Microbiome mapping in dairy industry reveals new species and genes for probiotic and bioprotective activities.

The resident microbiome in food industries may impact on food quality and safety. In particular, microbes residing on surfaces in dairy industries may actively participate in cheese fermentation and ripening and contribute to the typical flavor and texture. In this work, we carried out an extensive microbiome mapping in 73 cheese-making industries producing different types of cheeses (fresh, medium and long ripened) and located in 4 European countries. We sequenced and analyzed metagenomes from cheese samples, raw materials and environmental swabs collected from both food contact and non-food contact surfaces, as well as operators' hands and aprons. Dairy plants were shown to harbor a very complex microbiome, characterized by high prevalence of genes potentially involved in flavor development, probiotic activities, and resistance to gastro-intestinal transit, suggesting that these microbes may potentially be transferred to the human gut microbiome. More than 6100 high-quality Metagenome Assembled Genomes (MAGs) were reconstructed, including MAGs from several Lactic Acid Bacteria species and putative new species. Although microbial pathogens were not prevalent, we found several MAGs harboring genes related to antibiotic resistance, highlighting that dairy industry surfaces represent a potential hotspot for antimicrobial resistance (AR) spreading along the food chain. Finally, we identified facility-specific strains that can represent clear microbial signatures of different cheesemaking facilities, suggesting an interesting potential of microbiome tracking for the traceability of cheese origin.

Fermentation with Lactic Acid Bacteria for Bean Flour Improvement: Experimental Study and Molecular Modeling as Complementary Tools.

Pulses are considered superfoods for the future world due to their properties, but they require processing to reduce antinutritional factors (ANFs) and increase bioactivity. In this study, bean flour (Phaseolus vulgaris L.) was fermented under different conditions (addition of Lactiplantibacillus plantarum CRL 2211 and/or Weissella paramesenteroides CRL 2182, temperature, time and dough yield) to improve its nutri-functional quality. Fermentation for 24 h at 37 °C with the mixed starter increased the lactic acid bacteria (LAB) population, acidity, polyphenol content (TPC) and ANF removal more than spontaneous fermentation. Statistical and rep-PCR analysis showed that fermentation was mainly conducted by Lp. plantarum CRL 2211. Metabolic modeling revealed potential cross-feeding between Lp. plantarum and W. paramesenteroides, while the molecular docking and dynamic simulation of LAB tannases and proteinases involved in ANF removal revealed their chemical affinity to gallocatechin and trypsin inhibitors. Fermentation was better than soaking, germination and cooking for enhancing bean flour properties: it increased the free amino acids content by 50% by releasing glutamine, glutamic acid, arginine, leucine and lysine and modified TPC by increasing gallic acid and decreasing caffeic, ferulic and vanillic acids and quercetin-3-glucoside. The combination of experimental and simulation data may help us to understand fermentation processes and to design products with desirable features.

Thickness Determination and Control in Protein-Based Biomaterial Thin Films.

Controlling the thickness and uniformity of biomaterial films is crucial for their application in various fields including sensing and bioelectronics. In this work, we investigated film assemblies of an engineered repeat protein─specifically, the consensus tetratricopeptide repeat (CTPR) protein ─a system with unique robustness and tunability. We propose the use of microreflectance spectroscopy and apparent color inspection for the quick assessment of the thickness and uniformity of protein-based biomaterial films deposited on oxidized silicon substrates. Initially, we characterized the thickness of large, uniform, spin-coated protein films and compared the values obtained from microreflectance spectroscopy with those obtained from other typical methods, such as ellipsometry and atomic force microscopy. The excellent agreement between the results obtained from the different techniques validates the effectiveness of microreflectance as a fast, noninvasive, and affordable technique for determining the thickness of biomaterial films. Subsequently, we applied microreflectance spectroscopy to determine the thickness of drop-casted CTPR-based films prepared from small protein solution volumes, which present a smaller surface area and are less uniform compared to spin-coated samples. Additionally, we demonstrate the utility of apparent color inspection as a tool for assessing film uniformity. Finally, based on these results, we provide a calibration of film thickness as a function of the protein length and concentration for both spin-coated and drop-casted films, serving as a guide for the preparation of CTPR films with a specific thickness. Our results demonstrate the remarkable reproducibility of the CTPR film assembly, enabling the simple preparation of biomaterial films with precise thickness.

Tracing the origin and authenticity of Spanish PDO honey using metagenomics and machine learning.

The Protected Designation of Origin (PDO) indication for foods intends to guarantee the conditions of production and the geographical origin of regional products within the European Union. Honey products are widely consumed due to their health-promoting properties and there is a general interest in tracing their authenticity. In this regard, metagenomics sequencing and machine learning (ML) have been proposed as complementary technologies to improve the traceability methods of foods. Therefore, the aim of this study was to analyze the metagenomic profiles of Spanish honeys from three different PDOs (Granada, Tenerife and Villuercas-Ibores), and compare them with non-PDO honeys using ML models (PLS, RF, LOGITBOOST, and NNET). According to the results obtained, non-PDO honeys and Granada PDO showed higher beta diversity values than Tenerife and Villuercas-Ibores PDOs. ML classification of honey products allowed the identification of different microbial biomarkers of the geographical origin of honeys: Lactobacillus kunkeei, Parasaccharibacter apium and Lactobacillus helsingborgensis for PDO honeys and Paenibacillus larvae, Lactobacillus apinorum and Klebsiella pneumoniae for non-PDO honeys. In addition, potential microbial biomarkers of some honey varieties including L. kunkeei for Albaida and Retama del Teide varieties, and P. apium for Tajinaste variety, were identified. ML models were validated on an independent set of samples leading to high accuracy rates (above 90 %). This work demonstrates the potential of ML to differentiate different types of honey using metagenome-based methods, leading to high performance metrics. In addition, ML models discriminate both the geographical origin and variety of products corresponding to different PDOs and non-PDO products. Results here presented may contribute to develop enhanced traceability and authenticity methods that could be applied to a wide range of foods.

Exploring the modulatory effect of trehalose-derived galactooligosaccharides on key gut microbiota groups.

Trehalose (α-d-glucopyranosyl-(1-1)-α-D-glucopyranoside) has found applications in diverse food products as a sweetener, stabilizer, and humectant. Recent attention has focused on trehalose due to its contradictory effects on the virulence of Clostridium difficile. In this study, we investigate the impact of novel trehalose-derived galactooligosaccharides (Treh-GOS) on the human gut microbiota using in vitro fecal fermentation models. Distinct Treh-GOS structures elicit varying taxonomic responses. For instance, ß-Gal-(1-4)-trehalose [DP3(1-4)] leads to an increase of Bifidobacterium, comparable to results observed with commercial GOS. Conversely, ß-Gal-(1-6)-trehalose [DP3(1-6)] prompts an increase in Lactobacillus. Notably, both of these trisaccharides yield the highest concentrations of butyric acid across all samples. On the other hand, Treh-GOS tetrasaccharide mixture (DP4), featuring a novel trehalose galactosylation in both glucose units, fosters the growth of Parabacteroides. Our findings underscore the capacity of novel Treh-GOS to modulate the human gut microbiota. Consequently, these innovative galactooligosaccharides emerge as promising candidates for novel prebiotic applications.

A methyl esterase from Bifidobacterium longum subsp. longum reshapes the prebiotic properties of apple pectin by triggering differential modulatory capacity in faecal cultures.

Pectin structures have received increasing attention as emergent prebiotics due to their capacity to promote beneficial intestinal bacteria. Yet the collective activity of gut bacterial communities to cooperatively metabolize structural variants of this substrate remains largely unknown. Herein, the characterization of a pectin methylesterase, BpeM, from Bifidobacterium longum subsp. longum, is reported. The purified enzyme was able to remove methyl groups from highly methoxylated apple pectin, and the mathematical modelling of its activity enabled to tightly control the reaction conditions to achieve predefined final degrees of methyl-esterification in the resultant pectin. Demethylated pectin, generated by BpeM, exhibited differential fermentation patterns by gut microbial communities in in vitro mixed faecal cultures, promoting a stronger increase of bacterial genera associated with beneficial effects including Lactobacillus, Bifidobacterium and Collinsella. Our findings demonstrate that controlled pectin demethylation by the action of a B. longum esterase selectively modifies its prebiotic fermentation pattern, producing substrates that promote targeted bacterial groups more efficiently. This opens new possibilities to exploit biotechnological applications of enzymes from gut commensals to programme prebiotic properties.

Study of the intestinal microbiota composition and the effect of treatment with intensive chemotherapy in patients recovered from acute leukemia.

A dataset comprising metagenomes of outpatients (n = 28) with acute leukemia (AL) and healthy controls (n = 14) was analysed to investigate the associations between gut microbiota composition and metabolic activity and AL. According to the results obtained, no significant differences in the microbial diversity between AL outpatients and healthy controls were found. However, significant differences in the abundance of specific microbial clades of healthy controls and AL outpatients were found. We found some differences at taxa level. The relative abundance of Enterobacteriaceae, Prevotellaceae and Rikenellaceae was increased in AL outpatients, while Bacteirodaceae, Bifidobacteriaceae and Lachnospiraceae was decreased. Interestingly, the abundances of several taxa including Bacteroides and Faecalibacterium species showed variations based on recovery time from the last cycle of chemotherapy. Functional annotation of metagenome-assembled genomes (MAGs) revealed the presence of functional domains corresponding to therapeutic enzymes including L-asparaginase in a wide range of genera including Prevotella, Ruminococcus, Faecalibacterium, Alistipes, Akkermansia. Metabolic network modelling revealed potential symbiotic relationships between Veillonella parvula and Levyella massiliensis and several species found in the microbiota of AL outpatients. These results may contribute to develop strategies for the recovery of microbiota composition profiles in the treatment of patients with AL.

Gut microbiota modulation and effects of a diet enriched in apple pomace on inflammation in a DSS-induced colitis mouse model.

Certain types of soluble dietary fibre, such as pectin and pectic oligosaccharides from different sources, have demonstrated protective effects against inflammation in DSS-induced colitis mouse models. In this work, we have evaluated the impact of a diet enriched in apple pomace (AP-diet), an agricultural by-product with a significant content of pectin and that previously demonstrated prebiotic properties in human fecal batch fermentation models, on the gut microbiota composition, intestinal damage and inflammation markers in a DSS-induced colitis model. We found that the apple pomace enriched diet (AP-diet), providing a significant amount of pectin with demonstrated prebiotic properties, was associated with a slower increase in the disease activity index, translating into better clinical symptomatology of the animals. Histological damage scoring confirmed less severe damage in those animals receiving an AP-diet before and during the DSS administration period. Some serum inflammatory markers, such as TNFα, also demonstrated lower levels in the group receiving the AP-diet, compared to the control diet. AP-diet administration is also associated with the modulation of key taxa in the colonic microbiota of animals, such as some Lachnospiraceae genera and Ruminococcus species, including commensal short chain fatty acid producers that could play a role in attenuating inflammation at the intestinal level.

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments.

Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.

Mouse intestinal microbiome modulation by oral administration of a GABA-producing Bifidobacterium adolescentis strain.

IMPORTANCE: The gut microbiome-brain communication signaling has emerged in recent years as a novel target for intervention with the potential to ameliorate some conditions associated with the central nervous system. Hence, probiotics with capacity to produce neurotransmitters, for instance, have come up as appealing alternatives to treat disorders associated with disbalanced neurotransmitters. Herein, we further deep into the effects of administering a gamma-aminobutyric acid (GABA)-producing Bifidobacterium strain, previously demonstrated to contribute to reduce serum glutamate levels, in the gut microbiome composition and metabolic activity in a mouse model. Our results demonstrate that the GABA-producing strain administration results in a specific pattern of gut microbiota modulation, different from the one observed in animals receiving non-GABA-producing strains. This opens new avenues to delineate the specific mechanisms by which IPLA60004 administration contributes to reducing serum glutamate levels and to ascertain whether this effect could exert health benefits in patients of diseases associated with high-glutamate serum concentrations.

Next-generation sequencing of the athletic gut microbiota: a systematic review.

Aim: There is growing evidence that physical activity modulates gut microbiota composition through complex interactions between diet and microbial species. On the other hand, next-generation sequencing techniques include shotgun metagenomics and 16S amplicon sequencing. These methodologies allow a comprehensive characterisation of microbial communities of athletes from different disciplines as well as non-professional players and sedentary adults exposed to training. This systematic review summarises recent applications of next-generation sequencing to characterise the athletic gut microbiome. Methods: A systematic review of microbiome research was performed to determine the association of microbiota composition profiles with sports performance. Results: Bibliographic analysis revealed the importance of a novel research trend aiming at deciphering the associations between individual microbial species and sports performance. In addition, literature review highlighted the role of butyrate-producing bacteria such as Anaerostipes hadrus, Clostridium bolteae, Faecalibacterium prausnitzii, Roseburia hominis and unidentified species belonging to Clostridiales, Lachnospiraceae and Subdoligranulum species in gut health and sports performance across several disciplines. Interestingly, metabolic activities of Prevotella copri and Veillonella atypica involved in branched amino acid and lactate metabolism may contribute to reducing muscular fatigue. Other microbial metabolic pathways of interest involved in carbohydrate metabolism showed increased proportions in athletes´ metagenomes. Conclusion: Future research will aim at developing personalised nutrition interventions to modulate key species associated with certain components of exercise.

Metabolic response of intestinal microbiota to guar gum consumption.

Background: Guar gum is used extensively as a thickening agent in food, but it remains uncertain whether and to what extent it is fermented by colonic microbiota and whether it has microbiota modulatory properties. Aim: To determine the metabolic response of intestinal microbiota to guar gum consumption, specifically, the extent of initial fermentation and subsequent adaptation. Methods: Single-center, single arm, open label, proof-of-concept study testing the effect of guar gum on microbiota metabolism and adaptation. Healthy male subjects (n = 12) were administered gum guar (8 g/day) for 18 days. Outcomes were measured before, at initial and late administration: (a) anal gas evacuations (number/day); (b) digestive sensations (daily scales); and (c) fecal gut microbiota taxonomy and metabolic functions by shotgun sequencing. Results: At initial consumption, guar gum induced a transient increase in anal gas evacuations and digestive sensations; gas evacuation completely reverted upon continuous administration, whereas sensations reverted only in part. Guar gum induced moderate changes in human microbiota composition at both taxonomic and functional levels. Positive associations between effects on microbiota (proliferation of Agathobaculum butyriciproducens and Lachnospira pectinoschiza) and hedonic sensations were detected. Conclusion: Guar gum is metabolized by intestinal microbiota, and, upon continuous consumption, induces a selective adaptation of microbial taxonomy and function. These data highlight the potential interest of guar gum for novel prebiotic ingredient formulation.

Production of GABA in milk fermented by Bifidobacterium adolescentis strains selected on the bases of their technological and gastrointestinal performance.

There is an increasing interest in producing foods enriched in gamma-aminobutyric acid (GABA), due to their purported health promoting attributes. GABA is the main inhibitor neurotransmitter of the central nervous system, and several microbial species are capable to produce it through decarboxylation of glutamate. Among them, several lactic acid bacteria species have been previously investigated as an appealing alternative to produce GABA enriched foods via microbial fermentation. In this work we report for the first time an investigation into the possibility of utilizing high GABA-producing Bifidobacterium adolescentis strains as a mean to produce fermented probiotic milks naturally enriched in GABA. To this end, in silico and in vitro analyses were conducted in a collection of GABA-producing B. adolescentis strains, with the main goal to scrutinize their metabolic and safety traits, including antibiotic resistance patterns, as well as their technological robustness and performance to survive a simulated gastrointestinal passage. One of the strains, IPLA60004, exhibited better survival to lyophilization and cold storage (for up to 4 weeks at 4 °C), as well as survival to gastrointestinal passage, as compared to the other strains under investigation. Besides, the elaboration of milk drinks fermented with this strain, yielded products with the highest GABA concentration and viable bifidobacterial cell counts, achieving conversion rates of the precursor, monosodium glutamate (GMS), up to 70 %. To our knowledge, this is the first report on the elaboration of GABA enriched milks through fermentation with B. adolescentis.

Arabinoxylan-based substrate preferences and predicted metabolic properties of Bifidobacterium longum subspecies as a basis to design differential media.

Arabinoxylan (AX) and arabinoxylo-oligosaccharides (AXOS) derived therefrom are emergent prebiotics with promising health promoting properties, likely linked to its capacity to foster beneficial species in the human gut. Bifidobacteria appear to be one taxa that is frequently promoted following AX or AXOS consumption, and that is known to establish metabolic cross-feeding networks with other beneficial commensal species. Therefore, probiotic bifidobacteria with the capability to metabolize AX-derived prebiotics represent interesting candidates to develop novel probiotic and synbiotic combinations with AX-based prebiotics. In this work we have deepen into the metabolic capabilities of bifidobacteria related to AX and AXOS metabolization through a combination of in silico an in vitro tools. Both approaches revealed that Bifidobacterium longum and, particularly, B. longum subsp. longum, appears as the better equipped to metabolize complex AX substrates, although other related subspecies such as B. longum subsp. infantis, also hold some machinery related to AXOS metabolization. This correlates to the growth profiles exhibited by representative strains of both subspecies in AX or AXOS enriched media. Based on these results, we formulated a differential carbohydrate free medium (CFM) supplemented with a combination of AX and AXOS that enabled to recover a wide diversity of Bifidobacterium species from complex fecal samples, while allowing easy discrimination of AX metabolising strains by the appearance of a precipitation halo. This new media represent an appealing alternative to isolate novel probiotic bifidobacteria, rapidly discriminating their capacity to metabolize structurally complex AX-derived prebiotics. This can be convenient to assist formulation of novel functional foods and supplements, including bifidobacterial species with capacity to metabolize AX-derived prebiotic ingredients.

Evaluation of a Shotgun Metagenomics Approach for Detection of ESBL- and/or Carbapenemase-Producing Enterobacterales in Culture Negative Patients Recovered from Acute Leukemia.

Patients diagnosed with acute leukemia (AL) have a weakened immune system. Infections acquired by these patients are cause for concern and especially worrisome when Gram-negative multidrug-resistant (MDR) bacteria are involved, as they are difficult to treat, especially in the case of ESBL- and/or carbapenemase-producing Enterobacterales. Culture-based approaches have been relied on over the past decades as the method of choice for the early detection of gut colonization by MDR Gram-negative bacteria. However, various studies have indicated its limited sensitivity, underlining the need for new screening procedures in onco-hematological patients. Here, we evaluated a shotgun metagenomics approach to detect ESBL- and/or carbapenemase-producing Enterobacterales in the gut of 28 patients who had recovered from AL, which were previously colonized by these bacteria but cured at the time of sampling, as judged by culture-based methods. No ESBL or carbapenemase determinants were detected among the many resistance genes found by the metagenomics approach, supporting that patients were truly decolonized, with considerable consequences for their future clinical management. Due to the relatively low number of patients available for the present investigation, further studies should be conducted to support the utility and applicability of metagenomics for the routine screening of MDR bacteria in onco-hematological patients.

"Masato de Yuca" and "Chicha de Siete Semillas" Two Traditional Vegetable Fermented Beverages from Peru as Source for the Isolation of Potential Probiotic Bacteria.

In this work, two Peruvian beverages "Masato de Yuca," typical of the Amazonian communities made from cassava (Manihot esculenta), and "Chicha de Siete Semillas," made from different cereal, pseudo-cereal, and legume flours, were explored for the isolation of lactic acid bacteria after obtaining the permission of local authorities following Nagoya protocol. From an initial number of 33 isolates, 16 strains with different RAPD- and REP-PCR genetic profiles were obtained. In Chicha, all strains were Lactiplantibacillus plantarum (formerly Lactobacillus plantarum), whereas in Masato, in addition to this species, Limosilactobacillus fermentum (formerly Lactobacillus fermentum), Pediococcus acidilactici, and Weissella confusa were also identified. Correlation analysis carried out with their carbohydrate fermentation patterns and enzymatic profiles allowed a clustering of the lactobacilli separated from the other genera. Finally, the 16 strains were submitted to a static in vitro digestion (INFOGEST model) that simulated the gastrointestinal transit. Besides, their ability to adhere to the human epithelial intestinal cell line HT29 was also determined. Following both procedures, the best probiotic candidate was Lac. plantarum Ch13, a robust strain able to better face the challenging conditions of the gastrointestinal tract and showing higher adhesion ability to the intestinal epithelium in comparison with the commercial probiotic strain 299v. In order to characterize its benefit for human health, this Ch13 strain will be deeply studied in further works.

Effect of Resistant Dextrin on Intestinal Gas Homeostasis and Microbiota.

Previous studies have shown that a resistant dextrin soluble fibre has prebiotic properties with related health benefits on blood glucose management and satiety. Our aim was to demonstrate the effects of continuous administration of resistant dextrin on intestinal gas production, digestive sensations, and gut microbiota metabolism and composition. Healthy subjects (n = 20) were given resistant dextrin (14 g/d NUTRIOSE®, Roquette Frères, Lestrem, France) for four weeks. Outcomes were measured before, at the beginning, end, and two weeks after administration: anal evacuations of gas during daytime; digestive perception, girth, and gas production in response to a standard meal; sensory and digestive responses to a comfort meal; volume of colonic biomass by magnetic resonance; taxonomy and metabolic functions of fecal microbiota by shotgun sequencing; metabolomics in urine. Dextrin administration produced an initial increase in intestinal gas production and gas-related sensations, followed by a subsequent decrease, which magnified after discontinuation. Dextrin enlarged the volume of colonic biomass, inducing changes in microbial metabolism and composition with an increase in short chain fatty acids-producing species and modulation of bile acids and biotin metabolism. These data indicate that consumption of a soluble fibre induces an adaptative response of gut microbiota towards fermentative pathways with lower gas production.

Arabinoxylan and Pectin Metabolism in Crohn's Disease Microbiota: An In Silico Study.

Inflammatory bowel disease is a chronic disorder including ulcerative colitis and Crohn's disease (CD). Gut dysbiosis is often associated with CD, and metagenomics allows a better understanding of the microbial communities involved. The objective of this study was to reconstruct in silico carbohydrate metabolic capabilities from metagenome-assembled genomes (MAGs) obtained from healthy and CD individuals. This computational method was developed as a mean to aid rationally designed prebiotic interventions to rebalance CD dysbiosis, with a focus on metabolism of emergent prebiotics derived from arabinoxylan and pectin. Up to 1196 and 1577 MAGs were recovered from CD and healthy people, respectively. MAGs of Akkermansia muciniphila, Barnesiella viscericola DSM 18177 and Paraprevotella xylaniphila YIT 11841 showed a wide range of unique and specific enzymes acting on arabinoxylan and pectin. These glycosidases were also found in MAGs recovered from CD patients. Interestingly, these arabinoxylan and pectin degraders are predicted to exhibit metabolic interactions with other gut microbes reduced in CD. Thus, administration of arabinoxylan and pectin may ameliorate dysbiosis in CD by promoting species with key metabolic functions, capable of cross-feeding other beneficial species. These computational methods may be of special interest for the rational design of prebiotic ingredients targeting at CD.

Fermentation of chickpea flour with selected lactic acid bacteria for improving its nutritional and functional properties.

AIMS: To improve the nutri-functional quality of chickpea flour by fermentation with selected lactic acid bacteria (LAB) to formulate functional legume-derived products. METHODS AND RESULTS: A Randomized Complete Block Design was carried out to assess the influence of experimental conditions (presence/absence of Lactiplantibacillus plantarum CRL2211 and/or Weissella paramesenteroides CRL2182, temperature, time and dough yield) on LAB population, acidification, antinutritional factors and total phenolic contents (TPCs) of chickpea flour. Fermentation with both strains for 24 h at 37°C produced an increase in LAB (up to 8.9 log CFU/g), acidity (final pH 4.06), TPC (525.00 mg GAE/100 g) and tannin and trypsin inhibitor removal (28.80 mg GAE/100 g and 1.60 mg/g, respectively) higher than the spontaneously fermented doughs. RAPD and Rep-PCR analysis revealed that fermentation was dominated by L. plantarum CRL2211. Molecular docking and dynamics simulations were useful to explain LAB enzyme behaviour during fermentation highlighting the chemical affinity of LAB tannases and proteinases to gallocatechin and trypsin inhibitors. Compared with other processing methods, fermentation was better than soaking, germination and cooking for increasing the techno-functional properties of chickpea flour. Fermented doughs were applied to the manufacture of crackers that contained 81% more TPC and 64% more antioxidant activity than controls. CONCLUSIONS: Fermentation for 24 h at 37°C with selected autochthonous LAB was the best method for improving the quality of chickpea flour and derived crackers type cookies. SIGNIFICANCE AND IMPACT OF STUDY: Chickpea is suitable for the development of novel functional foods. Fermentation with selected LAB would improve the final product quality and bioactivity. The combination of experimental and simulation approaches can lead to a better understanding of the fermentation processes to enhance the properties of a food matrix.