Lactiplantibacillus pentosus strain CECT 30896

Abstract

The present invention relates to a novel L. pentosus CF-6HA strain (CECT 30896), isolated from aloreña table olives and with a dual effect as probiotic bacteria for humans / animals and also for plants, and its use as an antimicrobial agent and for bioremediation of selenium.

Description

[0001]STRAIN LACTIPLANTIBACILLUS PENTOSUS CECT 30896 DESCRIPTION TECHNICAL FIELD The present invention falls within the field of Microbiology, specifically, it relates to the identification of new strains of microorganisms for various uses, including their use as probiotics, for the production of functional compounds, as well as their use in the bioremediation of soils contaminated with metals. PRIOR STATE OF THE ART The increasing emergence of multi-resistant (MDR) pathogenic microorganisms in humans, animals and plants currently represents a global health challenge, the objective being to limit the spread of these pathogens and their antimicrobial / virulent genes through different ecosystems. In this sense, the promotion and increase in the use of probiotics, especially those of plant origin, provide a good opportunity to optimize and balance the health of people, animals,Plants and ecosystems. Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. They include microorganisms from different origins such as soil; plants; food; the gastrointestinal, urogenital, and vaginal tracts; breast milk; and feces of human subjects. They correspond to microorganisms from very diverse taxonomic groups, which are present in the environment with different bioavailability, and which exhibit a wide variety of metabolic activities and distinct mechanisms of action. The common characteristic that groups them is their ability to promote health, whether human, animal, or plant. The functional characterization of probiotics depends largely on the specific strain and its origin. In this sense, genomics provides valuable and interesting insights into the mechanisms and functionality of probiotics.as well as their health-promoting functions. Among probiotics, Gram-positive lactic acid bacteria (LAB) constitute a phylogenetically heterogeneous and versatile group that holds high probiotic potential, especially LAB of plant origin such as Lactiplantibacillus pentosus and Lactiplantibacillus plantarum (previously known as Lactobacillus pentosus and Lactobacillus plantarum), which are commonly isolated from raw and fermented plant foods such as table olives, cucumber, kimchi, and pickles, among others. Specifically, Aloreña table olives, traditional green olives naturally fermented with a protected designation of origin (PDO), are considered a potential source of probiotic L. pentosus strains. The versatility of L. pentosus and L. plantarum is due to their genomic diversity,plasticity and functionality linked to their ecological niches that allow them to adapt to the gastrointestinal tract and changing environmental conditions (Pérez-Díaz et al., 2021). Previous studies have shown that L. pentosus strains exhibit good growth and survival capacity under simulated gastrointestinal conditions (acidic pH of 1.5, up to 4% bile salts and 5 mM nitrate), good self-aggregation and coaggregation capacity with pathogenic bacteria, adherence to intestinal and vaginal cell lines, antimicrobial activity and fermentation of prebiotics and lactose (Pérez Montoro et al., 2016). It is worth noting that L. pentosus strains isolated from Aloreña table olives such as L. pentosus MP-10 and L. pentosus CF2-10N are especially interesting due to their genetic diversity and functionality, which gives them a great capacity for adaptation (Abriouel et al.,2022). In silico analysis and some in vitro assays of these strains confirmed their safety with respect to their use as probiotic food supplements. A promising area of ​​research is the search for probiotics that can interact with different hosts and fulfill different functions at the same time. DESCRIPTION OF THE INVENTION This document describes a probiotic strain with dual effect as a beneficial bacterium for humans and other animals and also for plants, L. pentosus CF-6HA, isolated from Aloreña table olives, which has been deposited in the Spanish Type Culture Collection (CECT) under deposit number CECT 30896. Although this strain shares several phenotypic and genotypic characteristics with the strains L. pentosus MP-10 and CF2-10N, isolated from the same ecological niche, there are some characteristics linked to the strain that confer each of them a differentiated probiotic potential and functionality (Abriouel et al.,2012; Pérez Montoro et al., 2016). An in silico study of its genome confirmed the safety of L. pentosus CF-6HA (CECT 30896) due to the absence of virulence determinants and acquired antibiotic resistance genes in its genome. It also allowed the detection of a wide variety of genes encoding defense and adaptability to different lifestyles, in addition to functional properties, which give the strain its robustness and probiotic potential. Genes that code for the synthesis of interesting secondary metabolites were identified, such as: - bacteriocins; proteins or peptide toxins synthesized by bacteria that inhibit the growth of similar bacteria or strains close to those that produce them. Some of the bacteriocins identified are pediocin, used as a preservative in vegetable and meat products and with high activity against Listeria species, and plantaricin Y,characterized by a strong inhibitory activity against Listeria monocytogenes; - various exopolysaccharides; - terpenes; which perform different functions in plants, mainly protection against insects and herbivores and protection against high temperatures; - type III polyketide synthases (T3PKS), with important biological functions and pharmaceutical activities; - polyketides (PKS); which participate in the biosynthesis of some lipid compounds and several secondary metabolites, and have significant biological functions and important pharmaceutical activities; - non-ribosomal peptides (NRPs), with an extremely wide range of biological and pharmacological activities, being able to act as toxins, siderophores or pigments, or present antibiotic, cytostatic or immunosuppressive activity; - hybrid peptide-polyketide products (PK+NRPs),compounds with antimicrobial and anticancer activity; - post-translationally modified peptides (RiPP); - proteins involved in adhesion and other functions. Of particular interest is the role of this strain as a probiotic agent for humans and animals, maintaining or restoring the host's natural microbiota. Furthermore, in both humans and animals, as well as in plants, it can reduce the spread of pathogens and their resistance genes. Its antimicrobial activity against human and animal pathogens and plant pests, such as Pseudomonas syringae and Verticillium dahliae, is noteworthy. The strain of the invention can be used alone or in combination with the aforementioned L. pentosus MP-10 and L. pentosus CF2-10N. Finally,L. pentosus CF-6HA (CECT 30896) has the ability to biotransform selenite into nanoparticles (SeNPs), so it can be used in the bioremediation of selenium in natural media and as a starter culture for the bioenrichment of fermented foods with selenium. Therefore, a first aspect of the invention describes the strain Lactiplantibacillus pentosus CF-6HA deposited in the Spanish Type Culture Collection under accession number CECT 30896, hereinafter "strain of the invention". The invention also relates to the pure bacterial culture, supernatant, extract or bioactive compound isolated from said strain, as well as to the composition comprising said culture, supernatant, extract or bioactive compound. A second aspect of the invention describes the non-therapeutic use of the strain of the invention,the pure bacterial culture and / or the composition comprising it as a probiotic for plants. A third aspect of the invention describes the non-therapeutic use of the strain of the invention, the pure bacterial culture and / or the composition comprising it as an antimicrobial agent against Pseudomonas fragi. Preferably, this antimicrobial agent is also used simultaneously against one or more of the microorganisms selected from the list consisting of: Enterococcus faecalis, Bacillus cereus, Staphylococcus aureus, Listeria innoccua, Escherichia coli, Salmonella enterica, Candida albicans, Pseudomonas syringae and Verticillium dahliae. A fourth aspect of the invention is the non-therapeutic use of the supernatant, extract or bioactive compound isolated from the strain of the invention and / or the composition comprising it as an antimicrobial compound or composition against Pseudomonas syringae, Verticillium dahliae, Pseudomonas fragi, Staphylococcus aureus, Bacillus cereus,Escherichia coli and / or Candida albicans. A fifth aspect of the invention relates to the strain of the invention, the pure bacterial culture and / or the composition comprising it for use as a probiotic medicament for humans and non-human mammals. A sixth aspect of the invention relates to the strain of the invention, the pure bacterial culture and / or the composition comprising it for use as an antimicrobial medicament against Pseudomonas fragi. Preferably, it is used simultaneously as an antimicrobial medicament against one or more of the microorganisms selected from the list consisting of: Enterococcus faecalis, Bacillus cereus, Staphylococcus aureus, Listeria innoccua, Escherichia coli, Salmonella enterica, Candida albicans, Pseudomonas syringae and Verticillium dahliae. A seventh aspect of the invention relates to the supernatant,extract or bioactive compound isolated from the strain of the invention and / or the composition comprising it for use as an antimicrobial drug against Pseudomonas syringae, Verticillium dahliae, Pseudomonas fragi, Staphylococcus aureus, Bacillus cereus, Escherichia coli and / or Candida albicans. An eighth aspect of the invention relates to the use of the strain of the invention for obtaining pediocin and plantaricin Y. A ninth aspect of the invention describes the use of the strain of the invention for the bioremediation of media contaminated with selenium. A tenth aspect of the invention describes the use of the strain of the invention for the bioenrichment of fermented foods with selenium. Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects,Advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Results of the prediction made with BlastKOALA of the functional categories in the genome of Lactiplantibacillus pentosus CF-6HA (CECT 30896) and their frequencies. Figure 2. Phylogenetic relationships between the genomes of Lactiplantibacillus spp. A phylogenetic tree was constructed with ND Tree 1.2 by comparing the genome sequence of L. pentosus CF-6HA (CECT 30896) with the genome sequences of other Lactiplantibacillus spp. (L. pentosus, L. plantarum,L. paraplantarum) visualized using the NCBI Tree Viewer. Figure 3. Organization of predicted antimicrobial biosynthetic gene clusters in the Lactiplantibacillus pentosus CF-6HA (CECT 30896) genome. (A) Bacteriocin gene cluster predicted using the BAGEL4 web server. (B) T3PKS (Type III PKS, region 2.1) gene cluster predicted using the antiSMASH software tool. Genes are represented by arrows with different colors corresponding to different functions. Figure 4. Scanning electron micrographs (A, B, D, and F) and EDX (energy dispersive X-ray microanalysis) (C, E, and G) of Lactiplantibacillus pentosus CF-6HA (CECT 30896) cells grown in MRS (Man, Rogosa, and Sharpe) broth in the absence (AC) and presence of 5 ppm Se (DG) at 37°C for 24 h. Scanning electron microscope (SEM) imagesby scanning electron microscope) were taken at different magnifications and the arrows indicate the locations of the EDX spectra. DETAILED DESCRIPTION OF THE INVENTION Bacterial strains and growth conditions Lactiplantibacillus pentosus CF-6HA (deposited in the Spanish Type Culture Collection as CECT 30896), originally isolated from naturally fermented green Aloreña table olives. Lactiplantibacillus pentosus CF-6HA (CECT 30896) was routinely grown at 37°C on de Man, Rogosa, Sharpe (MRS) agar under aerobic (atmospheric) conditions for 24-48 h. MRS agar, thanks to its composition, allows the proper development of lactobacilli and other lactic acid bacteria: proteose peptone, meat extract, yeast extract and glucose provide nitrogen, carbon, vitamins and minerals; sorbitan monooleate, sodium salts,Magnesium and manganese provide cofactors for bacterial growth and can inhibit the development of some microorganisms; ammonium citrate acts as a growth inhibitor for Gram-negative bacteria. The bacterial and fungal pathogens were obtained from the Spanish Type Culture Collection (CECT), except for the Pseudomonas syringae strains, which were supplied by Dr. J.A. Gutiérrez (University of Málaga, Spain). The growth conditions of the pathogens are shown in Table 1. Table 1: Pathogenic strains and growth conditions used in this study. MHB / MHA: Mueller Hinton Broth / Mueller Hinton Agar; TSB / TSA: Tryptone Soy Broth / Tryptone Soy Agar; PDA: Potato Dextrose Agar. All strains were preserved in 20% glycerol at -80°C for long-term storage. DNA extraction, whole genome sequencing, assembly, annotation and comparative analysis. The genome sequence of L. pentosus CF-6HA (CECT 30896) was sequenced and analysed as an unambiguous diagnostic tool for its safety (in accordance with the indications of the EFSA Panel - Food Safety and Authorisation of Regulated Products and the FEEDAP Panel - Panel on Products or Substances used in Animal Feed, 2018). L. pentosus CF-6HA (CECT 30896) bacterial cells, grown for 18 h at 37°C under aerobic conditions in liquid medium, were harvested by centrifugation and total genomic DNA was extracted using the PureGene core B kit, according to the manufacturer's instructions (Qiagen,Spain) and as described by Abriouel et al. (2022). Library preparation, genome sequencing, assembly, and annotation were carried out as described by Abriouel et al. (2022). Briefly, purified genomic DNA was sheared into 10–20 kb fragments using the protocol designed for PacBio RS II System DNA library preparation (Pacific Biosciences, Menlo Park, CA), and the resulting libraries (22–24 kb) were purified and sequenced as described by Abriouel et al. (2022). Raw sequence data were filtered (Q20), and a total of 150,292 reads with an average length of 12,167 bp were obtained. The resulting reads were assembled de novo following the Hierarchical Genome Assembly Process (HGAP3.0) method (SMRT analysis version: 2.3.0, patch no. 4) for Pacific Bioscience, as described by Abriouel et al. (2022). The prediction of coding DNA sequences (CDS), tRNA genes,rRNA and mRNA and signal peptides in the assembled sequences following the methodologies detailed by Abriouel et al. (2022). Sequence annotation was performed using BLAST2GO version 4.1.9 (https: / / www.blast2go.com / ) now part of OmicsBox (https: / / www.biobam.com / omicsbox / ) (Conesa et al., 2005), followed by complementary domain-specific annotation for protein domains using HMMER 3.1 (July 2017) (http: / / hmmer.org / ) and the database formerly known as Pfam (currently InterPro) (https: / / www.ebi.ac.uk / interpro / ) (Paysan-Lafosse et al., 2022). Blast2GO is a bioinformatics software tool for the automatic, high-throughput functional annotation of new sequence data (genes, proteins). Use the BLAST algorithm to identify similar sequences and thentransfers the existing functional annotation of already characterized sequences to the new sequence. Functional information is represented using Gene Ontology (GO), a major bioinformatics initiative to unify the representation of gene and gene product attributes across species. HMMER is a program used to search for homologous sequences in databases and to perform sequence alignments. It implements methods that use probabilistic models called profile hidden Markov models (profile HMMs). HMMER is often used in conjunction with a profile database, such as Pfam or Interpro. Pfam is a database that brings together a large collection of multiple sequence alignments and hidden Markov models that cover a large number of common protein domains and families. For each family in Pfam, you can view multiple alignments, review the architectures and organization of protein domains,examine species distribution, follow links to other databases, and view known protein structures. In addition, gene blasting was performed against Clusters of Orthologous Groups (COGs) of proteins (http: / / www.ncbi.nlm.nih.gov / COG) using the WebMGA server (http: / / weizhongli-lab.org / metagenomic-analysis) (Wu et al., 2011) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The COGs database is an attempt at phylogenetic classification of the proteins encoded in entire genomes. Each COG includes proteins that are inferred to be orthologs (direct evolutionary homologs). The COG database is updated periodically as new genomes become available. COGs can be applied to the task of functional annotation of newly sequenced genomes. For its part, WebMGA,is a customizable web server for rapid metagenomic analysis that includes more than 20 commonly used tools such as sequence pooling, raw read quality control, sequencing artifact and contamination removal, taxonomic analysis, functional annotation, etc., providing rapid analysis of metagenomic data. KEGG is a collection of online databases of genomes, enzymatic pathways, and biological chemicals. Genome sequencing, assembly, and annotation were performed at Biopolis-ADM (Valencia, Spain). Comparative genomic analysis was performed using the available complete genome sequences of Lactiplantibacillus spp. in the NCBI database and of L. pentosus CF-6HA (the CECT 30896 strain that is the subject of the present invention). First, the phylogenic relationship between L. pentosus CF2-10N (accession number ERR11550479), L. pentosus MP-10 (accession number GCA_900092635.1),L. pentosus DSM 20314 (accession number GCA_003641185.1), L. pentosus KCA1 (accession number GCA_000271445.1) and L. pentosus CF-6HA (the CECT 30896 strain subject of the present invention). The analysis of synteny between species was performed using MAUVE alignment (DNASTAR LASERGENE v17.4.2) (Darling et al., 2004). Next, a phylogenetic tree was constructed using the maximum likelihood method: RAxML Randomized Axelerated Maximum Likelihood (Stamatakis, 2014) using the MAUVE alignment (DNASTAR LASERGENE v17.4.2). The statistical reliability of the tree was assessed using a bootstrap analysis of 1000 replicates. Bootstrapping is a resampling method used to approximate the sampling distribution of a statistic. It is frequently used to approximate the bias or variance of a statistical analysis, as well as to construct confidence intervals or perform hypothesis tests on parameters of interest. In addition, NDtree 1.2 was used (Qian et al.,2003) to construct phylogenetic trees from Single-End or Pair-End FASTQ files of Lactiplantibacillus spp. (last accessed March 2023) (Kaas et al., 2014; Leekitcharoenphon et al., 2014) and the NCBI Tree Viewer (Tree Viewer 1.19.4 available as an NCBI app) for graphical display of phylogenetic trees (https: / / www.ncbi.nlm.nih.gov / tools / treeviewer / ). The complete genome sequence of L. pentosus CF-6HA (CECT 30896) has a length of 4,148,739 bp and an estimated mol% G+C content of 45.7%. The genomic sequence contains a single circular chromosome of 3,764,590 bp and an estimated mol% G+C content of 46.19% similar to its homologous strains such as L. pentosus MP-10 (3,698,214 bp; GC content of 46.32%) and L. pentosus CF2-10N (3,645,747 bp; GC content of 46.42%) (Abriouel et al., 2016, 2022) and 6 plasmids of 38.5–74.5 kb, namely pLPE6-1 (38,502 bp), pLPE6-2 (73,263 bp), pLPE6-3 (72,520 bp), pLPE6-4 (62,787 bp),pLPE6-5 (74,498 bp) and pLPE6-6 (61,672 bp) unlike the five present in L. pentosus MP-10 (29–46.5 kb) and the four in L. pentosus CF2-10N (58–120 kb) (Abriouel et al., 2016, 2022). These plasmids may play a key role in the fermentation process and adaptation to the ecosystem (soil, plant and brine), including heavy metal detoxification (Abriouel et al., 2019). In addition, 16 rRNA genes and 84 tRNA coding sequences have been predicted in the L. pentosus CF-6HA genome (CECT 30896) using RNAmmer (version 1.2). The RNAmmer 1.2 server predicts 5s / 8s, 16s / 18s, and 23s / 28s ribosomal RNAs in whole genome sequences (https: / / services.healthtech.dtu.dk / services / RNAmmer-1.2 / ). Regarding functional classification, the annotated genome sequence revealed 3,960 open reading frames (ORFs), of which 73.3% (2,902) were attributed to a COG (Cluster of Orthologous Groups) family and / or received a functional description. Furthermore,The genome of L. pentosus CF-6HA (CECT 30896) contains 1,245 KEGG orthology (KO) assignments, mainly enzymes (699 genes) and transporters (212 genes). The KEGG functional annotation frequency obtained by BlastKOALA (KEGG tool; last update May 15, 2019; Kanehisa et al., 2016), which assigned approximately half (47.4%) of the genes to KEGG annotations corresponding to genetic information processing (248 genes), carbohydrate metabolism (244), cellular signaling and processing (214 genes), environmental information processing (193 genes), genetic information processing (165 genes), unclassified: metabolism (113 genes), unclassified: genetic information processing (106 genes), amino acid metabolism (100 genes), nucleotide metabolism (69 genes),metabolism of cofactors and vitamins (68 genes) and metabolism (49 genes) among others (Figure 1). Comparative genomic analysis of L. pentosus CF-6HA (CECT 30896) and other L. pentosus strains isolated from the same ecological niche (Aloreña table olive), such as L. pentosus MP-10 and CF2-10N strains, L. pentosus DSM 20314 (type strain) isolated from corn silage and L. pentosus KCA1 isolated from the vaginal tract, was carried out by sequence alignment using the MAUVE algorithm. The synteny linkage of L. pentosus CF-6HA (CECT 30896) with other L. pentosus strains revealed the presence of highly conserved syntenic blocks, although inversions and rearrangements occurred among all L. pentosus strains, even among those isolated from the same ecological niche (Aloreña table olive; L. pentosus strains CF-6HA (CECT 30896), MP-10 and CF2-10N). To highlight this relationship,A maximum likelihood (RaxML) core genome tree was constructed, and the results revealed that L. pentosus CF-6HA (CECT 30896) shared an overall 97% identity (evolutionary distance "ED", ED=0.02) with L. pentosus strains MP-10 (97.62%), CF2-10N (97.62%), and L. pentosus DSM 20314 (97.53%), while L. pentosus KCA1 only displayed 92.16% identity (ED=0.08). Phylogenetic analysis using the NDtree tool, based on the number of nucleotide differences between strains, and the NCBI tree viewer showed a clear distinction between the 48 Lactiplantibacillus spp. strains clustered into conspecific strains (Figure 2). Genomic analysis of safety and antimicrobial resistance aspects of Lactiplantibacillus pentosus CF-6HA (CECT 30896). In addition to global annotation using the BLAST2GO program,Antibiotic resistance genes (ARGs) from L. pentosus CF-6HA (CECT 30896) were also examined using the Resistance Gene Identifier (RGI 6.0.1) software as part of the CARD tools "The Comprehensive Antibiotic Resistance Database" (https: / / card.mcmaster.ca / ) (Alcock et al., 2023), employing CARD's curated AMR (antimicrobial resistance) detection models (last accessed February 2023). RGI is used to predict resistomes from protein or nucleotide data based on homology models and single nucleotide substitutions (SNPs). CARD is a database that collects and organizes reference information about genes,Antimicrobial resistance proteins and phenotypes. The database covers all types of drug classes and resistance mechanisms and structures its data based on an ontology. It allows the detection of potential antibiotic resistance genes in newly sequenced genomes. Prediction using BLAST2GO annotation revealed the presence of several non-specific antimicrobial resistance mechanisms that depend on efflux transporters or transmembrane proteins involved in the response to drugs, including antibiotics, such as the multidrug transporter MFS (encoded by the KCA1_0967 gene); TIGR00374 family protein (encoded by the mprF gene); multidrug transporter protein (encoded by the FD24_GL001667, LPENT_02485 and LPE_01285 genes); cation efflux protein (encoded by the FD24_GL002035 gene); ABC transporter, ATP-binding protein and permease (encoded by the genes LPENT_00563, LPENT_02775,LPENT_02344 and LPENT_02438); ATP-binding cassette transporter permease ABC superfamily MutG family lantibiotic protection (encoded by gene LPENT_00532); ATP-binding cassette transporter ABC superfamily, ABC protein (encoded by gene LPE_00726); ATP-binding protein of the ABC transporter (encoded by LPENT_00338, FD24_GL000723, LPENT_02848, N692_09400, LPE_02094, LPE_00791, FD24_GL003445, FD24_GL002415, LPENT_02562, LPE_01544, LPENT_02495, KCA1_2339, LPENT_02440, gen_2043, LPENT_02320, gen_2108, LPE_01165, FD24_GL000926, FC82_GL001371, FD47_GL001009, LPENT_01461 and FD24_GL002891 genes); ABC transporter permease protein (encoded by genes FD24_GL000827, LPENT_02885, FD24_GL003057, and LPE_01349); multidrug ABC transporter ATP-binding protein (encoded by gene_1153); multidrug ABC transporter permease (encoded by gene LPE_02342); putative ABC transporter,ATP-binding protein (encoded by the LPENT_03065 gene); integral membrane protein (encoded by the LPENT_02708, LPENT_02080, and LPENT_01044 genes); multidrug ABC transporter system, ATPase and permease component (encoded by the LPENT_02699 gene); transporter (encoded by the FD24_GL003458 gene); bacitracin ATP-binding protein ABC transporter (encoded by the FD24_GL002522 gene); DMT metabolite superfamily transporter (encoded by the LPENT_02520 gene); hypothetical protein (encoded by the gene_1979 and gene_2937 genes); membrane protein (encoded by the KCA1_2377 gene); EamA transporter family (encoded by the LPE_02249 gene); multicompound ABC transporter,ATP-binding and protein permease (encoded by gene LPENT_02375); ABC transporter (encoded by gene LPE_03175); SMR transporter of quaternary ammonium efflux compounds of the QacE family (encoded by gene FD24_GL003284); MULTISPECIES: quaternary ammonium (encoded by gene N692_04340); transporter protein (encoded by gene LPE_01191); MULTISPECIES: undecaprenyl diphosphatase (encoded by gene uppP); VanZ family protein (encoded by gene LPE_02604); EamA / RhaT family transporter (encoded by gene KCA1_0065); MATE family efflux transporter (encoded by gene LPE_00986); ABC superfamily ATP-binding cassette transporter, ABC protein (encoded by gene LPENT_01629); ABC transporter-related (encoded by genes LPENT_01583 and LPENT_01583); MULTISPECIES: ABC transporter ATP-binding protein (encoded by gene KCA1_0270); ABC transporter,ATPase and permease components (encoded by gene LPENT_01451); EamA family transporter (encoded by gene LPENT_01209); multidrug transporter protein, major facilitator superfamily (MSF) (encoded by gene KCA1_0714); groel suppressor protein suge (encoded by gene FD24_GL000282); and cell surface protein (encoded by genes gene_3573 and gene_3843). All genes are chromosomally located except gene_3843 which encodes the cell surface protein on plasmid pLPE6-1. Only one strict match was detected by RGI, which was defined within the similarity limits of the individual AMR detection models and represented likely homologs of AMR genes according to Alcock et al. (2023). In this sense,The RGI revealed that the genome of L. pentosus CF-6HA (CECT 30896) contained a vanY gene in a vanB cluster conferring resistance to glycopeptides (100% identity with the vanZ gene of other L. pentosus strains that was located on the chromosome) and several non-specific antimicrobial resistance mechanisms that depend on efflux transporters or transmembrane proteins involved in the response to drugs, including antibiotics. On the other hand, the search for acquired antibiotic resistance genes / antimicrobial resistance-mediating chromosomal mutations in the L. pentosus CF-6HA (CECT 30896) genome was performed using ResFinder software, an open online resource for the identification of antimicrobial resistance genes in next-generation sequencing data and the prediction of phenotypes from genotypes (https: / / cge.cbs.dtu.dk / services / ResFinder / ) version 4.1 (Bortolaia et al., 2020; Camacho et al.,2009; Zankari et al., 2020), with a selected % identity (ID) threshold of 90% and a selected minimum length of 60% (last accessed February 2023). ResFinder did not detect any antimicrobial resistance genes acquired by horizontal gene transfer for aminoglycoside, beta-lactam, colistin, disinfectant, fluoroquinolone, fosfomycin fusidic acid, glycopeptide, MLS series (macrolide, lincosamide, and streptogramin B), nitroimidazole, oxazolidinone, phenicol, pseudomonic acid, rifampicin, sulfonamide,tetracycline and trimethoprim. Virulence factors (VFs) screening was performed by annotating predicted coding regions (CDRs) using reciprocal BLAST against the Virulence Factors of Bacterial Pathogens Database (VFDB) (http: / / www.mgc.ac.cn / VFs / main.htm), with hits considered positive when reciprocal BLAST results were similar and a sequence similarity cutoff of 80% was used (Liu et al., 2019). No known virulence factors (VFs), including toxins, were identified. These data confirm the safety status of L. pentosus CF-6HA (CECT 30896). They also confirm that intrinsic antimicrobial resistance is not horizontally transferable, as required in probiotic strains, although it contributes to drug defense and fitness (Tóth et al.,In silico analysis of safety and antimicrobial resistance of Lactiplantibacillus pentosus CF-6HA (CECT 30896) Once the safety of L. pentosus CF-6HA (CECT 30896) was confirmed, the genome sequence was explored for determinants involved in the robustness and functionality that allow this strain to withstand various stresses in its habitat, during fermentation and also in the gastrointestinal tract. Mobilome analysis The mobilome (set of mobile genetic elements) of L. pentosus CF-6HA (CECT 30896) was analyzed for the presence of conjugative plasmids, transposases, transposons, insertion sequences (IS) and prophage-encoding genes in the annotated genome sequence, as detailed by Abriouel et al. (2022). For this purpose, the search for insertion sequences (IS) was performed using the ISfinder search tool (https: / / isfinder.biotoul.fr / ), a database dedicated to bacterial insertion sequences (IS),which is the repository of ISs that is indexed along with their DNA sequence and open reading frames or possible coding sequences, the sequence of the element ends and target sites, their origin and distribution, etc. (Zhang et al., 2004). Regarding IS elements, 17 CDSs were detected in the L. pentosus CF-6HA (CECT 30896) genome belonging to nine families and distributed in 12 different bacteria, with IS3 as the most original family from Acinetobacter baumannii / A. lwoffii. For the analysis of prophages within the L. pentosus CF-6HA (CECT 30896) genome, the PHASTER version (https: / / phaster.ca / ; PHAge Search Tool Enhanced Release, last update March 2016; corresponding to the updated PHAST prophage / virus database "PHAge Search Tool") was used. PHASTER allows the identification and annotation of prophage sequences in bacterial genomes and plasmids (Arndt et al., 2016; Zhou et al., 2011). In addition,Complementary information was obtained from the annotated genome sequence for the prophage and the transposon / transposase. Regarding transposases, the L. pentosus CF-6HA genome (CECT 30896) harbored 98 transposases, of which 31 are putative transposases and 67 belong to 10 IS families (2 IS3, 1 IS4 / IS5, 8 IS5, 23 IS30, 25 ISL3, 1 IS200 / IS605, 2 IS256, 1 IS1380, 2 Mutator family, 2 mulE superfamily). Transposases from the ISL3 and IS30 families were the most prevalent. These transposases were equally distributed on both chromosomes (50 CDs) and plasmids (48 CDs in pLPE6-3, pLPE6-4, pLPE6-5 and pLPE6-6), mainly in pLPE6-6, and appeared as multiple copies. The results of the prophage search showed the presence of 13 temperate phage regions within the L. pentosus CF-6HA genome (CECT 30896) using the PHASTER bioinformatics tool. Seven of them were intact (Regions 1, 2, 3, 7, 8, 9 and 13; score > 90),one was doubtful (Region 12; score 70±90) and the other five were incomplete (Regions 4, 5, 6, 10 and 11; score < 70). The intact prophage regions of L. pentosus CF-6HA (CECT 30896) were mainly related to Lactob_Sha1_NC_019489 (Regions 1, 2, 7, 8 and 9; GC content, 30.30–42.12%; region length, 27.1–46 kb), whereas the incomplete prophage regions were associated with different phages such as Lactob_Sha1_NC_019489 (Region 6, 17.2 Kb), Lactob_phig1e_NC_004305 (Region 4, 15.7 Kb), Erwini_pEp_SNUABM_01_NC_048807 (Region 10, 12 Kb), Photob_PDCC_1_NC_048821 (Region 5, 7.6 Kb) and Paenib_Tripp_NC_028930 (Region 11, 5.5 Kb). We determined that Lactob_Sha_1 showed the highest protein match among the identified prophages. Regarding the questionable regions of the prophages, we only identified one that showed similarity to Staphy_SPbeta_like_NC_029119 (Region 12,4.9 Kb). Only five prophage regions have attL / attR and integrase sequences, such as regions 1, 2, 4, 6 and 7 located in the bacterial chromosome, and identified in intact and incomplete regions. CRISPR On the other hand, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) encoding genes were examined in the annotated genomic sequence of L. pentosus CF-6HA (CECT 30896) and the location of CRISPR RNA targets was determined using the CRISPRDetect version 2.4 program (http: / / crispr.otago.ac.nz / CRISPRDetect / predict_crispr_array.html) (Biswas et al., 2016). In the genome of L. pentosus CF-6HA (CECT 30896) the CRISPR I and II systems (both genes characteristic of the type I "cas3" and type II "cas9" systems) were identified as defense mechanisms against mobile genetic elements (i.e. viruses, transposable elements and conjugative plasmids). In this sense, 12 genes were detected,five of them exclusive to L. pentosus CF-6HA (CECT 30896). These are responsible for the synthesis of CRISPR-associated proteins, and are organized into two operons (CRISPR-I and CRISPR-II). Regarding the CRISPR arrays (CR), we identified three unquestionable CRISPR arrays using the CRISPRDetect program, located dispersed on the chromosome (678102-2943655). Regarding the lifestyle adaptation of L. pentosus CF-6HA (CECT 30896) to different stresses, in silico exploration of the genome sequence shows the frequent genetic diversification inferred by the mobilome (17 ISI elements, 98 transposases, 13 temperate phage regions) and CRISPR elements (CRISPR systems I and II). The high number of mobile genetic elements in the genome of L. pentosus CF-6HA (CECT 30896) is comparable to that of other strains of L. pentosus also isolated from Aloreña table olives, i.e. L. pentosus MP-10 (29 transposases,5 temperate phage regions) and L. pentosus CF2-10N (66 transposases, 45 IS elements and 8 temperate phage regions) (Abriouel et al., 2012, 2017, 2022). However, the number far exceeded other L. pentosus strains isolated from other ecological niches, such as L. pentosus IG1 (Spanish-style green olive fermentations; 5 genes encoding transposases), L. pentosus KCA1 (vagina; 25 genes encoding transposases) and L. pentosus DSM 20314 (corn silage; 14 genes encoding transposases) (Abriouel et al., 2017). These characteristics indicated that L. pentosus strains isolated from Aloreña table olives possess a large number of strain-specific elements with adaptive functions that play an important role in their habitat through chromosomal rearrangements, with particular attention to L. pentosus CF-6HA (CECT 30896). Furthermore,Other selective advantages for the survival of L. pentosus CF-6HA (CECT 30896) are conferred by 13 prophage regions detected in its genome, seven of which were intact and are mainly lactobacilli prophages, although the other dubious and incomplete prophages were related to other bacteria (Erwinia, Photobacterium, Paenibacillus or Staphylococcus). Indeed, L. pentosus CF-6HA (CECT 30896) possesses the machinery capable of coping with the risk of phage infection in different ecosystems such as some lactobacilli (Sun et al., 2015). The CRISPR system (CRISPR-I and CRISPR-II) as another defense mechanism was also detected in the L. pentosus CF-6HA (CECT 30896) genome. In particular, 12 genes were detected that provide protection against mobile genetic elements similar to L. pentosus CF2-10N and L. pentosus MP-10. However, both the genes and their organization were strain-specific,Probiotic functionalities of Lactiplantibacillus pentosus CF-6HA (CECT 30896) In silico analysis of antimicrobial activity The annotated genome sequence of L. pentosus CF-6HA (CECT 30896) was analyzed for the presence of genes encoding antimicrobial proteins / peptides and genes encoding bacteriocin production were examined using the BAGEL 4 web server (http: / / bagel4.molgenrug.nl / ), which allows mining bacterial (meta)genomic DNA for bacteriocins and ribosomally synthesized and post-translationally modified peptides (RiPPs) by providing databases and a BLAST against major peptide databases (van Heel et al., 2018). Each member of the predicted gene clusters was then confirmed using Blastp (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi),which finds regions of similarity between biological sequences by comparing nucleotide or protein sequences to sequence databases and calculating statistical significance. In addition, antiSMASH version 7.0 (https: / / antismash.secondarymetabolites.org / #! / start) (Blin et al., 2021) was used to search for secondary metabolite gene clusters. antiSMASH allows for the rapid identification,Annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial genomes. It integrates and interrelates with a large number of previously published in silico secondary metabolite analysis tools. Both BAGEL and antiSMASH were used for the detection of open reading frames (ORFs) involved in the production of antimicrobial compounds. RiPPs and bacteriocins (unmodified) were matched in the L. pentosus CF-6HA genome (CECT 30896) against the BAGEL database (BAGEL4). The results obtained showed the gene cluster responsible for bacteriocin biosynthesis and consists of 26 genes with a total length of approximately 20 kb located on the chromosome (Figure 3A). In this gene cluster, only one biosynthetic bacteriocin gene encoding pediocin, class IIa bacteriocin (core peptide, pediocin; location 2,511,611-2,511,967 bp) was detected (Figure 3A),However, no pediocin immunity / transport genes were identified, although several genes encoding other transporters or resistance genes were detected, such as the MFS transporter, the P-family cadmium-transporting ATPase, or the MerR family transcriptional regulator. Furthermore, in silico analysis of the L. pentosus CF-6HA genome (CECT 30896) using the BLAST2GO program showed the presence of a putative gene (LPENT_01393) encoding the biosynthesis of Plantaricin Y, a class IId bacteriocin (PlnY; location 3,248,967-3,249,260 bp). Furthermore, genome analysis using the antiSMASH software tool showed the presence of genes involved in the synthesis of secondary metabolites with antimicrobial potential related to T3PKS (type III polyketide synthase) (Figure 3B). In this sense,a region 2.1 (location: 1,169,377–1,210,546 nt): T3PKS was identified within the chromosome. This cluster was represented by a core biosynthetic gene (ctg2_1084) encoding hydroxymethylglutaryl-CoA synthase; six additional biosynthetic genes (ctg2_1067, ctg2_1074, ctg2_1076, ctg2_1079, ctg2_1087 and ctg2_1105) encoding peptidase_M50, SMCOG1115:HAD superfamily hydrolase, SMCOG1072 dehydrogenase, SMCOG1141:acyltransferase, SMCOG1123:polyprenol-monophosphomannose synthase ppm1 and SMCOG1001:short chain dehydrogenase / reductase SDR, respectively; a transporter gene (ctg2_1100) encoding an MFS transporter and other genes (Figure 3B). AntiSMASH blastp results indicate the closest compound from MiBIG database and showed the best matches corresponding to terpene, type III polyketide synthase (T3PKS), polyketides (PKS), non-ribosomal peptide (NRP), peptide-polyketide hybrid products (PK+NRP),post-translationally modified peptide (RiPP) and others, with NRP and PKS being the most abundant classes of biosynthetic gene clusters (Figure 3B). The interest of these secondary metabolites, including bacteriocins and exopolysaccharides, was based on their diverse structure and specific bioactivities that enhance the promising probiotic potential of L. pentosus CF-6HA (CECT 30896) as a source of antimicrobial and bioactive compounds. It is worth highlighting the ability of some lactic acid bacteria to produce a wide range of beneficial secondary metabolites such as bacteriocins, diacetyl, acetoin, exopolysaccharides, vitamins (group B), cyclic dipeptides, mevalonic acid, mevalonolactone and butanediol, among others, with antimicrobial, antioxidant, host interaction, immunomodulatory, etc. activity (Crowley et al., 2013; Mora-Villalobos et al., 2020). However, only a few strains are able to produce secondary metabolites,In vitro analysis of antimicrobial activity To complement the results obtained through in silico analysis, the antimicrobial activity against the pathogens listed in Table 1, including phytopathogens (responsible for plague), was examined in vitro. The expression of genes encoding bacteriocins in L. pentosus CF-6HA (CECT 30896) was thus evaluated, to confirm the presence of bacteriocins in the cell-free supernatant and their antimicrobial activity against several pathogens. To this end, 10 µl of an overnight culture of L. pentosus CF-6HA (CECT 30896) was spread on MRS agar and incubated for 24 h; The plates were then covered with soft buffered Brain Heart Infusion Agar (SF-BHA; pH 7.0, 0.1 M) inoculated with indicator strains (Table 1) grown for 24 h. In addition,The antimicrobial activity of the supernatant produced by L. pentosus CF-6HA (CECT 30896) in MRS broth was tested using agar well diffusion as described above. Antimicrobial activity was assessed by measuring the diameter of the inhibition zones from the edge of the well. In the case of V. dahliae, the antifungal activity of L. pentosus CF-6HA (CECT 30896) was performed on potato dextrose agar (PDA) plates according to Mohamad et al. (2018) evaluating the growth inhibition using the following formula: 100×[CT] / C, where, C is the radial growth of the fungus in the control and T is the same as in the dual culture (Aeron et al., 2011). The results of the antimicrobial activity of L. pentosus CF-6HA (CECT 30896) against phytopathogens were promising (Table 2). Table 2: Antimicrobial activity of Lactiplantibacillus pentosus CF-6HA (CECT 30896) against phytopathogens and other pathogens. and (+), the diameter of the inhibition zone from the growing edge is < 10 mm. (++), the diameter of the inhibition zone from the growing edge is in the range of 10-13 mm. (+++), the diameter of the inhibition zone from the growing edge is > 14 mm. * % growth inhibition according to Aeron et al. (2011). In this sense, the strains P. syringae subsp. syringae UMAF0291 and UMAF0158 and the strains Verticillium dahliae CECT 2694 and CECT 2884 were sensitive to the antimicrobial substances produced ex situ by L. pentosus CF-6HA (CECT 30896), since the supernatant of this strain was active against bacterial and fungal phytopathogens. This effect was not observed in Verticillium dahliae strain CECT 2884, possibly because it requires larger amounts of the antimicrobial present in the supernatant than does strain CECT 2694 of the same species. Furthermore, other pathogens were also sensitive to the antimicrobials produced by L.pentosus CF-6HA (CECT 30896), such as the spoilage bacterium P. fragi T81 and other human / animal pathogens, i.e. Bacillus cereus strains, Staphylococcus aureus strains, Enterococcus faecalis strains, Listeria innocua, Escherichia coli, Salmonella enterica and Candida albicans, however no activity was detected against Saccharomyces cerevisiae CECT 1171. Although P. fragi is not a human pathogen, it represents a reservoir of antibiotic resistance genes that can be transferred to other pathogenic bacteria, thus increasing their danger. Therefore, it is an agent of disruption of the food chain and also a reservoir of antibiotic resistance genes according to Elbehiry et al. (2022). This antimicrobial activity allows this strain to successfully overcome undesirable and pathogenic microbial species in its habitat and also in the intestine.Search for other probiotic properties To determine the probiotic characteristics related to adhesins, exopolysaccharides, enzymes, antioxidants and selenium metabolism, the annotated genome sequence of L. pentosus CF-6HA (CECT 30896) was examined for the corresponding coding genes. In silico analysis of the annotated genome sequence of L. pentosus CF-6HA (CECT 30896) revealed the presence of an arsenal of genes encoding proteins related to probiotic functions. Some of these proteins are multifunctional, also involved in other functions such as survival, competitive exclusion of the pathogen to epithelial cells, stress response, drug efflux and others. Although Abriouel et al. (2016, 2017, 2022) reported that L.pentosus MP-10 and CF2-10N possess in their genome several multipurpose proteins responsible for their functionality; these are not the same proteins and in the cases where they are the same protein, the functions performed are different in each strain and depending on the environmental conditions. Regarding the proteins involved in adhesion, 20 genes encoding adhesion proteins were identified, 7 cell surface proteins; 4 mucosal binding proteins; 1 adhesion protein; 1 metal ABC transporter permease; 1 manganese ABC transporter substrate-binding protein; 1 ABC transporter, ATP-binding protein and permease; 1 ATP-binding cassette transporter of the ABC superfamily, binding protein; 1 metal ABC transporter substrate-binding protein; 1 beta-fructosidase and 2 hypothetical proteins. Regarding exopolysaccharide biosynthesis, it was associated with four coding genes.Regarding the enzymes involved in key probiotic properties (such as oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases and translocases) the presence of 1 gene encoding bile salt hydrolase, 2 genes encoding tannase, 1 gene encoding alpha-amylase, 1 gene encoding amylopullulanase, 1 gene encoding catalase and 23 different genes encoding lactase (beta-galactosidase or beta-glucosidase) were identified. With reference to antioxidants, 11 genes encoding glutathione biosynthesis, 4 genes encoding 4-carboxymeuconolactone decarboxylase, 1 gene encoding 2-Cys peroxiredoxin, 1 gene encoding NAD(FAD)-dependent dehydrogenase, 1 gene encoding putative iron-dependent peroxidase, and 1 gene encoding Halo peroxidase were identified. Finally, the genome of L.pentosus CF-6HA (CECT 30896) in search of genes involved in selenium metabolism and 4 coding genes were identified: serine-tRNA ligase, homocysteine ​​methyltransferase, cysteine ​​sulfinate desulfinase and MULTISPECIES: serine-tRNA ligase. In vitro analysis of the ability to grow and biotransform selenite In addition, the ability to grow and biotransform selenite (Na2 SeO3) of L. pentosus CF-6HA (CECT 30896) was tested in vitro by spectrophotometrically measuring the residual Se in the supernatant after 24 h of growth in MRS broth supplemented with Se. The cells were grown in 5 ml of MRS or MRS-Se broth supplemented with 20 ppm Se (as Na2 SeO3) at 37ºC for 24 h as described by Martínez et al. (2020). The concentration of SeO. 2−3 remaining in the supernatants was determined spectrophotometrically by the modified method of Brown and Watkinson (Kessi et al., 1999) using a microplate assay (Martínez et al., 2020). The results obtained showed its growth capacity producing a 100% removal of Se since no detectable Se was recorded. On the other hand, the selenium biosorption capacity on the surface of L. pentosus CF-6HA (CECT 30896) in the form of nanoparticles (SeNPs) was examined by scanning electron microscope (SEM) coupled to X-ray energy dispersive spectroscopy in the presence and absence of Se in culture broth. For this purpose, L. pentosus CF-6HA (CECT 30896) was grown in MRS or MRS-Se broth supplemented with 5 ppm Se (as Na2SeO3) at 37°C for 24 h (Martínez et al., 2020).A drop of the bacterial pellet was spotted onto SEM sample stubs (ANAME, Spain), dried, and then dehydrated in a series of 20, 40, 60, 80, and 100% ethanol solutions (15 min each) before being suspended in acetone for 1 h. The stubs were then subjected to critical point drying prior to SEM imaging (FESEM, MERLIN by Carl Zeiss, Oxford) equipped with an energy-dispersive X-ray (EDX) analyzer. The presence of Se on the cell surface of L. pentosus CF-6HA (CECT 30896) in the form of white nanoparticles (SeNPs) was visualized and confirmed (Figure 4). Furthermore, no cell shape transformation was observed upon growth in the presence of Se and subsequent SeNP formation.This strain tolerates selenium in the culture broth and metabolizes selenium according to the presence of relevant coding genes (serine-tRNA ligase, homocysteine ​​methyltransferase, cysteine ​​sulfinate desulfinase and MULTISPECIES: serine-tRNA ligase) for proteins capable of biotransforming and reducing toxic selenite (SeO3. 2- ) into elemental selenium (Se 0). Thus, the strain can completely remove Se from the culture supernatant. Other lactobacilli belonging to other species isolated from plants or other fermented foods (sauerkraut) were able to reduce selenite, such as L. plantarum CRL 2030 (Martínez et al., 2020) and L. casei ATCC 393 (Qiao et al., 2023). This important and effective approach for the bioremediation of selenium contamination using L. pentosus CF-6HA (CECT 30896) presents an additional attraction that could be exploited in food, agriculture and the environment where the accumulation of this highly soluble and bioavailable micronutrient can be a great challenge. Furthermore, as a starter culture, L. pentosus CF-6HA (CECT 30896) could also be used for the biofortification of fermented foods with selenium. Statistical analysis All analyses were performed in triplicate.Statistical analyses were performed using Excel 2016 (Microsoft Corporation, Redmond, WA, USA) to determine means and standard deviations. Bioinformatics analysis was performed using the R package. References Abriouel et al., 2012. Characterization of lactic acid bacteria from naturally-fermented Manzanilla Aloreña green table olives. Food Microbiol. 32, 308–316. https: / / doi.org / 10.1016 / j.fm.2012.07.006 Abriouel et al., 2016. Complete genome sequence of a potential probiotic, Lactobacillus pentosus MP-10, isolated from fermented Aloreña table olives. Genome Announc. 4, e00854–e00816. https: / / doi.org / 10.1128 / genomea.00854-16 Abriouel et al., 2017. Insight into potential probiotic markers predicted in Lactobacillus pentosus MP-10 genome sequence. Front. Microbiol. 8, 891. https: / / doi.org / 10.3389 / fmicb.2017.00891 Abriouel et al. 2019. New insights into the role of plasmids from probiotic Lactobacillus pentosus MP-10 in Aloreña table olive brine fermentation. Sci.Rep. 9, 10938. https: / / doi.org / 10.1038 / s41598-019-47384-1 Abriouel et al., 2022. In silico genomic analysis of the potential probiotic Lactiplantibacillus pentosus CF2-10N reveals promising beneficial effects with health promoting properties. Front. Microbiol.13, 989824. https: / / doi.org / 10.3389 / fmicb.2022.989824 Aeron et al., 2011. Multifarious activity of bioformulated Pseudomonas fluorescens PS1 and biocontrol of Sclerotinia sclerotiorum in Indian rapeseed (Brassica campestris L.). Eur. J. Plant Pathol.131, 81–93. https: / / doi.org / 10.1007 / s10658-011-9789-z Alcock et al., 2023. CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res.51(D1), D690-D699. https: / / doi.org / 10.1093 / nar / gkac920 Arndt et al., 2016. PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res.44, W16–W21. https: / / doi.org / 10.1093 / nar / gkw387 Darling et al. 2004.Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004 Jul;14(7):1394-403. doi: 10.1101 / gr.2289704 https: / / doi.org / 10.1016 / j.foodres.2012.10.004 Benítez-Cabello, A., Torres-Maravilla, E., Bermúdez-Humarán, L., Langella, P., Martín, R., Jiménez-Díaz, R., Arroyo-López, F. N., 2020. Probiotic Properties of Lactobacillus strains isolated from Table Olive Biofilms. Probiotics and Antimicrob. Proteins, 12(3), 1071-1082. https: / / doi.org / 10.1007 / s12602-019-09604-y Biswas et al., 2016. CRISPR Detect: a flexible algorithm to define CRISPR arrays. BMC Genom.17, 356. https: / / doi.org / 10.1186 / s12864-016-2627-0 Blin et al.., 2021. antiSMASH 6.0: improving cluster detection and comparison capabilities. Nucleic Acids Res.49(W1), W29-W35. https: / / doi.org / 10.1093 / nar / gkab335 Bortolaia et al., 2020. ResFinder 4.0 for predictions of phenotypes from genotypes. J. Antimicrob. Chemother.75, 3491–3500. https: / / doi.org / 10.1093 / jac / dkaa345 Camacho et al., 2009.BLAST+: architecture and applications. BMC Bioinform. 10(1), 421. https: / / doi.org / 10.1186 / 1471-2105-10-421 Chen, Y. S., Wang, Y. C, Chow, Y. S., Yanagida, F., Liao, C. C., Chiu, C. M., 2014. Purification and characterization of plantaricin Y, a novel bacteriocin produced by Lactobacillus plantarum 510. Arch. Microbiol. 196(3), 193-9. https: / / doi.org / 10.1007 / s00203- 014-0958-2 Conesa et al., 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinform. 21, 3674–3676. https: / / doi.org / 10.1093 / bioinformatics / bti610 Crowley et al., 2013. Current perspectives on antifungal lactic acid bacteria as natural bio- preservatives. Trends Food Sci. Technol. 33, 93–109. https: / / doi.org / 10.1016 / j.tifs.2013.07.004 Elbehiry et al., 2022. Pseudomonas species prevalence, protein analysis, and antibiotic resistance: an evolving public health challenge. AMB Expr 12, 53. https: / / doi.org / 10.1186 / s13568-022-01390-1 Kaas et. al., 2014.Solving the Problem of Comparing Whole Bacterial Genomes across Different Sequencing Platforms. PLoS ONE 9(8), e104984. https: / / doi.org / 10.1371 / journal.pone.0104984 Kanehisa et al.., 2016. BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J. Mol. Biol. 428, 726-731. https: / / doi.org / 10.1016 / j.jmb.2015.11.006 Kessi et al.1999. Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum. Appl. Environ. Microbiol. 65, 4734–4740. https: / / doi.org / 10.1128 / AEM.65.11.4734-4740.1999 Leekitcharoenphon et al., 2014. Evaluation of Whole Genome Sequencing for Outbreak Detection of Salmonella enterica. PLoS ONE 9(2), e87991. https: / / doi.org / 10.1371 / journal.pone.0087991 Liu et al., 2019. VFDB 2019: a comparative pathogenomic platform with an interactive web interface. Nucleic Acids Res.47, D687–D692. https: / / doi.org / 10.1093 / nar / gky1080 Martínez et al., 2020.Biotransformation of Selenium by Lactic Acid Bacteria: Formation of Seleno-Nanoparticles and Seleno-Amino Acids. Front. Bioeng. Biotechnol. 8, 506. https: / / doi.org / 10.3389 / fbioe.2020.00506 Mohamad et al., 2018. Evaluation of the antimicrobial activity of endophytic bacterial populations from chinese traditional medicinal plant licorice and characterization of the bioactive secondary metabolites produced by Bacillus atrophaeus against Verticillium dahliae. Front. Microbiol.9, 924. https: / / doi.org / 10.3389 / fmicb.2018.00924 Mora-Villalobos et al., 2020. Multi-Product Lactic Acid Bacteria Fermentations: A Review. Ferment.6, 23. https: / / doi.org / 10.3390 / fermentation6010023 Paysan-Lafosse, et al., 2022 InterPro in 2022 Nucleic Acids Research, Nov, (doi: 10.1093 / nar / gkac993 Pérez-Díaz, et al., 2021. Genotypic and phenotypic diversity among Lactobacillus plantarum and Lactobacillus pentosus isolated from industrial scale cucumber fermentations. Food Microbiol.94, 103652. https: / / doi.org / 10.1016 / j.fm.2020.103652 Pérez Montoro, et al., 2016. Fermented Aloreña table olives as a source of potential probiotic Lactobacillus pentosus strains. Front. Microbiol. 7, 1583. https: / / doi.org / 10.3389 / fmicb.2016.01583 Qian at al., 2003, The ND-Tree: A Dynamic Indexing Technique for Multidimensional Non- ordered Discrete Data Spaces, Proceedings 2003 VLDB Conference, Morgan Kaufmann, 620-631,https: / / doi.org / 10.1016 / B978-012722442-8 / 50061-6 Qiao et al.2023. Selenite Bioremediation by Food-Grade Probiotic Lactobacillus casei ATCC 393: Insights from Proteomics Analysis. Microbiol. Spectr. e00659-23. https: / / doi.org / 10.1128 / spectrum.00659-23 Stamatakis, A., 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinform.30(9), 1312-3. https: / / doi.org / 10.1093 / bioinformatics / btu033 Sun et al., 2015. Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera. Nat. Commun. 6, 8322. https: / / doi.org / 10.1038 / ncomms9322 Tenea, G. N., Ascanta, P., 2022. Bioprospecting of Ribosomally Synthesized and Post- translationally Modified Peptides Through Genome Characterization of a Novel Probiotic Lactiplantibacillus plantarum UTNGt21A Strain: A Promising Natural Antimicrobials Factory. Front. Microbiol.13, 868025. https: / / doi.org / 10.3389 / fmicb.2022.868025 Tóth et al., 2021. Mobile Antimicrobial Resistance Genes in Probiotics. Antibiotics 10(11), 1287. https: / / doi.org / 10.3390 / antibiotics10111287 van Heel et al., 2018. BAGEL4: a user-friendly web server to thoroughly mine RiPPs and bacteriocins. Nucleic Acids Res.46(W1), W278-W281. https: / / doi.org / 10.1093 / nar / gky383 Wu et al., 2011. WebMGA: a customizable web server for fast metagenomic sequence analysis. BMC Genom.12, 444. https: / / doi.org / 10.1186 / 1471-2164-12-444 Zankari et al., 2020. PointFinder: a novel web tool for WGS-based detection of antimicrobial resistance associated with chromosomal point mutations in bacterial pathogens. J.Antimicrob. Chemother.72(10), 2764-2768. https: / / doi.org / 10.1093 / jac / dkx217 Zhang et al., 2004. A greedy algorithm for aligning DNA sequences. J. Comput. Biol.7, 203– 214. https: / / doi.org / 10.1089 / 10665270050081478 Zhou et al., 2011. PHAST: a fast phage search tool. Nucleic Acids Res. 39, W347–W352. https: / / doi.org / 10.1093 / nar / gkr485.

Claims

1. Lactiplantibacillus pentosus CF-6HA strain deposited in the Spanish Type Culture Collection under accession number CECT 30896.

2. Pure bacterial culture or supernatant isolated from the strain of claim 1.

3. Composition comprising the culture or supernatant according to claim 2.

4. Non-therapeutic use of the strain according to claim 1, the pure bacterial culture according to claim 2, and / or the composition comprising the culture according to claim 3 as a probiotic for plants.

5. Non-therapeutic use of the strain according to claim 1, the pure bacterial culture according to claim 2 and / or the composition comprising the culture according to claim 3 as an antimicrobial agent against Pseudomonas fragi. 6.Non-therapeutic use according to the preceding claim, characterized in that it also acts simultaneously against one or more of the microorganisms selected from the list consisting of: Enterococcus faecalis, Bacillus cereus, Staphylococcus aureus, Listeria innoccua, Escherichia coli, Salmonella enterica, Candida albicans, Pseudomonas syringae and Verticillium dahliae.

7. Non-therapeutic use of the isolated supernatant according to claim 2 and / or of the composition comprising the supernatant according to claim 3 as an antimicrobial compound or composition against Pseudomonas syringae, Verticillium dahliae, Pseudomonas fragi, Staphylococcus aureus, Bacillus cereus, Escherichia coli and / or Candida albicans.

8. The strain of claim 1, the pure bacterial culture of claim 2, and / or the composition comprising the culture of claim 3 for use as a probiotic medicament for humans and non-human mammals. 9.Strain according to claim 1, the pure bacterial culture according to claim 2 and / or the composition comprising the culture according to claim 3 for use as an antimicrobial medicament against Pseudomonas fragi.

10. Strain, culture and / or composition for use according to the preceding claim, characterized in that it is also used simultaneously as an antimicrobial medicament against one or more of the microorganisms selected from the list that.

11. The isolated supernatant according to claim 2 and / or the composition comprising the supernatant according to claim 3 for use as an antimicrobial medicament against Pseudomonas syringae, Verticillium dahliae, Pseudomonas fragi, Staphylococcus aureus, Bacillus cereus, Escherichia coli and / or Candida albicans.

12. Use of the strain according to claim 1 for obtaining pediocin and Plantaricin Y.

13. Use of the strain according to claim 1 for bioremediation of media contaminated with selenium.

14. Use of the strain according to claim 1 for the bioenrichment of fermented foods with selenium.

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