Efficacy of a new nutraceutical formulation: L-tryptophan, probiotics, charcoal, chamomile, mint, and licorice (COLONIR®) in the improvement of gastrointestinal symptoms in subjects with irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders. IBS is characterized by recurrent chronic abdominal pain and altered bowel habits in the absence of organic damage. Although there are reviews and guidelines for treating IBS, the complexity and diversity of IBS presentation make treatment difficult. Treatment of IBS focuses on relieving symptoms as mild signs and symptoms can often be controlled by managing stress and by making changes in diet and lifestyle. The use of nutraceutical compounds has been advocated as a possible alternative treatment in patients with IBS. COLONIR® (Omega Pharma Srl, Milan, Italy) may be an alternative or adjuvant treatment in patients with gastrointestinal symptoms. This study aimed to evaluate the effect of this new nutraceutical formulation in inducing symptoms remission and improve gastrointestinal habits. METHODS: An initial cohort of 1004 consecutive patients referred to 25 different Units of Internal Medicine a/o Gastroenterology in Italy to perform colonoscopy for intestinal symptoms was asked to participate. Patients were treated for 2 months with two doses of nutraceuticals/day during meals namely COLONIR®. Patients were assessed at baseline and after 2 months to evaluate the frequency and severity of gastrointestinal symptoms in the past seven days with a questionnaire based on ROMA IV criteria. RESULTS: After 2 months, 899 patients completed the follow-up. COLONIR® achieved a statistically significant reduction of severity of symptoms in the study population without any documented side effects. CONCLUSIONS: These promising results, here reported, need to be confirmed, valuating the efficacy of COLONIR® in relieving gastrointestinal symptoms in IBS patients in further studies.

Effects of a Low FODMAP Diet and Specific Carbohydrate Diet on Symptoms and Nutritional Adequacy of Patients with Irritable Bowel Syndrome: Preliminary Results of a Single-blinded Randomized Trial.

BACKGROUND AND OBJECTIVES: IBS is the most common functional disease of the low gastrointestinal tract. Recently, the interest towards a diet approach has increased, for example, a diet with low content of fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs). The aim of the present study is to evaluate the efficacy of a low FODMAP diet and a specific carbohydrate diet (SCD) conducted for 3 months on symptoms and to evaluate the deficiencies of vitamin D and folic acid in patients affected by IBS, matching the Rome IV criteria. METHODS: We evaluated 73 patients divided into 2 groups: one submitted to low FODMAP diet and one to SCD, for 3 months. Patients were assigned to one of the 2 groups randomly and blinded. All the patients filled a visual analogue scale (VAS) to evaluate the severity of symptoms and a diary to evaluate the number of days with symptoms, and this was repeated after 3 months. Final evaluation was made by a blinded investigator. RESULTS: In the end, the patients with low FODMAP diet had a significant improvement in bloating and distension (P = 0.000); the group with SCD instead had a low but not a significant improvement. One way ANOVA showed comparable severity of symptoms in the 2 groups pre-diet (P = 0.215), but a difference in the same symptoms after 12 days (P = 0.000). Tukey test showed a significant improvement in the low FODMAP diet group and only a trend of improvement in the second group of SCD. The vitamin D mean value in both groups at the time of enrollment was 38 ng/mL; in the end, the mean value in the low FODMAP diet group was 32 ng/mL and in the SCD group was 22 ng/mL, with a statistically significant difference. The folic acid mean value at the time of enrollment was 18 mg/dL; in the end, the mean value in the low FODMAP diet group was 15 mg/dL and in the SCD group was 8 mg/dL, with a statistically significant difference. CONCLUSION: Patients affected by IBS seem to have benefitted from a low FODMAP diet but not from an SCD, and a low FODMAP diet doesn't seem to cause vitamin D and folic acid deficiencies.

Gluten-free sourdough wheat baked goods appear safe for young celiac patients: a pilot study.

OBJECTIVES: This study was aimed at showing the safety, for young patients with celiac disease (CD), of sweet baked goods made of wheat flour, which was rendered gluten-free during sourdough fermentation. METHODS AND RESULTS: As shown by R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay (ELISA), selected lactobacilli and fungal proteases, routinely used in bakeries, degraded gluten to <10 ppm during sourdough fermentation. The resulting flour was mainly a mixture of water-/salt-soluble low-size peptides and free amino acids. Gliadin and glutenin fractions extracted from the pepsin-trypsin (PT) digest of the fermented wheat flour induced the expression of interferon (IFN)-γ at the level comparable with the negative control. After fermentation, the wheat flour was spray dried and used for making sweet baked goods. Eight patients with CD in remission were enrolled for the clinical challenge, and they daily consumed 200 g of sweet baked goods equivalent to 10 g of native gluten. Hematology, serology (total serum IgA, IgG and IgA antigluten, endomysial and tissue transglutaminase IgA antibodies), and intestinal permeability analyses were carried out over time. One patient interrupted the trial after 15 days and another after 30 days only due to difficulties in the compliance of the daily consumption. All of the other patients showed normal values of hematology, serology, and intestinal permeability during 60 days of challenge. CONCLUSIONS: This study showed that a wheat flour-fermented product, having gluten completely degraded, is not toxic for patients with CD. Nevertheless, these foods should not be recommended for patients with celiac disease until a formal trial has been done.

Quorum sensing in sourdough Lactobacillus plantarum DC400: induction of plantaricin A (PlnA) under co-cultivation with other lactic acid bacteria and effect of PlnA on bacterial and Caco-2 cells.

This work aimed at showing the effect of pheromone plantaricin A (PlnA) by Lactobacillus plantarum DC400 towards other sourdough lactic acid bacteria and the potential of PlnA to protect the function of the human intestinal barrier. Growth and survival of sourdough lactic acid bacteria were differently affected by co-cultivation with L. plantarum DC400. Compared to mono-cultures, Lactobacillus sanfranciscensis DPPMA174 and Pediococcus pentosaceus 2XA3 showed growth inhibition and decreased viability when co-cultured with L. plantarum DC400. L. sanfranciscensis DPPMA174 induced the highest synthesis of PlnA. Survival of strain DPPMA174 only slightly varied by comparing the addition of PlnA to the culture medium and the co-cultivation with L. plantarum DC400. Compared to mono-culture, the proteome of L. sanfranciscensis DPPMA174 grown in co-culture with L. plantarum DC400 showed the variation of expression of 58 proteins (47 over expressed and 11 repressed). Thirty-four of them were also over expressed or repressed during growth of DPPMA174 with PlnA. Fifty-one of the above 58 proteins were identified. They had a central role in stress response, amino acid, energy and nucleotide metabolisms, membrane transport, regulation of transcription, and cell redox homeostasis. PlnA markedly increased the viability of human Caco-2/TC7 cells and the transepithelial electrical resistance.

Two prolamin peptides from durum wheat preclude celiac disease-specific T cell activation by gluten proteins.

PURPOSE: Celiac disease (CD) is a permanent intolerance to wheat prolamins and related proteins displayed by genetically susceptible individuals. Blocking or modulation of CD-specific T cell response by altered prolamin peptides are currently considered as a potential alternative to the only effective therapy of CD based on a life-long gluten-free diet. Two prolamin peptides, the 9-mer ASRVAPGQQ and the 10-mer GTVGVAPGQQ sequences, were identified by mass spectrometry in the peptic/tryptic digest of prolamins (PTP) from durum wheat (Triticum turgidum ssp. durum) cv. Adamello, and investigated for their ability to preclude the stimulation of CD-specific mucosal T cells by gluten proteins. METHODS: Gluten-specific polyclonal intestinal T cell lines from five CD children (mean age 5 years) were exposed to 50 microg/ml of a deamidated PTP from whole flour of common wheat (T. aestivum) cv. San Pastore, and tested for proliferation and production of interferon-gamma (INF-gamma) and interleukin 10 (IL-10). The same experiment was performed in the presence of 20 microg/ml of the 9-mer or the 10-mer peptide. RESULTS: T cells exposed to PTP showed a threefold increase in proliferation and INF-gamma production, and a significant (P

T-cell response to different cultivars of farro wheat, Triticum turgidum ssp. dicoccum, in celiac disease patients.

BACKGROUND & AIMS: Celiac disease is a gluten sensitive disorder that occurs in genetically susceptible individuals. The present study deals with variation in the immune response of mucosal T-cells from celiac children to prolamins extracted from nine landraces of farro wheat (Triticum turgidum ssp. dicoccum) with contrasting storage protein compositions. METHODS: The prolamin fraction from nine 'dicoccum' wheat landraces was subjected to peptic-tryptic digestion and supplied to T-cells from mucosal explants of four celiac patients. Immune reactions in terms of cell proliferation and INF-gamma secretion by intestinal T lymphocytes were then determined. RESULTS: T-cell lines exposed to digested prolamins from landraces L5563, L5558 and L5540 showed negligible proliferative responses and released INF-gamma amounts similar to that of untreated control cells. By contrast, landraces Ersa 6, Ersa 8, Leonessa 4 and Leonessa 5 proved to be very active in triggering the immune responses, whereas landraces Filosini and Prometeo exhibited an intermediate behavior. One-dimensional fractionations by A-PAGE or SDS-PAGE revealed distinctive prolamin patterns amongst the landraces analysed. CONCLUSIONS: 'Dicoccum' wheat represents a heterogeneous species showing a wide variation in both prolamin composition and T-cell immunological activation, some 'dicoccum' landraces being poor in or devoid of noxious gluten proteins.

Toxic, immunostimulatory and antagonist gluten peptides in celiac disease.

Celiac disease (CD) is an increasingly diagnosed, permanent autoimmune enteropathy, triggered, in susceptible individuals, by the ingestion of gluten, the alcohol - soluble protein fraction of some cereals, such as wheat, rye and barley. The main protein of wheat gluten is called gliadin, the similar proteins of rye and barley are secalin and hordein, respectively. Approximately 96% of CD patients express the HLA molecule DQ2, while the remainder mostly express the less common haplotype DQ8, reflecting the pivotal role of these molecules in the pathogenesis of CD. Because of their aminoacid sequence and tri-dimensional structure, gluten peptides selectively bind to these HLA alleles present on the surface of antigen presenting cells and then they are presented to the T lymphocytes in intestinal mucosa, thus starting the inflammatory immune response. CD is defined by the characteristic histological changes of small bowel mucosa: villous atrophy, crypts hyperplasia and T cells infiltration of the lamina propria, along with the increase of the number of intra-epithelial lymphocytes. The withdrawal of the gluten- containing food from the diet determines a complete recovery of the intestinal mucosa, whereas the reintroduction causes a relapse of the disease. This review focuses on the description of gluten peptides that elicit the mucosal immune response via the activation of innate and adaptive immunity in CD. It also describes the antagonist gluten peptides, obtained by artificial modification of gluten T epitopes or naturally occurring in the alcohol protein fraction of a cultivar of durum wheat, able to immuno-modulate the pathogenic immune response of CD.

Antagonist peptides of the gliadin T-cell stimulatory sequences: a therapeutic strategy for celiac disease.

Celiac disease (CD) is a T helper 1-driven autoimmune permanent enteropathy, triggered in susceptible individuals by the ingestion of gluten, the alcohol-soluble protein fraction of some cereals, such as wheat, rye, and barley. The only available treatment for CD is the life-long withdrawal of gluten-containing foods from the diet. Complying with gluten-free diet is difficult and affects the quality of life. Therefore, alternative therapies are being investigated. In this paper, we review a new therapeutic strategy for CD, relying upon peptides that are analogs of gliadin T-cell epitopes that show the ability to down-modulate the immune response pathogenic of CD. These peptides have been obtained artificially by amino acids substitution of gliadin T-cell stimulatory sequences and an immunomodulatory sequence has been identified in the alcohol-soluble protein fraction of cultivars of durum wheat.

Distribution, proliferation, and function of Paneth cells in uncomplicated and complicated adult celiac disease.

Paneth cells, granulated epithelial cells located at the base of small bowel crypts, have a crucial role in innate immunity. Because controversies remain concerning Paneth cell numbers and function in celiac disease (CD), we quantified Paneth cells and human alpha-defensin (HD)-5 and HD-6 in 28 patients with uncomplicated CD, 8 patients with complicated CD (3 with ulcerative jejunoileitis, 2 with refractory sprue, and 3 with enteropathy-associated T-cell lymphoma), and 14 control subjects. Paneth cell numbers and proliferation did not differ in uncomplicated untreated and treated CD and control cases. However, the number of Paneth cells was significantly reduced in complicated CD. Mucosal HD-5 and HD-6 were comparable in uncomplicated untreated and treated CD and control cases. Ex vivo gliadin challenge of treated CD biopsy specimens had no effect on mucosal HD-5 and HD-6 transcripts. Paneth cell numbers and alpha-defensins are unchanged in the mucosa in uncomplicated CD. Further studies are needed to clarify the implications of reduction of numbers of Paneth cells in complicated CD.

A 10-residue peptide from durum wheat promotes a shift from a Th1-type response toward a Th2-type response in celiac disease.

BACKGROUND: Celiac disease (CD) is a Th1-driven autoimmune permanent enteropathy that is triggered by dietary gluten. Molecules able to shift the immune response from a Th1- to a Th2-type response have been suggested as therapeutic agents for Th1 autoimmune diseases. OBJECTIVE: We sought to investigate the possibility that a decapeptide from durum wheat (p10mer, QQPQDAVQPF), which was previously shown to prevent the activation of celiac peripheral lymphocytes, may promote a shift from a Th1- to a Th2-type immune response in gluten-specific intestinal T cells of CD patients. DESIGN: Intestinal T lymphocyte lines derived from 8 children with CD were incubated with gliadin peptides both alone and simultaneously with p10mer. Cell proliferation and the production of interferon-gamma and interleukin-10 by these T cells were measured. RESULTS: The incubation of celiac intestinal T cells with deamidated gliadin peptides resulted in a significant (P < 0.008) increase in cell proliferation and interferon-gamma release, whereas the simultaneous exposure to p10mer totally abolished the cell proliferation and cytokine release. Moreover, incubation with p10mer maintained an elevated release of interleukin-10, whereas exposure of the cells to culture medium only did not. The replacement of the residues of aspartic acid in position 5 or those of alanine in position 6 in the sequence of p10mer resulted in peptides with no activity in the activation experiments. CONCLUSION: In vitro, p10mer showed the ability to shift the pathogenic immune response of a CD patient from a Th1- to a Th2-type response.

Effect of a gluten-free diet on the risk of enteropathy-associated T-cell lymphoma in celiac disease.

Patients with celiac disease have an increased rate of enteropathy-associated T-cell lymphoma, but conflicting data are available about the protective role of a gluten-free diet with regard to the development of this malignancy. We followed 1,757 celiac patients for a total period of 31,801 person-years, collecting data about the frequency of gluten intake and the incidence of the enteropathy-associated T-cell lymphoma. Out of the nine celiac patients who developed an intestinal lymphoma [standard morbidity ratio of 6.42 (95% CI = 2.9-12.2; P < 0.001)], only two kept a strict gluten-free diet after the diagnosis of celiac disease and developed the malignancy after the peridiagnosis period of 3 years, dropping therefore the standard morbidity ratio to 0.22 (95%CI = 0.02-0.88; P < 0.001). The risk of developing an intestinal lymphoma for the celiac patients that used to have dietary gluten was significant (X(2 )= 4.8 P = 0.01). These results show that a strict gluten-free diet is protective towards the development of enteropathy-associated T-cell lymphoma.

Environmental factors of celiac disease: cytotoxicity of hulled wheat species Triticum monococcum, T. turgidum ssp. dicoccum and T. aestivum ssp. spelta.

BACKGROUND AND AIM: In the present paper, the toxicity of prolamines derived from three cereals with a different genome was investigated in human colon cancer Caco-2/TC7 and human myelogenous leukemia K562(S) cells. The purpose of this study was to investigate if species from ancient wheat could be considered as healthy food crops devoid or poor in cytotoxic prolamines for celiac disease. METHODS: Cytotoxicity was measured in terms of inhibition of cell growth, activation of apoptosis, release of nitric oxide (NO), detection of tissue transglutaminase (TG II) and alteration of transepithelial electrical resistance (TEER) on Caco-2/Tc7 and K562 (S) cell agglutination. Peptic-tryptic (PT) digest from bread wheat (T. aestivum S. Pastore) was used as a positive control. RESULTS: PT digests of prolamins from spelt wheat (T. aestivum ssp. spelta) were found to exert toxic effects on Caco-2/TC7 cells and to agglutinate K562(S) cells. Increased amounts of NO and TG II expression were observed in Caco-2/TC7 cells exposed to 1 mg/mL of spelt prolamins, suggesting that spelt wheat can induce cellular mechanisms implicated in the pathogenesis of celiac disease. By contrast, the PT digests from monoccum wheat (Triticum monococcum) and farro wheat (T. turgidum ssp. dicoccum) did not exhibit any negative effects on Caco-2/TC7 and K562(S) cells. CONCLUSIONS: The results have shown a constant and significant toxic effect of spelt wheat which is not shared by the two other ancient cereals. Future studies on celiac intestinal organ cultures are needed to increase the prospects of breeding programs aimed at developing wheat cultivars potentially tolerated by most celiac patients.

Avenins from different cultivars of oats elicit response by coeliac peripheral lymphocytes.

OBJECTIVE: The avoidance of oats in coeliac patients is still controversial. If oats is confirmed to be safe, it would be a valuable component and offer more variation in a gluten-free diet. The aim of this work was to evaluate whether avenins from different varieties of oats show different abilities in the activation of coeliac peripheral lymphocytes. MATERIAL AND METHODS: In order to assess whether the immunogenic effect of oats varies according to the cultivar, peripheral lymphocytes from 10 coeliac children were exposed to avenins from four different oats varieties: Lampton, Astra, Ava and Nave. Lymphocyte proliferation and interferon-gamma (IFN-gamma) release in the culture medium were measured as indexes of immune activation. RESULTS: All the varieties of oats tested were immunogenic, with Lampton and Ava avenins inducing lymphocyte activation similar to that activated by wheat gliadin, while Astra and Nave avenins showed less immunogenicity, but still with a measurable effect. CONCLUSIONS: There are still concerns about the suitability of including oats in a gluten-free diet. Coeliac patients consuming oats-containing food should be carefully monitored, until there is more evidence to show the safety of oats and varieties of low-toxicity oats.

Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: new perspectives for celiac disease.

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-electrospray 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 demonstrated 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.

Delayed diagnosis of coeliac disease increases cancer risk.

BACKGROUND: The association between coeliac disease (CD) and neoplasms has been long established, but few data are available about the risk factors. The aim of this paper is to estimate the risk of developing a neoplasm among non diagnosed coeliac patients and to evaluate if this risk correlates with the age of patients at diagnosis of coeliac disease. METHODS: The study population consists of patients (n = 1968) diagnosed with CD at 20 Italian gastroenterology referral Centers between 1st January 1982 and 31st March 2005. RESULTS: The SIR for all cancers resulted to be 1.3; 95% CI = 1.0-1.7 p < 0.001. The specific SIRs for non Hodgkin lymphoma was 4.7; 95% CI = 2.9-7.3 p < 0.001, for the small bowel carcinoma 25; 95% CI = 8.5-51.4 p < 0.001, for non Hodgkin lymphoma 10; 95% CI = 2.7-25 p = 0.01, finally for the stomach carcinoma 3; 95% CI = 1.3-4.9 p < 0.08. The mean age at diagnosis of CD of patients that developed sooner or later a neoplasm was 47,6 +/- 10.2 years versus 28.6 +/- 18.2 years of patients who did not. CONCLUSION: Coeliac patients have an increased risk of developing cancer in relation to the age of diagnosis of CD. This risk results higher for malignancies of the gastro-intestinal sites. An accurate screening for tumors should be performed in patients diagnosed with CD in adulthood and in advancing age.

In vitro tests indicate that certain varieties of oats may be harmful to patients with coeliac disease.

BACKGROUND: The presence of oats in gluten-free diet is controversial. The aim of this work is to evaluate if different varieties of oats exert different toxicity in coeliac disease. METHODS: Three varieties of oats were tested by two in vitro assay based on the known ability of peptic-tryptic digests of coeliac-active proteins to agglutinate K562 cells and to disrupt lysosomes, respectively. RESULTS: Avenins from the Italian variety Astra and the Australian variety Mortlook were much more active than the Australian variety Lampton. Gliadin, digested in the same way, certainly displayed more activity than all three avenins, but rice (var. Roma) did not have measurable activity. CONCLUSIONS: The results indicate that some varieties of oats may be potentially harmful to individuals with coeliac disease and therefore should be excluded from the gluten-free diet required to maintain good health in coeliac disease. It is important to realize that constant, small amounts of active proteins in the diet, such as certain avenins, may prevent complete recovery of the intestinal mucosa in this disease.

A decapeptide from durum wheat prevents celiac peripheral blood lymphocytes from activation by gliadin peptides.

Identifying antagonist peptides able to inhibit the abnormal immune response triggered by gliadin peptides in celiac disease (CD) is an alternative therapeutic strategy for CD. The aim of this study was to evaluate the antagonist effect of 10mer, a decapeptide (sequence QQPQDAVQPF) from alcohol-soluble protein fraction of durum wheat, assessing its ability to prevent celiac peripheral blood lymphocytes from activation by gliadin peptides. Peripheral blood mononuclear cells (PBMC) were obtained from DQ2-positive untreated coeliac children and from healthy controls and incubated with the peptic-tryptic digest of bread wheat gliadin (GLP) and peptide 62-75 from alpha-gliadin both alone and with 10mer simultaneously. PBMC proliferation, release of pro-inflammatory Th1 cytokines interferon-gamma and tumor necrosis factor-alpha, release of immunoregulatory cytokine IL-10, and analysis of CD25 expression as indexes of lymphocytes activation were carried out. Enhanced lymphocytes activation was seen after exposure to GLP and p62-75, whereas the simultaneous incubation with 10mer inhibits the lymphocytes response. These data indicate that a peptide naturally occurring in durum wheat exerts in vitro an antagonist effect against gliadin toxicity and could have a protective effect in CD disease.

VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for Celiac Sprue.

The native structure and distribution of gliadin epitopes responsible for Celiac Sprue (CS) may be influenced by cereal food processing. This work was aimed at showing the capacity of probiotic VSL#3 to decrease the toxicity of wheat flour during long-time fermentation. VSL#3 (10(9) cfu/ml) hydrolyzed completely the alpha2-gliadin-derived epitopes 62-75 and 33-mer (750 ppm). Two-dimensional electrophoresis, immunological (R5 antibody) and mass spectrometry analyses showed an almost complete degradation of gliadins during long-time fermentation of wheat flour by VSL#3. Gliadins non-hydrolyzed during fermentation by VSL#3 were subjected to peptic-tryptic (PT) digestion and analyzed by CapLC-ESI-Q-ToF-MS (Capillary Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry). Search for several epitopes showed the only presence of alpha2-gliadin-fragment 62-75 at a very low concentration (sub-ppm range). Compared to IEC-6 cells exposed to intact gliadins extracted from the chemically acidified dough (control), VSL#3 pre-digested gliadins caused a less pronounced reorganization of the intracellular F-actin which was mirrored by an attenuated effect on intestinal mucosa permeability. The release of zonulin from intestinal epithelial cells treated with gliadins was considerably lower when digested with VSL#3. Agglutination test on K 562 (S) cells showed that the PT-digest of wheat flour treated with VSL#3 increased the Minimal Agglutinating Activity of ca. 100 times. Wheat proteins were extracted from doughs and subjected to PT digestion. Compared to PT-digest from chemically acidified dough, celiac jejunal biopsies exposed to the PT-digest from the dough fermented by VSL#3 did not show an increase of the infiltration of CD3(+) intraepithelial lymphocytes. Proteolytic activity by probiotic VSL#3 may have an importance during food processing to produce pre-digested and tolerated gliadins for increasing the palatability of gluten-free products.

Pasta made from durum wheat semolina fermented with selected lactobacilli as a tool for a potential decrease of the gluten intolerance.

A pool of selected lactic acid bacteria was used to ferment durum wheat semolina under liquid conditions. After fermentation, the dough was freeze-dried, mixed with buckwheat flour at a ratio of 3:7, and used to produce the "fusilli" type Italian pasta. Pasta without prefermentation was used as the control. Ingredients and pastas were characterized for compositional analysis. As shown by two-dimensional electrophoresis, 92 of the 130 durum wheat gliadin spots were hydrolyzed almost totally during fermentation by lactic acid bacteria. Mass spectrometry matrix-assisted laser desorption/ionization time-of-flight and reversed phase high-performance liquid chromatography analyses confirmed the hydrolysis of gliadins. As shown by immunological analysis by R5-Western blot, the concentration of gluten decreased from 6280 ppm in the control pasta to 1045 ppm in the pasta fermented with lactic acid bacteria. Gliadins were extracted from fermented and nonfermented durum wheat dough semolina and used to produce a peptic-tryptic (PT) digest for in vitro agglutination tests on cells of human origin. The whole PT digests did not cause agglutination. Affinity chromatography on Sepharose-6-B mannan column separated the PT digests in three fractions. Fraction C showed agglutination activity. The minimal agglutinating activity of fraction C from the PT digest of fermented durum wheat semolina was ca. 80 times higher than that of durum wheat semolina. Pasta was subjected to sensory analysis: The scores for stickiness and firmness were slightly lower than those found for the pasta control. Odor and flavor did not differ between the two types of pasta. These results showed that a pasta biotechnology that uses a prefermentation of durum wheat semolina by selected lactic acid bacteria and tolerated buckwheat flour could be considered as a novel tool to potentially decrease gluten intolerance and the risk of gluten contamination in gluten-free products.