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Probiotics are essential for promoting health, with lactic acid bacteria (LAB) from traditional fermented foods like Ethiopian kocho offering valuable benefits. The objective of this study was to systematically analyse the genomic characteristics, bacteriocin production, and probiotic potential of LAB strains isolated from fermented Ethiopian kocho. LAB were isolated from kocho and screened for probiotic properties following standard methods. For the potent isolates, whole-genome sequencing (WGS) was conducted to investigate genetic relatedness. Out of 150 LAB isolates, 7 (4.67%) exhibited remarkable acid tolerance by surviving at rates between 50.52–74.05% and 33.33–62.40% after 3 and 6 hours of exposure to pH 2, respectively. These seven acid-tolerant isolates also demonstrated exceptional resistance to 0.3% bile salt, maintaining survival rates ranging from 88.96% to 98.10% over 24 hours. In addition, the isolates displayed inhibitory effects against several important foodborne pathogenic bacteria, underscoring their potential as natural antimicrobial agents. Antibiotic susceptibility testing revealed that all the isolates were susceptible to ampicillin, tetracycline, and erythromycin, whereas the most potent isolates exhibited significant resistance to kanamycin. The WGS analysis revealed that the isolates belonged to the Lactobacillus genus, including six Lactiplantibacillus plantarum strains and one Levilactobacillus brevis strain. Genomic analysis using the Bayesian Analysis of Gene Essentiality (BAGEL) tool predicted the presence of two class II bacteriocins across all the seven strains, further supporting their potential as functional probiotic candidates. Analysis of the isolates using Abricate with the Virulence Factor Database (VFDB) showed that none of the strains carried putative virulence factors. Moreover, screening for antibiotic resistance genes revealed no resistance determinants, suggesting a low risk of resistance gene transfer. Overall, these results confirm the favorable safety profile of the probiotic properties of Lactobacillus strains and support their suitability for industrial and dietary applications.

Probiotic microorganisms are increasing their interest today due to the benefits they provide to humans. Vinegar is the process of processing foods containing carbohydrates that can be fermented by acetic acid bacteria and yeasts. Hawthorn vinegar is also important in terms of amino acids, aromatic compounds, organic acids, vitamins and minerals it contains. Depending on the variety of microorganisms in it, the content of hawthorn vinegar changes, especially its biological activity. Bacteria were isolated from handmade hawthorn vinegar obtained in this study. After performing its genotypic characterization, it has been tested that it can grow in low pH environment, survive in artificial gastric and small intestinal fluid, survive against bile acids, surface adhesion characteristics, antibiotic susceptibility, adhesion, and degrade various cholesterol precursors. According to the results obtained, the studied isolate is Levilactobacillus brevis, it can reproduce best at pH 6.3, survives 72.22% in simulated gastric juice, 69.59% in small intestinal fluid, and 97% adhesion to HTC-116. Partially reproduces even in the presence of 2% ox-bile, surface hydrophobicity is 46.29% for n-hexadecane. It has been determined that it can degrade 4 different cholesterol precursors except for Sodium thioglycolate and is generally resistant to antibiotics except for CN30 and N30. Considering the experimental findings of Levilactobacillus brevis isolated from hawthorn vinegar for the first time, it can be said that Levilactobacillus brevis has probiotic properties.

Levilactobacillus brevis KU15006, isolated from kimchi, exhibits pathogen-antagonistic and anti-diabetic activities; however, the safety of this strain has not been assessed. In the present study, L. brevis KU15006 was evaluated to elucidate its safety as a probiotic strain using phenotypic and genotypic analyses. Its safety was assessed using a minimum inhibitory concentration test comprising nine antibiotics, 26 antibiotic resistance genes, a single conjugative element, virulence gene analysis, hemolysis, cell cytotoxicity, mucin degradation, and toxic metabolite production. L. brevis KU15006 exhibited equal or lower minimum inhibitory concentration for the nine antibiotics than the cut-off value established by the European Food Safety Authority. It did not harbor antibiotic resistance and virulence genes. L. brevis KU15006 lacked β-hemolysis, mucin degradation, cytotoxicity against Caco-2 cells, gelatin liquefaction, bile salt deconjugation, and toxic metabolite production abilities. Based on the results, L. brevis KU15006, which has antagonistic and anti-diabetic effects, could be marketed as a probiotic in the future.

Although rare, the ability to produce surface S-layer proteins is beneficially associated with particular Lactobacillus strains being investigated as probiotics. Therefore, this work aimed to study specific probiotic functionalities of selected Levilactobacillus brevis strains MB1, MB2, MB13 and MB20, isolated from human milk microbiota, and to assess the contribution of S-proteins. Firstly, Rapid Annotation using Subsystem Technology revealed that cell wall-related genes were abundant in analysed L. brevis genomes. Furthermore, the results demonstrated that S-proteins mediate aggregation capacity and competitive exclusion of selected pathogens by L. brevis strains. The improvement of Caco-2 epithelial monolayer barrier function was demonstrated by the increase in JAM-A and occludin expressions when L. brevis strains or S-proteins were added, with the effect being most pronounced after treatment with MB2 and S-proteins of MB1. L. brevis strains, especially MB20, exerted the potential to adhere to recombinant human ZG16. Strain MB2 and MB20-S-proteins improved the barrier function of HT29 epithelial monolayer, as evidenced by increased ZG16 expression. Analysed L. brevis strains and S-proteins differentially affected the protein expression of IL-1β, IL-6 and IL-8, and IL-10 cytokines. The most prominent effect was observed by S-proteins of MB20, since IL-1β production was decreased while IL-10 production was significantly increased.

Isolation and characterization of probiotics from traditional fermented food have contributed many beneficial strains to the field of health and nutritional sciences. Handia, a traditional fermented alcoholic beverage popular in different parts of Odisha, was our source of isolation. This study characterizes one such potential bacteria, Levilactobacillus brevis ILSH3 (H3) isolated from Handia. The investigation for the probiotic attributes as per ICMR-DBT guidelines qualified the checkpoint assays like acid and bile tolerance, bile salt hydrolase activity, antimicrobial properties, and pathogen exclusion ability. The whole genome sequence of H3 (2,460,966 bp in size with GC content of 45.62%) was subjected to comparative genome analysis for its taxonomic identification and validation of probiotic potential. Various genes pertaining to its probiotic potential were identified in the genome and it showed zero matches against any pathogenic families. Metabolite profiling of cell-free supernatant using liquid chromatography-mass spectrometry revealed the presence of essential amino acids, short-chain fatty acids, antimicrobial molecules, immunomodulatory molecules, and flavor/aroma-enhancing compounds. Immunomodulatory property investigation using Bioplex and qRT-PCR showed a reduction in the levels of pro-inflammatory cytokines in L. brevis ILSH3-treated Caco-2 cells. Collectively, the results demonstrate that this Handia-origin bacteria Levilactobacillus brevis ILSH3 possesses desirable attributes of a probiotic, which is now open for nutritional and health biologists to explore. This new probiotic strain may show promising results when utilized in healthcare or functional foods.

Plant cell wall polysaccharides (PCWPs) serve as an abundant but recalcitrant carbon source for many microbes living in the gut of humans and animals. An adhesion to PCWPs is common in gut bacteria and can even be observed in the lactobacilli, which are supposed to promote the growth competence of these non-PCWP degraders because of the facilitated acquisition of newly released oligosaccharides. Nevertheless, the binding of molecules of lactobacilli to PCWPs and the underlying mechanisms remain largely unknown. By analyzing the transcriptome of grown in xylan supplemented with a xylanase, a gene was identified to encode a putative S-layer PCWP-binding protein (Lb1145). Lb1145 was predicted to have four domains, among which domains 1 and 2 were responsible for binding PCWPs. The binding was nonspecific, since structurally distinct PCWPs, e.g., cellulose, xylan, mannan, and chitin, and even lignin, were all bound by Lb1145. Both of the two N-terminal domains have a high pI, and we demonstrated that a non-enzymatic glycosylation-like process plays an important role in binding. Compared with another surface protein, i.e., the WxL protein Lb630, Lb1145 displayed a binding preference for the phloem sieve tube in the wheat stem section. Moreover, Lb1145 could bind ten strains within the , , , and genera among the seventeen selected gut bacterial species. An analysis of the reported S-layer proteins from the Gram-positive bacteria (lactobacilli and bifidobacteria) and outer membrane proteins from the Gram-negative ( and ) indicated that bacterial cell surface proteins with high pI values are not rare. The high pI-based and non-enzymatic glycosylation-like process-mediated binding represents a new paradigm and may be popular in gut bacterial surface proteins binding to PCWPs, with important physiological implications in growth competition in the gut microbiota.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid playing a significant role in the central nervous system and the gut–brain axis. This study investigated the potential to produce GABA by lactic acid bacteria (LAB) isolated from different varieties of organic tomatoes. The isolated LAB were taxonomically identified by 16S rRNA gene sequencing, the presence of the gadB gene (glutamate decarboxylase) was detected, and GABA production was quantified using HPLC. Levilactobacillus brevis CRAI showed the highest GABA production under optimised fermentation conditions with 4% monosodium glutamate (MSG). The genome sequencing of L. brevis CRAI revealed the presence of gadA and gadB isoforms and assessed the strain’s safety profile. The gene expression analysis revealed that the gadA and gadB genes were upregulated in the presence of 4% MSG. The probiotic potential of L. brevis CRAI was also assessed by functional assays. The strain showed strong antimicrobial activity against representative enteropathogens, i.e., Escherichia coli ETEC, Salmonella choleraesuis, and Yersinia enterocolitica, and anti-inflammatory effect, reducing nitric oxide production in LPS-stimulated RAW264.7 macrophages. In addition, its ability to adhere to intestinal epithelial Caco-2 cells was demonstrated. These results highlight L. brevis CRAI as a promising candidate for the development of GABA-enriched functional foods or probiotic supplements with the perspective to modulate the gut-brain axis.

This study evaluated the effects of Lactic acid bacteria and cellulase, individually and in combination, on fermentation quality and microbial community dynamics of alfalfa silage. Six treatments were tested, including control, cellulase alone and two lactic acid bacteria species ( Pediococcus pentosaceus , Levilactobacillus brevis ) applied individually or in combination with cellulase. The results showed that Levilactobacillus brevis in combination with cellulase, producing higher lactic acid concentrations, lower pH (< 4.2) to the other treatments. The microbiome analysis revealed that Lactiplantibacillus was dominant, while undesirable bacterium Achromobacter was suppressed. Functional prediction of microbial communities analysis indicated a higher predicted abundance of sequences associated with pyruvate metabolism, glycolysis/gluconeogenesis and starch and sucrose metabolism pathways. These findings provide insights into optimizing alfalfa silage quality through synergistic use of cellulase and lactic acid bacteria silage inoculants with high metabolic stability. Graphical Abstract

γ-Aminobutyric acid (GABA) is a bioactive metabolite valued in functional foods, but its microbial production is strongly influenced by nutrient availability. Levilactobacillus brevis CRL 2013 is an efficient GABA producer; however, its biosynthesis depends on culture medium composition. In this study, integrated physiological, proteomic, and transcriptional analyses were applied to assess the influence of nitrogen source composition and concentration on GABA production. No extracellular GABA was detected in a chemically defined medium containing all amino acids and glutamate (CDMg), whereas supplementation with yeast extract or Casitone restored high-level production. The highest GABA accumulation (~250 mM) was obtained in CDMg supplemented with 1% yeast extract or 2% Casitone, and a clear peptide dose-dependent effect was observed. In contrast, other protein hydrolysates or free amino acids alone did not stimulate GABA synthesis. Proteomic analysis revealed overexpression of the key enzyme GadB and changes in nucleotide and fatty acid pathways. Transcriptional analysis confirmed that peptide supplementation was accompanied by increased transcription of the gadRCB–gltX operon, in agreement with GABA accumulation. Overall, these results demonstrated that peptide composition and availability are critical determinants of GABA biosynthesis in Lv. brevis CRL 2013and provide a basis for optimizing peptide-based media to enhance GABA formation in food fermentations.

Background A growing number of in vitro and in vivo studies suggest the application of probiotics as a natural approach to maintaining oral health. This double-blind, randomized controlled trial aimed to evaluate the efficacy of Levilactobacillus brevis CD2 (CNCM I-5566), a multifunctional probiotic frequently used in oral medicine, in preserving or improving several recognized oral health indicators. Methods Thirty consenting healthy adults were randomized to receive four lozenges per day of L. brevis CD2 probiotic ( n  = 15) or placebo ( n  = 15) over four weeks. Clinical parameters (full-mouth bleeding on probing (BoP) and plaque index (PI) scores) were recorded. Unstimulated saliva was collected to measure salivation rate, pH, and buffer capacity. Salivary biomarkers were analyzed, including glucose, D-lactate, and secretory immunoglobulins A (sIgA). Clinical and salivary parameters were assessed at baseline, after four weeks of intervention, and two weeks post-intervention. Wilcoxon rank-sum test and robust regression analysis were used for statistical comparisons. The possible mediating effect of PI on BoP changes was assessed. Results After four weeks, the probiotic group showed significant improvements in BoP and PI compared to baseline and placebo. The probiotic group had a higher salivation rate than baseline and placebo after four weeks of treatment and washout. While changes in salivary pH were not significant, buffering capacity increased in the probiotic group after four weeks of treatment and washout. Salivary glucose and D-lactate levels were lower in the probiotic group post-treatment and after washout. sIgA values increased and remained stable after washout in the probiotic group. No adverse effects were reported. Conclusions The treatment with L. brevis CD2 significantly improved clinical and salivary parameters, supporting its efficacy as a probiotic for oral health. Trial registration ClinicalTrials.gov , NCT06457724; Registered 7 June 2024 - Retrospectively registered; https://clinicaltrials.gov/study/NCT06457724?viewType=Table&page=452&rank=4512#study-overview .