 showed hydrolysis factors which
ranged from 95 to 100% and 24 and 9 demonstrated hydrolysis
of 50 to 70% and 30 to 50%, respectively.
MALDI-TOF MS. CAD and S2E12 were subjected to ex-
traction by 60% ethanol and analyzed by MALDI-TOF MS
(Fig. 3). Gliadin peaks corresponding to the European gliadin
standard and the CAD completely disappeared for S2E12. To
look for small polypeptides, the m/z range used to analyze
S2E12 was extended. A few peaks corresponding to molecular
masses of less than ca. 8 kDa were found.
Protein and peptide concentrations. Spray-dried flour from
CAD contained 0.29% water/salt-soluble proteins (mainly
albumins and globulins; 16.3% of the total organic nitrogen
content), 0.66% gliadins (37.1% of the total organic nitrogen
content), and 0.83% glutenins (46.6% of the total organic
nitrogen content), with a total organic nitrogen content of
1.78% that approximately corresponded to the protein concen-
tration (the organic nitrogen content multiplied by 5.70 is
10.15%, versus a protein concentration of 10.3%) of the wheat
flour used. The spray-dried flour from S2E12 showed a marked
increase in the water/salt-soluble fraction to 1.66% (90.7% of
the total organic nitrogen content), which corresponded to the
accumulation of water/salt-soluble low-molecular-mass pep-
tides and amino acids derived from the hydrolysis of all wheat
proteins. Indeed, no organic nitrogen was detectable in the
gliadin fraction, and the level of glutenins decreased to 0.17%
(9.3%). The concentrations of free amino acids confirmed this
difference: the CAD had ca. 1,050 mg/kg, while S2E12 con-
tained ca. 14,622 mg/kg. Leu, Val, Glu, Ile, and Pro were
present at the highest concentrations.
The Tricine-SDS-PAGE of the 60% ethanol-soluble fraction
of S2E12 confirmed the presence of a few polypeptides with
molecular masses of less than ca. 8 kDa. No immunogenic
polypeptides were detected by the R5 monoclonal antibody in
Tricine-SDS-PAGE. Both the 60% ethanol-soluble and the
water/salt-soluble fractions of S2E12 were further analyzed
using SCX-LC/CapLC followed by ESI-Q-TOF MS. Fractions
were collected from the SCX-LC eluate whenever a peak with
significant absorbance was observed; each of the fractions was
then subjected to CapLC-ESI-Q-TOF MS analysis. Mass
spectra related to each peak detected in the relevant total ion
current traces were elaborated in order to search for m/z of
[M ? nH]n?(where n is a variable) ions arising from gliadin
peptides. No peak could be related to such species. One of the
SCX-LC fractions from the water/salt-soluble fraction was
spiked with 1 ppm of the synthetic epitope spanning residues
62 to 75 of ?-gliadin (1) and analyzed by CapLC-ESI-Q-TOF
MS. A very intense peak was found, and the corresponding
mass spectra confirmed the attribution of the epitope to the
fragment comprising residues 62 to 75. Therefore, if present,
gliadin peptides with molecular masses lower than 3 kDa (the
detectable molecular mass limit for the ESI-Q-TOF MS anal-
ysis) had concentrations well below 1 ppm.
Kinetics of hydrolysis of the 33-mer. To elucidate the mech-
anism of the hydrolysis of gliadin epitopes, lactobacillus pools
FIG. 1. R5-based Western blot analyses of the European gliadin
reference (St), CAD (lane 1), and S2E12 (lane 2) fermented for 48 h
at 37°C. After one-dimensional SDS-PAGE, proteins were electro-
transferred for R5-based Western blot analysis. S2E12 ingredients are
described in Materials and Methods.
FIG. 2. 2DE analysis of glutenin polypeptides of CAD (A) and
S2E12 (B) fermented for 48 h at 37°C. S2E12 ingredients are described
in Materials and Methods. Mr, molecular mass.
4502 RIZZELLO ET AL.APPL. ENVIRON. MICROBIOL.
1 and 2 were incubated in a buffer system with 2 mM 33-mer.
The concentrations of the 33-mer peptide both in the extra-
cellular environment and the cell cytoplasm were monitored
for 48 h using HPLC-ESI-ion trap MS. The 33-mer was found
in the cell cytoplasm as soon as the incubation started, al-
though its concentration could be estimated to be 100-fold
lower than the one in the extracellular environment (Fig. 4).
Remarkable decreases in the peptide concentrations in both
media were also observed, although the decrease in the cyto-
plasm was sharper. About 70% of the 33-mer was degraded in
6 h, and its hydrolysis was completed after 18 h. No traces of
the 33-mer were detectable in the cell cytoplasm. Potential
33-mer hydrolysis products were searched for in both samples,
after each of the incubation periods. No evidence of their
presence, at least at the minimum detectable concentration (20
ppm), was found. This minimum concentration was estimated
by injecting each sample with the synthetic analogue of peptide
QLQPFPQPQLPY, a potential hydrolytic product of the
PBMC proliferation. Polypeptides soluble in 60% ethanol
and glutenins were extracted from CAD and the sourdoughs,
subjected to PT hydrolysis, and used to treat PBMCs. The
MTT assay was used to asses the lymphocyte proliferation,
which decreased in proportion to the activation of apoptosis in
FIG. 3. MALDI-TOF mass spectra of the European gliadin reference (st), CAD (1), and S2E12 (2) fermented for 48 h at 37°C. S2E12
ingredients are described in Materials and Methods.
FIG. 4. Time dependence of the peak areas obtained for the 33-mer in the selected ion monitoring mode in the HPLC-ESI-ion trap MS analysis
of the extracellular environment (A) and lactobacillus cell cytoplasm (B).
VOL. 73, 2007 LACTOBACILLI AND ENZYMES: NEW PERSPECTIVES FOR CD4503
the presence of the antigen. The proliferation rate of PBMCs
subjected to PT digests of polypeptides soluble in 60% ethanol
increased according to the intensity of gluten hydrolysis in the
sourdoughs (Fig. 5A and Table 1). The rate of proliferation in
the presence of the PT digest from S2E12 was the same as that
in the negative control. The same result was found for the PT
digest of glutenins from S2E12 (Fig. 5B).
IFN-? production by PBMCs and iTCLs. The exposure of
lymphocytes to PT digests from different sourdoughs resulted
in a progressive decrease in the cytokine production (Fig. 6A).
The release of IFN-? by PBMCs treated with the PT digest
from S2E12 was the same as that in the negative control. No
differences in glutenins between the negative control and
PBMCs treated with the PT digest from S2E12 were found
IFN-? production in the culture supernatants was measured
after 48 h of gliadin stimulation of long-term iTCLs established
from intestinal mucosae of CD patients and highly responsive
to tTG-PT-gliadin. iTCLs produced significant levels of IFN-?
in response to gliadins from cv. Sagittario and albumin-globu-
lin, gliadin, and glutenin fractions from the CAD (Fig. 7). The
IFN-? level induced by the PT digest of CAD-derived gliadins
was comparable to that in the positive control (cv. Sagittario).
PT-glutenin stimulation resulted in lower levels of IFN-? pro-
duction than stimulation with albumin-globulin and gliadin
fractions from CAD. On the contrary, no IFN-? production
was found following stimulation with any of the PT digests
corresponding to S2E12.
Bread making. After the fermentation of S2E12, the water
was removed and the pretreated wheat flour was used for
bread making by using baker’s yeast and structuring agents.
This bread (sourdough bread) was compared to baker’s yeast
bread made with untreated flour and without structuring
agents. The specific loaf volume (vol/wt) of the sourdough
bread was similar to that of the baker’s yeast bread (1.9 ? 0.06
cm3/g versus 2.06 ? 0.06 cm3/g, respectively).
Although new and attractive molecular treatments are under
investigation (21, 25, 32, 37), presently, a GFD remains the
only effective therapy for CD. Nevertheless, a GFD also has
some drawbacks. It is expensive, gluten-free products have
poor sensory and shelf life properties, and the diet is hard to
follow and needs continuous monitoring by dieticians, also due
to occasional malnutrition problems (18, 41). On the other
hand, the development of wheat varieties that are free of toxic
polypeptide sequences is considered unlikely because of the
high degree of sequence homology existing among members of
FIG. 5. Effects of the PT digests of polypeptides soluble in 60% ethanol (A) and glutenins (B) extracted from CAD and sourdoughs S1, S2,
S2E1, S2E2, and S2E12 on PBMC proliferation. Cn, negative control. The proliferative response in the presence of the culture medium alone
(negative control) was taken as 100%, and the percentage of inhibition in response to each PT digest was calculated. Sourdough ingredients are
described in Materials and Methods. Data are the means ? standard deviations from three independent fermentations each analyzed twice.
FIG. 6. Effects of the PT digests of polypeptides soluble in 60% ethanol (A) and glutenins (B) extracted from CAD and sourdoughs S1, S2,
S2E1, S2E2, and S2E12 on IFN-? production by PBMCs. Cn, negative control. The IFN-? production in the presence of the culture medium alone
was taken as 100% and used as a standard to assess the responses of PBMCs treated with PT digests. Sourdough ingredients are described in
Materials and Methods. Data are the means ? standard deviations from three independent fermentations each analyzed twice.
4504 RIZZELLO ET AL.APPL. ENVIRON. MICROBIOL.
the cereal protein family and because cereals like wheat are
Novel proteomic technologies to eliminate toxic epitopes
during food processing were pursued in this study. Food-grade
sourdough lactobacilli, as cell factories for multiple and com-
plementary proteolytic enzymes, supplemented with fungal
proteases were used. This approach conceptually resembles
that of other studies (24, 29, 34, 35), which used microbial
PEPs but as an oral supplement to the diet. Flavobacterium
meningosepticum PEP-catalyzed cleavage activity was shown to
be complementary to intestinal brush border enzymes. The
pretreatment of gluten with PEP of F. meningosepticum pre-
vented the development of fat or carbohydrate malabsorption
in the majority of those patients who were subjected to a
2-week gluten challenge (30). A newly identified PEP from A.
niger efficiently degrades T-cell-stimulatory peptides as well as
a PT digest of gluten and intact gluten (39). In our study, the
addition of fungal proteases routinely used in bread making
was useful to start the primary proteolysis of wheat proteins.
After primary hydrolysis, medium-sized polypeptides, includ-
ing the 33-mer and analogue epitopes, are efficiently trans-
ported into lactobacillus cytoplasm and subjected to peptidase
Previous in vivo, ex vivo, and in vitro assays showed that
a few of the sourdough lactobacilli (pool 1) considered in
this study, although markedly decreasing the toxicity of glu-
ten epitopes (6, 7, 10, 11), were helpful only in eliminating
the risk of gluten cross contamination. Aiming at manufac-
turing a bread made of wheat flour alone that could be
tolerated by CD patients, further efforts were made in this
study to increase the hydrolyzing capacity during sourdough
fermentation (7, 10, 11). Besides adding fungal proteases,
we employed a new pool (pool 2) of selected L. sanfranci-
scensis strains (8). These strains are characterized by
marked peptidase activity towards Pro-rich peptides. As re-
quired by the Codex Alimentarius Commission, the hydro-
lyzed wheat flour contained ?20 ppm of gluten. This thresh-
old was also confirmed by the R5-based competitive ELISA
(15) that allows the quantification of hydrolyzed gliadins,
the level of which may be underestimated in some cases by
the R5-based sandwich ELISA, in foods that have been
processed by enzymatic procedures. The gluten concentra-
tion was also consistent with the recommendation of the
Prolamins Working Group, which suggested a lower limit
than the previous threshold of 200 ppm (40). After hydro-
lysis, S2E12 spray-dried flour was a mixture of mainly water/
salt-soluble low-molecular-mass peptides and amino acids.
By using several complementary immunological, electro-
phoresis, and MS techniques, it was shown that gliadin and
albumin-globulin were completely degraded, as were the
glutenins for the most part. As determined by SCX-LC/
CapLC-ESI-Q-TOF MS and R5-based Western blot analy-
ses, no epitopes with molecular masses of ca. ?8 kDa were
detectable in the 60% ethanol-soluble and water/salt-solu-
ble fractions. About 20% of the glutenin fraction persisted
in the sourdough S2E12. Glutenins may contain sequences
(e.g., glt04 residues 707 to 742) that activate T cells from the
small intestine and result in the secretion of large amounts
of IFN-? (38, 44). High-molecular-mass glutenin subunits
stimulate T-cell lines from some CD patients and exacerbate
CD in vivo (9). For these reasons, the present diagnostic
value of the R5 antibody for detecting toxic epitopes may be
in part limited, and in vitro assays (e.g., those involving
iTCLs and PBMCs) are imposed to confirm the absence of
Although peripheral lymphocytes cannot reflect the cyto-
kine profile of the tissue under analysis, some studies (1, 2)
have shown the suitability of PBMCs as a model system.
Besides, the in vitro incubation of PBMCs from CD patients
with PT digests provides a noninvasive system to screen the
toxicity of sourdough samples. As expected, the PT digests
from the CAD caused a marked decrease in PBMC prolif-
eration due to the related activation of apoptosis that occurs
when highly activated lymphocytes again encounter the an-
tigen (47). Responses to PT digests from various sourdoughs
differed and culminated with the response to the PT digest
from S2E12, which activated PBMCs to the level in the
negative control. IFN-? production is a Th1-specific event
(2), whereas proliferation is an index of T-cell activation.
Indeed, the proliferation of PBMCs of CD patients is inhib-
ited by anti-DR antibody, indicating that class II molecules
are involved in the presentation of gliadin peptides to pe-
ripheral T cells (28). IFN-? coordinates many aspects of the
innate and adaptive immune responses, and it is the princi-
pal cytokine produced by ?? CD4?reactive T cells upon
gluten activation (26, 27, 32, 45). The PT digests of 60%
ethanol-soluble polypeptides and glutenins from sourdough
S2E12 had the same capacity to induce IFN-? as the nega-
tive control. To exclude immunoreactivity, the immune re-
sponses of iTCLs to the PT digest of S2E12 was also assayed
(3, 13, 20, 36, 42). Six gluten-specific iTCLs obtained from
jejunal explants increased IFN-? production when stimu-
lated by protein fractions from the CAD. None of the iTCLs
used in this study demonstrated immunoreactivity when
treated with the PT digest of S2E12.
Technologically, an expected perplexity concerns the suit-
ability of such wheat flour to ensure bread quality given the
complete degradation of gluten. Spray-dried flour from S2E12
was used as an ingredient in bread made by conventional
short-time fermentation with baker’s yeast and structuring
agents. The specific loaf volume of this bread was just slightly
FIG. 7. Effect of tTG-deamidated PT digests of proteins extracted
from CAD (columns 1, 2, and 3) and S2E12 (columns 4, 5, and 6) on
IFN-? production by iTCLs. Columns: Cn, negative control (medium
alone); 1 and 4, albumins and globulins; 2 and 5, gliadins; 3 and 6,
glutenins; Sg, PT digest of gliadins from cv. Sagittario. The IFN-? level
in 48-h cell culture supernatant was measured by a sandwich ELISA.
S2E12 ingredients are described in Materials and Methods. Data are
the means ? standard deviations from two independent fermentations
each analyzed twice.
VOL. 73, 2007 LACTOBACILLI AND ENZYMES: NEW PERSPECTIVES FOR CD4505
lower than that of baker’s yeast bread, and the bread had the
typical flavor of sourdough wheat bread as judged by an inter-
nal panel test. Given the encouraging results of this study,
research including a long-term in vivo challenge of CD patients
is in progress.
This work was supported by the Italian Ministry of University and
Research, project no. 12819, D.D. 1801 (31 December 2004).
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