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Background: The histopathologic diagnosis of celiac disease (CD) may be challenging when the duodenal biopsies mucosal injury is limited. Intraepithelial T-lymphocytes (IELs) can be useful to characterize the degree of mucosal inflammation. A small fraction of IELs expresses the γδ T-cell receptor (named γδ-IELs), whose density, determined by flow cytometry or frozen section immunohistochemistry (IHC), is a specific marker for CD. Aim: To establish a new IHC assay for γδ-IELs applicable to formalin-fixed paraffin-embedded (FFPE) duodenal biopsies. Methods: We analyzed γδ-IELs using IHC in 138 duodenal biopsies using a standard IHC staining protocol with a new monoclonal antibody H-41. IELs were quantitated with digital image analysis. Results: Compared to those in non-celiac controls (n = 51), γδ-IEL density was significantly increased in newly diagnosed celiac disease patients (n = 22, p < 0.0001). In ROC-curve analysis, the cutoff of 6.5 γδ-IELs/100 enterocytes distinguished optimally active CD patients from non-celiac controls (sensitivity 96%, specificity 95%). γδ-IEL density in CD patients on a gluten-free diet (n = 53) were also higher than in controls (p < 0.0001), but lower than those in newly diagnosed CD (p < 0.0001). The diagnostic value of γδ-IELs outperformed that of CD3 + IELs in both patient groups. γδ-IELs were better than CD3 + IELs distinguishing between celiac disease and conditions histologically mimicking celiac disease (n = 12). Conclusions: Intraepithelial γδ T-lymphocytes can be stained and quantitated reliably in FFPE duodenal biopsies. The results showed excellent specificity and sensitivity for celiac disease. The new IHC method of detection of γδ-IELs is a promising addition to the routine histopathologic assessment methodology of celiac disease. publishedVersion

Gluten Friendly™ (GF) is a new gluten achieved through a physicochemical process applied to wheat kernels. The goal of this research was to assess the in vivo effects of Gluten Friendly™ bread on celiac gut mucosa and microbiota. In a double-blind placebo-controlled intervention study, 48 celiac disease (CD) patients were randomized into 3 groups to eat 100 g of bread daily, containing different doses (0; 3 g; 6 g) of GF for 12 weeks. The small-bowel morphology (VH/CrD), intraepithelial densities of CD3+, celiac serology, MUC2, CB1, gut permeability, proinflammatory cytokines, gluten in stools, symptoms, and gut microbial composition were assessed. All 48 CD subjects experienced no symptoms. K-means analysis evidenced celiac subjects clustering around unknown parameters independent of GF dosage: K1 35%; K2 30%; K3 35%. VH/CrD significantly decreased in K1 and K2. VH/CrD did not correlate with IEL increase in K2. 33-mer was not detected in 47% and 73% of patients in both K1 and K2, respectively. VH/CrD and IEL did not change significantly and strongly correlated with the absence of 33-mer in K3. Inflammation and VH/CrD decrease are strongly related with the presence of proinflammatory species at the baseline. A boost in probiotic, butyrate-producing genera, is strongly related with GF tolerance at the end of the trial. Our research suggests that a healthy and proinflammatory ecology could play a crucial role in the digestion and tolerance of the new gluten molecule in celiac subjects. However, GF can be completely digested by gut microbiota of CD subjects and shapes it toward gut homeostasis by boosting healthy butyrate-producing populations. The clinical trial registry number is NCT03137862 (https://clinicaltrials.gov). publishedVersion

Celiac disease (CD) is an autoimmune disease with the destruction of small intestinal villi, which occurs in genetically predisposed individuals. At the present moment, a gluten-free diet (GFD) is the only way to restore the functionality of gut mucosa. However, there is an open debate on the effects of long-term supplementation through a GFD, because some authors report an unbalance in microbial taxa composition. Methods: For microbiome analysis, fecal specimens were collected from 46 CD individuals in GFD for at least 2 years and 30 specimens from the healthy controls (HC). Data were analyzed using an ensemble of software packages: QIIME2, Coda-lasso, Clr-lasso, Selbal, PICRUSt2, ALDEx2, dissimilarity-overlap analysis, and dysbiosis detection tests. Results: The adherence to GFD restored the alpha biodiversity of the gut microbiota in celiac people but microbial composition at beta diversity resulted as different to HC. The microbial composition of the CD subjects was decreased in a number of taxa, namely Bifidobacterium longum and several belonging to Lachnospiraceae family, whereas Bacteroides genus was found to be more abundant. Predicted metabolic pathways among the CD bacterial communities revealed an important role in tetrapyrrole biosynthesis. Conclusions: CD patients in GFD had a non-dysbiotic microbial composition for the crude alpha diversity metrics. We found significant differences in beta diversity, in certain taxon, and pathways between subjects with inactive CD in GFD and controls. Collectively, our data may suggest the development of new GFD products by modulating the gut microbiota through diet, supplements of vitamins, and the addition of specific prebiotics.

The histopathologic diagnosis of celiac disease (CD) may be challenging when the duodenal biopsies mucosal injury is limited. Intraepithelial T-lymphocytes (IELs) can be useful to characterize the degree of mucosal inflammation. A small fraction of IELs expresses the γδ T-cell receptor (named γδ-IELs), whose density, determined by flow cytometry or frozen section immunohistochemistry (IHC), is a specific marker for CD.To establish a new IHC assay for γδ-IELs applicable to formalin-fixed paraffin-embedded (FFPE) duodenal biopsies.We analyzed γδ-IELs using IHC in 138 duodenal biopsies using a standard IHC staining protocol with a new monoclonal antibody H-41. IELs were quantitated with digital image analysis.Compared to those in non-celiac controls (n = 51), γδ-IEL density was significantly increased in newly diagnosed celiac disease patients (n = 22, p 0.0001). In ROC-curve analysis, the cutoff of 6.5 γδ-IELs/100 enterocytes distinguished optimally active CD patients from non-celiac controls (sensitivity 96%, specificity 95%). γδ-IEL density in CD patients on a gluten-free diet (n = 53) were also higher than in controls (p 0.0001), but lower than those in newly diagnosed CD (p 0.0001). The diagnostic value of γδ-IELs outperformed that of CD3 + IELs in both patient groups. γδ-IELs were better than CD3 + IELs distinguishing between celiac disease and conditions histologically mimicking celiac disease (n = 12).Intraepithelial γδ T-lymphocytes can be stained and quantitated reliably in FFPE duodenal biopsies. The results showed excellent specificity and sensitivity for celiac disease. The new IHC method of detection of γδ-IELs is a promising addition to the routine histopathologic assessment methodology of celiac disease.

The main goal of this paper was to design a synbiotic yogurt containing Bifidobacterium infantis and Gluten Friendly FlourTM; the proposed approach relies upon milk fermentation through the classical starter of yogurt (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) to avoid a strong production of acetic acid by bifidobacterial and inoculum of B. infantis after the fermentation. The research was divided in 3 steps. The aim of the first step was the optimization of fermentation kinetic by L. delbureckii and S. thermophilus, by combining the amount of flour (either Gluten Friendly Flour-GF- or Control Flour-CF) in milk, temperature and inoculum level; the factors were combined through a mixture design. As a result of this step, the best combination was pointed out: flour at 2.5 g/l; L. delbrueckii subsp. bulgaricus at 6 log cfu/ml; temperature at 37-40°C. The goal of the second step was to study the effect of flour (2.5 g/l) on the viability of B. infantis. GF prolonged the viability of the probiotic for 14 days. In the last step, a synbiotic yogurt, supplemented with GF and fermented with L. delbureckii and S. thermophilus, and then inoculated with B. infantis, was produced. The product was stored at 8 and 15°C. A positive effect of GF was found at 15°C, with B. infantis at 7.0 log cfu/g in GF sample and 5.5.5.7 log cfu/g in CF sample.

The main goal of this paper was to design a synbiotic yogurt containing Bifidobacterium infantis and Gluten Friendly FlourTM; the proposed approach relies upon milk fermentation through the classical starter of yogurt (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) to avoid a strong production of acetic acid by bifidobacterial and inoculum of B. infantis after the fermentation. The research was divided in 3 steps. The aim of the first step was the optimization of fermentation kinetic by L. delbureckii and S. thermophilus, by combining the amount of flour (either Gluten Friendly Flour-GF- or Control Flour-CF) in milk, temperature and inoculum level; the factors were combined through a mixture design. As a result of this step, the best combination was pointed out: flour at 2.5 g/l; L. delbrueckii subsp. bulgaricus at 6 log cfu/ml; temperature at 37–40°C. The goal of the second step was to study the effect of flour (2.5 g/l) on the viability of B. infantis. GF prolonged the viability of the probiotic for 14 days. In the last step, a synbiotic yogurt, supplemented with GF and fermented with L. delbureckii and S. thermophilus, and then inoculated with B. infantis, was produced. The product was stored at 8 and 15°C. A positive effect of GF was found at 15°C, with B. infantis at 7.0 log cfu/g in GF sample and 5.5.5.7 log cfu/g in CF sample.

Recently, an innovative gluten detoxification method called Gluten FriendlyTM (GF) has been developed. It induces structural modifications, which abolish the antigenic capacity of gluten and reduce the in vitro immunogenicity of the most common epitopes involved in coeliac disease, without compromising the nutritional and technological properties. This study investigated the in vitro effects of GF bread (GFB) on the faecal microbiota from healthy and coeliac individuals by a three-stage continuous fermentative system, which simulates the colon (vessel 1, proximal colon; vessel 2, transverse colon; vessel 3, distal colon), as well as on the production of short chain fatty acids (SCFA, acetate, propionate, butyrate). The system was fed with GFB and the changes in microbiota through fluorescence in situ hybridization (FISH) and in SCFA content were assessed. GFB exerted beneficial modulations such as bifidogenic effects in each compartment of the model both with healthy and coeliac derived samples, as well as growth in Clostridium clusters XIVa+b in coeliac derived samples. Furthermore, increased levels of acetic acid were found in vessel 1 inoculated with the faecal microbiota of healthy individuals, as well as acetic and propionic in vessel 1 and 2 with coeliac derived samples. In addition, the use of multivariate approaches showed that the supplementation of GFB could result in a different modulation of the faecal microbiota and SCFA, as a function of initial equilibrium.

The gluten-free diet is, to date, the only efficacious treatment for patients with Celiac Disease. In recent years, the impressive rise of Celiac Disease incidence, dramatically prompted changes in the dietary habit of an increasingly large population, with a rise in demand of gluten-free products. The formulation of gluten-free bakery products presents a formidable challenge to cereal technologists. As wheat gluten contributes to the formation of a strong protein network, that confers visco-elasticity to the dough and allows the wheat flour to be processed into a wide range of products, the preparation of cereal-based gluten-free products is a process somehow difficult process. This review focuses on nutritional and technological quality of products made with gluten-free cereals available on the market. The possibility of using flour from naturally low toxic ancient wheat species or detoxified wheat for the diet of celiacs is also discussed.

The main aim of this paper was to assess the impact of Gluten-Friendly™ (GF) technology (Italian priority patent n° 102015000084813 filed on 17th December 2015) on wheat kernel endosperm morphology and gluten protein structure, using SEM, light and immunofluorescent microscopy. Microscopy was combined with immunodetection with specific antibodies for gliadins, γ-gliadins, LMW subunits and antigenic epitopes to gain a better understanding of the technology at a molecular level. The results showed significant changes to gluten proteins after GF treatment; cross-reactivity towards the antibodies recognizing almost the entire range of gluten proteins as well as the antigenic epitopes through the sequences QQSF, QQSY, PEQPFPQGC and QQPFP was significantly reduced. The present study confirms the results from our previous work and shows, for the first time, the mechanism by which a chemical-physical treatment abolishes the antigenic capacity of gluten.

The aim was to study the effects of toasting of durum wheat kernels on the properties of dough and spaghetti produced with semolina and increasing amounts of the meal deriving from toasted kernels. The replacement of semolina with the toasted meal caused a decrease in the dough strength and an increase of its tenacity already at a substitution level of 100 g kg−1. Pasta made with 100% semolina or with a mixture of semolina and 200 g kg−1 of toasted durum wheat meal appeared as the best, being the less compressible and showing the highest values of cohesiveness. Water sorption curves were successfully described through the Peleg's equation. The application of response surface regression followed by stepwise regression allowed to write one equation for each of the textural properties measured. Gluten index was the best predictor of compressibility, cohesiveness, and gumminess whereas elasticity was better predicted on the basis of both gluten content and gluten index. Observed and predicted values were high...

Spaghetti produced in a pilot plant were made from semolina and semolina blended with 10%, 15%, 25% or 50% of defatted soy flour (DSF) or toasted soy flour (TSF). Proteins of spaghetti were characterized by size exclusion-high-performance liquid chromatography (SE-HPLC). Results showed that soy globulins interact with semolina proteins during pasta making, forming polymers of high molecular weight. Of these, the sodium dodecyl sulphate-unextractable components were significantly higher (p

The influence of different ageing conditions on lees, and in particular of lees obtained with and without a rough raking, on polysaccharide glycosyl residues of Chardonnay wine is addressed in this work. Methanolysis and derivatization procedures of ethanol precipitated polysaccharides, followed by gas-chromatography coupled to mass spectrometry, allowed the determination of all the main sugars known to constitute wine polysaccharides. The aging on lees led to an increase of the contents of all the glycosyl residues, except for both galacturonic acid, whose concentration slightly decreased, and glucose and myo-inositol, which were not affected by the ageing process. Results suggest that the use of aging on lees without a rough raking enriched wine of mannoproteins meanwhile contributing to the solubilization of grape polysaccharides more than aging on lees with a rough raking.

Durum wheat kernels were subjected to a toasting process and the proteins characterised by size exclusion-high performance liquid chromatography (SE-HPLC) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis. With this physical process, albumins and globulins, as well as glutenins and gliadins, polymerised as seen by a shift of the SE-HPLC profile to lower elution times. The polymerisation seemed to happen mainly through disulphide bonds, even though the participation of ω-gliadins to the aggregation suggested the involvement of other kinds of interactions. It led to the revelation of a new peak originated by thermal aggregation of small polymeric proteins. The changes in the chromatographic profile were accompanied by increasing amounts of total unextractable polymeric proteins. The replacement of semolina with toasted durum wheat flour (5%, 10%, 15%, 20% and 30%) for the production of pasta in the shape of spaghetti significantly ( p p > 0.05) change the unextractable polymeric proteins (UPP) when compared with spaghetti made with 100% durum semolina. On the other hand, the replacements of semolina with 15–30% TDWF showed significant ( p

The aim was to study the effects of toasting of durum wheat kernels on the physical and mechanical properties of bread containing increasing amounts of toasted wholemeal. Two toasting procedures were considered: heating of the kernels arranged on tin cans (TDW1) and the stubble burning (TDW2). The addition of toasted flour determined a decrease in volume (greater when TDW2 was used) and detrimental effects on mechanical properties caused by the thermal denaturation of the gluten-forming proteins and the interference of bran. Volume was negatively correlated with compressibility, gumminess, masticability and positively correlated with elasticity (0.824 R

The effects of the following additives on the amaranth (A), quinoa (Q) and oat (O) dough rheological properties and the extruded tagliatelle dough mechanical characteristics were evaluated: carboxymethylcellulose of sodium (CMC), whey protein isolate (WPI), casein (CAS), chitosan (CHIT) and pregelatinized starch (PS). The amaranth, quinoa and oat rheological dough properties and amaranth, quinoa and oat tagliatelle mechanical characteristics were compared to those of their respective controls (ACTRL, QCTRL and OCTRL) and of the SEMOLINA sample. The storage modulus (G′) and loss modulus (G″) values of the quinoa and oat doughs with PS were similar to those of the semolina dough. For all tagliatelle samples, WPI reduced the elastic modulus or Young's modulus towards that of the semolina tagliatelle. Moreover, the additives did not have particular influence on the tenacity with the exception of the amaranth tagliatelle added with WPI.

Summary The characterisation of traditional Italian pasta obtained by mixing amounts of toasted whole meal with re-milled semolina and other ingredients was obtained by means of physico-chemical, rheological, mechanical, sensory and image analyses. The toasted meal showed higher ash, fibre and protein contents than re-milled semolina. The replacement of percentages of re-milled semolina with the toasted meal and soft flour increased tenacity and decreased extensibility and strength, making the dough less suitable for pasta-making. The P/L values were indices of high starch damage. The replacement of part of re-milled semolina and water with toasted whole wheat meal, soft flour and eggs increased the optimal cooking time and the amount of water absorbed during cooking but made the other cooking parameters worse. The image analysis provided evidence of the changes induced by the use of toasted wholemeal, soft flour and eggs in the microscopic structure of pasta protein and starch.

Spaghetti produced in a pilot plant was dried using three different drying conditions: 60 °C for 7.5 h; 75 °C for 5.5 h; 90 °C for 5 h. Proteins of spaghetti were characterized by size-exclusion chromatography (SEC) and size-exclusion–high-performance liquid chromatography (SE-HPLC). As the temperature of the drying cycles increased, a progressive decrease of the small and large monomeric proteins and an increase in molecular size of the large polymeric proteins (LPPs) were observed. Drying temperature at 60 °C already induces a significant increase in the molecular size of the LPPs and a significant decrease of the other protein fractions. At 70 and 90 °C, large and small monomeric proteins as well as LPPs polymerized as seen by a progressive shift towards higher molecular weights in the SEC profile as temperature increased. The changes in the chromatographic profiles were accompanied by increasing amount of total unextractable polymeric protein (UPP). A decrease in stickiness and an increase in firmness corresponded to the formation of large and insoluble protein aggregates.

Wheat kernels were subjected to microwave treatment, and the proteins were characterized by size exclusion high-performance liquid chromatography (SE-HPLC) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Using this process, the proteins polymerize, forming intermolecular bonds among the same classes of proteins. Furthermore, the polymerization occurs only through disulphide bonds. Although SDS–PAGE did not show any differences for either the number or intensity of protein bands between flour samples before and after microwave treatment, gliadins from treated flours showed significantly reduced cross-reactivity with the R5 antibody. Moreover, the gluten became soluble in an aqueous saline solution, and it was not possible to isolate it using the Glutomatic apparatus. However, the treated flour, in the presence of water, was able to form dough and leaven and produce bread.

Genetic manipulation of durum wheats by tobacco rab-1 genes influence the trafficking of gluten proteins through the secretory system by up- or down-regulating the transport step from the ER to the Golgi apparatus which may in turn modify functional performance of the grain. Gluten proteins were extracted from two genetically manipulated lines - Svevo B730 1-1 and Ofanto B688 1-2 - and their control lines and were analyzed by two dimensional gel electrophoresis. When the two-dimensional maps were compared by image analysis no significant differences between the GM line with an up-regulated trafficking containing the wild type tobacco rab1 (Svevo B730 1-1) and its control (Svevo control). By contrast, significant differences were found between the GM line with a down-regulated trafficking due to the tobacco rab1 mutant form (Ofanto B688 1-2) and its control (Ofanto control). Of the new protein spots detected in the down-regulated Ofanto B688 1-2 map, only a beta-amylase was identified. The remaining spots were susceptible to chymotripsin action but not to trypsin one, as in the case of the gluten protein. Rheological measurements showed that gluten quality was enhanced in the down-regulated Ofanto B688 1-2 without an increase in the amount of gluten. Proteomics is a useful and powerful tool for investigating protein changes in GMOs and in understanding events in food science and technology.

To evaluate process-induced protein modifications in cooked ham and emulsion sausages, the proteomes of whole-cut (Parma and “Praga” cooked hams) and comminuted pork (mortadella and wurstel) products were compared to raw pork using two-dimensional gel electrophoresis (2-DE) coupled to image analysis and mass spectrometry (MS). Other than heat-induced breakdown of part of the myosin heavy chains, the 2-DE pattern of cooked ham was substantially similar to that of raw pork. However, the MS-based analysis showed minor modifications, including the extensive oxidation of methionines. In contrast, likely due to emulsification, comminuted sausages were characterized by an abundant insoluble protein fraction (IPF). Interestingly, tropomyosin and myosin light chains in comminuted sausages were exclusively found in the IPF. Our results indicate that the protein aggregation systems of cooked hams and emulsion sausages reflect the processing conditions and are definitely different, the former being characterized mainly by disulphide bridges and the latter by additional covalent inter-protein links.

The characterization of the promoter of a wheat (Triticum aestivum) cv. Cheyenne high molecular weight glutenin subunit (HMW subunit) gene, Glu-1D-1 is reported. The nucleotide sequence of the promoter from position -1191 to -650 with respect to the transcription start site was determined, to add to that already determined. Analysis of this region of the promoter revealed the presence of an additional copy of part of the primary enhancer sequence and sequences related to regulatory elements present in other wheat seed protein genes. A chimaeric gene was constructed comprising the 5' flanking region of the Glu-1D-1 gene from position -1191 to +58, the coding region of the UID:A (Gus) gene, and the nopaline synthase (Nos) gene terminator. This chimaeric gene was introduced into wheat (Triticum durum cv. Ofanto) by particle bombardment of inflorescence explants. Two independent transgenic lines were produced, and both showed expression of the Gus gene specifically in the endosperm during mid-development (first detected 10-12 d after anthesis). Histochemical analysis of homozygous T(2) seed confirmed this pattern of expression, and showed that expression was initiated first in the central lobes of the starchy endosperm, and then spread throughout the endosperm tissue, while no expression was detected in the aleurone layer. Native HMW subunit protein was detectable by Western analysis 12-14 d after anthesis, consistent with concurrent onset of activity of the native and introduced HMW subunit gene promoters.

Two red wines (Primitivo and Uva di Troia) treated with oak chips inoculated with Penicillium purpurogenum were analysed in order to assess their contents of furfural, cis-β-methyl-γ-octalactone, syringol, eugenol, vanillin and 4-vinylguaiacol. Two different sizes of oak chips (small and big, of length 2 and 8 mm respectively) and two different degrees of toasting (low and high) were used in the study. Aroma compounds were analysed by gas chromatography/mass spectrometry to determine differences among samples after 15 days of chip contact time.Big oak chips inoculated with P. purpurogenum increased the level of 4-vinylguaiacol, while small oak chips inoculated with P. purpurogenum, in some conditions, increased the level of eugenol. Chip size and degree of toasting also played an important role in the content of eugenol.The use of oak chips inoculated with mould might be a promising alternative to barrel aging. Moreover, different fungal inocula could contribute to the enrichment of wine with specific compounds (e.g. 4-vinylguaiacol and eugenol).

A study on the use of natural antimicrobial compounds to improve the microbiological stability of refrigerated amaranth-based homemade fresh pasta is presented in this work. In particular, the antimicrobial activity of thymol, lemon extract, chitosan and grapefruit seed extract (GFSE) has been tested against mesophilic and psychrotrophic bacteria, total coliforms, Staphylococcus spp., yeasts and moulds. A sensory analysis on both fresh and cooked pasta was also run. Results suggest that chitosan and GFSE strongly increase the microbial acceptability limit of the investigated spoilage microorganisms, being the former the most effective. Thymol efficiently reduces the growth of mesophilic bacteria, psychrotrophic bacteria and Staphylococcus spp., whereas it does not affect, substantially, the growth cycle of total coliforms. Lemon extract is the less effective in preventing microbial growth. In fact, it is able to delay only total mesophilic and psychrotrophic bacterial evolution. From a sensorial point of view no significant differences were recorded between the control samples and all the types of loaded amaranth-based pasta.