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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.

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.

The cellular NADH/NAD + ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis  ( Lb )NOX 1 , a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD + ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD + ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance 2 , insulin resistance 3 and mitochondrial disease 4 , and are associated with a common genetic variant in GCKR 5 , which has previously been associated with many seemingly disparate metabolic traits. Using Lb NOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD + ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as Lb NOX to empower studies of ‘causal metabolism’. The authors identify an increased hepatic NADH/NAD + ratio as an underlying metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases.

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.

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.

The probiotic fermentation of the bioactive substance gamma-aminobutyric acid (GABA) is an attractive research topic. There is still room for further improvement in reported GABA fermentation methods based on a single substrate (l-glutamic acid or l-monosodium glutamate). Here, we devised a pH auto-buffering strategy to facilitate the fermentation of GABA by Levilactobacillus brevis CD0817. This strategy features a mixture of neutral monosodium l-glutamate plus acidic l-glutamic acid as the substrate. This mixture provides a mild initial pH; moreover, the newly dissolved l-glutamic acid automatically offsets the pH increase caused by substrate decarboxylation, maintaining the acidity essential for GABA fermentation. In this study, a flask trial was first performed to optimize the GABA fermentation parameters of Levilactobacillus brevis CD0817. The optimized parameters were further validated in a 10 L fermenter. The flask trial results revealed that the appropriate fermentation medium was composed of powdery l-glutamic acid (750 g/L), monosodium l-glutamate (34 g/L [0.2 mol/L]), glucose (5 g/L), yeast extract (35 g/L), MnSO4·H2O (50 mg/L [0.3 mmol/L]), and Tween 80 (1.0 g/L). The appropriate fermentation temperature was 30 °C. The fermenter trial results revealed that GABA was slowly synthesized from 0–4 h, rapidly synthesized until 32 h, and finally reached 353.1 ± 8.3 g/L at 48 h, with the pH increasing from the initial value of 4.56 to the ultimate value of 6.10. The proposed pH auto-buffering strategy may be popular for other GABA 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 .

Bedsores impose an important challenge to the healthcare system. Se-baring probiotics are considered effective agents in wound healing and inflammation reduction via several pathways. The present study focused on the administration of a Se-enriched probiotic, originally obtained from a traditional dairy product for bedsore healing. Daily doses of the probiotic were administered to 20 ICU patients for 14 days and the wound healing criteria were compared with those of the same group of ICU patients as control, both groups suffering from stages I and II bedsore (a randomized, double-blind, controlled clinical trial). The administered Se-enriched probiotic decreased the bedsore healing period significantly (on average by 2.4 days, P -value: 0.039), as well as bedsore size (on average by 7 mm 2 , nonsignificant) and bedsore grade (10%, nonsignificant) in the treatment group more efficiently than the control group. Some key laboratory parameters associated with inflammation were also improved in patients receiving the Se-supplemented probiotic. The limitations of this study include the low number of patients meeting inclusion criteria within the timeframe of the study, and the impossibility of following up patients after discharge from the ICU. In summary, this study revealed the effectiveness of the Se-enriched probiotic in bedsore improvement, suggesting consideration of the enriched probiotic as an auxiliary agent in bedsore management.

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.

Excessive cholesterol levels can lead to hypercholesterolemia, which is related to cardiovascular diseases (CVDs), and CVDs are a serious threat to human health. Therefore, lowering cholesterol levels is necessary, and diet intervention is safer than drugs are. The cholesterol-lowering effect of Levilactobacillus brevis M-10 isolated from spontaneously fermented millet sour porridge was investigated in fifty C57BL/6N male mice. After a 4-week intervention, the food intake, weight gains and organ indices were calculated; the lipid contents in the serum, liver, and feces were determined; the histopathology of the liver tissues was observed; the expression of metabolism-related genes was determined; and short-chain fatty acid (SCFA) levels in the droppings were monitored. The results showed that administration of a high dose of L. brevis M-10 (1 × 10  CFU/mL) significantly reduced food intake, suppressed weight gain; prevented excessive liver growth; and reduced the total serum cholesterol, triglycerides, low-density lipoproteins; and total hepatic cholesterol and triglyceride contents (P < 0.05) in high-cholesterol mice. Moreover, a high dose of L. brevis M-10 significantly promoted the fecal excretion of cholesterol and triglycerides (P < 0.05) and alleviated liver damage induced by a high-cholesterol diet. Furthermore, a high dose of L. brevis M-10 significantly downregulated the cholesterol metabolism-related gene expression of NPC1L1, ACAT2, HMG-CoA, and SREBP2 but upregulated the gene expression of ABCG5, CYP7A1, and LXR-α (P < 0.05). Additionally, a high dose of L. brevis M-10 significantly increased SCFA contents, including those of acetic acid, propionic acid and n-butyric acid (P < 0.05). These findings could provide support for the use of L. brevis M-10 in the application of functional foods to alleviate hypercholesterolemia.