Background: Epidemiology of celiac disease (CD) is increasing. CD mainly presents in early childhood with small intestinal villous atrophy and signs of malabsorption. Compared to healthy individuals, CD patients seemed to be characterized by higher numbers of Gram-negative bacteria and lower numbers Gram-positive bacteria.Results: This study aimed at investigating the microbiota and metabolome of 19 celiac disease children under gluten-free diet (treated celiac disease, T-CD) and 15 non-celiac children (HC). PCR-denaturing gradient gel electrophoresis (DGGE) analyses by universal and group-specific primers were carried out in duodenal biopsies and faecal samples. Based on the number of PCR-DGGE bands, the diversity ofEubacteriawas the higher in duodenal biopsies of T-CD than HC children.Bifidobacteriawere only found in faecal samples. With a few exceptions, PCR-DGGE profiles of faecal samples forLactobacillusandBifidobacteriadiffered between T-CD and HC. As shown by culture-dependent methods, the levels ofLactobacillus,EnterococcusandBifidobacteriawere confirmed to be significantly higher (P= 0.028;P= 0.019; andP= 0.023, respectively) in fecal samples of HC than in T-CD children. On the contrary, cell counts (CFU/ml) of presumptiveBacteroides,Staphylococcus,Salmonella,ShighellaandKlebsiellawere significantly higher (P= 0.014) in T-CD compared to HC children.Enterococcus faeciumandLactobacillus plantarumwere the species most diffusely identified. This latter species was also found in all duodenal biopsies of T-CD and HC children. Other bacterial species were identified only in T-CD or HC faecal samples. As shown by Randomly Amplified Polymorphic DNA-PCR analysis, the percentage of strains identified as lactobacilli significantly (P= 0.011) differed between T-CD (ca. 26.5%) and HC (ca. 34.6%) groups. The metabolome of T-CD and HC children was studied using faecal and urine samples which were analyzed by gas-chromatography mass spectrometry-solid-phase microextraction and¹H-Nuclear Magnetic Resonance. As shown by Canonical Discriminant Analysis of Principal Coordinates, the levels of volatile organic compounds and free amino acids in faecal and/or urine samples were markedly affected by CD.Conclusion: As shown by the parallel microbiology and metabolome approach, the gluten-free diet lasting at least two years did not completely restore the microbiota and, consequently, the metabolome of CD children. Some molecules (e.g., ethyl-acetate and octyl-acetate, some short chain fatty acids and free amino acids, and glutamine) seems to be metabolic signatures of CD.

This study aimed to investigate the salivary microbiota and metabolome of 13 children with celiac disease (CD) under a gluten-free diet (treated celiac disease [T-CD]). The same number of healthy children (HC) was used as controls. The salivary microbiota was analyzed by an integrated approach using culture-dependent and -independent methods. Metabolome analysis was carried out by gas chromatography-mass spectrometry–solid-phase microextraction. Compared to HC, the number of some cultivable bacterial groups (e.g., total anaerobes) significantly (P< 0.05) differed in the saliva samples of the T-CD children. As shown by community-level catabolic profiles, the highest Shannon's diversity and substrate richness were found in HC. Pyrosequencing data showed the highest richness estimator and diversity index values for HC. Levels ofLachnospiraceae,Gemellaceae, andStreptococcus sanguiniswere highest for the T-CD children.Streptococcus thermophiluslevels were markedly decreased in T-CD children. The saliva of T-CD children showed the largest amount ofBacteroidetes(e.g.,Porphyromonassp.,Porphyromonas endodontalis, andPrevotella nanceiensis), together with the smallest amount ofActinobacteria. T-CD children were also characterized by decreased levels of someActinomycesspecies,Atopobiumspecies, andCorynebacterium durum.Rothia mucilaginosawas the onlyActinobacteriaspecies found at the highest level in T-CD children. As shown by multivariate statistical analyses, the levels of organic volatile compounds markedly differentiated T-CD children. Some compounds (e.g., ethyl-acetate, nonanal, and 2-hexanone) were found to be associated with T-CD children. Correlations (false discovery rate [FDR], <0.05) were found between the relative abundances of bacteria and some volatile organic compounds (VOCs). The findings of this study indicated that CD is associated with oral dysbiosis that could affect the oral metabolome.

Pyrosequencing of the 16S rRNA targeting RNA, community-level physiological profiles made with Biolog EcoPlates, proteolysis, and volatile component (VOC) analyses were mainly used to characterize the manufacture and ripening of the pasta filata cheese Caciocavallo Pugliese. Plate counts revealed that cheese manufacture affected the microbial ecology. The results agreed with those from culture-independent approaches. As shown by urea-PAGE, reverse-phase high pressure liquid chromatography (RP-HPLC), and free-amino-acid (FAA) analyses, the extent of secondary proteolysis mainly increased after 30 to 45 days of ripening. VOCs and volatile free fatty acids (VFFA) were identified by a purge-and-trap method (PT) and solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS), respectively. Except for aldehydes, the levels of most of VOCs and VFFA mainly increased from 30 to 45 days onwards. As shown through pyrosequencing analysis, raw cows' milk was contaminated by Firmicutes (53%), Proteobacteria (39%), Bacteroidetes (7.8%), Actinobacteria (0.06%), and Fusobacteria (0.03%), with heterogeneity at the genus level. The primary starter Streptococcus thermophilus dominated the curd population. Other genera occurred at low incidence or sporadically. The microbial dynamics reflected on the overall physiological diversity. At 30 days, a microbial succession was clearly highlighted. The relative abundance of Streptococcus sp. and especially St. thermophilus decreased, while that of Lactobacillus casei , Lactobacillus sp., and especially Lactobacillus paracasei increased consistently. Despite the lower relative abundance compared to St. thermophilus , mesophilic lactobacilli were the only organisms positively correlated with the concentration of FAAs, area of hydrophilic peptide peaks, and several VOCs (e.g., alcohols, ketones, esters and all furans). This study showed that a core microbiota was naturally selected during middle ripening, which seemed to be the main factor responsible for cheese ripening.

Pasta represents a dominant portion of the diet worldwide and its functionalization with high nutritional value ingredients, such as legumes, is the most ideal solution to shape consumers behavior towards healthier food choices. Aiming at improving the nutritional quality of semolina pasta, semi-liquid dough of a Mediterranean black chickpea flour, fermented with Lactiplantibacillus plantarum T0A10, was used at a substitution level of 15% to manufacture fortified pasta. Fermentation with the selected starter enabled the release of 20% of bound phenolic compounds, and the conversion of free compounds into more active forms (dihydrocaffeic and phloretic acid) in the dough. Fermented dough also had higher resistant starch (up to 60% compared to the control) and total free amino acids (almost 3 g/kg) contents, whereas antinutritional factors (raffinose, condensed tannins, trypsin inhibitors and saponins) significantly decreased. The impact of black chickpea addition on pasta nutritional, technological and sensory features, was also assessed. Compared to traditional (semolina) pasta, fortified pasta had lower starch hydrolysis rate (ca. 18%) and higher in vitro protein digestibility (up to 38%). Moreover, fortified cooked pasta, showing scavenging activity against DPPH and ABTS radicals and intense inhibition of linoleic acid peroxidation, was appreciated for its peculiar organoleptic profile. Therefore, fermentation technology appears to be a promising tool to enhance the quality of pasta and promote the use of local chickpea cultivars while preventing their genetic erosion.