Lactic acid bacterial strain

Abstract

The present invention refers to a lactic acid bacterium capable of producing gamma-aminobutyric acid (GABA), and more in particular the strain Levilactobacillus brevis CRAI with enhanced GABA production and probiotic properties. Furthermore, the invention relates to the medical uses of the bacterium and a dietary supplement comprising the bacterium.

Description

[0001] DESCRIPTION

[0002] Lactic acid bacterial strain

[0003] Technical field

[0004] The present invention relates to a lactic acid bacterium capable of producing gamma-aminobutyric acid (GABA), and more in particular a strain of Levilactobacillus brevis with enhanced GABA production and probiotic properties.

[0005] Prior art

[0006] Gamma-aminobutyric acid (GABA) is a non-protein amino acid widely distributed in nature among microorganisms, plants and animals. GABA is one of the main inhibitory neurotransmitters in the central nervous system of mammals. It exhibits numerous physiological and pharmacological effects on various processes, including brain development, stress management, control of blood pressure and diabetes. GABA also plays a role in gut-brain signaling through various pathways involving enteric neurons, enteroendocrine cells and immune cells.

[0007] Lactic acid bacteria (LAB) are an important group of Gram-positive bacteria with physiological activities that have been widely used in the food industry. Many LAB strains also demonstrate properties that are beneficial for health, making them potential probiotics. Among LAB, the majority of the GABA-producing strains belong to Lactobacillus and related genera. The biosynthesis of GABA in these bacteria takes place through the decarboxylation of L-glutamate, catalyzed by the enzyme glutamate decarboxylase (GAD).

[0008] Although GABA-producing LAB strains have shown interesting potentialities, commercial and therapeutic applications still represent a challenge, given that the efficiency of GABA production may vary significantly among strains. Furthermore, maintaining the viability and metabolic activity of probiotic strains during processing and storage posesdifficulties. There are also limitations in our understanding of the precise mechanisms whereby probiotic GABA producers may provide health benefits.

[0009] In fact, the safety and effectiveness of probiotic GABA-producing strains must be thoroughly evaluated before they can be used in foods or as therapeutics. Potential problems such as antibiotic resistance and unintentional physiological effects require careful consideration and adequate scientific documentation. Finally, there are numerous technological obstacles to optimizing fermentation conditions and increasing GABA production to industrial levels.

[0010] Thus, there is a greatly felt need to select, by exploiting natural biodiversity, a bacterium that may overcome one or more of these problems.

[0011] Summary of the invention

[0012] In a first aspect, the invention relates to a bacterium capable of producing GABA. Preferably, the bacterium is a lactic acid bacterium comprising a DNA sequence coding for 16S rRNA having at least 80% homology with SEQ ID NO. 1.

[0013] This bacterium provides a novel and efficient source for GABA production, which has important applications in the food, pharmaceutical and nutraceutical industries thanks to the various physiological functions and health benefits of GABA.

[0014] Preferably, the bacterium is the strain Levilactobacillus brevis CRAI deposited with the International Depositary Authority: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on February 6, 2025, under accession number DSM 35345.

[0015] In a second aspect, the invention concerns a composition comprising the above-described bacterium.Said composition enables convenient storage, handling and application of the GABA-producing bacterium in various contexts, including food production, probiotic formulations or research settings.

[0016] In a third aspect, the invention relates to the above-described bacterium or composition for use as a medicament.

[0017] A fourth aspect relates to the bacterium or composition for use in the treatment or prevention of an infection and / or disease caused by a pathogenic bacterium. Preferably, the infection or pathology is of a gastrointestinal nature.

[0018] A fifth aspect of the present invention relates to the bacterium or composition for use in the treatment or prevention of a neurogastrointestinal pathology.

[0019] A sixth aspect of the present invention relates to the bacterium or composition for use in restoring and / or maintaining the balance of the gut microbiota in an individual.

[0020] The bacterium or composition may be administered in association or combination with at least one compound or molecule useful for the treatment of a gastrointestinal disease.

[0021] In a seventh aspect, the invention relates to a non-medical use of the above-described bacterium or composition as a dietary supplement.

[0022] Finally, in an eighth aspect, the invention refers to a process for obtaining GABA, wherein said process comprises at least one step of using the above-described bacterium.

[0023] Brief description of the drawings

[0024] Figure 1 shows the gene organization of the gad operon in Levilactobacillus brevis CRAI; gadR, transcriptional regulator; gadC, glutamate / gamma-aminobutyric acid antiporter gene; gadA, glutamate decarboxylase A gene; gltX, glutamate-tRNA ligase gene; gadB, glutamate decarboxylase B gene. The arrows indicate the gene orientation.Figure 2 shows the anti-inflammatory activity of the filtered culture supernatant (cell-free supernatant, CFS) and of the viable cells of L. brevis ORAL The RAW264.7 cells stimulated with LPS were treated with CFS or cells of L. brevis CRAI in a 1:100 ratio (100 bacteria per RAW264.7 cell). The NO levels are expressed as a percentage of inflammation compared to the control (100%). ****p<0.0001.

[0025] Detailed description of preferred embodiments of the invention

[0026] A first aspect of the present invention relates to a bacterium capable of producing gamma-aminobutyric acid (GABA).

[0027] In one embodiment, the bacterium belongs to a genus selected from: Levilactobacillus, Lactiplantibacillus and Lactococcus.

[0028] In one embodiment, the bacterium was isolated / selected from at least one fruit and / or at least one vegetable. Preferably, the bacterium is isolated from at least one vegetable, more preferably selected from: cucumber, eggplant, bell pepper, tomato, zucchini, beet, carrot, turnip, radish, celeriac, asparagus, fennel, celery, potato, sunchoke, onion, leek and shallot.

[0029] In a preferred embodiment, the bacterium was isolated from tomato.

[0030] Preferably, the bacterium belongs to the genus Levilactobacillus.

[0031] In one embodiment, the bacterium belonging to the genus Levilactobacillus belongs to the species L. brevis.

[0032] In a preferred embodiment, the bacterium comprises a DNA sequence coding for 16S rRNA with at least 70% identity, preferably at least 80%, even more preferably 90% identity with SEQ ID NO. 1.

[0033] Preferably, the bacterium comprises a DNA sequence coding for 16S rRNA having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO.1.

[0034] 16S ribosomal RNA sequences have been widely used in the classification and identification of bacteria and archaea. The comparison of 16S rRNAgene sequences has been widely used to establish taxonomic relationships between strains of prokaryotes, with a similarity of 98.65% currently recognized as a cut-off for delimiting species. Comparing the 16S rRNA genetic sequence of an isolated strain with sequences of standard strains of all prokaryotic species provides a precise and convenient way to classify and identify prokaryotes. Bacterial 16S ribosomal RNA (rRNA) genes contain nine “hypervariable regions” (V1-V9) which demonstrate a considerable sequence diversity among different bacteria.

[0035] In accordance with a preferred embodiment of the invention, the genome of the bacterium is characterized by a DNA sequence comprising SEQ ID NO: 2.

[0036] Preferably, the genome of the bacterium is characterized by a DNA sequence having at least 60%, preferably 70%, more preferably 80%, even more preferably 90% identity with SEQ ID NO. 2.

[0037] Preferably, the genome of the bacterium is characterized by a DNA sequence having 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with SEQ ID NO. 2.

[0038] Preferably, the bacterium is characterized by a DNA sequence coding for the gadA gene and / or a sequence coding for the gadB gene.

[0039] In a preferred embodiment, the bacterium is a strain of Levilactobacillus brevis; preferably, the bacterium is the strain Levilactobacillus brevis CRAI deposited with the International Depositary Authority: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on February 6, 2025, under accession number DSM 35345.

[0040] The bacterium of the present invention comprises both the gadA gene and the gadB gene, which are involved in GABA production. These genes code for glutamate decarboxylase enzymes which catalyze the conversion of glutamate into GABA.

[0041] The Applicant has surprisingly found that, in addition to GABA production, the bacterium shows other beneficial characteristics. In fact, the bacterium demonstrates antimicrobial activity against pathogenic bacteria such as,for example, Escherichia coli ETEC, Salmonella choleraesuis and Yersinia enterocolitica. This antimicrobial activity may contribute to the potential probiotic properties of the bacterium.

[0042] The bacterium also shows anti-inflammatory activity. As shown in the example, in an in vitro model using RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS), the bacterium reduces nitric oxide production, indicating a potential ability to modulate inflammatory responses.

[0043] The GABA-producing bacterium described herein can be used in various applications, including the production of functional foods, dietary supplements and pharmaceutical formulations. The bacterium can also have potential probiotic applications combining GABA production with other beneficial effects on human health.

[0044] A genomic analysis of the bacterium reveals the presence of genes coding for bile salt hydrolases and enzymes involved in riboflavin synthesis. These genetic characteristics may contribute to the ability of the bacterium to survive and function in the gastrointestinal environment.

[0045] The claimed bacterium, in particular the strain L. brevis CRAI, is capable of producing 180 mM of GABA in 48 hours, thus showing a good conversion capacity (75.7%). Compared to other strains of L. brevis described in the literature, such as L. brevis HYE1 (18.8 mM of GABA), the claimed bacterium, in particular the CRAI strain, produces larger amounts of GABA under optimal conditions. Other strains, such as L. brevis K203 (430 mM), L. brevis RK03 (606 mM) and L. brevis NCL912 (1005.81 mM), reach higher production levels, but with longer fermentation times and larger amounts of glutamate. From an industrial viewpoint, though the use of high concentrations of glutamate and extended fermentation times may lead to a larger GABA production, these factors could entail limitations in terms of costs and availability of raw materials.A further aspect of the invention refers to mutants and / or modified strains derived / obtainable from the bacterium, preferably from the strain Levilactobacillus brevis CRAI of the present invention.

[0046] As used here, “mutant” means any microorganisms obtained / obtainable by direct mutation, selection or genetic recombination of Levilactobacillus brevis CRAI. Mutant strains can be obtained using any method known in the art for this purpose, such as mutant selection, chemical mutagenesis, physical mutagenesis (radiation), genetic manipulation and biological mutagenesis.

[0047] “Modified” preferably means gene-edited strains in which at least one gene of interest is edited / modified using the common biological tools useful for that purpose, in particular biological tools based on the use of nuclease, such as “zinc finger” nucleases (ZFNs), “transcription activator-like effector” nucleases (TALEN) and “clustered regularly-interspaced short palindromic repeats (CRISPR)ZCRISPR-associated (Cas)” nucleases. A second aspect of the present invention relates to a composition comprising the bacterium described above in detail and at least one excipient and / or at least one pharmaceutically acceptable and / or food grade substance.

[0048] Preferably, the composition is formulated for oral administration, more preferably in the form of pills, capsules, tablets, granular powder, hard-shelled capsules, orodispersible granules, sachets, lozenges or drinkable vials or lyophilized powder.

[0049] Alternatively, the composition of the invention is formulated as a liquid, for example as a syrup or as a beverage, or else it is added to a food, for example to a yogurt, a cheese, or a fruit juice. Alternatively, the composition of the invention is formulated in a form capable of exerting an action topically, for example as an enema.

[0050] For the lyophilized powders, cryoprotectants such as trehalose or sucrose may be added to improve stability during the lyophilization process. The liquid suspensions can include appropriate buffers and stabilizers.Capsules and pills can incorporate binders, disintegrating agents and other common pharmaceutical excipients.

[0051] In one embodiment, the composition further comprises at least one additional probiotic strain. The additional probiotic strain can be selected from genera such as Lactobacillus, Bifidobacterium, Streptococcus or other beneficial bacteria. The inclusion of several probiotic strains may provide complementary, synergistic health benefits.

[0052] The composition can also include a prebiotic substance. Prebiotics are non-digestible food ingredients that selectively stimulate the growth and / or activity of beneficial microorganisms in the intestine. Examples of prebiotic substances that can be included in the composition comprise inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS) and resistant starch.

[0053] A third aspect of the present invention relates to the above-described bacterium or the composition comprising it for use as a medicament.

[0054] A fourth aspect of the present invention refers to the above-described bacterium or the composition comprising it for use in the treatment or prevention of an infection and / or pathology caused by a pathogen.

[0055] In one embodiment, the infection or pathology is of a gastrointestinal nature.

[0056] Preferably, the bacterium or composition is administered to treat or prevent gastrointestinal infections caused by pathogenic bacteria such as Escherichia coli, Salmonella or Yersinia. The bacterium may help to restore the balance of the gut microbiome and inhibit the growth of harmful bacteria.

[0057] According to one embodiment, the bacterium or composition is administered in association or combination with at least one compound or additional molecule useful for the treatment of gastrointestinal diseases. The additional compound may include, for example, antibiotics, antiinflammatory agents or other probiotic strains.The ability of the bacterium to produce gamma-aminobutyric acid (GABA) can contribute to its therapeutic effects. GABA has been associated with various physiological benefits, including modulation of the immune response and the regulation of gastrointestinal function.

[0058] In one embodiment, the bacterium or composition is used prophylactically to help prevent gastrointestinal infections in individuals at risk. Preferably, the individuals at risk are travelers, individuals undergoing antibiotic therapy or those with a compromised immune system.

[0059] The therapeutic applications of the bacterium are not limited to gastrointestinal conditions. In accordance with one embodiment, the bacterium or composition is administered to treat or prevent infections in other areas of the body where the probiotic bacteria can exert beneficial effects.

[0060] According to a further embodiment, the bacterium or composition is used to prevent or reduce the signs and / or symptoms of inflammation in an individual.

[0061] A fifth aspect of the present invention relates to the bacterium or composition for use in the treatment or prevention of a neurogastrointestinal pathology.

[0062] In one embodiment, the neurogastrointestinal pathology is selected from: esophageal disorders, functional disorders of the stomach and duodenum, intestinal disorders, disorders mediated by gastrointestinal pain, anorectal disorders, and functional disorders in children and adolescents.

[0063] A sixth aspect of the present invention relates to the bacterium or composition for use in restoring and / or maintaining the balance of the microbiota in an individual. In other words, the above-described bacterium is used as a probiotic for a correct balance of the microbiota or to treat a dysbiosis in an individual, preferably an intestinal dysbiosis.

[0064] A seventh aspect of the present invention relates to a use of the bacterium or composition described above in detail as a dietary supplement. In fact, the bacterium or composition can be used tosupplement an individual’s diet, thereby helping to maintain the balance of the gut microbiota and preventing symptoms or pathologies correlated with an imbalance of the microbiota.

[0065] An eighth aspect of the present invention relates to a process for obtaining GABA, wherein said process comprises at least one step of using the bacterium described above in detail. Preferably, said process comprises a fermentation step and at least one step of purifying the GABA from the bacterial culture medium.

[0066] In a preferred embodiment, the bacterium is cultured in the presence of at least one salt, preferably the sodium salt of glutamic acid (MSG).

[0067] Preferably, the bacterium is cultured with an MSG concentration comprised between 150 and 300 mM, more preferably between 180 and 270 mM.

[0068] EXAMPLE

[0069] Isolation of lactic acid bacteria from a tomato source

[0070] Lactic acid bacteria (LAB) were isolated from different varieties of organic tomatoes collected in the hills of Bologna. The tomato pulp was mixed in a De Man, Rogosa and Sharp (MRS) broth (Difco, Detroit, Ml, USA), supplemented with 0.05% L-cysteine (Merck, Milan, Italy). Different concentrations of the mixture were cultured on MRS plates and incubated under anaerobiosis for 72 hours at 37°C, in jars rendered anaerobic through the insertion of GasPak™ sachets (GasPak™ EZ Anaerobe Container System, Sparks, MD, USA). Various colonies which, based on observation under a microscope, were formed by cells with a rod-like morphology, were selected for the preparation of glycerol stock. The isolates were cultured in MRS medium supplemented with 0.05% L-cysteine, at 37°C, under anaerobiosis.

[0071] Taxonomic identification of isolates by means of 16S rRNA gene sequence

[0072] The isolates were cultured overnight; then 2 mL were centrifuged (10 mina 10,000 x g) and the genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the “Pretreatment for Gram-positive bacteria” protocol

[0026] . The complete 16S ribosomal RNA (rRNA) gene was amplified with the universal 27F and 1492R primers

[0027] and sequenced. The sequences obtained were compared with the ones available in BLASTn (https: / / blast.ncbi.nlm.nih.gov) to achieve a taxonomic identification of the isolates.

[0073] Molecular detection of gad B gene

[0074] The genomic DNA samples obtained from LAB isolated from tomatoes were used to verify the presence of the gadB gene, which codes for glutamate decarboxylase. The PCR primers, designed on highly conserved regions of the gadB sequence, were the following: CoreF 5'-CCTCGAGAAGCCGATCGTTAGTTCG-3' (SEQ ID NO. 3), CoreR 5'-TCATATTGACCGGTATAAGTGATGCCC-(SEQ ID NO. 4) for the strains of L. brevis and L. plantarum 3'

[0028] , Forward 5'-CAACATGATCGCTGACCTTTGG (SEQ ID NO. 5) - 3' Reverse 5' -GCCATTCCACCAAGCATACAAG-3' (SEQ ID NO. 6) for the strains of L. lactis

[0029] .

[0075] Quantitative analysis of GABA production by high-performance liquid chromatography (HPLC)

[0076] The LAB strains positive for the gadB gene were cultured under optimal fermentation conditions to obtain the maximum yield of GABA, following parameters reported in the literature [18,22,30-32]. In particular, the strains were cultured in MRS broth supplemented with 0.5% L-cysteine at 32°C for 48h under anaerobic conditions; the cultures had different concentrations of L-glutamic acid monosodium salt (MSG, ThermoFisher, Kandel, Germany) added to them in the range of 0 mM (0%) - 236.53 mM (4%), the initial pH was adjusted to 5 and the initial inoculum was 107CFU / mL. After 48 hours of incubation, the culture supernatants werefiltered with 0.22 m membrane filters and then used to quantify the GABA. The GABA was derivatized by combining 1.75 mL of borate buffer 1M (pH 9), 750 pL of methanol, 1 mL of sample and 20 pL of diethyl ethoxymethylenemalonate (DEEMM). The mixture thus obtained was subjected to an ultrasound treatment for a duration of 30 minutes

[0033] . The GABA derivative was analyzed and quantified by HPLC-DAD using the binary gradient described by Gomez-Alonso et al., 2007 (phase A, acetate buffer 25mM pH 5.8 with 0.02% sodium azide; phase B, 80:20 mixture of acetonitrile and methanol; flow rate of 0.9 mL / min)

[0034] . As a minor modification, the injection volume was reduced to 1 pL and the chromatography run was shortened to 40 minutes versus the original 80 minutes using an isocratic eluent A (5 minutes) to re-equilibrate the column between samples. The standard concentration of GABA was subjected to derivatization in order to construct the calibration curve (from 0.96 to 29.09 mM, R2=0.999).

[0077] The strain L. brevis CRAI, cultured in MRS with 236 mM of glutamate, produces 180 mM of GABA in 48 hours, thus showing a good conversion capacity (75.7%). Compared to other strains of L. brevis described in the literature, such as L. brevis HYE1 (18.8 mM of GABA) (19), the strain L. brevis CRAI produces larger amounts of GABA under optimal conditions. Other strains, such as L. brevis K203 (430 mM), L. brevis RK03 (606 mM) and L. brevis NCL912 (1005.81 mM) (50), reach higher production levels, but with longer fermentation times and larger amounts of glutamate. From an industrial viewpoint, though the use of high concentrations of glutamate and extended fermentation times may lead to a larger GABA production, these factors could entail limitations in terms of costs and availability of raw materials.

[0078] Sequencing of Levilactobacillus brevis CRAI genome

[0079] The DNA of L. brevis CRAI extracted as described above was quantified with the Qubit dsDNA BR assay kit using a Qubit 2.0 fluorimeter (LifeTechnologies, Monza, Italy) and then sequenced with Oxford Nanopore Whole Genome Sequencing technology. Briefly, the genomic library was prepared using the SQK-LSK109 kit with EXP-NBD196 (Oxford Nanopore Technologies, Oxford, United Kingdom) and sequenced in an R9.4.1 flow cell (MIN106D, Oxford Nanopore Technologies, Oxford, United Kingdom) with the Grid ION X5 Nanopore device (Oxford Nanopore Technology, Oxford, United Kingdom). The genome was de novo-assembled using a Canu assembler v2.2

[0035] and annotated with Bakta v1.4.1

[0036] , with a genomic coverage of 39.47. The L. brevis CRAI genome was analyzed for the presence of antibiotic resistance genes and virulence genes using three different tools, namely AMR Finder Plus software (https: / / www.ncbi.nlm.nih.gov / pathogens / antimicrobial-resistance / AMRFinder / , updated to September 15, 2024), the ResFinder database 4.6.0 (https: / / cge.food.dtu.dk / services / ResFinder / , identity threshold: 90.0%, minimum length: 60.0%) and the Resistance Gene Identifier (RGI 6.0.3) using the Comprehensive Antibiotic Resistance Database (CARD, version 3.3.0; https: / / card.mcmaster.ca / ).

[0080] Analysis of gadA and gadB gene expression by RT PCR

[0081] L. brevis CRAI was cultured under optimized conditions as described above with the addition of 0% and 4% MSG to the medium. After 48 hours of culture, the total RNA was extracted with the RNeasy kit following the “Enzymatic Lysis, Proteinase K Digestion and Mechanical Disruption of Bacteria” protocol (Qiagen, Hilden, Germany)

[0037] . 1 g of extracted RNA was subjected to reverse transcription using the QuantiTect® Reverse Transcription kit (Qiagen, Hilden, Germany); the cDNA was used in realtime quantitative PCR (RT PCR) with a LightCycler 2.0 instrument (Roche, Mannheim, Germany) and LightCycler® FastStart DNA Master SYBR Green I (Roche, Mannheim, Germany). At the end of every reaction, the specificity of the amplicon was verified by analyzing the melting curve and the integrity of the PCR product was evaluated by electrophoresis onagarose gel. The relative expression of the gadA gene (primer 5'-CAGGTTACAAGACGATCATGC-3'(SEQ ID NO. 7), 5'- ATACTTAGCCAGCTCGGACTC-3'

[0038] (SEQ ID NO. 8)) and of the gadB gene (Core F and Core R primers) was normalized with respect to the 16S rRNA gene (HDA1 5'-ACTCCTACGGGAGGAGCAGT-3' (SEQ ID NO. 9), HAD2 5'- GTATTACCGCGGCTGCTGGCAC-3'

[0039] (SEQ ID NO. 10)) considering the cycle threshold (Ct) values, and the expression level of the gadB gene was calculated according to the 2-AACTmethod

[0040] .

[0082] Evaluation of in vitro antimicrobial activity of cell culture supernatants of L. brevis CRAI

[0083] The cell-free supernatant (CFS) of L. brevis CRAI was collected by centrifugation of an overnight culture at 5,000 x g for 10 minutes at 4°C and filtered through a 0.22 pm membrane; the pH value was measured with a pH meter. The antimicrobial activity of the CFS was assayed against the following pathogens: Escherichia coli ETEC H10407 and Salmonella choleraesuis serovar typhimurium, cultured twice in Nutrient Broth (NB) (Difco, Detroit, Ml), and Yersinia enterocolitica cultured twice in Tryptic Soy Broth (TSB) (Difco, Detroit, Ml), before every experiment. The experiment was carried out in flat-bottom 96-well plates filled with 0.1 mL of L. brevis CFS and subsequently inoculated with 0.1 mL of a suspension of pathogens (1x106CFU / mL). A growth control well contained 0.1 mL of sterile MRS medium and 0.1 mL of the same suspension of pathogens. The plates were incubated at 37°C for 24 hours. Pathogen growth was analyzed by reading absorbance at 600 nm (EnSpire Multimode Plate Reader, PerkinElmer Inc., Waltham, MA, USA). The residual growth in the presence of L. brevis CFS was calculated as a percentage compared to the absorbance of the corresponding control. Every experiment was repeated in at least six replicates, in two independent series.

[0084] Evaluation of antagonist activity of L. brevis CRAI cells againstgastrointestinal pathogens

[0085] In order to evaluate the ability of L. brevis CRAI cells to antagonize the growth of intestinal pathogens, the protocol described by Siroli et al. (2017)

[0041] was used. The pathogens utilized are the ones already mentioned (E. coli ETEC H10407, S. choleraesuis serovar typhimurium, Y. enterocolitica). Briefly, 5 pL of overnight cultures of L. brevis CRAI (about 5x108CFU / mL) were inoculated onto the surface of MRS plates, and the strain was allowed to grow under anaerobic conditions at 37°C for 24 hours. Then, 0.1 mL (corresponding to 108CFU / mL) of an overnight culture of pathogens were inoculated into 10 mL of NB (E. coli ETEC H 10407, S. choleraesuis serovar typhimurium) or TSB (Y. enterocolitica) soft agar (containing 0.7% agar) and poured onto the plates where L. brevis CRAI had grown. After 24 hours at 37°C, the plates were checked to evaluate the zones of growth inhibition. The inhibition haloes were measured from the outer perimeter of the spots in four directions, and the mean radius was considered. Based on the radius of inhibition, the antagonist activity shown by the strain was expressed as: +, radius between 14-18 mm; ++, radius > 18 mm.

[0086] Inhibition of NO production in LPS-stimulated RAW 264.7 cells

[0087] A murine macrophage cell line (RAW264.7) was cultured in Dulbecco’s minimum essential medium (DMEM) (EuroClone, Pero, Italy), supplemented with 10% fetal bovine serum (Gibco Life Technologies Corporation, Segrate, Ml, Italy) and 1% L-glutamine (Merck, Saint Louis, MO, USA) and incubated in a 5% CO2 atmosphere at 37°C. Nitrite production was measured in the cell culture medium using the Griess Reagent Nitrite Measurement Kit (Cell Signaling Technology, Inc., Trask Lane, Danvers, MA)

[0042] . The Griess assay offers an indirect method for detecting nitric oxide (NO) by quantifying nitrites, nitrates and nitrosating agents. The RAW264.7 cells were seeded in 24-well plates with a density of 1x105cells / well and cultured for 48 hours in complete medium. Theinflammatory process was induced by stimulating the cells with 1 pg / mL of bacterial LPS (LPS from E. coli, Enzo Life Sciences, serotype TLRGRADE) and the RAW264.7 cells were also treated with bacterial cells or L. brevis CRAI CFS for 24 hours. After the treatment period, 0.1 mL of cell culture medium were removed and added to 0.1 mL of Griess reagent in a 96-well plate; then a spectrophotometric measurement was made at 550 nm (EnSpire Multimode Plate Reader, PerkinElmer Inc., Waltham, MA, USA). The inflammation was calculated as a percentage compared to the absorbance of the control, in which the RAW 264.7 cells were stimulated with LPS alone (considered as 100% inflammation). Every experiment was repeated in at least three replications, in two independent series.

[0088] Adhesion of L. brevis CRAI to Caco-2 cells

[0089] In order to determine the adhesion capacity of the strain L. brevis CRAI, the human intestinal Caco-2 cell line was used, following the method previously described by

[0043] . The Caco-2 cells were cultured on cover glasses, seeded at a concentration of 5x104cells / mL in Dulbecco’s minimum essential medium (DMEM) (EuroClone, Pero, Italy), supplemented with 10% fetal bovine serum (Gibco Life Technologies Corporation, Segrate, Ml, Italy) and 1% L-glutamine and incubated in a 5% CO2 atmosphere at 37°C. After 48 hours (about 70% confluence), the Caco-2 cells were washed twice in PBS. An overnight culture of L. brevis CRAI was washed twice in PBS and resuspended in DMEM at a concentration of 108CFU / mL. The Caco-2 cells were incubated with the suspension of L. brevis CRAI in a 1:100 ratio (eucaryotic cell: bacteria) for 3 hours at 37°C with 5% CO2. Subsequently, the cell monolayers were washed several times with PBS, fixed with May-Grunwald and stained with Giemsa. The results were obtained by counting, using an optical microscope (1000x), the number of bacterial cells of L. brevis attached to 200 randomly selected Caco-2 cells.Data and statistical analysis

[0090] The data related to HPLC quantification are presented as the means of two independent experiments ± standard deviation (SD). The antimicrobial and anti-inflammatory activities were expressed as the mean ± SD of two independent experiments and one-way ANOVA, followed by the Bonferroni correction, was used for the multiple comparisons. The differences were deemed significant with p<0.05. All the statistical analyses were carried out using GraphPad Prism version 9.5.1 for Windows.

[0091] Taxonomic characterization of LAB isolated from tomatoes and gadB gene Thirteen LAB isolates were obtained from organic tomatoes of 6 different varieties. The LAB isolates were taxonomically identified at the species level by 16S rRNA gene sequencing and alignment of the sequence with the BLAST database; 5 strains of Levilactobacillus brevis (DRBA2, DVBA22, CRAI, CRAR), 3 strains of Lactiplantibacillus plantarum (DRBA1, DVBA1, PRA) and 5 strains of Lactococcus lactis (CRAC, IBA, IBB, PBMD, PBMC) were detected (Table 1). In order to identify which isolates were potential GABA producers, the LAB strains were subjected to screening for the presence of the gadB gene by PCR amplification. The gadB gene was detected in all thirteen LAB strains (Table 1).

[0092] Table 1. Taxonomic characterization of LAB isolates by 16S rRNA gene sequencing and detection of gadB gene

[0093]

[0094] HPLC quantification of GABA and selection of the strain L. brevis CRAI All the LAB strains with the gadB gene were tested for their ability to produce GABA. Based on the information reported in the literature

[0030] , the fermentations were set by adding 236.53 mM (4%) of MSG to the culture medium to maximize GABA production, which was then quantified by HPLC analysis. Among the strains of L. brevis, the largest amount of GABA was produced by CRAI (179.15 mM) and CRAR (165.26 mM)(Table 2). In contrast, a smaller amount of GABA was produced by strains belonging to the species L. plantarum and L. lactis. The CRAI and CRAR strains showed a conversion yield of 75.7% and 69.9%, respectively (Table 2). These data highlight that L. brevis CRAI is the best GABA producer, with the highest rate of conversion of MSG into GABA.

[0095] Table 2. GABA quantification by HPLC and conversion yield. Fermentation was conducted in the presence of 236.53 mM MSG (4%).

[0096]

[0097] GABA production when L. brevis CRAI was cultured with different concentrations of MSG

[0098] The selected strain, Levilactobacillus brevis CRAI, was cultured under optimized conditions

[0030] and different concentrations of MSG, comprised between 0 and 4% (0-236.5 mM), were added to the MRS medium. GABA production showed to be dependent on the concentration of MSG. L.brevis CRAI produced the highest amount of GABA (179.15 ± 15.14 mM) when the monosodium glutamate was 236.5 mM (4%) and produced the lowest amount of GABA (6.94±0.48 mM) when no monosodium glutamate was added to the MRS medium (0%) (Table 3).

[0099] Table 3. GABA production and conversion by L. brevis CRAI in the presence of different concentrations of MSG. The data are expressed as the mean of two independent experiments ±SD.

[0100]

[0101] Characteristics of the L. brevis CRAI genome

[0102] The genome sequence of the CRAI strain comprises a circular chromosome of 2 550 312 bp with a mean GC content of 45.9%. The annotation revealed the presence of two genes coding for glutamate decarboxylase, corresponding to two GAD isoforms known as gadA and gadB genes

[0038] . The two genes show a sequence identity of 64% and are located in two distinct loci of the chromosome. GadA is part of a gene cluster in which the transcriptional regulator gadR is positioned at the upstream end, followed by the glutamate / gamma-aminobutyric acid antiporter gene gadC, the glutamate decarboxylase gene gadA, and the gadX gene, which codes for glutamate-tRNA ligase (Figure 1). These results are consistent with those reported in the literature for other strains of L. brevis

[0044] . The gadB gene is not included in the gene cluster and is located at a distance of 1.7 Mbp from gadA. The genome annotation alsoshowed the presence of genes that may be correlated to beneficial properties: the bsh gene codes for cholylglycine hydrolase, a bile salt hydrolase (BSH) which catalyzes the hydrolysis of the amide bond and its activity contributes to reducing the cholesterol level

[0045] . Furthermore, the presence of important genes involved in riboflavin synthesis was detected: in particular, the rib operon, containing the riboflavin synthesis genes ribG, ribB, ribA, ribE, and ribH was identified, with a regulator element (RFN) at the end of the operon

[0046] . The safety profile of the strain was analyzed using various tools, including AMRFinderPlus, ResFinder and CARD. Whereas no genes associated with antibiotic resistance or virulence were identified with the first two tools, CARD detected the vanT and nimA genes. The vanT gene codes for an enzyme, alanine racemase, whilst nimA codes for a protein of the pyridoxamine 5'-phosphate oxidase superfamily involved in the metabolism of vitamin B6. However, these genes are not associated with antimicrobial resistance.

[0103] DNA sequencing of L. brevis CRAI further revealed the presence of two sequences attributable to natural plasmids. As revealed by RAST analysis, the plasmid carries genes involved in potassium homeostasis and bile hydrolysis, whereas genes linked to virulence factors or antibiotic resistance are absent.

[0104] Transcriptional analysis of gadB and gadA genes in L. brevis CRAI

[0105] In order to study the impact of adding MSG to the culture medium on the expression level of the gadA and gadB genes, RT-PCR experiments were conducted on mRNA extracted from L. brevis CRAI cells cultured under optimized conditions (inoculation of 107CFU / mL, pH 5, 32°C, 48h of incubation) in the presence of 236.53 mM (4%) of MSG. The expression of the gadA and gadB genes was compared with the baseline condition (MSG concentration: 0). The data indicate that supplementation of the MRS medium with 236.53 mM (4%) of MSG led to a 52.52-times increase in the mRNA of gadA and a 4.44-times increase in the mRNA of gadB.Antimicrobial activity of L. brevis

[0106] The selection of potential probiotic strains to be used as dietary supplements entails an evaluation of various activities beneficial for human health, including the ability to counter gastrointestinal infections

[0047] . Therefore, enterotoxigenic E. coli, S. choleraesuis and Y. enterocolitica were chosen as representative pathogenic strains responsible for intestinal infections. We tested the ability of the cell-free supernatant of L. brevis CRAI to inhibit the planktonic growth of these intestinal pathogens. The CRAI strain showed to be highly effective in significantly inhibiting the intestinal pathogens: the percentages of inhibition were 97.25±1.53% and 97.54±0.74% for E. coli ETEC and S. choleraesuis, and 99.13±0.67% for Y. enterocolitica (Table 4). The antagonist activity of viable cells of L. brevis CRAI was also evaluated by means of an overlay assay. We found that viable CRAI cells possess considerable inhibitory activity, with a zone of inhibition larger than 16 mm against E. coli ETEC and S. choleraesius and larger than 20 mm against Y. enterocolitica (Table 4).

[0107] Table 4. Inhibition of pathogens by CFS and viable cells of L. brevis CRAI. The enteropathogens tested were E. coli ETEC, S. choleraesuis serovar typhimurium, and Y. enterocolitica. The % of inhibition was evaluated by comparing the turbidity (OD600) of the sample treated with CFS with the untreated control (0%). The antagonist activity shown by the viable cells of the strain was expressed as: +, radius between 14-18 mm; ++, radius> 18 mm.

[0108]

[0109]

[0110] Anti-inflammatory activity of L. brevis CRAI

[0111] An unbalanced gut microbiota, or dysbiosis, is often associated with inflammation, which can be considered one of the consequences thereof. Since recent strategies aim to restore the intestinal balance using probiotics

[0048] , the anti-inflammatory activity of L. brevis CRAI was investigated. The RAW264.7 cell line is commonly used to study immune responses, in particular in relation to inflammation. It is well known that lipopolysaccharide (LPS) activates macrophages, triggering the release of various pro-inflammatory cytokines and inflammation mediators such as nitric oxide (NO). In order to evaluate the production of nitric oxide (NO), which is indicative of the inflammatory response in macrophages, we conducted a Griess assay, in which RAW 246.7 cells were stimulated with LPS, and the NO level was evaluated in the presence or absence of L. brevis CRAI. As illustrated in Figure 2, treatment of the LPS-induced macrophages with the cell-free supernatant of L. brevis CRAI or with the bacterial cells brought about a highly significant reduction in inflammation, 16.50 ± 1.24% and 32.33 ± 2.69%, respectively, compared to the control (100%) (Figure 2).

[0112] Capacity of L. brevis CRAI to adhere to the intestinal epithelium

[0113] The adhesion of L. brevis CRAI to the Caco-2 cell line, taken as an in vitro model of the intestinal epithelium, was evaluated. A quantitative method based on a count of adherent cells was applied. L. brevis CRAI showed a capacity to adhere to Caco-2 cells: the ratio between adherent viable L. brevis cells and Caco-2 cells was 3.06± 0.71 :1.

[0114] Discussion

[0115] The aim of this study was to isolate from a plant matrix, select andcharacterize a strain of lactobacillus with a demonstrated ability to produce GABA. The best GABA producer was selected, namely the strain Levilactobacillus brevis CRAI, which showed a 75.75% monosodium glutamate (MSG) conversion yield and a GABA production of 179.15 mM starting from an initial MSG concentration of 236.53 mM (4%). The results showed that GABA production was maximum in the presence of 236.53 mM MSG (4%).

[0116] The genome sequence was studied in order to predict the functional characteristics of the strain and the genes involved in GABA production. Genome annotation revealed that L. brevis CRAI contains both the gadA and gadB genes. The gadA gene is located in the gad operon, whereas gadB is located separately from the operon (Figure 1). This arrangement has also been found in L. brevis ATCC367, where gadA is located in the operon distant from gadB

[0044] , unlike in the majority of strains of L brevis, in which the gadCB operon is more common

[0053] . Furthermore, the genomic analysis revealed important genes responsible for probiotic and functional activities, such as the genes associated with the hydrolysis of bile salts and the riboflavin synthesis pathway. The gene coding for the enzyme cholylglycine hydrolase is responsible for the hydrolysis of bile salts. It is involved in bacterial tolerance to bile salts, in that it facilitates the use of amino acids as sources of carbon and nitrogen, thus favoring bile detoxification, with cholesterol reducing effects

[0054] . In the genome of L. brevis CRAI, this operon was identified together with the RFN regulatory region. The ability to resist stress due to bile salts and to synthesize vitamin B represents an added value which highlights the probiotic potential of the strain. Furthermore, various tools were used to analyze the safety profile of L. brevis CRAI. The CARD Resistance Gene Identifier (RGI) software identified two putative genes of antimicrobial resistance (AMR), vanT and nimA, which both show a low sequence identity (32% and 48%, respectively). These genes have also been identified in the strain L. brevis CRL 2013, in which the vanT gene, member of theracemase family, has been indicated as essential for the growth of lactic acid bacteria (LAB) and has not been linked to the vanG cluster, associated with vancomycin resistance. DNA sequencing revealed the presence of two sequences correlated with natural plasmids which have been found in other strains of L. brevis [57,58]. Gene annotation showed that the two plasmids carry genes involved in potassium homeostasis and bile hydrolysis, whereas no genes of virulence factors or antibiotic resistance were found.

[0117] The genomic analysis of L. brevis CRAI identified two genes, gadA and gadB, involved in GABA production. In order to verify whether the differences observed in GABA production were linked to variations in gene expression, the expression of these genes in response to two concentrations of MSG, 0 and 236.53 mM (4%), added to the culture medium was examined. The expression of the mRNA of the gadA and gadB genes increased respectively 52.52 and 4.44 times in the presence of 4% MSG (236.53 mM), compared to the conditions without MSG. These results suggest that both genes are induced by the presence of glutamate. In particular, the gadA gene shows a stronger, more marked induction, probably because of its position within an operon, which contains a regulatory region upstream. In conclusion, we may hypothesize that the transcriptional factor upstream of the operon may be activated by the presence of glutamate.

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Claims

CLAIMS1. A bacterium capable of producing gamma-aminobutyric acid (GABA), wherein said bacterium is the strain Levilactobacillus brevis CRAI deposited with the International Depositary Authority: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on February 6, 2025, under accession number DSM 35345.

2. A composition comprising the bacterium according to claim 1 and at least one excipient and / or at least one pharmaceutically acceptable and / or food grade substance.

3. The composition according to claim 2, formulated for oral administration.

4. The composition according to claim 3, formulated in the form of pills, capsules, tablets, granular powder, hard-shelled capsules, orodispersible granules, sachets, lozenges or drinkable vials or lyophilized powder.

5. The composition according to claim 2, formulated as a syrup or as a beverage, or else it is added to a food.

6. The composition according to claim 5, added to a yogurt, a cheese, or a fruit juice.

7. The composition according to any one of claims 2-6, further comprising at least one additional probiotic strain selected from the genera Lactobacillus, Bifidobacterium, and Streptococcus.

8. The composition according to any one of claims 2-7, further comprising a prebiotic substance selected from inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), and resistant starch.

9. The bacterium according to claim 1 or the composition according to any one of claims 2-8, for use as a medicament.

10. The bacterium according to claim 1 or the composition according to any one of claims 2-8, for use in the treatment or prevention of an infection and / or pathology caused by a pathogen.

11. The bacterium or composition for use according to claim 10, wherein the infection or pathology is of a gastrointestinal nature.

12. The bacterium or composition for use according to claim 10 or 11, administered in association or combination with at least one compound or molecule useful for the treatment of a gastrointestinal pathology.

13. The bacterium according to claim 1 or the composition according to any one of claims 2-8, for use in the treatment or prevention of a neurogastrointestinal pathology.

14. The bacterium according to claim 1 or the composition according to any one of claims 2-8, for use in restoring and / or maintaining the balance of the microbiota in an individual.

15. Non-medical use of the bacterium according to claim 1 or the composition according to any one of claims 2-8 as a dietary supplement.

16. A process for obtaining GABA, wherein said process comprises at least one step of using the bacterium according to claim 1.

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