Novel probiotic preparation with in vivo gluten-degrading activity and potential modulatory effects on the gut microbiota.

Gluten possesses unique properties that render it only partially digestible. Consequently, it exerts detrimental effects on a part of the worldwide population who are afflicted with celiac disease (1%) or related disorders (5%), particularly due to the potential for cross-contamination even when adhering to a gluten-free diet (GFD). Finding solutions to break down gluten during digestion has a high nutritional and social impact. Here, a randomized double-blind placebo-controlled in vivo challenge investigated the gluten-degrading activity of a novel probiotic preparation comprising lactobacilli and their cytoplasmic extracts, Bacillus sp., and bacterial protease. In our clinical trial, we collected feces from 70 healthy volunteers at specific time intervals. Probiotic/placebo administration lasted 32 days, followed by 10 days of wash-out. After preliminary GFD to eliminate residual gluten from feces, increasing amounts of gluten (50 mg-10 g) were administered, each one for 4 consecutive days. Compared to placebo, the feces of volunteers fed with probiotics showed much lower amounts of residual gluten, mainly with increased intakes. Probiotics also regulate the intestinal microbial communities, improving the abundance of genera pivotal to maintaining homeostasis. Quantitative PCR confirmed that all probiotics persisted during the intervention, some also during wash-out. Probiotics promoted a fecal metabolome with potential immunomodulating activity, mainly related to derivatives of branched-chain amino acids and short-chain fatty acids. IMPORTANCE: The untapped potential of gluten-degrading bacteria and their application in addressing the recognized limitations of gluten-related disorder management and the ongoing risk of cross-contamination even when people follow a gluten-free diet (GFD) emphasizes the significance of the work. Because gluten, a common protein found in many cereals, must be strictly avoided to stop autoimmune reactions and related health problems, celiac disease and gluten sensitivity present difficult hurdles. However, because of the hidden presence of gluten in many food products and the constant danger of cross-contamination during food preparation and processing, total avoidance is frequently challenging. Our study presents a novel probiotic preparation suitable for people suffering from gluten-related disorders during GFD and for healthy individuals because it enhances gluten digestion and promotes gut microbiota functionality.

Exploring the Gut Microbiome and Metabolome in Individuals with Alopecia Areata Disease.

In recent years, heightened attention has been devoted to unravelling the intricate interplay between genetic and environmental factors shaping the gut microbiota and its significance for human health. This study delves into exploring the plausible connection between Alopecia Areata (AA), an autoimmune disease, and the dynamics of the gut microbiome. Examining a cohort of healthy adults and individuals with AA, both the gut microbiota composition and volatile organic compound (VOC) metabolites from faeces and urine were analysed. While overall microbiota composition showed no significant differences, intra-individual variability revealed distinctions related to age, gender, and pathology status, with AA individuals exhibiting reduced species richness and evenness. Differential abundance analysis identified microbial biomarkers for AA, notably Firmicutes, Lachnospirales, and Blautia, while Coprococcus stood out for healthy individuals. The Data Integration Analysis for Biomarker discovery using Latent Components (DIABLO) method further supported these findings including metabolite biomarkers, such as esters of branched chain fatty acids and branched chain amino acids as predictors for AA, suggesting potential links to oxidative stress. Despite certain limitations, the study highlights the complexity of the gut microbiome and its metabolites in the context of AA, while the biomarkers identified could be useful starting points for upcoming studies.

Comparative analysis of microbial succession and proteolysis focusing on amino acid pathways in Asiago-PDO cheese from two dairies.

In this study, a comprehensive and comparative analysis was conducted on Italian Asiago-PDO cheese obtained from two different dairies named Dairy I and Dairy II using industrial and natural fermented milk, respectively. The analysis encompassed the evaluation of chemical composition, the succession of the microbiota during manufacture and ripening, and proteolysis mainly focusing on free individual amino acid (FAA) profiles. A metagenomic approach was used to investigate the cheese microbiome functionality. Differences in gross chemical composition were more evident during ripening, with Dairy II showing higher variability within batches. The microbiota varied significantly between the two dairies and ripening stages. The choice of starter culture shaped the microbiota during production and affected the microbial diversity of non-starter lactic acid bacteria (NSLAB) originated from the raw milk during ripening. Peptide chromatographic profiles and FAA concentrations increased as ripening progressed, with Dairy I showing higher production of FAA. Functional analysis of the metagenomes linked species to specific amino acid metabolism/catabolism pathways. The amino acid metabolism pathways, particularly those related to aromatic amino acids, lysine, and branched-chain amino acids, were affected by the presence of specific NSLAB species, which differed between the two dairies. The results obtained in this study reveal the impact of starter culture on peculiar cheese microbiota assemblies, which selectively targets amino acid pathways, providing insights into the potential flavor and aroma characteristics of Asiago-PDO cheese.

How starter cultures affect the peptidomic profile and bioactive activities of the Asiago-PDO cheese throughout ripening.

Our study investigated the chemical, microbiological, and bioactive peptide profiles of Asiago Protected Designation of Origin (PDO) cheese from two dairies (Dairy I and II) produced over two consecutive days (batches) and analysed during three months of ripening. The effect of different starter cultures was evaluated. The microbiome varied between the dairies and batches, with curds post-salting dominated by the starter culture-associated genera. During ripening, there was an increasing trend in the Lactobacillus genus, especially for Dairy I, which used an industrial starter. Bioactive peptide intensities differed throughout ripening due to the extent of proteolysis, and their intensity or concentration evolved, modifying, and differentiating profiles. The industrial starter used in Dairy I had the highest relative intensity (average value 76.50%) of bioactive peptides after three months of ripening. In contrast, the cheeses made with natural milk starter (Dairy II) had lower total relative intensity (average value 47.75%) but produced ACE-inhibitory peptides through sub-dominant strains and non-starter lactic acid bacteria. The importance of autochthonous strains of each micro-region even within a delimited PDO production area was highlighted.

The Effect of Hydrolyzed and Fermented Arabinoxylan-Oligo Saccharides (AXOS) Intake on the Middle-Term Gut Microbiome Modulation and Its Metabolic Answer.

Although fermentation and hydrolyzation are well-known processes to improve the bioavailability of nutrients and enable the fortification with dietary fibers, the effect of such pre-treatments on the prebiotic features of arabinoxylan-oligosaccharides (AXOS) had not been explored. The middle-term in vitro simulation through the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) demonstrated that the feeding with different formulations (namely oat bran, rye bran and wheat bran) containing hydrolyzed AXOS fermented by lactic acid bacteria significantly increased the synthesis of short-chain fatty acids (SCFA) by colon microbiota, with hydrolyzed and fermented rye bran displaying the highest effect. After two weeks from the interruption of intake, SCFA concentrations significantly decreased but remained still significantly higher compared to the original condition. The microbiome was also affected, with a significant abundance increase in Lactobacillaceae taxon after feeding with all fermented and hydrolyzed formulates. Hydrolyzed and fermented rye bran showed the highest changes. The fungal community, even if it had a lower variety compared to bacteria, was also modulated after feeding with AXOS formulations, with an increase in Candida relative abundance and a decrease in Issatchenkia. On the contrary, the intake of non-hydrolyzed and non-fermented wheat bran did not produce relevant changes of relative abundances. After two weeks from intake interruption (wash out period) such changes were mitigated, and the gut microbiome modulated again to a final structure that was more like the original condition. This finding suggests that hydrolyzed AXOS fermented by lactic acid bacteria could have a more powerful prebiotic effect compared to non-hydrolyzed and non-fermented wheat bran, shaping the colon microbiome and its metabolic answer. However, the intake should be continuous to assure persistent effects. Opening a window into the ecological evolutions and plausible underlying mechanisms, the findings reinforce the perspective to explore more in depth the use of hydrolyzed and fermented AXOS as additional ingredient for bread fortification.

Metabolic framework of spontaneous and synthetic sourdough metacommunities to reveal microbial players responsible for resilience and performance.

BACKGROUND: In nature, microbial communities undergo changes in composition that threaten their resiliency. Here, we interrogated sourdough, a natural cereal-fermenting metacommunity, as a dynamic ecosystem in which players are subjected to continuous environmental and spatiotemporal stimuli. RESULTS: The inspection of spontaneous sourdough metagenomes and transcriptomes revealed dominant, subdominant and satellite players that are engaged in different functional pathways. The highest microbial richness was associated with the highest number of gene copies per pathway. Based on meta-omics data collected from 8 spontaneous sourdoughs and their identified microbiota, we de novo reconstructed a synthetic microbial community SDG. We also reconstructed SMC-SD43 from scratch using the microbial composition of its spontaneous sourdough equivalent for comparison. The KEGG number of dominant players in the SDG was not affected by depletion of a single player, whereas the subdominant and satellite species fluctuated, revealing unique contributions. Compared to SMC-SD43, SDG exhibited broader transcriptome redundancy. The invariant volatilome profile of SDG after in situ long-term back slopping revealed its stability. In contrast, SMC-SD43 lost many taxon members. Dominant, subdominant and satellite players together ensured gene and transcript redundancy. CONCLUSIONS: Our study demonstrates how, by starting from spontaneous sourdoughs and reconstructing these communities synthetically, it was possible to unravel the metabolic contributions of individual players. For resilience and good performance, the sourdough metacommunity must include dominant, subdominant and satellite players, which together ensure gene and transcript redundancy. Overall, our study changes the paradigm and introduces theoretical foundations for directing food fermentations. Video Abstract.

Effect of Seasonality on Microbiological Variability of Raw Cow Milk from Apulian Dairy Farms in Italy.

Raw cow milk is one of the most complex and unpredictable food matrices shaped by the interaction between biotic and abiotic factors. Changes in dairy farming conditions impact the quality and safety of milk, which largely depend on seasonality. Changes in microbiome composition and relative metabolic pathways are derived from microbial interactions, as well as from seasonality, mammary, and extramammary conditions (e.g., farm management and outdoor environment). Breeding data from >600 Apulian farms were examined, and the associated physicochemical parameters were processed by a reductionist approach to obtain a raw cow milk sample subset. We investigated the microbiological variability in cultivable and 16S rRNA sequencing microbiota as affected by seasonal fluctuations at two time points (winter and summer seasons). We identified families (Xanthomonadaceae, Enterobacteriaceae, and Pseudomonadaceae) whose increased abundance during winter may cause a shift toward a pathobiont microbial niche that leads to lower milk quality. Apulian summer season conditions were advantageous to the presence of specific taxa, i.e., Streptococcaceae (i.e., Lactococcus) and Limosilactobacillus fermentum, which in turn may favor better milk preservation. IMPORTANCE The strength of this study lies in the microbiological characterization of a wide range of farm management data to achieve a more comprehensive framework of Apulian milk. Specific regional pedoclimatic and management conditions impact the taxa present and their abundances within this ecological food niche. The obtained results lay the groundwork for comparison with other worldwide extensive farming areas.

How cereal flours, starters, enzymes, and process parameters affect the in vitro digestibility of sourdough bread.

Digestibility of leavened baked goods relies on multiple factors: starch bioavailability and protein hydrolysis during food processing and digestion, presence of antinutritional factors, and satiety and gastrointestinal symptoms after intake. Several studies highlighted that bread digestibility might be positively affected by long-time sourdough fermentation. Nevertheless, most research is focused on single factors and their effect on digestibility, excluding the potential complementary effects of more than one factor. In this work, a multitude of factors influencing the the in vitro starch and protein digestibility and predicted glycemic index were assessed simultaneously. Forty-six different breads made with various raw material/ingredients (flour, enzymes, lactic acid bacteria cytoplasmic extracts and gluten), type of sourdoughs (fresh or commercial liquid or dried), strains of lactic acid bacteria and yeasts, and time and temperature of fermentation, were preliminarily investigated. Further selection of optimal conditions was based on statistical analysis and final breads were further characterized for their peptide profiles, total free amino acids and quality indexes of the digestible protein fraction. Among the factors considered, results identified a claimed in vitro digestibility for breads made with whole wheat, spelt, and rye flours, obtained with lactic acid bacteria strains selected for the peptidase activity, added of fungal proteases, and fermented at the optimal temperature of 37 °C.

Effects of Dietary Fibers on Short-Chain Fatty Acids and Gut Microbiota Composition in Healthy Adults: A Systematic Review.

There is an increasing interest in investigating dietary strategies able to modulate the gut microbial ecosystem which, in turn, may play a key role in human health. Dietary fibers (DFs) are widely recognized as molecules with prebiotic effects. The main objective of this systematic review was to: (i) analyze the results available on the impact of DF intervention on short chain fatty acids (SCFAs) production; (ii) evaluate the interplay between the type of DF intervention, the gut microbiota composition and its metabolic activities, and any other health associated outcome evaluated in the host. To this aim, initially, a comprehensive database of literature on human intervention studies assessing the effect of confirmed and candidate prebiotics on the microbial ecosystem was developed. Subsequently, studies performed on DFs and analyzing at least the impact on SCFA levels were extracted from the database. A total of 44 studies from 42 manuscripts were selected for the analysis. Among the different types of fiber, inulin was the DF investigated the most (n = 11). Regarding the results obtained on the ability of fiber to modulate total SCFAs, seven studies reported a significant increase, while no significant changes were reported in five studies, depending on the analytical methodology used. A total of 26 studies did not show significant differences in individual SCFAs, while the others reported significant differences for one or more SCFAs. The effect of DF interventions on the SCFA profile seemed to be strictly dependent on the dose and the type and structure of DFs. Overall, these results underline that, although affecting microbiota composition and derived metabolites, DFs do not produce univocal significant increase in SCFA levels in apparently healthy adults. In this regard, several factors (i.e., related to the study protocols and analytical methods) have been identified that could have affected the results obtained in the studies evaluated. Future studies are needed to better elucidate the relationship between DFs and gut microbiota in terms of SCFA production and impact on health-related markers.

Sourdough performances of the golden cereal Tritordeum: Dynamics of microbial ecology, biochemical and nutritional features.

The novel cereal 'Tritordeum' was employed in sourdough fermentation for bread making using a traditional backslopping procedure over 10 days. Culture-dependent and culture-independent approaches were used to characterize microbial ecology during sourdough preparation and propagation. Sourdough reached the highest microbial diversity after three days of propagation. Microbial diversity decreased as sourdough reached maturity (day 5). Microbiota dominance shifted from Weissella to Lactiplantibacillus genera after 5 days of propagation. Lactic acid bacteria (LAB) showed a constant increase throughout the propagations starting from 3.9 ± 0.24 log CFU g-1 on day 0 up to 8.0 ± 0.39 log CFU g-1 on day 5. Weissella confusa/cibaria and Weissella paramesenteroides were the most prevalent LAB species until day 5 of propagation, while Lactiplantibacillus plantarum was the most prevalent thereafter. Yeasts were present in low cell density (2.0 ± 0.11 log CFU g-1) until the fourth backslopping (day 4) and then gradually increased until day 10 (5.0 ± 0.29 log CFU g-1), with Saccharomyces cerevisiae being the most prevalent and dominant species. Lactic and acetic acid concentrations increased throughout Tritordeum sourdough propagations, indicative of a proportional decrease of fermentation quotient (lactic acid/acetic acid) from 13.54 ± 1.29 to 4.08 ± 0.15. Utilization of glucose, fructose and sucrose was observed, followed a progressive increase in mannitol concentrations beginning from day 4. The nutritional potential (total phenol content, antioxidant activity, dietary fiber content and total free amino acids) remained elevated during sourdough propagations. Antinutritional factors (phytic acid and raffinose) were reduced to minimal concentrations by day 10. Finally, texture analysis of Tritordeum sourdough bread was demonstrated to have better cohesiveness, resilience and firmness compared to baker's yeast bread, confirming its potential to improve functionality and use in sourdough biotechnology.

Date Seeds Flour Used as Value-Added Ingredient for Wheat Sourdough Bread: An Example of Sustainable Bio-Recycling.

Our study proposed date seeds flour (DSF) as an innovative ingredient for sourdough bread production through sustainable bio-recycling. We isolated autochthonous lactic acid bacteria and yeasts from DSF and DSF-derived doughs to build up a reservoir of strains from which to select starters ensuring rapid adaptation and high ecological fitness. The screening based on pro-technological criteria led to the formulation of a mixed starter consisting of Leuconostoc mesenteroides, Lactiplantibacillus plantarum, and Saccharomyces cerevisiae strains, which allowed obtaining a mature type I sourdough after consecutive refreshments, in which an aliquot of the durum wheat flour (DWF) was replaced by DSF. The resulting DSF sourdough and bread underwent an integrated characterization. Sourdough biotechnology was confirmed as a suitable procedure to improve some functional and sensory properties of DWF/DSF mixture formulation. The radical scavenging activity increased due to the consistent release of free phenolics. Perceived bitterness and astringency were considerably diminished, likely because of tannin degradation.

Colonization Ability and Impact on Human Gut Microbiota of Foodborne Microbes From Traditional or Probiotic-Added Fermented Foods: A Systematic Review.

A large subset of fermented foods act as vehicles of live environmental microbes, which often contribute food quality assets to the overall diet, such as health-associated microbial metabolites. Foodborne microorganisms also carry the potential to interact with the human gut microbiome via the food chain. However, scientific results describing the microbial flow connecting such different microbiomes as well as their impact on human health, are still fragmented. The aim of this systematic review is to provide a knowledge-base about the scientific literature addressing the connection between foodborne and gut microbiomes, as well as to identify gaps where more research is needed to clarify and map gut microorganisms originating from fermented foods, either traditional or added with probiotics, their possible impact on human gut microbiota composition and to which extent foodborne microbes might be able to colonize the gut environment. An additional aim was also to highlight experimental approaches and study designs which could be better standardized to improve comparative analysis of published datasets. Overall, the results presented in this systematic review suggest that a complex interplay between food and gut microbiota is indeed occurring, although the possible mechanisms for this interaction, as well as how it can impact human health, still remain a puzzling picture. Further research employing standardized and trans-disciplinary approaches aimed at understanding how fermented foods can be tailored to positively influence human gut microbiota and, in turn, host health, are therefore of pivotal importance.

How multiple farming conditions correlate with the composition of the raw cow's milk lactic microbiome.

Questionnaires on farming conditions were retrieved from 2129 dairy farms and clustered, resulting in 106 representative raw cow's milk samples analysed in winter and summer. Substantiating the efficiency of our survey, some farming conditions affected the milk physicochemical composition. Culturing identified several species of lactic acid bacteria (LAB) per milk, whose number increased through 16S ribosomal RNA (rRNA) gene sequencing and shotgun metagenome analyses. Season, indoor versus outdoor housing, cow numbers, milk substitutes, ratio cattle/rest area, house care system during lactation, and urea and medium-chain fatty acids correlated with the overall microbiome composition and the LAB diversity within it. Shotgun metagenome detected variations in gene numbers and uniqueness per milk. LAB functional pathways differed among milk samples. Focusing on amino acid metabolisms and matching the retrieved annotated genes versus non-starter lactic acid bacteria (NSLAB) references from KEGG and corresponding to those identified, all samples had the same gene spectrum for each pathway. Conversely, gene redundancy varied among samples and agreed with NSLAB diversity. Milk samples with higher numbers of NSLAB species harboured higher number of copies per pathway, which would enable steady-state towards perturbations. Some farming conditions, which affected the microbiome richness, also correlated with the NSLAB composition and functionality.

Role prediction of Gram-negative species in the resistome of raw cow's milk.

Extended use of antibiotics in dairy farming for therapeutic and prophylactic reasons, but also the higher prevalence of antibiotic resistant bacteria (ARB) in the farm environment raised the concern of consuming raw cow's milk and its derived products. The aim of this study was to predict by shotgun metagenomic analyses the presence of antibiotic resistance genes (ARGs) mainly correlated with Gram-negative bacteria in antibiotic residue free raw cow's milk derived exclusively from healthy animal from South Tyrol (Northern Italy), chosen as a model system. Assessment of shotgun metagenomic data of reconstructed scaffolds, revealed the existence of Pseudomonas spp. as the most abundant Gram-negative species in the raw cow's milk samples bearing ARGs. Besides, ARGs also linked to lactic acid bacteria such as Lactococcus sp. and Lactobacillus sp. ARGs correlated to microbiome found in milk samples conferred resistance towards aminoglycoside-streptothricin, beta-lactamase, macrolide, tetracycline, carbapenem, cephalosporin, penam, peptide, penem, fluoroquinolone, chloramphenicol and elfamycin antibiotics. Further bioinformatic processing included de-novo reassembly of all metagenomic sequences from all milk samples in one, to reconstruct metagenome assembled genomes (MAGs), which were further used to investigate mobile genetic elements (MGE). Analyses of the reconstructed MAGs showed that, MAG 9 (Pseudomonas sp1.) contained the oriT gene (origin of transfer gene) needed for transferring virulent factors. Although the presence of Pseudomonas is common in raw cow's milk, pasteurization treatment reduces their survivability. Nevertheless, attention should be paid on Pseudomonas spp. due to their intrinsic resistance to antibiotics and their capability of transferring virulent factors to other bacteria.