Lactobacillus salivarius strain and uses thereof

Abstract

The present invention relates to an isolated strain of Ligilactobacillus salivarius deposited at the "Colección Española de Cultivos Tipo (CECT)" under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

Description

[0001] LACTOBACILLUS SALIVARIUS STRAIN AND USES THEREOF

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to the prevention and / or treatment of mental disorders.

[0004] BACKGROUND OF THE INVENTION

[0005] Depression is a disorder in which negative emotions arise due to the change in brain function for controlling emotions. Over 300 million persons are suffering from depression all over the world, a total of 5.0% of adults and 5.7% of adults older than 60 have depression. Suicide, which is driven by depression, is the fourth leading cause of death in 15- to 29-year-olds. Depression as well as anxiety and mood disorder are not only a medical concern but also a social problem.

[0006] Representative antidepressants used in depression treatment are a tricyclic antidepressant such as imipramine and a selective serotonin reuptake inhibitor (SSRI). However, SSRI-based drugs such as trazodone and mirtazapine have a problem in stability, and particularly, when the elderly take these drugs, a possibility of increased risk of stroke, fractures and death is raised.

[0007] Previous studies have shown that the composition of the microbiota in the host gut might promote or alleviate the pathogenesis of depression. Moreover, probiotics could counteract gut dysbiosis and produce anti-depressant-like effects. Among different probiotics, Lacticaseibacillus seems to alleviate Escherichia co / / -induced depression and reduce cognitive impairment in mice by regulating IL-6 expression (Yun S-W. et al., Benef Microbes 2021 Nov 16;12(6):541-551). In addition, it has been known that Lactobacillus plantarum PS128 can reduce the severity of anxiety and depression symptoms, adjust autonomic nervous system function, and improve the sleep quality (H. Y-T. et. al., Nutrients 2021 Aug 17;13(8):2820).

[0008] There are no biomarkers for depression and mood disorders, which makes drug development more difficult. Therefore, there is a current need for alternative treatments of depression.

[0009] SUMMARY OF THE INVENTION

[0010] The inventors of the present invention have found that Ligilactobacillus salivarius deposited at the “Coleccibn Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 has an effect in the central nervous system for the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, anxiety disorder and depression. A first aspect of the present invention relates to an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0011] In a second aspect, the invention relates to a pharmaceutical composition comprising a therapeutic effective dose of an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or variant thereof and an acceptable excipient for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0012] In a third aspect, the invention relates to a combination comprising a) a therapeutic effective dose of an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof or a pharmaceutical composition comprising said strain or bacterial culture and an acceptable excipient and b) a compound suitable for the prevention and / or treatment of mood disorder, an anxiety disorder and / or depression.

[0013] In a fourth aspect, the invention relates to the combination of the invention for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1. Latency period in the splash test (ST).

[0016] Figure 2. Number of colony forming units (CFU) in spleen and liver induced during chronic mild stress (CMS) and subsequent treatment with the L salivarius (CECT 30632) strain.

[0017] Figure 3. Amount of lipopolysaccharides (LPS) and lipopolysaccharide binding proteins (LBP) in plasma, induced due to chronic moderate stress (CMS) and after treatment with the L. salivarius strain (CECT 30632). CT: Control

[0018] Figure 4. Total oxidant capacity (T-AOC) and oxidized glutathione (Oxidized GSH) induced after chronic moderate stress (CMS) and after treatment with the L. salivarius strain (CECT 30632).

[0019] Figure 5. Rate of conversion of tryptophan into kynurenine due to CMS and after treatment with the L. salivarius strain (CECT 30632). Figure 6. Plasma levels of Tryptophan, kynurenine and serotonine precursor 5- HT after chronic mild stress (CMS) and after treatment with the strain of L. salivarius (CECT 30632).** p<0.01 ; *** p<0.001

[0020] Figure 7. Kynurenic acid (KYNA) and quinolinic acid (QUINA) plasma levels and ratio between both levels after chronic moderate stress and after treatment with the L. salivarius strain. * p<0.05; ** p<0.01; *** p<0.001 ; **** p<0.0001

[0021] Figure 8. IL-6 levels in plasma after chronic mild stress (CMS) and after treatment with the strain of L. salivarius (CECT 30632).

[0022] Figure 9. TNF-alpha levels in frontal cortex, after chronic mild stress (CMS) and after treatment with the L. salivarius strain. * p<0.05

[0023] Figure 10. Tryptophan, kynurenine and 5-HT levels in the frontal cortex after CMS and after treatment with the L. salivarius strain (CECT 30632). * p<0.05; ** p<0.01

[0024] Figure 11. Indolamine 2,3-dioxygenase (IDO) conversion ratio (kynurenine vs tryptophan levels), in frontal cortex, after chronic moderate stress (CMS) and treatment with the strain of L. salivarius (CECT 30632).

[0025] Figure 12. Levels of kynurenic acid (KYNA) and quinolinic acid (QUINA) in the frontal cortex and ratio between them (excitotoxicity risk) after chronic moderate stress (CMS) and treatment with the L. salivarius strain (CECT 30632) * p<0.05.

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] The inventors of the present invention have found that Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 has antidepressant effect.

[0028] In a first aspect, the invention relates to an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0029] Alternatively, the invention relates to a method for preventing and / or treating a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression which comprises administering an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof to a subject in need thereof. Alternatively, the invention relates to the use of an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof for the preparation of a medicament for preventing and / or treating a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0030] As used herein the term “isolated strain” refers to a bacterial strain that has been removed from its natural milieu. The term “isolated” does not necessarily reflect the extent to which the microbe has been purified. In contrast, the phrase “substantially pure culture” refers to a microbial culture that contains substantially no other microbes than the desired strain or strains, and is substantially free of other contaminants, which can include microbial contaminants as well as unwanted chemical contaminants.

[0031] Methods for the isolation of bacteria are widely known in the art. One such method consists in spreading samples obtained from the tissue of interest, e.g. a sample of the vaginal mucosa, onto selective agar plates followed by incubation in aerobic or anaerobic conditions as to isolate bacteria. Follow isolation, bacteria can be identified by 16S rDNA sequencing (Kullen et al., J. Appl. Microbiol. 2000;89:511-518).

[0032] The term "Ligilactobacillus salivarius” or "Lactobacillus salivarius” as used herein refers to an isolated strain of the species Ligilactobacillus salivarius having identifying characteristics that render the strain suitable for use with the present invention. Exemplary non-limiting identifying characteristics can include an anti-inflammatory response, a detoxifying effect of the blood from bacterial translocation, increased membrane permeability, an antioxidant capacity and a neuroprotective balance. A particular strain suitable for use with the present invention can exhibit one or more identifying characteristics. The anti-inflammatory response can be determined by any method known in the art, for example by measuring the levels of IL-6 and TNF-alpha as shown in Example 2d) or 3a) of the application. The detoxifying effect of the blood from bacterial translocation can be determined by any method known in the art, for example by measuring the LPS transport as shown in Example 2a). The membrane permeability can be determined by any method known in the art, for example by measuring the levels of bacterial debris in plasma, such as lipopolysaccharides as shown in Example 2a). The antioxidant capacity can be determined by any method known in the art, for example by measuring the antioxidant capacity (T-AOC) or oxidized glutathione (Oxidized GSH X) as shown in Example 2 b). The neuroprotective balance can be determined by any method known in the art, for example by studying the metabolism of tryptophan and kynurenines both in plasma and in the prefrontal cortex, as shown in Example 2c) or 3 of the application. In a particular embodiment, the variant of the isolated strain for use according to the invention is an isogenic mutant, that is, a strain that has a genome that only differs from the L. salivarius CECT 30632 strain by the presence of a mutation.

[0033] In another particular embodiment, the degree of identity between a variant of the isolated strain for use according to the invention and the parental strain is determined as the average nucleotide identity (ANI), which detects the conservation of the DNA of the central genome (Konstantinidis K and Tiedje JM , 2005, Proc. Natl. Acad. Sci. USA 102: 2567-2592). In some embodiments, the ANI between the variant of the isolated strain of the invention and the L. salivarius CECT 30632 is about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.5 %, of about 99.6%, of about 99.7%, of about 99.8%, of about 99.9%, of about 99.99%, of about 99.999%, of about 99.9999%, of about 99.99999%, about 99.999999% or more but less than 100%.

[0034] In another embodiment, the degree of relationship between the variant of the isolated strain for use according to the invention and the L. salivarius CECT 30632 is determined as the tetranucleotide signature frequency correlation coefficient, which is based on oligonucleotide frequencies (Bohlin J. et al. 2008, BMC Genomics, 9: 104). In some embodiments, the tetranucleotide signature frequency correlation coefficient between the mutant and L salivarius CECT 30632 is approximately 0.99, 0.999, 0.9999, 0.99999, 0.999999, 0.999999 or more, but less than 1.

[0035] In another embodiment, the degree of relationship between the variant of the isolated strain for use according to the invention and the L. salivarius CECT 30632 is determined as the degree of similarity obtained by analysing the genomes of the parent and the variant strain by pulsed field gel electrophoresis (PFGE) using one or more endonucleases of restriction. The degree of similarity obtained by PFGE can be measured by the coefficient of similarity of dice. In some embodiments, the given similarity coefficient between the variant of the isolated strain for use according to the invention and the L salivarius CECT 30632 is about 95%, about 96%, about 97%, about 98%, about 99%, about 99, 1%, of about 99.5%, of about 99.6%, of about 99.7%, of about 99.8%, of about 99.9%, of about 99.99%, of about 99.999%, from about 99.9999%, about 99.99999%, about 99.999999% or more but less than 100%.

[0036] In another embodiment, a strain is considered a variant of a given parental strain when both strains have the same ribotype, as obtained using any of the methods known in the art and described, for example, by Bouchet et al. (Clin. Microbiol. Rev., 2008, 21 : 262-273).

[0037] In another embodiment, the degree of relationship between the variant of the isolated strain for use according to the invention and the L. salivarius CECT 30632 is the Pearson correlation coefficient obtained by comparing the genetic profiles of both strains obtained by repetitive PCR based on extragenic palindromic elements (REP-PCR) (see, for example, Chou and Wang, Int J Food Microbiol. 2006, 110: 135-48). In some embodiments, the Pearson correlation coefficient obtained by comparing the REP-PCR profiles of the mutant and the isolated strain of the invention is approximately 0.99, 0.999, 0.9999, 0.99999, 0,999999, 0,999999 or more, but less than 1.

[0038] In another embodiment, the degree of relationship between the variant of the isolated strain for use according to the invention and the L. salivarius CECT 30632 is the bonding distance obtained by comparing the genetic profiles of both strains obtained by Multilocus sequence sequencing (MLST) (see, for example, Maiden, MC, 1998 , Proc. Nati. Acad Sci. USA 95: 3140-3145). In some embodiments, the MLST binding distance of the mutant and L salivarius CECT 30632 is about 0.99, 0.999, 0.9999, 0.99999, 0,999999, 0,999999 or more but less than 1.

[0039] In another embodiment, the degree of relationship between the variant of the isolated strain for use according to the invention and the L. salivarius CECT 30632 is the distance of the variant to the isolated strain of the invention obtained by determining the dendogram of the PCR fingerprinting profiles obtained by random amplification of polymorphic DNA (RAPD), wherein the dendogram distances are calculated using the unweighted pair group method with arithmetic mean (see for example Sharm et al., Food Sci. Biotechnol. 2016 25(6), 1651-1655). In a particular embodiment, the variant of the isolated strain for use according to the invention has a dendrogram distance to the isolated strain of the invention of less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, less than 1 , less than 1, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1 , less than 0.1 , but more than 0.

[0040] In a particular embodiment, the degree of relationship between the variant of the isolated strain for use according to the invention and the L salivarius CECT 30632 is determined by PCR fingerprinting profiles obtained by RAPD, wherein the profiles are obtained through a PCR amplification reaction using a generic primer followed by gel electrophoresis of the PCR amplification product. The term “PCR amplification reaction” as used herein refers to the process of performing a copy of a region of nucleic acids, wherein said copy is multiplied exponentially using oligonucleotides, known usually as “primers”, which are complementary and hybridize with the extremes of said region being amplified. In a particular embodiment the amplification reaction is performed in a thermocycler using the following conditions:

[0041] Initial denaturation of 3 minutes at 96°C,

[0042] First amplification phase comprising at least 10 cycles of the following steps:

[0043] • Denaturation at 94°C for 30 seconds; • Annealing at 30°C for 30 seconds; and

[0044] • Extension at 72°C for 60 seconds;

[0045] Second amplification phase comprising at least 20 cycles of the following steps:

[0046] • Denaturation at 94°C for 30 seconds;

[0047] • Annealing at 30°C for 60 seconds; and

[0048] • Extension at 72°C for 85 seconds;

[0049] The term “generic primers” as used herein refers to primers which are shorter, between 8 to 12 base pairs of random sequence, which serve as both forward and reverse primer, and are usually able to amplify fragments at least 5 genomic sites simultaneously.

[0050] In a particular embodiment the generic primer is selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, preferably SEQ ID NO: 4

[0051] Once the PCR amplification product is obtained the PCR profile is created by running the amplification product in a gel electrophoresis. In a particular embodiment, the electrophoresis is carried out in a 2% agarose gel for 90 minutes at 90 volts.

[0052] The isolated strain for use according to the invention L. salivarius CECT 30632 is herein described equally and interchangeably with the acronym “MP98” or “ES27”.

[0053] In another particular embodiment, the variant of the isolated strain for use according to the invention has a genome that shares a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the genome of the L. salivarius CECT 30632. In another particular embodiment, the variant of the of the L. salivarius CECT 30632 is characterized by the presence of a 16S rRNA gene that has at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.7 %, or at least 99.8 %, or at least 99.9% sequence identity with SEQ ID NO: 1 .

[0054] SEQ ID NO: 1

[0055] GACAGTTACTCTCACTCGTGTTCTTCTCTAACAACAGAGTTTTACGATCCGAAGACCTTCTTCACTCACG CGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGG CCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATCACCTTGGTAGGCCGT TACCCCACCAACTAGTTAATACGCCGCGGGTCCATCTAAAAGCGATAGCAGAACCATCTTTCATCTAAGG ATCATGCGATCCTTAGAGATATACGGTATTAGCACCTGTTTCCAAGTGTTATCCCCTTCTTTTAGGCAGG TTACCCACGTGTTACTCACCCGTCCGCCACTCAACTTCTTACGGTGAATGCAAGCATTCGGTGTAAGAAA GTTTCGTTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGGNTCAAACTCTAN

[0056] In another particular embodiment, the variant of the L salivarius CECT 30632 is characterized by the presence of a 16S rRNA gene that has at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.7 %, or at least 99.8 % or at least 99.9% sequence identity with SEQ ID NO: 2.

[0057] SEQ ID NO: 2

[0058] TAGAAAGGAGGTGATCCAGCCGCAGGTTCTCCTACGGCTACCTTGTTACGACTTCACCCCAATCATCTGT CCCACCTTAGACGGCTGGCTCCTTGCGGTTACCCCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGA CGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATGCTGATTCGCGATTACTAGCGATTCCG ACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTAAACCTCGC GGTCTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTT GACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCGCCAGAGTGCCCAACTTAATGCTGGCAA CTGACAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCAT GCACCACCTGTCACTTTGTCCCCGAAGGGAAAGCCTAATCTCTTAGGTGGTCAAAGGATGTCAAGACCTG GTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCT TTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTATTGCGTTAGCTGCGGCACTGAAGGGC GGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTA CCCACGCTTTCGAACCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACTGGTGTTCTTCCATAT ATCTACGCATTTCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGTCTTCCAGTTTCCAA TGCACTACTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAAAAGACCGCCTGCGTTCCCTTTACGCCC AATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTGC TGGTTAGATACCGTCATCGAATGAACAGTTACTCTCACTCGTGTTCTTCTCTAACAACAGAGTTTTACGA TCCGAAGACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACT GCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTAC GTATCATCACCTTGGTAGGCCGTTACCCCACCAACTAGTTAATACGCCGCGGGTCCATCTAAAAGCGATA GCAGAACCATCTTTCATCTAAGGATCATGCGATCCTTAGAGATATACGGTATTAGCACCTGTTTCCAAGT GTTATCCCCTTCTTTTAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCAACTTCTTACGGTGA ATGCAAGCATTCGGTGTAAGAAAGTTTCGTTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCT GAGCCAGGATCAAACTCTCATTTTAAAGTTTGTGA

[0059] In a preferred embodiment, the sequence identity is determined throughout the whole length of the SEQ ID NO: 1 or 2. In another preferred embodiment, the sequence identity is determined throughout the whole length of the 16S rRNA gene of the variant. The terms “identity”, “identical” or “percent identity” in the context of two or more amino acid or nucleotide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotide or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. Publicly available software programs can be used to align sequences. Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percentage identity “X” of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y / Z), where Y is the number of nucleotide residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the second sequence is longer than the first sequence, then the global alignment taken the entirety of both sequences into consideration is used, therefore all letters and null in each sequence must be aligned. In this case, the same formula as above can be used but using as Z value the length of the region wherein the first and second sequence overlaps, said region having a length, which is substantially the same as the length of the first sequence.

[0060] For instance, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0061] The invention also relates to a bacterial culture composition comprising the isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0062] The term “bacterial culture” in the context of the present invention refers to a population of living bacterial cells. Several types of culture and culture conditions are part of the expert general knowledge, which would have any difficulty in setting up and cultivating the strain of the invention. Several types of culture are known in the art, such as liquid culture or broth culture, agar plate cultures, agar-based dipsticks and stab cultures. The cultures are grown according to their requirements. In a particular embodiment of the bacterial culture of the invention, the bacterial culture is grown on MRS agar medium. In another particular embodiment of the MRS agar is supplemented with L-cysteine or horse blood, preferably from about 1.5 g / L to about 3.5 g / L of L-cysteine or from about 2.5 % weight per volume (w / v) to about 7.5 % w / v horse blood. In another particular embodiment of the bacterial culture of the invention, the bacterial culture is incubated at a temperature from about 30°C to about 42 °C, preferably 37°C, during from about 24 h to about 72 h, preferably 48 h. In a particular embodiment of the bacterial culture of the invention, the culture is incubated in anaerobic conditions, wherein the incubation environment comprises from about 80% to about 90% nitrogen, 5% to about 15% hydrogen and 1% to about 10% carbon dioxide, wherein the sum of the gas percentages is 100%.

[0063] The bacterial culture for use according to the invention can be preserved and presented in a solution, suspension, powder, lyophilized, etc. In a particular embodiment of the bacterial culture of the invention, the bacterial culture is lyophilized or freeze-dried powder.

[0064] As used herein, the term “freeze dried / lyophilized” refers to a laboratory method where live microbes in aqueous suspension are rapidly frozen to <50° C, and then the majority of the frozen water content is forced to sublime under vacuum conditions, allowing this water to be efficiently removed in the gaseous phase. Protocols for the lyophilization of bacterial cultures are known in the art such as, without limitation, the protocol disclosed by (Shekh et al., Ann Microbiol 2020, 70, 16). In a particular embodiment of the culture for use according to the invention, the bacterial culture is lyophilized in the presence of wall materials selected from a group consisting of: fructooligosaccharide, inulin, lactulose, sucrose, fructooligosaccharide and skim milk, inulin and skim milk, lactulose and skim milk, sucrose and skim milk. In a more particular embodiment of the bacterial culture for use according to the invention, the bacterial culture is lyophilized in the presence of skim milk.

[0065] The term “wall material” as used herein refers to the material or substance used in the process of the lyophilization that is used to encapsulate or trap the microorganism to be encapsulated (in the present case the isolated strain of the invention). This material is also called the encapsulation material, cover membrane, shell, vehicle, or external phase matrix.

[0066] The invention also relates to a pharmaceutical composition comprising a therapeutic effective dose of an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof and an acceptable excipient for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0067] Alternatively the invention relates to a method for treating and / or preventing a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression which comprises administering a pharmaceutical composition comprising a therapeutic effective dose of an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or variant thereof and an acceptable excipient to a subject in need thereof.

[0068] Alternatively, the invention relates to the use of a pharmaceutical composition comprising a therapeutic effective dose of an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or variant thereof ad an acceptable excipient for the preparation of a medicament for treating and / or preventing a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0069] The expression “therapeutic effective dose” as used herein refers to an amount of an active agent (i.e. , an ingredient such as a bacterial strain, in the present invention is the strain L. salivarius CECT 30632 or a variant thereof) high enough to deliver the desired benefit, either the treatment or prevention of the illness, but low enough to avoid serious side effects within the scope of medical judgment. The particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations. In a particular embodiment of the pharmaceutical composition of the invention the therapeutic effective dose is from about 1 x 109cfu to about 1 x 1010cfu, preferably 0.8 x 1010cfu or 0.9 x1010cfu or 1 x1010cfu.

[0070] The expression “colony forming units” as used herein refers to an estimation of the number of bacteria or fungal cells in a sample that are viable, able to multiply via binary fission under the controlled conditions. The number of viable cells present in a sample can be multiplied by a diluting factor to obtain CFU / mL. In addition, the weight of sample can be measured to obtain a measurement in mg / mL.

[0071] The pharmaceutical composition can be formulated as a solution, a suspension, a tablet, a cream, etc. In a particular embodiment, the pharmaceutical composition for use according to the invention is formulated for oral, vaginal, intramammary, rectal, anal, intravenous, intraarterial, parenteral, intracranial, topical, respiratory or nasal administration. In a more preferred embodiment, the pharmaceutical composition for use according to the invention is formulated for sublingual administration, particularly in the form of disintegrating tablet, film, lollipop, toothpaste, sublingual drops or chewing gum. In another preferred embodiment, the pharmaceutical composition for use according to the invention is formulated for rectal administration, for example in the form of sugar pill or enema. In another preferred embodiment, the pharmaceutical composition for use according to the invention is formulated for nasointestinal administration, particularly in the form of suspension. In another preferred embodiment, the pharmaceutical composition for use according to the invention is formulated for nasogastric administration, particularly in the form of suspension. In another preferred embodiment, the pharmaceutical composition for use according to the invention is formulated for vaginal, topical or nasal administration.

[0072] In a preferred embodiment, the isolated strain, bacterial culture or pharmaceutical composition for use according to the invention is to be administered orally, more particularly in the form of tablet, capsule, effervescent powder or tablet, hydrogen solution or suspension. Therefore, the isolated strain, bacterial culture or pharmaceutical composition is formulated as oral composition.

[0073] Solid dosage forms for oral administration include conventional capsules, sustained release capsules, conventional tablets, sustained-release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules and gels. In the solid dosage forms, the active ingredient is admixed with at least one suitable excipient or carrier, such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, such as for example, starches, lactose, sucrose, mannitol, or silicic acid; (b) binders, such as for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, or acacia; (c) humectants, such as for example, glycerol; (d) disintegrating agents, such as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, or sodium carbonate; (e) solution retarding agents, such as for example, paraffin; (f) absorption accelerators, such as for example, quaternary ammonium compounds; (g) wetting agents, such as for example, cetyl alcohol or glycerol monostearate; (h) adsorbents, such as for example, kaolin or bentonite; and / or (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules and tablets, the dosage forms may also comprise buffering agents. Solid formulations of a similar type may also be used as fillers in soft or hard filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols, and the like. Solid dosage forms such as coated tablets, capsules and granules can be prepared with coatings or shells, such as enteric coatings and others known in the art. They may also contain opacifying agents, and can be formulated such that they release the active ingredient or ingredients in a delayed manner. Examples of embedding formulations that can be used are polymeric substances and waxes. The active ingredients can also be in micro-encapsulated form, if appropriate, with one or more of the aforementioned excipients

[0074] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing suitable excipients or carriers used in the art. In addition to the active ingredient, the liquid dosage form may contain one or more excipients or carriers commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, particular cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame seed oil, Miglyol®, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, or mixtures of these substances, and the like. In addition to said inert diluents, the formulation can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfuming agents. Suspensions, in addition to the active ingredient or ingredients, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol or sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, or tragacanth, or mixtures of these substances, and the like.

[0075] In another particular embodiment, the pharmaceutical composition of the invention can be formulated as a topical composition. Said topical pharmaceutical compositions can be adapted to apply to the skin and mucosa in the form of: a non-ionic vesicular dispersion, emulsion, cream, lotion, gel, aerosol, cream-gel, gel-cream, suspension, dispersion, powder, solid stick, wipe, poultice, foam, spray, oil, ointment, fluid, soap, sanitary towel, ovule, pessary tampon, rectal suppository, vaginal suppository or any other form that is known in the art of cosmetics and pharmacy.

[0076] The terms “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier”, refer to any compound or combination of compounds that is essentially nontoxic to the subject at the dosage and concentration employed, and is compatible with the other components of a pharmaceutical composition. Thus, an excipient is an inactive substance formulated alongside the active ingredient of a pharmaceutical composition, for the purpose of bulking-up compositions that contain said active ingredients. Bulking up allows convenient and accurate dispensation of a drug substance when producing a dosage form. Excipients also can serve various therapeutic-enhancing purposes, such as facilitating compound (drug) absorption or solubility, or other pharmacokinetic considerations. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. The selection of appropriate excipients depends upon the rout of administration and the dosage form, as well as the active ingredient and other factors. An excipient can be a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. Illustrative, non-limitative, examples of excipients or carriers include water, salt (saline) solutions, alcohol, dextrose, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, monoglycerides and diglycerides of fatty acids, fatty acid esters petroetrals, hydroxymethyl cellulose, polyvinylpyrrolidone and the like. In a particular embodiment of the pharmaceutical composition for use according to the invention, the composition further comprises a component selected from a group consisting of: prebiotic, vitamin, mineral, metal, oligoelement, plant-derived component, fungal-derived component, carotenoid, anti-oxidant, or any combination thereof.

[0077] As used herein, the term "prebiotic" includes substances or compounds that beneficially affect the host mammal by selectively promoting the growth and / or activity of one or more probiotic bacteria in the gastrointestinal tract of the host mammal, thus maintaining normal health or improving health of the host. Typically, prebiotics are carbohydrates, (such as oligosaccharides), but the term "prebiotic" as used herein does not preclude non-carbohydrates (e.g., polyphenols).

[0078] Non-limiting examples of prebiotics suitable for use in the pharmaceutical composition for use according to the invention include inulin, psyllium, fructooligosaccharides, oligofructose, galacto-oligosaccharides, isomalto-oligosaccharides xylo-oligosaccharides, soy-oligosaccharides, gluco-oligosaccharides, mannanoligosaccharides, arabinogalactan, arabinxylan, lactosucrose, gluconannan, lactulose, polydextrose, oligodextran, gentioligosaccharide, pectic oligosaccharide, xanthan gum, gum arabic, hemicellulose, resistant starch and its derivatives, and mixtures and / or combinations thereof.

[0079] The isolated strain, bacterial culture or pharmaceutical composition previously defined is for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0080] The term “prevention", as used herein, relates to the capacity to prevent, minimize, or hinder the onset or development of a disease or condition before its onset.

[0081] As used herein, the terms "treat", "treatment", "treatment", or "amelioration". The term refers to therapeutic treatment, the purpose of which is to reverse, reduce, suppress, delay or stop the progression or severity of the condition associated with the disease or disorder. The term "treatment" includes reducing or alleviating at least one adverse effect or condition of a condition, a disease or disorder. Treatment is usually "effective" when one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if disease progression is delayed or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the interruption of at least a condition that indicates the progression or worsening of symptoms that would be expected in the absence of treatment. The beneficial or desirable clinical outcome, whether detectable or not, is a reduction in one or more symptoms, a reduction in the extent of the disease, a stable (ie, not aggravated) condition of the disease. These include, but are not limited to, delayed or slowed progression, amelioration or alleviation of the disease state, and remission (partial or total). The term "treatment" of a disease also includes providing relief from symptoms or side effects of the disease (including symptomatic treatment).

[0082] The term "subject" or “patient”, as used herein, refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, e.g., human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats or rodents. Preferably, the subject is a male or female human of any age or race.

[0083] The isolated strain or pharmaceutical composition for use according to the invention may be dosed according to the requirements of the treatment and under medical guidance as to obtain the desired outcome. In a particular embodiment the isolated strain, the bacterial culture or the pharmaceutical composition for use according to the invention is administrated at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times a day. In another particular embodiment the isolated strain, the bacterial culture or the pharmaceutical composition for use according to the invention is administrated at least 1 time, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times a week. In another particular embodiment, the isolated strain, the bacterial culture or the pharmaceutical composition for use according to the invention is administered at least once a day, at least once a week, at least once a month.

[0084] The dosing of the isolated strain of the invention can combine several regimes of administration, having interval periods wherein no dose is administered between periods of administration.

[0085] Together with the dosing the isolated strain, the bacterial culture or the pharmaceutical composition for use according to the invention can be formulated in several forms depending on the desired use. In a particular embodiment, the isolated strain, the bacterial culture or the pharmaceutical composition is formulated for oral, sublingual, rectal, nasointestinal, nasogastric, vaginal, topical o nasal administration, preferably oral.

[0086] In another preferred embodiment of the medical use of the invention, the isolated strain of the invention or the bacterial culture of the invention is in the form of a food product in combination with an appropriate amount of an edible ingredient.

[0087] The term "food product" as used herein includes both solid and liquid ingestible materials for man or animals, which materials usually do, but need not, have nutritional value. Thus, food products includes meats, gravies, soups, convenience foods, malt, alcoholic, and other beverages, milk and dairy products, seafoods including fish, crustaceans and mollusks, candies, vegetables, cereals, soft drinks, snacks, dog and cat foods, other veterinary products.

[0088] Within the context of the present invention, the term "edible ingredient" means any kind of edible ingredient or mixture of ingredients that may be safely consumed by a human or animal.

[0089] In a particular embodiment of the food product for use according to the invention is selected from the group consisting of a milk product, a yogurt, a curd, a cheese, a fermented milk, a powdered milk, a milk based fermented product, a meat based fermented product, an ice-cream, a cereal based fermented product, a beverage, a snack, a flour, a chewing-gum, a sweet, a sweet food, a pet food, a dietary or food supplement, a functional food, a clinical nutrition formula, a nutritional complement, a formula for pregnant woman, a formula for the elderly and an infant formula.

[0090] In another preferred embodiment of the medical use of the invention, the isolated strain of the invention of the bacterial culture of the invention is in the form a nutraceutical in combination with a food acceptable adjuvant and / or food ingredient.

[0091] In the present invention, the term "nutraceutical" is understood to refer to a food that has a beneficial effect on health. Similarly, the term "nutraceutical" may be applied to chemical extracts or compounds derived from common foods. Examples of foods to which nutraceutical properties are attributed include olive oil, red wine, broccoli and soya, etc. Nutraceuticals are usually employed in nutritional mixtures and in the pharmaceutical industry. Like various food products, some nutritional supplements may also be classified as nutraceuticals, for example fatty acids such as omega-3 derived from fish oils and from some vegetables or antioxidants and vitamins.

[0092] The expression “food acceptable adjuvant and / or food ingredient’’ according to the present invention refers to protective hydrocolloids (such as gums, proteins, modified starches), binders, film-forming agents, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilising agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, tastemasking agents, weighting agents, jellyfying agents, gel-forming agents, antioxidants and antimicrobials.

[0093] Moreover, a multi-vitamin and mineral supplement may be added to nutraceutical compositions for use according to the present invention to obtain an adequate amount of an essential nutrient, which is missing in some diets. The multi-vitamin and mineral supplement may also be useful for disease prevention and protection against nutritional losses and deficiencies due to lifestyle patterns.

[0094] The nutraceutical compositions for use according to the present invention may be in any galenic form that is suitable for administering to the body, especially in any form that is conventional for oral administration, e.g. in solid forms such as (additives / supplements for) food or feed, food or feed premix, fortified food or feed, tablets, pills, granules, dragees, capsules and effervescent formulations, such as powders and tablets, or in liquid forms, such as solutions, emulsions or suspensions as e.g. beverages, pastes and oily suspensions. The pastes may be incorporated in hard or soft shell capsules, whereby the capsules feature e.g. a matrix of (fish, swine, poultry, cow) gelatine, plant proteins or ligninsulfonate. Examples for other application forms are those for transdermal, parenteral or injectable administration. The dietary and pharmaceutical compositions may be in the form of controlled (delayed) release formulations.

[0095] In another preferred embodiment of the medical use of the invention, the isolated strain of the invention of the bacterial culture of the invention is in the form of medical device comprising an effective amount of the isolated strain of the invention or the bacterial culture of the invention as an active ingredient.

[0096] The term "medical device" refers to, as defined by the European Directive 2007 / 47 / EC, any product, instrument, device, apparatus, computer program, material or article used in the medico sanitary field and being regulated by the European Directive 90 / 385 / CEE of active implantable medical device, 98 / 79 / CE of medical devices for in vitro diagnostic, and 93 / 42 / CEE for general medical devices. The medical devices are included in the sanitary technologies. They are used in human beings for the diagnostic, prevention, control, treatment or alleviation of a disease, for the compensation of a deficiency; for investigating, substituting or modification of anatomy or a physiologic process; and for the regulation of contraception. Medical devices for use according to the present invention relate to compositions or formulations ("medical device compositions"), or to products or devices comprising said compositions or formulations, comprising an effective amount of the isolates strain of the invention (L. salivarius CECT 30632 or a variant thereof) as active ingredient. The term "medical device composition" is used herein to identify the compositions according to the invention, which compositions, as such (by their selves) are catalogued by the competent sanitary authorities as medical devices.

[0097] The medical device for use according to the present invention allows the application or administration of the strain of the invention to a desired tissue surface, for example, in order to pharmaceutically and / or via the medical device itself treat or take care of the surface to which it is applied. In some embodiments, the composition comprising the strain of the invention or any mutant or variant thereof is per se catalogued by the authorities as a medical device. In other particular embodiments of the medical devices, they correspond to sanitary articles that include woven or nonwoven fabrics, in which the strain of the invention is homogeneously distributed along the surface of the fabric. This distribution can be performed, optionally, with the aid of a solvent. The solvent may also optionally be dried after layer or coating of the composition comprising the effective amount of the strain, is totally or partially distributed onto the surface of the fabric, thus obtaining fabrics with the properties conferred by the strain according to the invention.

[0098] The invention relates to the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0099] “Mood disorder” also known as an affective disorder, refers to any of a group of conditions of mental and behavioral disorder where a disturbance in the person's mood is the main underlying feature. Mood disorders fall into seven groups, including; abnormally elevated mood, such as mania or hypomania; depressed mood, of which the best-known and most researched is major depressive disorder (MDD) (alternatively known as clinical depression, unipolar depression, or major depression); and moods which cycle between mania and depression, known as bipolar disorder (BD) (formerly known as manic depression). There are several sub-types of depressive disorders or psychiatric syndromes featuring less severe symptoms such as dysthymic disorder (similar to MDD, but longer lasting and more persistent, though often milder) and cyclothymic disorder (similar to but milder than BD).

[0100] “Anxiety disorder”, as used herein, Illustrative, non-limitatives examples of anxiety disorders are generalized anxiety disorder (GAD), panic disorder, phobias and separation anxiety.

[0101] “Depression”, as described in the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-V) published by the American Psychiatric Association, and the International Classification of Diseases, tenth edition (ICD-10), includes different subtypes of mental illnesses and disorders with: (1) depression and mood disorders, such as depression, mania, mixed mania, and hypomania; (2) depressive disorders, such as depressive disorder and bad mood; (3) other affective disorders, for example, caused by ill-health, which include different mental illnesses or subtypes of disorders with characteristics of depressive disorders and affective disorders caused by substances (for example, addictive drugs) or therapy (such as surgery, radiotherapy, or chemotherapy); (4) bipolar disorder or bipolar affective disorder, which contains two or more episodes of depression and hypomania, and alternating episodes of mania and depression.

[0102] In a preferred embodiment the disorder or disease is selected from the group consisting of major depressive disorder, dysthymic disorder, psychotic depression, persistent depressive disorder, melancholic depression, catatonic depression, menopause depression, premenstrual dysphoric disorder, antenatal depression, postpartum depression, seasonal affective disorder, bipolar disorder, bipolar depression, atypical depression, childhood depression, minor depressive disorder, depressions caused by chronic medical conditions such as cancer or chronic pain or chemotherapy, manic depressive disorder, endogenous depression, manic depression reactions, reactive depression, depressions accompanied by agoraphobia and panic attacks, depression accompanied by anxiety, mental depression, Persistent Depressive Disorder (dysthymia), cyclothymic disorder, chronic stress, post-traumatic stress disorders, acute stress, social anxiety disorder, disruptive mood dysregulation disorder and mental illness associated with chronic stress. More preferably, the disorder or disease is major depressive disorder. In another preferred embodiment, the disorder or disease is acute stress.

[0103] In a preferred embodiment, the particular disorders or diseases corresponds to the disorders or diseases defined according to The Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM5*). According to DSM5*, major depressive disorder, persistent depressive disorders or other depressive disorders not otherwise specified and those included in ethiological classifications (including dual pathologies, premenstrual and others). Similarly for anxiety disorders (including generalized, posttraumatic stress disorders, acute, adaptative and other stress related disorders).

[0104] “Major depressive disorder”, MDD, also known as clinical depression, is a mental disorder characterized by at least two weeks of pervasive low mood, low self-esteem, and loss of interest or pleasure in normally enjoyable activities.

[0105] “Acute stress”, as used herein relates to an intense, unpleasant, and dysfunctional reaction beginning shortly after an overwhelming traumatic event and lasting less than a month. If symptoms persist longer than a month, people are diagnosed as having posttraumatic stress disorder (PTSD). In another preferred embodiment, the disease or disorder is severe, mild or moderate depression. The exact classification is based on many factors which include the types of symptoms experience, their severity, and how often they occur.

[0106] “Severe depression” is used to refers when a person's depression symptoms are very intense, often enough to interfere with many daily functions.

[0107] “Mild depression” involves depressive symptoms that are considered low-grade.

[0108] A person suffering “moderate depression” may experience symptoms that are more serious in terms of severity and duration than someone with mild depression. They may also experience more symptoms than a person with mild depression does.

[0109] In another preferred embodiment, the subject to be treated according to the invention suffers anhedonia.

[0110] “Anhedonia”, as used herein relates to a diverse array of deficits in hedonic function, including reduced motivation or ability to experience pleasure, near-complete absence of enjoyment, motivation, and interest. Among other types, anhedonia may be social anhedonia or physical anhedonia.

[0111] In another preferred embodiment, the mood disorder, anxiety disorder or depression is a single episode, recurrent, persistent, refractory or treatment resistant disease

[0112] “Single episode”, refers to a type of depression that is characterized by a single episode of major depression.

[0113] “Recurrent” Recurrent depressive disorder, also known as recurrent depression, dysthymic disorder, and high-functioning depression, is listed in the DSM-5 and refers to the depression after more than one episode.

[0114] “Persistent depression”, as used herein relates to depression that lasts for 2 years or more.

[0115] “Treatment resistant”, TRD or refractory depression, is a type of major depressive disorder ( DD). It happens when at least two different first-line antidepressants are not enough to manage the condition during a depressive episode. The two treatments must be of adequate dosage and duration (at least six to eight weeks) before a healthcare provider can diagnose TRD.

[0116] Combination of the invention and medical use thereof

[0117] In another aspect, the invention relates to a combination comprising a) a therapeutic effective dose of an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof or a pharmaceutical composition comprising said strain or bacterial culture and an acceptable excipient and b) a compound suitable for the prevention and / or treatment of mood disorder, an anxiety disorder and / or depression.

[0118] In another aspect, the invention relates to the combination of the invention for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression. Alternatively, the invention relates to a method for treating and / or preventing a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression which comprises administering a combination of the invention to a subject in need thereof. Alternatively, the invention relates to the use of a pharmaceutical composition comprising a therapeutic effective dose of combination of the invention for the preparation of a medicament for treating and / or preventing a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

[0119] “Combination", as used herein, stands for the various combinations of an isolated strain of Ligilactobacillus saliva us deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof or a pharmaceutical composition comprising said strain or bacterial culture and an acceptable excipient and a compound suitable for the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression in a composition, in a combined mixture composed from separate formulations of the single active compounds, such as a "tank-mix", and in a combined use of the single active ingredients when applied in a sequential manner, i.e. one after the other with a reasonably short period, such as a few hours or days or in simultaneous administration.

[0120] The compounds of the combination may be formulated for its simultaneous, separate or sequential administration. This has the implication that the combination of the two compounds may be administered:

[0121] - as a combination that is being part of the same medicament formulation, the two compounds being then administered always simultaneously.

[0122] - as a combination of two units, each with one of the substances giving rise to the possibility of simultaneous, sequential or separate administration.

[0123] “Compound suitable for the prevention and / or treatment of mood disorder, an anxiety disorder and / or depression”, as used herein relates to a compound to prevent the onset of the symptoms or complications, alleviate the symptoms or complications, or eliminate the mood disorder, an anxiety disorder and / or depression. In a preferred embodiment, the compound suitable for use in the the prevention and / or treatment of mood disorders, including anxiety disorder or depression is selected from the group consisting of selective serotonin reuptake inhibitors (SSRIs, i.e. escitalopram, fluoxetine, paroxetine, sertraline and others), serotonin-norepinephrine reuptake inhibitors (SNRIs, i.e. venlafaxine, desvenlafaxine, duloxetine, milnacipram and others), tricyclic antidepressants (i.e. amitriptiline, clomipramine, doxepine, imipramine, desipramine, maprotiline, nortriptyline and other) and monoamine oxidase inhibitors (MAOIs, i.e. moclobemide) and other antidepressants such as NA or DA reuptake inhibitors (i.e. reboxetine, bupropion), melatonin agonists (agomelatine), alpha2 blockers (mianserine, mirtazapine) and multimodal antidepressants (vortioxetine, vilazodone, trazodone and others). Also other antidepressant drugs based on specific BDNF approaches (s-ketamine) orallopregnanolone like compounds. Similarly, anxiolitics such as benzodiacepine compounds, 5HT1a partial agonists (i.e. buspirone class) or mood stabilizers (carbamacepine, valproate, lamotrigine and others).

[0124] All the terms and embodiments previously mentioned in relation to the medical use of the isolated strain or a variant thereof, or a bacterial culture or a pharmaceutical composition equally apply to the combination of the invention and the medical use thereof.

[0125] ***

[0126] The invention will be described by way of the following examples, which are to be considered as merely illustrative and not limitative of the scope of the invention.

[0127] EXAMPLES

[0128] MATERIALS AND METHODS

[0129] CHRONIC MILD STRESS ANIMAL MODEL

[0130] The chronic stress study (chronic mild stress model - CMS) is a behavioral test in vivo in rats, which analyses specific analytes in plasma and in prefrontal cortex. Young male adult rats Wistar Hannover (HsdRccHan:Wist), with initial weight between 200 and 225 grams and not consanguineous, were used throughout. Animals were housed in individual cages under standard humidity and temperature conditions, with a 12-hour light / dark cycle (switched on at 8 am) and with water and food ad libitum. They were kept under constant conditions for the 7 days prior to the onset of stress. All experimental protocols followed the guidelines of the Animal Experimentation Committee of the Complutense University of Madrid, Spain, in accordance with Spanish legislation (RD 53 / 2013, of 1 February) and European legislation in force (2010 / 63 / EU) and all necessary permits were obtained. In order to minimize animal suffering and the number of animals used, the three R principles (reduction, replacement and refinement) were applied wherever possible.

[0131] The depression model was adapted from that proposed by Willner (Willner et al., Psychopharmacology. 1987. 93:358-364) and known as "chronic mild stress" (CMS). It is a chronic stress of mixed physical-psychological type, which is unpredictable, inescapable and uncontrollable for animals, which results in the induction of depressive type behavior, in addition to sharing biochemical and structural alterations related to this psychiatric disease. These characteristics give the model sufficient robustness and potential for experimental approaches to the study of depression as it is endorsed by numerous studies that support its validity of content, prediction and construct (Willner, Neurobiol Stress. 2016. 6:78-93, Neurobiol Sress. 2016. 6:68-77):

[0132] 1. Content validity: CMS induces in animals many of the typical symptoms of human depression, validated through corresponding behavioral tests, such as anhedonia, apathy and lack of motivation, anxiety or weight loss, even biochemical alterations such as the decrease in the bioavailability of serotonin, inflammatory markers in plasma and the increase of the main stress hormone (in humans cortisol, in rodents corticosterone).

[0133] 2. Predictive validity: the main pharmacological treatments used in humans against depression (tricyclic antidepressants, selective serotonin reuptake inhibitors, selective serotonin-noradrenaline reuptake inhibitors) are capable of reversing or attenuating the behavioral changes following CMS.

[0134] 3. Construct validity: chronic stress has been identified as one of the main triggers of depressive processes (Leonard and Song, Pharmacology, Biochemistry and Behavior. 54:299-304, 1996). The etiology of the disease is thus confirmed in this model.

[0135] As previously indicated, the protocol used is a modification of the original used by Willner (Willner et al., cited supra). The total duration of chronic stress was 21 days after having verified in previous studies of our research that it was sufficient to induce depressive behaviour and biochemical alterations related to depression (Garate et al., J Neuroinflammation. 2011. 8:151 , Bravo et al., Anesthesiology 2012. Vol 117, No 3). Each animal was individually housed and subjected to two different stressors changed daily every 12 hours, with two mornings of rest a week, following an irregular pattern that was unpredictable for the animals. The application of stressors over each of the three weeks is summarized in Table 1:

[0136] Table 1 : Chronic mild stress protocol.

[0137] The different stressors used constitute a stress of moderate intensity. The description is as follows:

[0138] • Matching animals: represents the invader-host paradigm. They also pair the animals after a period of deprivation of food and drink, which is an additional stress for animals.

[0139] • Flash lighting: changing light / dark cycle every 2 hours.

[0140] • Wet sawdust: moisten sawdust with 350 ml_ of water. At the end of the stress, it was changed to new sawdust.

[0141] • Food and drink deprivation: withdrawal of free access to food and drink.

[0142] • Tilted cage: inclination of the cages at a 45 degrees angle.

[0143] • Strobe: placement of an LED spotlight with a lighting frequency of 150 flashes / minute.

[0144] The report compares control / vehicle (placebo)Zstrain of L. salivarius with the group of rats that were stressed and received either placebo or L. salivarius strain.

[0145] Each rat received skim milk (1 mL) via intragastric route (control group), or a single intragastric dose of 1x 101° colony-forming units (cfu) of the tested strain dissolved in 1 mL of skim milk (treated group).

[0146] BEHAVIORAL STUDY

[0147] The behavioural study was carried out under controlled conditions of humidity, temperature, lighting and acoustics and minimizing as much as possible the time of the researcher in the behavioural room during their performance. The behaviour to be studied was recorded using a video camera, and the videos were later analysed with the researcher, who was unaware of the research conditions.

[0148] The behavioural testconsisted of Splash test (ST) (anhedonia-related behaviours and degree of personal self-care)

[0149] The "splash" test is another behavioural test about parameters related to depressive type behaviour and was performed by adapting a protocol previously used (Yalcin et al.,Eur J Pharmacol 2005 May 9;514(2-3):165-74.). The evaluation is carried out after day 21 of stress (day 22 of the experiment) through the grooming behaviour of the animal after spraying a solution of 10% sucrose in the dorsal coat during the activity phase and under dark conditions. Grooming is considered a personal hygiene index, as well as an indirect measure of appetite for a sweet solution related to anhedonic characteristics. Behaviours are analysed / quantified with the help of a camcorder for a period of 5 minutes: o Latency period (time until the start of grooming) o Grooming (duration of grooming)

[0150] SAMPLE COLLECTION AND PROCESSING

[0151] Biological sampling in rats was performed in a staggered and balanced manner between the different groups, to minimize possible interference by circadian variations of corticosterone levels. After an overdose of intraperitoneal anesthesia (320 mg / kg) with sodium pentobarbital (Vetoquinol), the absence of reflexes, both in foot and in tail, was verified, the chest cavity was opened after sterilization with 70% ethanol (v / v) and the removal of tissues. a) Extraction process:

[0152] • Plasma: blood was extracted by cardiac puncture after the opening of the chest cavity. Using sterile syringes loaded with 0.1 ml of ethylenediaminetetra-acetic acid (EDTA) 0.5 M as an anticoagulant. The separation of the different blood fractions was performed by centrifugation at 1500 r.p.m. for 15 minutes, at room temperature, and following a separation protocol based on Ficoll. Plasma and peripheral blood mononuclear cells (PBMCs) were stored at -80 °C until further use for the various tests.

[0153] • Brain: brain was extracted from skull separating and collecting the frontal cortex divided into 6 segments. They were immediately frozen in dry ice and stored at -80 °C for later use.

[0154] The choice of the frontal cortex is due to its involvement in depression (Drevets et al., J. Neurosci. 1992. 12,9:3628-3641 , Bremner et al., The American Journal of Psychiatry. 2000. 157,1:115-118) as it plays a crucial role in the so-called executive function that includes the processing of emotions and decision making and planning (Damasio et al., Nature volume 386, pages769-770 (1997)). It is a structure with special sensitivity towards pro and anti-inflammatory mediators induced by stress, the etiological factor of depression that emulates the animal model (de Pablos et al., J Neurosci. 2006 May 24;26(21):5709-19, Garcia-Bueno et al., Neuroscience & Biobehavioral Reviews 32(6):1136-5 2008a). In addition, previous studies have shown that TLR-4, the innate immunity receptor, is activated in the prefrontal cortex after CMS (Garate et al., J Neuroinflammation . 2011 Nov 3:8:151.).

[0155] • Mesenteric lymph nodes, spleen and liver: under sterile conditions, the same amount of each tissue type was weighed and homogenized in 2 ml_ of sterile saline for the subsequent microbiological cultures to be performed within the study of colon bacterial translocation.

[0156] • Descending colon: 2 cm were taken from the distal part of the colon (between the splenic angle and the rectum) and, after the cleaning of the feces, the extraction of proteins was carried out, preserving them at -80 °C for further analysis. This tissue area was chosen because the research focuses on the TLR-4 pathway and this area is the one with the highest amount of expression of this receptor (Wang et al., 2010 PLOS ONE 5(10): e13607 ). b) Sample processing:

[0157] • Cytosolic and nuclear fraction

[0158] The method described by Schreiber (Schreiber et al., Nucleic Acids Res Aug 11 ;17(15):64191989) was followed for the separation of cytosolic and nuclear cell fractions with some modifications. The entire protocol was performed at 4 °C. The tissue was homogenized (TyssueLyser, QUIAGEN) and a frequency of 50 oscillations / s in 600 pL of a buffer solution composed of: HEPES 10 mM at pH 7.9, EDTA 1 mM, EGTA 1 mM, KCI 10 mM, DTT 1 mM, 0.5 NaF 5 mM, NaVC 1 mM and N2MOO4 10 mM and a protease inhibitor cocktail composed of pancreas extract, pronase, thermolysine, chymotrypsin, trypsin and papain (complete, Roche). After 5 minutes on ice, the detergent Nonidet p-40 (Roche) was added to 0.05% and vigorously stirred with the vortex (VELP scientifica, Italy) to keep them again 5 minutes in ice. Samples were centrifuged for 5 minutes at 1620 g. The supernatant was collected and centrifuged for 3 minutes at 19083 g. The supernatant was collected, which constitutes the cytosolic fraction. The pellet was resuspended in 70 to 100 pl (depending on the amount of the pellet) of the initial buffer to which 20% glycerol and KCI were added to a final concentration of 0.4 M and was shaken vigorously for 30 minutes. A final centrifugation of 5 minutes was carried out at 19083 g and the supernatant was collected, that corresponds to the nuclear fraction. Both nuclear and cytosolic fractions were stored at -80 °C for further processing. This method is commonly used and has been shown to provide a high purity nuclear extract with virtually no cytosolic contamination (Garate et al., J Neuroinflammation Nov 3:8:151. 2011).

[0159] • RNA Extraction

[0160] Tissue (brain or colon) was homogenized under sterility conditions (TyssueLyser, QUIAGEN) in 600 pl of Trizol (Invitrogen, Life Technologies) for 4 minutes at 4 °C and a frequency of 50 oscillations / s. The supernatant was collected and 200 pl of chloroform were added and, after vortex and incubation of 2-3 minutes at room temperature, centrifuged at 12000 rpm for 15 minutes in cold. From the two resulting phases (aqueous and organic), the aqueous phase was collected and the extraction was repeated with 200 pl of Trizol and 40 pl of chloroform. The aqueous phase was collected and 400 pl of isopropanol added, leaving to stand after mixing in vortex overnight at 4 °C. Next, it was centrifuged at 16260 g for 10 minutes in cold, obtaining a pellet of RNA which was washed three times with 600 pl of 80 % ethanol, using water with diethylpyrocarbon free of RNAases and DNAases, (DEPC water, Invitrogen). Once the ethanol was evaporated, the pellet was resuspended in 50 pl of DEPC water and shaken with the vortex to ensure dilution. Samples were stored at -40 °C until further use.

[0161] • Preparation of total tissue homogenate

[0162] In order to study intercellular mediators and global protein quantities within the tissue, a procedure was performed to obtain total tissue homogenate. To do this, the portion of colon or brain was homogenized in 600 pl (TyssueLyser, QUIAGEN) to 50 oscillations / s and 4 °C for 4 minutes. Total homogenization was obtained by centrifuging at 12000 rpm for 10 minutes in cold and collecting the supernatant. Samples were stored at -80 °C until the corresponding tests were performed.

[0163] • Preparation for microbiology studies

[0164] Bacterial translocation was measured by the growth of colony-forming units (CFUs) in different organs. Mesenteric lymph nodes (GLMs), liver and spleen were selected, in addition to the blood itself. The entire procedure was performed under sterility conditions. Each tissue was weighed and homogenized in 2 mL of sterile saline 9% (v / v) by performing two serial dilutions of each sample and inoculating them in microbial culture media.

[0165] BIOCHEMICAL TECHNIQUES a) Microbiology studies

[0166] The culture media used were Blood Agar and MacConkey Agar 5% for aerobic bacteria count and Blood Agar Brucella supplemented with vitamin K1 for anaerobic bacteria. After 24 hours of incubation at 37 °C for aerobic bacteria and 48 hours for anaerobics, CFUs were counted as CFUs / mg of tissue. The identification of the different bacterial species was done by Gram staining and standard biochemical tests. In the case of aerobic microorganisms the following assays were used: the oxidase assays (Mast Diagnostics®), antibiotic discs of optoquine, bacitracin and novobiocin (Mast®), coagulase (Beckton Dickinson®), catalase, and the biochemical identification system BBLTM Enterotubo I ITM (Difco Laboratories, Detroit, Ml, USA). The API 20 NE system (Biomerieux®) was used for non fermenter Gram negative bacilli, and the API 20 Strep for enterococcus. For anaerobes, the identification system Rapid ID 32 A (Biomerieux®) was used. b) Determination of the corticosterone, lipopolysaccharide (LPS), Lipopolysaccharide binding protein (LSP), tryptophan (Trp), kynurenine (Kyn), serotonine (5-HT), quinolinic acid (QUINA) and kynurenic acid (KYNA)

[0167] LPS is a component of the cell wall of Gram-negative bacteria and a potent TLR- 4 activator. It can be used to measure bacterial translocation from the gut into the body after CMS, as measured by elevated levels of plasma lipopolysaccharide.

[0168] Commercial ELISA kits were used: Corticosterone ELISA Kit from ENZO Life Sciences; LAL Chromogenic Endpoint Assay and LBP ELISA kit from Hycult® Biotechnology, USA; Trp, Kyn, 5-HT ELISA kits from LDN; QUINA, KYNA ELISA kits from Cloud-Clone Corp., USA. Absorbance was measured with the Synergy 2 microplate reader (BioTek®, USA) and data processing was performed with the built-in Gen5 Data Analysis Software (BioTek®, USA). c) Quantitative polymerase chain reaction with reverse transcriptase or reverse transcription (RT)-qPCR

[0169] The determination of mRNA gene expression was carried out by the polymerase chain reaction technique after the conversion of the extracted RNA in the different tissues to complementary DNA (cDNA) by retrotranscription or reverse transcription.

[0170] • Reverse transcription (RT)

[0171] The amount of RNA extracted was quantified and its purity checked by spectrophotometry using NanoDrop 1000 (Thermo Fisher Scientific Inc., USA). RNA concentrations were normalized to 100 ng / I diluting with DEPC water in a 10 pl final volume and 4.5 pl of a mixture with Random primers, deoxynucleotide triphosphate (dNTPs) and DEPC water were added. The solution was subjected to a sequential heating of 10 minutes in the My Cycler thermocycler (Bio-Rad, USA): 1 min at 95 °C, 8 min at 70 °C and 1 min at 42 °C. This step allows the denaturing of the double strand of DNA and the random hybridization of the primers to the monocatenary chains. Then, 5.5 pl of a second mix formed by the retrotranscriptase enzyme, its corresponding buffer and RNAse inhibitors were added, and the samples were heated for 60 minutes at 42 °C for reverse transcription. Finally, 80 pl of DEPC water were added to the 20 pl reaction, and the cDNA was stored at -20 °C until further use.

[0172] • Quantitative-Polymerase Chain Reaction (qPCR)

[0173] The quantitative or real-time polymerase chain reaction (qPCR or real time-PCR) is a variant of conventional PCR that has the ability to amplify and, simultaneously, comparatively quantify the mRNA of the protein we intend to determine. The detection is possible thanks to the use of a non-specific intercalant fluorophore, in our case SYBR Green, excited by blue light (Amax = 488 nm) and emitting green light (Amax = 522 nm) collected by the Rotor-Gene 6000 thermocycler (Corbett Life Science, Germany).

[0174] For each sample, 2pl of cDNA and 18 ml of a mixture consisting of Quantimix Easy Master Mix (Biotools B&M Labs, Spain), water-DEPC and the first / primer specific for the protein sequence to be detected were mixed for each sample. After an initial heating of 3 minutes at 95 °C, the cycles to which the samples were subjected on the PCR were 10 s to 95 °C for denaturing, 15 s to 60 °C for alignment and 20 s to 72 °C for extension, repeating this process for a total of 35 amplification cycles. Relative mRNA concentrations were performed from the PCR take-off point using the built-in commercial software and normalized with tubulin levels.

[0175] Oligonucleotides used as primers / primers in qPCR were designed with the Primer3 tool (Untergasseret al., NucleicAcids Res Aug;40(15):e1152012). Its specificity for the target protein was tested through an in silico PCR available in USCS GenomeBrowser (Kent et al., Genome Res. 2002 Jun;12(6):996-1006.) and an alignment of Blast sequences (NCBI) against complementary DNA (cDNA) and genomic DNA (gDNA), selecting those primers / primers capable of hybridizing only with the target sequence. d) Western blot

[0176] • Protein quantification and sample preparation

[0177] Western blot protein expression assays require prior normalization of the total protein content in samples. The total protein content was measured according to the Bradford assay (Bradford, 1976) based on the change of absorbance of Coomassie® brilliant blue dye under acidic conditions which acquires a bluish tone when binding to proteins. The mechanism by which this happens is due to two types of interactions that the dye establishes with proteins. Its reddish form donates its free electron to ionizable protein groups causing exposure of hydrophobic zones present in the tertiary structure of the protein. This allows its interaction with the non-polar regions of Coomassie by forces of van der Waals, also allowing the binding of the dye to the protein to be strengthened by ionic interaction by approximating charges of different polarity. Changes resulting from this binding stabilize the bluish form of the dye and can be used as an indirect measure of the total amount of protein present in the samples. Absorbance is read at 595 nm with the microplate reader Synergy 2 (BioTek, USA) after 5 minutes of incubation, and the amount of protein is estimated by interpolation with a calibration line created from known concentrations of bovine serum albumin (Sigma) between 0 and 400 g / ml after prior dilution of the samples concerned. Known the protein concentration, samples were normalized to 1 pg / pl and the buffer Laemmli (BioRad, USA) was added with 5 % [3-mercaptoethanol as denaturing agent.

[0178] • Western blot

[0179] Wells were loaded with 15-20 g of each protein, reserving one for the molecular weight marker Precision Plus Protein (BioRad, USA), of polyacrylamide electrophoresis gels (SDS-PAGE) in acrylamide / bisacrylamide percentage (BioRad, USA) variable according to the molecular weight of the protein under study (Table 2). Electrophoresis was carried out at 120 V until complete migration of the front through the gel and the proteins were transferred to a nitrocellulose membrane Trans-Blot Midi Nitrocellulose (BioRad, USA) through the semi-dry transfer system T rans-Blot® T urbo T ransfer System (BioRad®, USA) for 10 minutes at 2.5 A and 2.5 V.

[0180] Membranes with transferred proteins were blocked for 1 hour with a 5% solution of skimmed milk or bovine serum albumin (PanReac, Germany) to avoid non-specific binding to the antibody, reducing background noise. Membranes were then incubated overnight at 4 °C with the protein-specific primary antibody to be detected at the appropriate dilution of TBS-Tween. After three 10-minute washes with Tris-saline buffer with Tween 20 (BioRad, USA) at 0.1% (TBS-Tween), the membranes were incubated with the appropriate dilution in TBS-Tween of the corresponding specific secondary antibody: mouse, rabbit or goat.

[0181] After eliminating the secondary antibody by performing three new washes with TBS-Tween, the development was performed by chemiluminescence applying the Amersham ECL Prime kit (GE Healthcare, United Kingdom) which allows catalyzed oxidation of luminol by action of peroxidase coupled to secondary antibodies. The detection of the emitted chemiluminescence was recorded with the Odyssey team Fc Imager (LI-COR, USA) and the quantification of the bands was carried out with ImageJ software (NIH, USA). The signal obtained was optimized by analyzing different temporary exposures to ensure the linearity of the intensity of the bands, always based on the available literature.

[0182] Beta-actin (p-actin) was used as load controls for total homogenates or cytosolic samples and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for nuclear samples. e) Statistical analysis

[0183] Data were expressed as the mean standard error (EMS). The ROUT test was performed for the detection of outliers, the Saphiro-Wilks test to evaluate the normality of distribution and the Brown- Forsythe test to check the homogeneity of variances. If the data presented a normal distribution and equality of variances, a one-way ANOVA was performed followed by a Tukey post-hoc for multiple comparisons between groups. If the distribution was normal but equal variances could not be assumed, a Brown- Forsythe ANOVA followed by a Tamhane T2 post-hoc was applied for multiple comparisons between groups. If the data did not show a normal distribution, a nonparametric Kruskal- Wallis test with a post-hoc Dunn was performed for multiple comparisons between groups. A value of p 0.05 was considered statistically significant. The data was analysed ing the GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA).

[0184] Example 1 -Behavioral studies

[0185] Treatment with L. salivarius strain (CECT 30632) significantly reduced the latency period (p<0.05) in the group of stressed mice (Fig. 1) showing ability to improve anhedonia and the degree of personal self-care. This is directly related with the hypothesis of a potential antidepressant effect of the CECT 30632strain of L. salivarius.

[0186] Example 2- Biochemical Results In Plasma

[0187] Several parameters were studied in plasma. One of the most interesting parameters was the bacterial translocation, where it was seen that the rats that had been treated with the strain of L. salivarius (CECT 30632) were able to reduce the amount of bacterial toxins (see below). a) Biochemical results in plasma - Bacterial translocation

[0188] The gut-brain axis (GBA) is a bilateral communication network between the gastrointestinal (Gl) tract and the central nervous system (CNS). The essential amino acid tryptophan contributes to the normal growth and health of both animals and humans and, importantly, exerts modulatory functions at multiple levels of the GBA. Tryptophan is used for protein synthesis, affecting the growth and health of animals and humans. Changes in the gut microbiota composition affect the GBA by modulating the tryptophan metabolism. Most tryptophan from ingested protein is absorbed in small intestine. The remaining tryptophan reaches the large intestine, where it is degraded by commensal microbes. Tryptophan is the sole precursor of serotonin [5-hydroxytryptamine (5-HT)], which is a key neurotransmitter involved in the modulation of emotional control, food intake, sleep and pain. Tryptophan can be metabolized into kynurenine, tryptamine and indolic compounds, thereby modulating neuroendocrine and intestinal immune responses and having profound effects on the interaction between gut microbiota and the GBA. Tryptamine, a monoamine that is structurally similar to serotonine, can be generated from the decarboxylation of tryptophan by tryptophan decarboxylases (T rpD) from commensal bacteria. Bacteria belonging to Clostridium, Ruminococcus, Blautia, and Lactobacillus have been identified as being able to convert tryptophan to tryptamine in a TrpD-dependent manner [Gao et al., Adv Nutr. 2020. 11 :709-723], When there is a depressive process, there is an increase in intestinal permeability which leads to the translocation of bacteria from the intestine to the blood, causing a generalized inflammatory process in the body that can be communicated to the brain, producing neuro inflammation and leading to depressive processes.

[0189] When bacterial translocation occurs and bacteria pass into the blood, it is expected to find high levels of bacterial debris in plasma, such as lipopolysaccharides (LPSs). LPSs are found in the plasma membrane of GRAM-negative bacteria. Many of these LPSs act as toxins and cause inflammation. If there are bacteria in the body it is expected to find LPSs in blood. Lipopolysaccharide binding proteins (LBPs) bind to LPSs by removing substances toxic to the body (LPSs), cleaning the blood and reducing the level of inflammation.

[0190] After CMS, treatment with CECT 30632strain of L. salivarius does not induce a statistically significant decrease of the presence of bacteria (measured in CFUs) in any of the organs analysed, although it seems to slightly decrease those present in the liver (Fig.2).

[0191] No significant differences were found between the LPS levels in the group that had not been stressed and in the CMS stressed group that had been treated with the L. salivarius strain (CECT 30632). However, LBP levels increased significantly after CMS and further treatment with the L. salivarius strain (CECT 30632) (p< 0.05) (Fig. 3). The first hypothesis proposed is that the increase in the LBP levels induced by the L. salivarius strain (CECT 30632) decreases LPS levels, possibly preventing generalized inflammation. However, LBP forms a complex with LPS with two almost antagonistic objectives: (1) to transport LPS for detoxification and (2) to form a complex together with the Cluster of Differentiation 14 (CD14) and the Myeloid Differentiation factor 2 (MD2) to activate the Toll Like Receptor 4 (TLR4) and then activate transcription factors like the Nuclear Factor kappa B (NK-kB) and the proteins which regulate interferon, which induce the production of proinflam matory cytokines like IL-1 , IL-6 and TNF-a. Therefore, the changes detected must be put into context with other data that give us information either on the detoxification of LPS (in which case the compounds could have a protective effect) or on the activation of TLR4 (increased inflammation). b) Biochemical results in plasma - Oxidative stress

[0192] The total antioxidant capacity (T-AOC) and oxidized glutathione (Oxidized GSH X) indicate the amount of free radicals present in the body. It is known in the literature that in the depressive process, free radicals are triggered in the body and vice versa, in a two-way relationship. The consumption of the strain of L. salivarius (CECT 30632) produced a close to significant increase (p=0.0511) in the T-AOC of the organism and therefore its ability to keep free radicals at bay (Fig. 4). Based on the current results, it can be affirmed that L. salivarius reduces this oxidative capacity when administered to animals subjected to a stress protocol that induces depressive-like behaviour. Additionally, animals treated with L. salivarius show a trend towards reducing levels of oxidized GSH, indicating that the administration of L. salivarius is decreasing oxidative stress levels in animals subjected to an experimental model of depression, thus demonstrating a certain antioxidant capacity. c) Biochemical analysis in plasma (and prefrontal cortex) - Kynurenines pathway

[0193] The metabolism of tryptophan and kynurenines has been studied both in plasma and in the prefrontal cortex.

[0194] More than 90% of total tryptophan is oxidized, via the kynurenine pathway, into kynurenine in the liver. This kynurenine pathway exerts a primary role in affecting tryptophan availability by the clearance of excess tryptophan. The tryptophan metabolism along the kynurenine pathway is mainly initialized by the induction of either of the rate limiting enzyme indoleamine-2,3-dioxygenase (IDO) or by tryptophan 2,3- dioxygenase (TDO). While IDO exists in various organs, such as the brain, the gastrointestinal (Gl) tract, and liver, TDO is almost entirely expressed in the liver. IDO can be activated in response to immune stimuli, with interferon-gamma being the most efficient inducer. Among other inflammatory conditions, overexpression of IDO can be observed in the colonic mucosa of patients with inflammatory bowel disease. TDO activity is regulated by tryptophan’s availability, with its activity being relatively stable, while stress-induced changes in the expression of TDO in the liver are primarily influenced by the activation of the hypothalamic-pituitary-adrenal axis through the action of glucocorticoids (Gao et al., Adv Nutr. 2020. 11:709-723).

[0195] In case of CMS, the production of indolamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) is induced, the TDO being expressed only in the liver. The production of IDO reduces the bio-availability of tryptophan, since it passes to the kynurenine pathway. In the absence of IDO, tryptophan is a precursor to serotonin (5- HT), which is a basic neurotransmitter, thanks to tryptophan hydrolase (TPH) and aromatic L-amino acid decarboxylase (DDC).

[0196] An indirect calculation was performed using the ratio of kynurenine and tryptophan levels to try to infer the possible degree of IDO activity due to CMS (Fig. 6). No changes were detected beyond a slight increasing trend in all groups compared to the control.

[0197] Plasma levels of Tryptophan, kynurenine and serotonine precursor 5-HT after chronic mild stress (CMS) and after treatment with the strain of L salivarius are shown in Fig. 6. When IDO is produced and tryptophan enters into the kynurenine pathway the following kynurenine metabolites are found:

[0198] 1) Kynurenic acid (KYNA), which is neuroprotective, since it is an antagonist of the glutamate N-methyl D-aspartate receptor (NMDA receptor antagonist). Typically, KYNA levels do not increase in cases of induced IDO production in CMS.

[0199] 2) Quinolinic acid (QUINA), which is neurotoxic, since it is an agonist of the glutamate receptor called N-methyl D-aspartate (NMDA agonist). It normally increases in cases of CMS-induced IDO production.

[0200] The ratio between the two metabolites (QUINA / KYNA) is what is measured. This ratio increases in the case of CMS, with levels returning to normal after the administration of antidepressants (AD).

[0201] No variation in the levels of tryptophan was seen with respect to the controls after CMS and a decrease in the levels was seen after CMS and treatment with the strain of L. salivarius (Fig 6). However, the levels of serotonin are stable in all the groups, indicating that the fluctuation of tryptophan is possibly due to the consumption of tryptophan to enter the kynurenines pathway to provide a neuroprotective balance QUINA / KYNA thanks to the tryptophan conversion to KYNA (Figure 7).

[0202] In groups treated with a strain of L. salivarius (CECT 30632), the QUINA:KYNA ratio obtained in plasma and in frontal cortex is neuroprotective, that is: KYNA is produced in a larger proportion that QUINA (Fig. 7). As seen before, both metabolites bind to the same brain receptor: the N-methyl-D-aspartate (NMDA) receptors, which regulate brain arousal. As the amount of KYNA, NMDA receptor antagonist, is greater, we say that excitotoxicity is reduced.

[0203] The results of kynurenine levels in the prefrontal cortex (see corresponding section) support what is observed in plasma, as they are the same or very similar. Furthermore, a statistically significant decrease in the TN Fa factor was seen in the prefrontal cortex, which again denotes the anti-inflammatory nature of the L salivarius strain (CECT 30632). d) Biochemical analysis in plasma - Inflammation

[0204] The effect of the treatment with the L. salivarius strain (CECT 30632) in modulating the levels of inflammatory cytokines IL-6 and TNF-alpha were studied.

[0205] IL-6 is related to an inflammatory process caused by CMS. IL-6 works controlling the extent of the inflammatory responses of the tissue. IL-6 helps affected cells to induce cellular immune responses directed against infection In the case under study, the L. salivarius strain did not show an effect over the increase produced in the IL-6 levels due to CMS (Fig. 8). The Lactobacillus strain diminishes the TNF-a levels in the frontal cortex of treated mice, particularly after CMS (Figure 9). However, CMS does not induce an increase of this parameter with respect to controls.

[0206] The results obtained for the parameters implicated in inflammation indicate that the administration of L. salivarius does not induce an inflammatory response at the peripheral level, as it does not affect IL-6 levels. This, along with the results obtained regarding TNF-alpha levels in the cerebral cortex, indicates that such administration is not only safe from the perspective of inflammation induction, but also has antiinflammatory properties at the CNS level, which has potentially significant therapeutic implications, considering the potential role of neuroinflammation in the pathophysiology of stress and depression.

[0207] Example 3- Biochemical Results In The Frontal Cortex

[0208] The objective is to determine parameters relative to inflammation (TNF-a) and to the kynurenine pathway (tryptophan, kynurenine, serotonin, quinolinic acid, kynurenic acid, IDO-1 mRNA and enzymes of KATs and Haao by mRNA synthesis). a) Frontal Cortex - Inflammation

[0209] TNF-alpha levels in the frontal cortex are measured in a CMS situation. TNF- alpha is a key cytokine in inflammatory reactions. Its neutralization is very important for the treatment of chronic inflammatory illnesses.

[0210] Treatment with L. salivarius strain (CECT 30632) was able to decrease the TNF- alpha levels in the frontal cortex, particularly under the CMS depression model (Fig. 9). CMS does not induce an increase in the TNF-alpha levels when compared with the controls. b) Frontal Cortex - Kynurenine pathway

[0211] The metabolism of tryptophan and kynurenines has been studied both in plasma and in the frontal cortex.

[0212] Immune stimulation [major depressive disorder (MDD) or chronic mild stress (CMS)] induces indolamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxigenase (TDO), which is expressed only in liver. IDO production decreases the bio-availability of tryptophan, as it moves to the IDO pathway [Martin-Hernandez et al, Molecular Neurobiology (2019), 56(1):490-501 , de 20192019], In the absence of IDO, tryptophan is a precursor of serotonin (5-HT), which is a basic neurotransmitter, thanks to the enzymes tryptophan hydrolase (TPH) and aromatic L- amino acid decarboxylase (DDC). Treatment with the L. salivarius strain (CECT 30632) decreased tryptophan levels and tended to decrease kynurenine levels in control groups (Figure 10). In the group under CMS, there is also a decrease in tryptophan and kynurenine levels in the untreated groups. On the other hand, no changes in IDO activity were detected by indirect mathematical calculation (Fig. 11).

[0213] Treatment with the strain of L. salivarius (CECT 30632) increased the levels of KYNA in the frontal cortex, with maintained QU I NA levels (Fig. 12). This decreases the risk of excitotoxicity (damage or death of nerve cells when levels of otherwise necessary and safe neurotransmitters become pathologically high). In the frontal cortex, no effects have been registered for the kynurenine metabolites QUINA and KYNA in the group exposed to CMS.

[0214] (Original in Electronic Form)

[0215] (This sheet is not part of and does not count as a sheet of the international application)

[0216] FOR RECEIVING OFFICE USE ONLY

[0217] FOR INTERNATIONAL BUREAU USE ONLY -5 This form was rec international Bure -5- Authorized officer

Claims

1. CLAIMS1. An isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

2. A pharmaceutical composition comprising a therapeutic effective dose of an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or variant thereof and an acceptable excipient for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

3. A combination comprising a) a therapeutic effective dose of an isolated strain of Ligilactobacillus salivarius deposited at the “Coleccion Espanola de Cultivos Tipo (CECT)” under the accession number CECT 30632 or a variant thereof, or a bacterial culture composition comprising said strain or a variant thereof or a pharmaceutical composition comprising said strain or bacterial culture and an acceptable excipient and b) a compound suitable for the treatment of mood disorder, an anxiety disorder and / or depression.

4. A combination according to claim 3 for use in the prevention and / or treatment of a disorder or disease selected from the group consisting of mood disorder, an anxiety disorder and depression.

5. The pharmaceutical composition for use according to claim 2 or the combination for use according to claim 4 wherein the therapeutic effective dose is from about 0.8x1010colony forming units (CFU) to about 1x101° CFU.

6. The pharmaceutical composition for use according to claim 5 or the combination for use according to claim 5, wherein the therapeutic effective dose is 0.9 x1010CFU.

7. The isolated strain or bacterial culture for use according to claim 1 , or the combination for use according to any one of claims 4 to 6, or the pharmaceutical composition for use according to any one of claims 2, 5 or 6, wherein the strain, bacterial culture or pharmaceutical composition is to be administered as a freeze-dried powder.

8. The isolated strain or bacterial culture for use according to any one of claims 1 or 7 or the combination for use according to any one of claims 4 to 7, or the pharmaceuticalcomposition for use according to any one of claims 2, 5 to 7, wherein the isolated strain, bacterial culture or pharmaceutical composition is to be administered by oral, sublingual, rectal, nasointestinal, nasogastric, vaginal, topical or nasal route..

9. An isolated strain or bacterial culture for use according to any one of claims 1,7 or 8 , or the combination for use according to any one of claims 4 to 8, or the pharmaceutical composition for use according to any one of claims 2, 5 to 8, wherein the disorder or disease is selected from the group consisting of major depressive disorder, dysthymic disorder, psychotic depression, persistent depressive disorder, melancholic depression, catatonic depression, menopause depression, premenstrual dysphoric disorder, antenatal depression, postpartum depression, seasonal affective disorder, bipolar disorder, bipolar depression, atypical depression, childhood depression, minor depressive disorder, depressions caused by chronic medical conditions such as cancer or chronic pain or chemotherapy, manic depressive disorder, endogenous depression, manic depression reactions, reactive depression, depressions accompanied by agoraphobia and panic attacks, depression accompanied by anxiety, mental depression, Persistent Depressive Disorder (dysthymia), cyclothymic disorder, chronic stress, post- traumatic stress disorders, acute stress, social anxiety disorder, disruptive mood dysregulation disorder and mental illness associated with chronic stress..

10. The isolated strain or bacterial culture for use according to claim 9 or the combination for use according to claim 9, or the pharmaceutical composition for use according to claim 9, wherein the disorder is major depressive disorder.

11. The isolated strain or bacterial culture for use according to any one of claims 1 , 7 to 10, or the combination for use according to any one of claims 4 to 10 or a pharmaceutical composition for use according to any one of claims 2 to 8, wherein the disease is severe, mild or moderate depression.

12. An isolated strain or bacterial culture for use according to any of claims claim 1 , 7 to 11 or the combination for use according to any one of claims 4 to 11 or a pharmaceutical composition for use according to any one of claims 2, 5 to 11 , wherein the use is for treating a subject suffering anhedonia.

13. An isolated strain or bacterial culture for use according to any one of claims 1 , 7 to 12, or the combination for use according to any one of claims 4 to 12, or a pharmaceutical composition for use according to any one of claims 2, 5 to 12, wherein the mood disorder, anxiety disorder or depression is a single episode, recurrent, persistent, refractory or treatment resistant disease.

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