Serotonin-producing bacteria

Abstract

The present invention relates to the selection of a bacterial strain for use in treating serotonin deficiency and / or a disease associated with serotonin deficiency by culturing a lactic acid-producing bacterial strain in a medium containing tryptophan under aerobic conditions, and detecting any serotonin produced by the lactic acid-producing bacterial strain in the medium. If serotonin is detected in the medium, the lactic acid-producing bacterial strain is selected as being effective in treating serotonin deficiency and / or a disease associated with serotonin deficiency in a subject.

Description

[Technical field]

[0001] The present invention relates generally to lactic acid producing bacteria, in particular to screening of such lactic acid producing bacteria for the production of serotonin, and to the use of such lactic acid producing bacteria in the treatment of serotonin deficiency and disorders and diseases associated with serotonin deficiency. [Background technology]

[0002] It is well established that interactions between the host and the gut microbes are fundamental to the health and well-being of the host. The gut microbiota provides nutrients to the host, but also produces metabolic products that may be involved in immune responses and the regulation and development of the host's immune system.

[0003] Lactobacillus and other lactic acid-producing bacteria, such as bifidobacteria, are commonly used as probiotics in various types of food, such as yogurt. The growth and colonization of harmful microorganisms can be prevented by such lactic acid-producing bacteria colonizing themselves in the intestinal ecosystem, for example by the formation of biofilms, by competition for available nutrients, and / or by the production of certain substances that lower the intestinal pH, such as hydrogen peroxide, bacteriocins and / or organic acids, including lactic acid and acetic acid.

[0004] Serotonin, or 5-hydroxytryptamine (5-HT), is a bioamine synthesized from the aromatic amino acid tryptophan and has several physiological functions including behavior, platelet activation and coagulation, intestinal transit, enteric nervous system (ENS) maturation, development, homeostasis and activity, intestinal immune system activity, energy metabolism (fasting adaptation, adipose tissue browning and lipolysis, hepatic glucose production, insulin secretion), gut-brain axis activity and bone metabolism and homeostasis. In humans, serotonin is found primarily in the gastrointestinal (GI) tract, platelets, and the central nervous system (CNS). In mammalian cells, serotonin production is enabled by the rate-limiting enzyme tryptophan hydroxylase (TPH), which produces 5-hydroxytryptophan (5-HTP). 5-HTP is the immediate precursor of serotonin and is converted to serotonin by aromatic L-amino acid decarboxylase (AADC). Two isoforms of TPH exist. TPH1 is primarily expressed in peripheral tissues, whereas TPH2 is primarily expressed in brain (CNS) and ENS neurons. TPH1 is the enzyme responsible for the majority of endogenous serotonin production, and this enzyme is expressed by highly specialized endocrine cells in the GI tract, called enterochromaffin (EC) cells. It has been clearly demonstrated that the gut microbiota participates in regulating endogenous host serotonin production by metabolic signaling to EC cells. Metabolites derived from the gut microbiota, such as short-chain fatty acids and secondary bile acids, have been shown to induce the expression of TPH1 in mouse EC cells, and correspondingly, the levels of endogenously produced serotonin are directly regulated by these bacteria. As a result, serum serotonin levels are substantially reduced in mice raised without microbial colonization, e.g., germ-free (GF) mice, compared to conventionally colonized control animals with a standard microbiota.

[0005] Serotonin deficiency occurs when the body does not have enough serotonin and can occur for several reasons. Localized serotonin deficiency in the gut or the rest of the body can cause pathological conditions including depression, anxiety, obsessive-compulsive disorder, irritable bowel syndrome, cardiovascular disease, osteoporosis, abnormal gastrointestinal motility, abnormal platelet aggregation, abnormal platelet activation, and abnormal immune response. Serotonin deficiency therefore presents a health risk. Various drugs have been developed to treat serotonin deficiency, such as selective serotonin reuptake inhibitors (SSRIs) and monoamine oxidase (MAO) inhibitors. However, these drugs are often accompanied by more or less severe side effects such as diarrhea, constipation, insomnia, dizziness, and dry mouth. Furthermore, these drugs are not suitable for everyone, for example SSRIs should not be taken by pregnant women, breastfeeding women, those under the age of 18, or those with diabetes, epilepsy, or kidney disease. In addition, both SSRI and MAO inhibitors may interact with other drugs, such as painkillers, other antidepressants, and cold and allergy medicines, and thus may cause serious adverse reactions.MAO inhibitors may also interact with some foods and drinks, causing dangerously high blood pressure.Due to these characteristics, known serotonin deficiency drugs are not suitable for a large group of patients, and therefore there is a great need to provide alternative, effective treatments for serotonin deficiency or disorders related to serotonin deficiency that have no or few side effects.

[0006] Taken together, there is a need for safe methods that can increase serotonin levels in patients with serotonin deficiency and that can be used to treat serotonin deficiency and diseases or disorders associated with serotonin deficiency. Summary of the Invention [Problem to be solved by the invention]

[0007] The general objective is to screen lactic acid producing bacterial strains for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0008] A specific objective is to screen for lactic acid-producing bacterial strains capable of producing serotonin locally in the gastrointestinal tract in the more oxygenated niches of the gut, such as near the mucosa. [Means for solving the problem]

[0009] These and other objectives are met by the embodiments disclosed herein.

[0010] The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0011] One aspect of the embodiment relates to a method for selecting a bacterial strain for use in treating serotonin deficiency and / or a disease associated with serotonin deficiency. The method includes culturing bacteria of a lactic acid-producing bacterial strain in a medium containing tryptophan under aerobic conditions. The method also includes detecting any serotonin produced by the bacteria of the lactic acid-producing bacterial strain in the medium or a sample thereof. The method further includes selecting the lactic acid-producing bacterial strain as effective for treating serotonin deficiency and / or a disease associated with serotonin deficiency in a subject if serotonin is detected in the medium or a sample thereof.

[0012] Another aspect of the embodiment relates to a method for selecting a bacterial strain for use in treating serotonin deficiency and / or a disease associated with serotonin deficiency. The method includes providing a germ-free Tph1- / - subject with bacteria of a lactic acid-producing bacterial strain. The method also includes detecting any serotonin produced by the bacteria of the lactic acid-producing bacterial strain in a body sample taken from the germ-free Tph1- / - subject. The method further includes selecting the lactic acid-producing bacterial strain as effective for treating serotonin deficiency and / or a disease associated with serotonin deficiency in the subject if serotonin is detected in the body sample.

[0013] Another aspect of the embodiment relates to a lactic acid producing bacterial strain capable of producing and releasing serotonin extracellularly under aerobic conditions for use in treating serotonin deficiency and / or a disease associated with serotonin deficiency in a subject.

[0014] Further aspects of the embodiment include Lactobacillus mucosae DSM 33291, Lactobacillus mucosae DSM 33292, Lactobacillus mucosae DSM 33293, Lactobacillus mucosae DSM 33661, Lactobacillus reuteri DSM 33509, Lactobacillus reuteri DSM 33632, Lactobacillus reuteri DSM 33633, Lactobacillus reuteri DSM 33634, Lactobacillus reuteri DSM 33635, Lactobacillus plantarum DSM 33295 and Lactobacillus plantarum DSM 33662 and their use as medicines and in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0015] The lactic acid-producing bacterial strain selected according to the embodiment can produce serotonin in the gastrointestinal system of a mammal. In particular, the lactic acid-producing bacterial strain selected according to the present invention can produce serotonin in the conditions of the more oxygenated niche of the intestine, for example near the epithelial lining of the intestine where serotonin receptors and / or serotonin transporters (SERT) are expressed and where enteroendocrine cells such as enterochromaffin cells are located. This microbially produced serotonin is bioactive and has effects locally in the gastrointestinal tract, but can also have effects peripherally with respect to the gastrointestinal system. Thus, the serotonin produced by the enterobacteria strain can be used to treat serotonin deficiency and / or diseases related to serotonin deficiency in a subject.

[0016] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken together with the accompanying drawings, in which: [Brief description of the drawings]

[0017] [Figure 1]Evaluation of serotonin production by lactic acid-producing bacterial strains. (A) Serotonin levels were measured in the culture medium (broth) of Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus acidophilus bacterial strains after culturing them under anaerobic and aerobic conditions in tryptophan-modifying decarboxylase (DECT) broth. All L. reuteri bacterial strains tested were found to produce serotonin under anaerobic conditions, and even more so under aerobic conditions, while no serotonin production was observed in L. plantarum 36E, L. casei LMG 6904 and L. acidophilus ATCC 4365 bacterial strains under any of the corresponding conditions in vitro. The ratio of aerobic to anaerobic serotonin production was calculated for each bacterial strain. All L. reuteri bacterial strains investigated produced serotonin better under aerobic conditions in vitro compared to anaerobic conditions. (B) Serum serotonin concentrations were measured in blood samples obtained from the vena cava of germ-free (GF) Tph1- / - mice and from GF Tph1- / - mice fed (inoculated) with either L. reuteri DSM 17938, L. reuteri DSM 27131 or L. casei LMG 6904 (n=3–6 / group). Data are presented as box plots in the graph, with each dot representing one mouse, and maximum, minimum, median, and interquartile range are shown. The graph shows that serotonin levels, which are very low in GF Tph1- / - mice, are restored in these mice after inoculation with L. reuteri bacterial strains. However, no increase in serotonin levels was observed in serum samples obtained from mice inoculated with L. casei LMG 6904, indicating that this bacterial strain does not produce serotonin in vivo.Each group was analyzed for normality before statistical significance was assessed by Student's t-test (if data were normally distributed) or Mann-Whitney test. When comparing more than two groups, data were analyzed using one-way ANOVA followed by post hoc Tukey's multiple comparison test if data were normally distributed, otherwise Kruskal-Wallis test followed by post hoc Dunn's multiple comparisons. Significance level was set at *p<0.05. [Diagram 2]Bacterially produced serotonin has local effects in the gastrointestinal tract and is important for enteric nervous system (ENS) maturation and crypt innervation in the proximal colon. Graphs show serotonin-positive area in the colonic longitudinal myenteric plexus (LMMP) (A,D), PGP 9.5-positive area in colonic crypts (B,E), and TUJ1-positive area in colonic crypts (C,F). (A-C) GF in the graphs shows Tph1- / - germ-free animals. CONV-D 3d and CONV-D 14d show Tph1- / - germ-free animals inoculated with Tph1+ / + mouse cecum for 3 or 14 days, respectively. CONV-R shows Tph1- / - animals with normal microbiota. DECT colonization 14d shows Tph1- / - germ-free animals inoculated with mouse cecal contents cultured in DECT for 48 h prior to inoculation (n=3-6 / group). Results D-F represent data obtained from GF Tph1- / - mice 14 days after inoculation with L. reuteri DSM 27131 or L. casei LMG 6904 (n=3-6 / group). (A) Data show that inoculation with mouse cecal microbiota increased the levels of serotonin in enteric neurons of the proximal colon compared to the levels of serotonin in the ENS of GF Tph1- / - mice. Serotonin levels in the ENS after 3 days are restored to nearly the same levels as in CONV-R mice. (B,C) Data also show that the presence of mouse cecal microbiota in the gastrointestinal tract increases well-established markers of mature enteric neurons (PGP9.5 and Tuj1). Data in D show that serotonin levels are also increased in enteric neurons of the proximal colon following inoculation with a single probiotic bacterial strain, L. reuteri DSM 27131, whereas L. casei LMG 6904 has no effect on enteric neuron serotonin levels. Furthermore, inoculation with L. reuteri DSM 27131 increased enteric neuron maturation as visualized with markers PGP9.5 and Tuj1 (E,F), whereas L. casei LMG 6904 had no effect on these two markers of enteric neuron maturation. Data are presented as box plots showing maximum, minimum, median, and interquartile range. Each dot represents one mouse.Each group was analyzed for normality before statistical significance was assessed by Student's t-test (if data were normally distributed) or Mann-Whitney test. When comparing more than two groups, data were analyzed using one-way ANOVA followed by post hoc Tukey's multiple comparison test if data were normally distributed, otherwise Kruskal-Wallis test followed by post hoc Dunn's multiple comparisons. Significance levels were set at *p<0.05, **p<0.01. [Diagram 3] Serotonin production by bacterial strains isolated from human donors. Of the total 199 isolates obtained from two human donors, 24 isolates were found to produce serotonin under anaerobic and / or aerobic conditions in vitro, as shown in the graph. A total of six isolates from donor #1 and one isolate from donor #2 were found to produce more than 1 nM serotonin in DECT broth in vitro under anaerobic conditions, while all six isolates from donor #1 and 15 isolates from donor #2 produced more than 1 nM serotonin under aerobic conditions. Interestingly, the majority of these strains were found to produce almost twice as much serotonin under aerobic conditions compared to anaerobic conditions. Sequencing analysis identified the serotonin-producing isolates as strains of L. mucosae (n = 13), L. plantarum (n = 6), and Enterococcus (n = 1). Four isolates could not be identified, but these isolates aligned closely with Escherichia, Shigella, or Brenneria bacterial strains. [Figure 4]Selected isolated human bacterial strains produce functional serotonin in vivo. Two of the bacterial strains isolated from human donors that were found to produce serotonin in vitro under both anaerobic and aerobic conditions, L. plantarum DSM 33662 and L. mucosae DSM 33661, were selected and used to inoculate germ-free Tph1- / - mice. The graph shows that these bacterial strains increased serotonin levels in vivo both locally in the gut lumen (A) and in the systemic circulation (serum samples) (B) compared to levels seen in germ-free Tph1- / - mice. Furthermore, both strains increased the serotonin-positive area of ​​the ENS of the proximal colon (C). Thus, these bacterial strains can elevate serotonin levels locally in the intestinal lumen (cecum) in vivo, and this locally produced serotonin is bioactive and can be transported to the systemic circulation (serum) and to neurons of the enteric nervous system in the proximal colon. Data are presented as box plots showing maximum, minimum, median, and interquartile range. Each dot represents one mouse. [Diagram 5] 1 is a flow chart illustrating a method for selecting a bacterial strain according to one embodiment. [Figure 6] 6 is a flow chart illustrating an optional additional step to the method shown in FIG. 5, according to one embodiment. [Figure 7] 6 is a flow chart illustrating an optional additional step to the method shown in FIG. 5, according to one embodiment. [Figure 8] 1 is a flow chart illustrating a method for selecting a bacterial strain according to another embodiment. [Figure 9]Two Lactobacillus reuteri strains, strains A and B, that do not grow well under aerobic conditions, were challenged with oxygen during cultivation and evolved into new strains with better oxygen tolerance. This method identified two new strains, L. reuteri DSM 33632 and DSM 33634, derived from strains A and B, respectively. Growth by the bacterial strains was quantified by measuring the optical density (OD) after 24 hours of incubation, and both new strains had significantly improved OD under aerobic conditions compared to strains A and B (A). This improvement in growth was stable for up to 100 generations (B,C). The new, more oxygen-tolerant bacterial strains were further challenged by several rounds of oxygen stress and further evolved into further strains with improved oxygen tolerance by additional freeze-drying steps and accelerated storage stress. These further stress steps led to the identification and selection of two further new bacterial strains, namely L. reuteri DSM 33633 derived from DSM 33632 and L. reuteri DSM 33635 derived from DSM 33634. These two new strains showed slightly improved growth properties under aerobic conditions compared to DSM 33632 and DSM 33634, and significantly improved growth properties under aerobic conditions compared to strains A and B (D). [Figure 10]Figure 2 shows the mucus adhesion ability of the serotonin-producing lactic acid bacteria strains L. reuteri DSM 27131, DSM 32465, DSM 33632, DSM 33633, DSM 33634, DSM 33635 and DSM 33509, L. mucosae DSM 33291, DSM 33292, DSM 33293 and DSM 33661, and L. plantarum DSM 33662. L. reuteri DSM 27131, which is capable of producing serotonin under aerobic conditions in vitro and contributing to the systemic pool of serotonin in vivo, is also able to adhere to mucus (A,B). Mucus adhesion was also observed for all other L. reuteri, L. mucosae and L. plantarum strains tested (A, B). The results indicate that these serotonin-producing bacterial strains are also able to adhere to the intestinal mucosa. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] TPH1 is the enzyme responsible for endogenous serotonin production in the gastrointestinal tract. Nevertheless, when the Tph1 gene is deleted in a mouse model (i.e., resulting in Tph1- / - animals), some serotonin remains, indicating that some of the serotonin is not produced by endogenous TPH1-mediated mechanisms. The inventors of the present invention surprisingly found that re-inducing Tph1- / - mice (i.e., animals lacking gastrointestinal microbiota) as germ-free resulted in almost complete depletion of serotonin. The next surprising finding was that inoculation of germ-free Tph1- / - mice with wild-type mouse cecal microbiota or a serotonin-producing Lactobacillus bacterial strain fully restored serotonin levels in the gut. These novel findings suggested that bacteria in the normal gastrointestinal tract can not only affect endogenous (host) serotonin production as previously known, but also surprisingly produce serotonin locally in the gastrointestinal tract by themselves. The inventors then found that certain strains of lactic acid bacteria can produce serotonin under both anaerobic and aerobic conditions in vitro. Moreover, for certain strains, serotonin production is greatly improved under aerobic conditions compared to the production of serotonin under anaerobic conditions. Thus, the present invention is based on the finding that certain strains of bacteria, in particular certain strains of lactic acid-producing bacteria that are normally anaerobic, can produce serotonin under aerobic conditions. Thus, the objective is to screen and select such bacteria that can produce and release serotonin extracellularly under aerobic conditions. These bacteria that can produce and release serotonin extracellularly under aerobic conditions are particularly adapted to produce and release serotonin locally in the gastrointestinal tract of a subject, for example in the more oxygenated niche of the intestine, close to the mucosa / epithelial lining of the intestine. In these more oxygenated niches of the intestine, enteroendocrine cells such as enterochromaffin cells are located and express serotonin receptors and / or serotonin transporters (SERTs).Any selected bacterial strain is preferably capable of adhering to mucus or at least being present in or near the mucosa, which is exposed to the relatively high oxygen tensions typical of the oxygenated niche of the gastrointestinal tract.

[0019] Screening of such bacteria can be carried out in vitro by culturing lactic acid-producing bacterial strains under aerobic conditions or under anaerobic and aerobic conditions, and selecting the bacteria identified as capable of producing serotonin only under aerobic conditions or under both anaerobic and aerobic conditions.When administered to a subject, the selected bacteria can then be used to produce serotonin locally in the gastrointestinal tract of the subject, thereby increasing the level of serotonin in subjects who have serotonin deficiency or suffer from diseases or disorders related to serotonin deficiency.

[0020] Screening of such bacteria can also be performed in vivo using germ-free (GF) Tph1- / - mammalian subjects fed with the lactic acid-producing bacterial strain. Serotonin production by the lactic acid bacterial strain in the gastrointestinal tract of the GF Tph1- / - subject can then be evaluated or analyzed in a body sample taken from the GF Tph1- / - subject to determine whether the lactic acid bacterial strain is effective in treating serotonin deficiency or a disease or disorder associated with serotonin deficiency.

[0021] Furthermore, microbially produced serotonin is bioactive and can not only have local effects in the gastrointestinal tract, but can also be transported beyond the epithelial lining of the gastrointestinal tract into the systemic circulation, in which case it can act peripherally with respect to the gastrointestinal system, i.e., have systemic effects.In order for the serotonin produced by bacteria to reach the systemic circulation, it is preferable that the production of serotonin occurs near the intestinal mucosa and intestinal epithelium, where oxygen levels are generally higher compared to the intestinal lumen.Therefore, in certain embodiments, the serotonin-producing bacteria selected according to the embodiment can also preferably adhere to mucus in the more oxygenated niche of the gastrointestinal tract, or at least be present at or near the mucosa, and / or can colonize the mucosa of the gastrointestinal tract, ensuring the proximity between the bacteria and the epithelial cells of the gastrointestinal tract.

[0022] Furthermore, serotonin produced by lactic acid-producing bacterial strains, when produced near the epithelial cells of the gastrointestinal tract, is capable of local effects on the gastrointestinal tract itself, such as affecting maturation of and communication within the gastrointestinal nervous system (ENS). The ENS consists of neuroglial and neuronal cells embedded in the lining of the gastrointestinal tract, and this nervous system is responsible, for example, for motility control of the gastrointestinal system and secretion of gastrointestinal enzymes. Furthermore, this nervous system communicates through many neurotransmitters, including via acetylcholine, dopamine, and serotonin, which are similar to communication in the central nervous system.

[0023] Thus, to treat serotonin deficiency and / or disorders and diseases associated with serotonin deficiency, lactic acid producing bacterial strains that produce serotonin in the gastrointestinal tract can be used locally in the gastrointestinal tract, but also in other parts of the body where the serotonin is transported across the gastrointestinal lining into the systemic circulation and even into peripheral tissues.

[0024] Lactic acid-producing bacterial strains, also called lactic acid bacteria strains, are Gram-positive, low GC, acid-tolerant, generally non-spore-forming, non-respiratory, rod-shaped or coccoid bacteria capable of producing lactic acid as the major metabolic end product of carbohydrate fermentation. Lactic acid-producing bacteria grow anaerobically, but unlike most anaerobes, they can also grow in the presence of oxygen as aerotolerant, or relatively oxygen-tolerant, anaerobes. Lactic acid-producing bacteria are generally recognized as safe (GRAS) and include genera in the order Lactobacillales, which includes the genera Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus, and Lactobacillus. In addition to the genera Lactobacillus, Weissella, and Lactobacillus sp., the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus are included, as well as the genus Bifidobacterium in the order Bifidobacteriales.

[0025] In the past decades, new analytical tools have allowed scientists to discover many new bacterial species, as well as to recognize that species historically grouped under the genus Lactobacillus were too different from each other. Therefore, to keep the probiotics population accurate and organized, the genus Lactobacillus was divided into 25 different genera. As a result, many probiotics have been given new genera in recent years. Therefore, the alternative genus name for Lactobacillus reuteri is Limosilactobacillus reuteri, the alternative genus name for Lactobacillus mucosae is Limosilactobacillus mucosae, the alternative genus name for Lactobacillus plantarum is Lactiplantibacillus plantarum, and the alternative genus name for Lactobacillus casei is Lacticaseibacillus casei.

[0026] FIG. 5 is a flow chart showing a method for selecting a bacterial strain for use in treating serotonin deficiency and / or a disease associated with serotonin deficiency. The method starts with step S1, which includes culturing bacteria of a lactic acid-producing bacterial strain in a medium containing tryptophan under aerobic conditions. A next step S2 includes detecting any serotonin produced by the bacteria of the lactic acid-producing bacterial strain in the medium or a sample thereof. The method also includes selecting the lactic acid-producing bacterial strain in step S3 as being effective for treating serotonin deficiency and / or a disease associated with serotonin deficiency in a subject if serotonin is detected in the medium or a sample thereof.

[0027] Such bacteria are therefore selected for their ability to produce serotonin in the more oxygenated niche conditions of the intestine, for example in the gastrointestinal mucosa and near the epithelial lining of the intestine where serotonin receptors and / or serotonin transporters (SERTs) are expressed and where certain enteroendocrine cells, such as enterochromaffin cells, are located.

[0028] The selection method shown in FIG. 5 involves culturing and testing lactic acid-producing bacterial strains in a medium containing tryptophan. Tryptophan is the starting material in the production of serotonin. More specifically, tryptophan hydroxylase (TPH) (EC 1.14.16.4), which exists in two isoforms, TPH1 and TPH2, synthesizes 5-hydroxytryptophan (5-HTP) from tryptophan. 5-HTP is then converted to serotonin (5-HT) by aromatic L-amino acid decarboxylase (AADC) (EC 4.1.1.28). Thus, the medium in which the bacteria of the lactic acid-producing bacterial strain to be tested are cultured contains tryptophan as the starting material for the production of any serotonin by the lactic acid-producing bacterial strain.

[0029] According to the present invention, bacteria of the lactic acid producing bacterial strain are inoculated into a tryptophan-containing medium under aerobic conditions. Therefore, the selected lactic acid producing bacterial strain must be able to produce serotonin at least under aerobic conditions. Differences in oxygen partial pressure can have a significant effect on bacterial metabolism in that they shift the bacterial metabolism of aromatic L-amino acids, such as tryptophan, to different metabolites. This means that a given lactic acid producing bacterial strain will produce different metabolites from tryptophan when cultured under different oxygen partial pressures, i.e., aerobic conditions vs. anaerobic conditions. In other words, lactic acid producing bacteria that can produce serotonin under aerobic conditions will not necessarily produce serotonin under anaerobic conditions, and conversely, lactic acid producing bacteria that can produce serotonin under anaerobic conditions will not necessarily produce serotonin under aerobic conditions.

[0030] The oxygen partial pressure in the intestine and stomach is generally higher closer to the intestinal mucosa compared to the inside of the small intestine (consisting of the duodenum, jejunum and ileum) and the large intestine (colon consisting of the cecum, rectum and anal canal). This means that the oxygen present in the intestinal epithelium corresponds to a gradient, with higher oxygen concentrations closer to the mucosa and lower oxygen concentrations towards the luminal side. Step S1 of FIG. 5 is therefore carried out under aerobic conditions, i.e. in the presence of oxygen, in order to mimic the aerobic conditions or oxygenated niche close to the intestinal mucosa. The lactic acid-producing bacterial strains selected according to the method shown in FIG. 5 are preferably capable of adhering and / or colonizing the intestinal mucosa or epithelial lining of the gastrointestinal tract, or at least being present at or near the intestinal mucosa, where they are exposed to a relatively high oxygen partial pressure, as will be further discussed in connection with FIG. 7. This means that in order to produce serotonin in vivo in the gastrointestinal system of a subject, and for the microbially produced serotonin to be efficiently taken up by the subject from the gastrointestinal tract into the systemic circulation, the lactic acid-producing bacterial strain selected in the method shown in Figure 5 must be capable of serotonin production under aerobic conditions that mimic those at or near the intestinal mucosa.

[0031] The terms "anaerobe" and "anaerobic" are used herein in reference to anaerobic culture conditions, which are characterized by the absence of oxygen (O 2 )-free conditions, i.e., 0% or 0 ppm dissolved oxygen and 0% or 0 ppm headspace in the culture tube. 2 , at least 0% or close to 0 ppm O 2 As an illustrative example, anaerobic culture may include culturing lactic acid-producing bacteria in a medium in a closed culture vessel, such as an airtight serum vial or a Hungate tube. The medium may be degassed prior to adding the lactic acid-producing bacteria. Additionally or alternatively, the culture vessel may be degassed prior to or after closing of the vessel, e.g., with N 2 The medium may be purged with 0.5% CO, or alternatively may be boiled for 15-20 minutes and cooled under a stream of nitrogen to remove any dissolved oxygen present in the medium or any trapped gas volume within the sealed culture vessel.

[0032] Correspondingly, the terms "aerobic bacteria" and "aerobic" are used herein in reference to aerobic culture conditions, which refer to the free oxygen (O) concentration in the headspace of the culture tube, which corresponds to 100% saturation or 8.87 ppm of dissolved oxygen. 2 The initial amount of dissolved oxygen in the growth medium can range from any detectable level, for example, from 2.41 ppm, corresponding to 29.3% saturation, preferably from 4.8 ppm, corresponding to 64% saturation, preferably up to 9 ppm, corresponding to about 103% saturation. The oxygen level in atmospheric air is about 21%. As an illustrative example, aerobic culture can include culturing the lactic acid-producing bacteria in a medium in an open culture vessel. Alternatively, the lactic acid-producing bacteria can be cultured in a closed culture vessel, with oxygen added to the closed culture vessel.

[0033] Step S1 of Figure 5 can be carried out for a predetermined period of time. For example, the bacteria of the lactic acid-producing bacterial strain can be incubated and cultured in the medium aerobically for at least 1 hour, at least several hours, i.e. at least 2 hours, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. The bacteria of the lactic acid-producing bacterial strain can also be cultured for a longer period of time, for example, 24 hours, 36 hours, 48 ​​hours or 60 hours, as illustrative but non-limiting examples.

[0034] In one embodiment, step S2 of FIG. 5 includes taking a sample from the medium and detecting any serotonin produced by the bacteria of the lactic acid producing bacterial strain in the sample. In one embodiment, the medium sample is a cell-free sample. In such an embodiment, any cells, particularly bacterial cells, present in the sample are first removed, for example by subjecting the sample to one or more centrifugations, for example at 10,000×g for 1-4 minutes, for example 2 minutes, in order to separate the sample into a cell-containing pellet and a supernatant that may contain serotonin. Step S2 of FIG. 5 then also includes detecting any serotonin produced by the lactic acid producing bacterial strain in the supernatant. Additionally or alternatively, the medium sample may be filtered through one or more filters configured to capture the bacteria but at the same time allow serotonin in the medium sample to pass through the filter. In such an embodiment, detection of serotonin can be performed in the filtrate.

[0035] Serotonin can be measured in the medium, sample or supernatant using any known serotonin measurement assay or technique. For example, serotonin can be measured using an enzyme-linked immunosorbent assay (ELISA) or mass spectrometry (MS) assay.

[0036] If the measurement of serotonin identifies serotonin in the medium, sample or supernatant from a particular lactic acid-producing bacterial strain, this will confirm that the lactic acid-producing bacterial strain is capable of producing and releasing serotonin extracellularly, and the tested lactic acid-producing bacterial strain will be selected in step S3 of Figure 5 as being effective in producing serotonin and thereby also effective in treating a disease associated with serotonin deficiency in the subject.

[0037] Therefore, detecting the presence of serotonin in the medium also includes detecting the presence of serotonin in a sample of the medium or a processed sample of the medium, such as the supernatant after centrifugation of the medium sample or the filtrate after filtration of the medium sample, so that in step S2, the bacteria of the lactic acid-producing bacterial strain can not only produce serotonin, but also release serotonin extracellularly into the medium.Such extracellular release of serotonin is important for the use of the lactic acid-producing bacterial strain for treating serotonin deficiency and / or diseases related to serotonin deficiency in a subject, because the bacterially produced serotonin needs to reach the subject's gastrointestinal tract locally and / or be absorbed by epithelium to reach the systemic circulation.

[0038] In one embodiment, the medium contains a carbon source in addition to tryptophan. Any carbon source that can be used by the lactic acid producing bacterial strain to be tested can be included in the medium, including but not limited to glucose. The medium also preferably contains a source of nitrogenous compounds, such as amino acids; manganese; sulfate; vitamins and growth factors, for example in the form of yeast extract and / or beef extract. An example of a medium that can be used in the method of FIG. 5 is a novel tryptophan modified decarboxylase (DECT) broth designed to promote tryptophan decarboxylation and microbial production of serotonin. Such a DECT broth or medium contains tryptophan, glucose, beef extract and yeast extract.

[0039] In one embodiment, the medium comprises at least 0.4 g / 100 ml tryptophan per 100 ml of medium, preferably at least 0.5 g / 100 ml, such as at least 0.6 g / 100 ml, at least 0.7 g / 100 ml, more preferably at least 0.8 g / 100 ml, such as at least 0.9 g / 100 ml or about 1 g tryptophan.

[0040] A currently preferred medium, DECT broth as mentioned above, preferably contains 1 g / 100 ml tryptophan, 0.05 g / 100 ml glucose, 0.5 g / 100 ml beef extract and 0.1 g / 100 ml yeast extract.

[0041] In one embodiment, the method includes additional steps as shown in FIG. 6. In such an embodiment, the method starts with step S10 or continues from step S2 of FIG. 5. Step S10 includes culturing bacteria of the lactic acid producing bacterial strain in a medium containing tryptophan under anaerobic conditions. The method then continues to step S11, which includes detecting any serotonin produced by the bacteria of the lactic acid producing bacterial strain in the medium or a sample thereof when cultured under anaerobic conditions. Step S10 is preferably performed in a similar manner as described above in connection with step S1 of FIG. 5, including the culture time, medium, etc., with the difference that in step S1 of FIG. 5, the bacteria of the lactic acid producing strain are cultured under aerobic conditions, whereas in step S10 of FIG. 6, the bacteria of the lactic acid producing strain are cultured under anaerobic conditions. Correspondingly, the detection of serotonin in step S11 of FIG. 6 could be performed in the same manner as discussed in connection with step S2 of FIG. 5.

[0042] This embodiment preferably also includes step S12 as shown in FIG. 6. This step S12 includes calculating the ratio or quotient between the concentration of serotonin detected in the medium or sample thereof when cultured under aerobic conditions (see step S2 in FIG. 5) and the concentration of serotonin detected in the medium or sample thereof when cultured under anaerobic conditions (see step S11 in FIG. 6). The method then continues to an optional step S13 of comparing the calculated ratio or quotient with a threshold value such as 1.0. If the ratio or quotient is equal to or greater than the threshold value, preferably greater than the threshold value, for example equal to or greater than 1.0, preferably greater than 1.0, the method continues to step S3 in FIG. 5. Thus, in this embodiment, step S3 includes selecting the lactic acid-producing bacterial strain as being effective for treating serotonin deficiency and / or a disease associated with serotonin deficiency in a subject if the ratio is equal to or greater than 1.0, preferably greater than 1.0.

[0043] In certain embodiments, the threshold against which the ratio or quotient is compared in optional step S13 is 1.5, preferably 2.0, such as 2.5, more preferably 3.0, such as 3.5, or even higher, such as 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or even 10.0. The threshold may in some embodiments be even higher, such as 15, 20, 25, 30, 40 or 50 or even more.

[0044] The efficiency of bacterial serotonin production of certain lactic acid producing bacterial strains is significantly improved when cultured under aerobic conditions compared to when cultured under anaerobic conditions. In some lactic acid producing bacterial strains, the amount of serotonin detected in the medium of the sample is very low, even below the detection limit of the assay used to detect serotonin, i.e., what can be accurately detected using the particular assay. Thus, such lactic acid producing bacterial strains have a very high ratio or quotient. Thus, in any embodiment, the method as shown in FIG. 6 includes an additional optional step (not shown in FIG. 6) of determining whether the concentration of serotonin in the medium or sample detected in step S11 when cultured under anaerobic conditions is below a threshold value, such as the detection limit of the assay used in step S11 to detect serotonin in the medium or sample and determine the concentration of serotonin detected. In this optional embodiment, the ratio or quotient is calculated in step S12 only if the concentration in the medium detected in step S11 when cultured under anaerobic conditions is equal to or greater than this threshold value.

[0045] If the lactic acid producing bacterial strain is capable of producing and releasing serotonin when cultured under aerobic conditions but not when cultured under anaerobic conditions, the lactic acid producing bacterial strain can still be selected in step S3 as being effective for treating serotonin deficiency and / or a disease associated with serotonin deficiency in a subject, according to the embodiment shown in FIG. 6, if the concentration of serotonin in the supernatant is at least 0.4 nM, preferably at least 0.5 nM, such as at least 0.75 nM or at least 1 nM, preferably at least 2 nM, more preferably at least 3 nM, such as at least 4 nM or at least 5 nM or at least 6 nM or at least 7 nM, as further described below.

[0046] Thereby, this embodiment of the present invention selects a lactic acid bacteria strain that produces serotonin particularly well under aerobic conditions, i.e. produces at least more, preferably more, serotonin under aerobic conditions compared to under anaerobic conditions. Most lactic acid producing bacterial strains are anaerobic, some of which may produce serotonin under anaerobic conditions. However, the present invention selects a lactic acid bacteria strain that is not only capable of surviving and growing under aerobic conditions, but also capable of producing and extracellularly releasing serotonin under such aerobic conditions. Moreover, the lactic acid bacteria strain selected according to the embodiment as shown in FIG. 6 actually produces at least the same amount, or even more, of serotonin under aerobic conditions compared to anaerobic conditions. Such a lactic acid bacteria strain can produce and secrete a biologically effective amount of serotonin when present in a niche of the subject's gastrointestinal system or tract where the oxygen level is relatively higher than in other parts of the gastrointestinal tract, and is therefore particularly suitable for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0047] In one embodiment, step S2 of Figure 5 comprises measuring the concentration of serotonin produced by the lactic acid-producing bacterial strain in the medium or a sample thereof. Step S3, in this embodiment, comprises selecting the lactic acid-producing bacterial strain as effective for treating serotonin deficiency in the subject if the concentration of serotonin in the medium or a sample thereof is at least equal to or greater than a defined threshold value.

[0048] The concentration of serotonin of a given lactic acid producing bacterial strain measured in the medium or a sample thereof will depend, at least in part, on the assay or technique used to analyze serotonin, e.g., ELISA vs. MS. Thus, the above threshold values ​​will depend on the particular assay used to measure the concentration of serotonin in the medium.

[0049] In a particular embodiment, the bacteria of the lactic acid-producing bacterial strain are cultured in a tryptophan-containing medium, preferably DECT medium, under aerobic conditions for 24 hours, followed by taking a medium sample and subjecting the medium sample to at least one centrifugation to obtain a supernatant and a pellet.In such a case, a conventional mass spectrometry assay is used to measure the concentration of serotonin produced by the bacterial lactic acid-producing bacterial strain in the supernatant.In this embodiment, step S3 comprises selecting the lactic acid-producing bacterial strain as being effective for treating serotonin deficiency and / or diseases related to serotonin deficiency in a subject if the concentration of serotonin in the supernatant is at least 0.4nM, preferably at least 0.5nM, such as at least 0.75nM or at least 1nM, preferably at least 2nM, more preferably at least 3nM, such as at least 4nM or at least 5nM or at least 6nM or at least 7nM.

[0050] In one embodiment, the method includes an additional step S15 as shown in Figure 7. The method starts with this step S15 or continues from step S2 of Figure 5 or indeed from step S13 of Figure 6. Step S15 includes determining whether the bacteria of the lactic acid-producing bacterial strain are capable of adhering to intestinal mucus and / or to the gastrointestinal mucosa. In this embodiment, step S3 of Figure 5 includes selecting the lactic acid-producing bacterial strain as effective for treating serotonin deficiency and / or diseases associated with serotonin deficiency in a subject if serotonin is detected in the medium or in the sample thereof and if the bacteria of the lactic acid-producing bacterial strain are capable of adhering to intestinal mucus and / or to the gastrointestinal mucosa.

[0051] This embodiment can also be combined with the embodiment shown in FIG. 6, i.e. the selection can be made based on a comparison of the ratio with a threshold value and based on whether the bacteria of the lactic acid producing bacterial strain are able to adhere to intestinal mucus and / or to the gastrointestinal mucosa.

[0052] Mucus, the main structural component of which is mucins (a family of highly glycosylated proteins of high molecular weight), is produced by the mucosa of mammals and birds, as well as amphibians, fish, hagfish, snails and slugs. Thus, by being able to adhere to intestinal mucus, bacteria of lactic acid-producing strains are able to adhere to the mucosa of the gastrointestinal tract.

[0053] For example, as disclosed in Example 6, the ability of bacteria of lactic acid-producing bacterial strains to adhere to the mucosa of the gastrointestinal tract can be examined. Briefly, gastrointestinal mucosa is extracted and plated on a surface. Bacteria of lactic acid-producing bacterial strains are then added to the plated mucus and allowed to adhere to the mucus. Unbound bacteria are then removed by one or more washes. The amount of adherent bacteria can then be quantified directly on the mucus or indirectly by first releasing the adherent bacteria by enzymatic treatment, such as trypsin treatment, and then quantifying the released bacteria.

[0054] In one embodiment, the lactic acid producing bacterial strain has a mucoadhesive property at least as good as that of Lactobacillus reuteri DSM 27131, as shown in Example 6. In such an embodiment, step S15 of FIG. 7 comprises determining whether the bacteria of the lactic acid producing bacterial strain have a mucoadhesive property equal to or greater than that of the bacteria of L. reuteri DSM 27131. In this embodiment, step S3 of FIG. 5 comprises selecting the lactic acid producing bacterial strain as being effective for treating serotonin deficiency and / or a disease associated with serotonin deficiency in a subject if serotonin is detected in the medium or a sample thereof and if the bacteria of the lactic acid producing bacterial strain have a mucoadhesive property equal to or greater than that of the bacteria of L. reuteri DSM 27131.

[0055] The above methods and embodiments thereof are in vitro methods for identifying and selecting lactic acid-producing bacteria effective in treating serotonin deficiency and / or diseases associated with serotonin deficiency in a subject.

[0056] However, the present invention is not limited thereto. Figure 8 is a flow chart showing a method implemented as an in vivo method of selecting a bacterial strain for use in treating serotonin deficiency and / or a disease associated with serotonin deficiency. The method comprises, in step S20, feeding or inoculating a germ-free (GR) Tph1- / - subject with bacteria of a lactic acid-producing bacterial strain. A next step S21 comprises detecting any serotonin produced by the bacteria of the lactic acid-producing bacterial strain in a body sample taken from the GF Tph1- / - subject. The method further comprises, in step S22, selecting the lactic acid-producing bacterial strain as effective for treating serotonin deficiency and / or a disease associated with serotonin deficiency in the subject if serotonin is detected in the body sample.

[0057] Thus, in this method, the test subject for the germ-free test subject lacking the production of the enzyme TPH1 is a Tph1 knockout subject. The GF Tph1- / - subject is a non-human GF Tph1- / - animal subject, preferably selected from the group consisting of mouse, rat, guinea pig, pig, swine, cat, dog, sheep, horse, non-human primate, monkey or bird, more preferably a mouse or a rat. The GF Tph1- / - mouse can be obtained as disclosed in Example 1.

[0058] Bacteria of the lactic acid-producing bacterial strain to be tested in the method shown in Figure 8 are provided, i.e., inoculated, e.g., orally administered by gavage, to a GF Tph1- / - subject, for example in the form of a solubilized powder, capsule or tablet containing the bacteria, or a solution containing the bacteria.

[0059] A body sample is taken from a GF Tph1- / - subject and analyzed for the presence of any serotonin in step S21. The subject is germ-free, i.e., lacks or is deficient in gastrointestinal microflora, and further lacks TPH1, the enzyme responsible for the majority of endogenous serotonin production. Thus, the serotonin detected in the body sample in step S21 is produced by the bacteria of the lactic acid-producing bacterial strain given to the subject in step S20, or by bacteria obtained from the inoculated bacteria.

[0060] The body sample can be any sample taken from a GF Tph1- / - subject, and if the subject is not Tph1- / -, i.e., is a non-GF Tph1+ / + subject of the same species, it will usually contain serotonin.For example, the sample can be a gastrointestinal sample, a tissue sample taken from the subject, for example, from gastrointestinal tissue, or a body fluid sample, for example, a blood sample, a plasma sample or a serum sample.In a particular embodiment, the body sample is a body fluid sample, preferably a blood sample, a plasma sample or a serum sample.Serotonin detected in such a body fluid sample not only indicates that the bacteria of the lactic acid-producing bacterial strain can produce serotonin in the gastrointestinal system of the GF Tph1- / - subject and produce it extracellularly, but also indicates that the bacterially produced serotonin is taken up and transported to the systemic circulation, i.e., blood system, of the GF Tph1- / - subject.

[0061] In one embodiment, the lactic acid producing bacterial strain is a Lactobacillus bacterial strain. In a particular embodiment, the Lactobacillus bacterial strain is selected from the group consisting of L. reuteri, L. mucosae and L. plantarum bacterial strains.

[0062] In one embodiment, a lactic acid-producing bacterial strain selected according to the present invention, such as that shown in any of Figures 5 to 8, is not only capable of producing and releasing serotonin extracellularly, but also capable of inducing endogenous serotonin production when administered to a subject.

[0063] In another embodiment, a lactic acid producing bacterial strain selected according to the present invention, such as that shown in any of Figures 5 to 8, can produce and release serotonin extracellularly, but cannot induce endogenous serotonin production when administered to a subject.

[0064] Another aspect of the present invention relates to a lactic acid-producing bacterial strain capable of producing and releasing serotonin extracellularly under aerobic conditions for use in treating serotonin deficiency and / or diseases associated with serotonin deficiency in a subject.

[0065] In one embodiment, the lactic acid-producing bacterial strain is also capable of producing and releasing serotonin extracellularly under anaerobic conditions, but the ratio between the amount of serotonin produced extracellularly by bacteria of the lactic acid-producing bacterial strain under aerobic conditions and the amount of serotonin produced extracellularly by bacteria of the lactic acid-producing bacterial strain under anaerobic conditions is equal to or greater than 1, preferably greater than 1.

[0066] In one embodiment, the lactic acid producing bacterial strain is further capable of adhering to intestinal mucus and / or to the gastrointestinal mucosa.

[0067] In one embodiment, the lactic acid producing bacterial strain is capable of producing serotonin in, or at least in association with, the intestinal mucosa of the subject.

[0068] As mentioned hereinbefore, it is generally preferred if the bacteria of the lactic acid-producing bacterial strain can adhere to mucus or at least be present near the gastrointestinal mucosa, particularly the intestinal mucosa, thereby facilitating the uptake of the serotonin produced and secreted by the bacteria of the lactic acid-producing bacterial strain by the intestinal mucosa, particularly by the intestinal epithelium.The serotonin taken up by the intestinal epithelium can then be transported peripherally or systemically to a specific location in the subject, where it exerts its biological effect.

[0069] As used herein, the term "treatment" can encompass both alleviating symptoms of a particular medical condition, disease or disorder, as well as preventing the onset of symptoms. Thus, the term includes prevention, risk reduction, attenuation, inhibition and prevention of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0070] In one embodiment, a lactic acid producing bacterial strain capable of producing serotonin under aerobic conditions is selected according to the method described above and shown in any of Figures 5-8 or an embodiment thereof.

[0071] Serotonin has several physiological functions, including behavior, platelet activation and coagulation, regulation of gut motility and enteric nervous system (ENS) homeostasis and activity, gut immune system activity, energy metabolism (fasting adaptation, adipose tissue browning and lipolysis, hepatic glucose production, insulin secretion), gut-brain axis activity, and bone metabolism and homeostasis.Therefore, imbalance of serotonin level, especially deficiency, can cause abnormalities in many biological processes and functions in subjects.

[0072] In one embodiment, the disease associated with serotonin deficiency, also referred to herein as a serotonin-dependent disease, is a disease or disorder selected from the group consisting of anxiety, depression, aggression, poor memory, eating disorders, obsessive-compulsive disorder, panic disorder, post-traumatic stress disorder, social anxiety disorder, gastrointestinal motility disorders (e.g., constipation), irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), cardiovascular disease, osteoporosis, autoimmune polyendocrine deficiency-candidiasis-ectodermal dystrophy (APECED), osteopenia, osteopenic conditions, and combinations thereof.

[0073] In one embodiment, the disease associated with serotonin deficiency is selected from the group consisting of IBS, IBD, cardiovascular disease, osteoporosis, gastrointestinal motility disorders, APECED, osteopenia and bone loss conditions.

[0074] In one embodiment, the disease associated with serotonin deficiency is a disease or disorder of the central nervous system, such as one selected from the group consisting of anxiety, depression, aggression, memory problems, eating disorders, obsessive-compulsive disorder, panic disorder, post-traumatic stress disorder, and social anxiety disorder.

[0075] In a preferred embodiment, the disease associated with serotonin deficiency is a disease or disorder of the gastrointestinal system, such as gastrointestinal motility disorder, IBS or IBD.

[0076] In one embodiment, the disease associated with serotonin deficiency is osteoporosis, osteopenia and bone loss conditions, such as diseases associated with glucocorticoid-associated bone loss.

[0077] In one embodiment, the lactic acid producing bacterial strain is a Lactobacillus bacterial strain. In a particular embodiment, the Lactobacillus bacterial strain is selected from the group consisting of L. reuteri, L. mucosae and L. plantarum strains.

[0078] In one embodiment, the L. reuteri strain is selected from the group consisting of L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 33509, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, L. reuteri ATCC PTA 5289, L. reuteri DSM 17938, L. reuteri ATCC PTA 6475, and L. reuteri DSM 32465.

[0079] In certain embodiments, the L. reuteri strain is selected from the group consisting of L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 33509, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, L. reuteri ATCC PTA 5289, and L. reuteri DSM 32465.

[0080] In another embodiment, the L. reuteri strain is selected from the group consisting of L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 33509, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, L. reuteri DSM 17938, and L. reuteri DSM 32465.

[0081] In a further embodiment, the L. reuteri strain is selected from the group consisting of L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 33509, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, L. reuteri ATCC PTA 6475, and L. reuteri DSM 32465.

[0082] Lactobacillus reuteri DSM 27131 was deposited in accordance with the Budapest Treaty at the Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on April 18, 2013.

[0083] Lactobacillus reuteri DSM 32846, DSM 32848 and DSM 32849 were deposited in accordance with the Budapest Treaty on July 4, 2018 at the Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0084] Lactobacillus reuteri DSM 33509 was deposited in accordance with the Budapest Treaty at the Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on April 23, 2020.

[0085] Lactobacillus reuteri DSM 32465 was deposited in accordance with the Budapest Treaty at the Leibniz Institute DSMZ-German collection of Microorganisms and Cell Cultures (Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on March 21, 2017.

[0086] Lactobacillus reuteri ATCC PTA 5289 was deposited in accordance with the Budapest Treaty on June 25, 2003 at the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, US.

[0087] Lactobacillus reuteri DSM 33632, DSM 33633, DSM 33634 and DSM 33635 were deposited in accordance with the Budapest Treaty on September 9, 2020 at the Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0088] Lactobacillus reuteri DSM 17938 was deposited in accordance with the Budapest Treaty on January 30, 2006 at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany.

[0089] Lactobacillus reuteri ATCC PTA 6475 was deposited in accordance with the Budapest Treaty on December 21, 2004 at the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, US.

[0090] In certain embodiments, the L. reuteri strain is selected from the group consisting of L. reuteri DSM 32846, L. reuteri DSM 32848, L. reuteri DSM 32849, L. reuteri DSM 27131, L. reuteri DSM 33509, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, and L. reuteri DSM 32465.

[0091] In another embodiment, the L. reuteri strain is selected from the group consisting of L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, and L. reuteri DSM 33509.

[0092] In a further embodiment, the L. reuteri strain is selected from the group consisting of L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, and L. reuteri DSM 33635. These L. reuteri strains have evolved to be more oxygen tolerant and stable viable for long-term storage, as described below.

[0093] Some Lactobacillus strains grow well under anaerobic conditions but not under aerobic conditions. Thus, two L. reuteri strains, strains A and B, with low oxygen tolerance, were challenged by oxygen (oxygen stress) and thereby forced to evolve into new strains, i.e., actively modified or adapted, in a laboratory-controlled environment. By this method, two new strains were identified, L. reuteri DSM 33632, which was identified as derived from parent strain A, and L. reuteri DSM 33634, which was identified as derived from parent strain B. Both of these new strains showed newly developed and improved properties, one of which was an increased oxygen tolerance, which allowed them to grow significantly better under aerobic conditions for up to 100 generations (Figure 9A) (Figures 9B and 9C).

[0094] Furthermore, there is a general need to improve the storage stability of bacterial cultures for use in probiotic products, including both increasing the resistance of bacteria to the freeze-drying process and increasing their stability during long-term storage to extend the shelf life of probiotic products. Thus, the new bacterial strains with improved oxygen tolerance were further challenged in a laboratory controlled environment with more rounds of oxygen stress to further increase their resistance to aerobic conditions, as well as additional freeze-drying and accelerated storage stress, thereby identifying and selecting two more bacterial strains, L. reuteri DSM 33633 derived from DSM 33632 and L. reuteri DSM 33635 derived from DSM 33634. These two additional new strains were not only resistant to the additional freeze-drying process and accelerated storage stress, but also showed slightly improved growth characteristics under aerobic conditions compared to DSM 33632 and DSM 33634 (Figure 9D), and showed significantly improved growth characteristics under aerobic conditions compared to parent strain A and parent strain B. All four engineered strains, L. reuteri DSM 33632, DSM 33633, DSM 33634 and DSM 33635, exhibited these adapted / evolved properties and were found to remain stable over at least 100 generations.

[0095] In a particular embodiment, the L. reuteri strain is L. reuteri DSM 33509. This L. reuteri strain has been challenged to evolve into a stable bacterial strain with the aim of increasing its survival during long-term storage and to improve its survival rate in the gastrointestinal tract. More specifically, the L. reuteri strain DSM 33509 has been modified from its parent strain in a multi-step selection process, which includes culturing and freeze-drying the bacteria and selecting colonies that survive the freeze-drying process, resulting in a strain with increased resistance to bile and increased adhesion to mucus.

[0096] In one embodiment, the L. mucosae strain is selected from the group consisting of L. mucosae DSM 33291, L. mucosae DSM 33292, L. mucosae DSM 33293 and L. mucosae DSM 33661.

[0097] L. mucosae DSM 33291, DSM 33292 and DSM 33293 were deposited in accordance with the Budapest Treaty at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on September 20, 2019.

[0098] L. mucosae DSM 33661 was deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on October 14, 2020 in accordance with the Budapest Treaty.

[0099] In one embodiment, the L. plantarum strain is selected from the group consisting of L. plantarum DSM 33295 and L. plantarum DSM 33662.

[0100] L. plantarum DSM 33295 was deposited in accordance with the Budapest Treaty at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on October 9, 2019.

[0101] L. plantarum DSM 33662 was deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on October 14, 2020 in accordance with the Budapest Treaty.

[0102] In certain embodiments, the lactic acid bacteria strain is selected from the group consisting of L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, L. reuteri DSM 33635, and L. reuteri DSM 33509, L. mucosae DSM 33291, L. mucosae DSM 33292, L. mucosae DSM 33293, L. mucosae DSM 33661, L. plantarum DSM 33295, and L. plantarum DSM 33662.

[0103] It is also possible to use a combination of bacteria from two or more lactic acid producing bacterial strains, such as those selected from the L. mucuosae, L. plantarum and L. reuteri strains mentioned above.

[0104] In one embodiment, the lactic acid producing bacterial strain is preferably a probiotic bacterial strain.

[0105] In one embodiment, when the disease associated with serotonin deficiency is selected from the group consisting of IBS, anxiety, depression, post-traumatic stress disorder, IBD and gastrointestinal motility disorders, the lactic acid producing bacterial strain is other than Lactobacillus reuteri DSM 17938. In another embodiment, the lactic acid producing bacterial strain is other than Lactobacillus reuteri DSM 17938.

[0106] In one embodiment, when the disease associated with serotonin deficiency is selected from the group consisting of osteoporosis, osteopenia, bone loss, depression, IBS and IBD, the lactic acid producing bacterial strain is other than Lactobacillus reuteri ATCC PTA 6475. In another embodiment, the lactic acid producing bacterial strain is other than Lactobacillus reuteri ATCC PTA 6475.

[0107] Further embodiments of the present invention relate to the Lactobacillus mucosae strain DSM 33291, such as the L. mucosae strain DSM 33291 in lyophilized or otherwise dried form, the L. mucosae strain DSM 33291 for use as a medicament, the L. mucosae strain DSM 33291 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency and the use of the L. mucosae strain DSM 33291 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0108] Further embodiments of the present invention relate to the Lactobacillus mucosae strain DSM 33292, such as the L. mucosae strain DSM 33292 in lyophilized or otherwise dried form, the L. mucosae strain DSM 33292 for use as a medicament, the L. mucosae strain DSM 33292 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency and the use of the L. mucosae strain DSM 33292 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0109] Embodiments of the present invention further relate to the Lactobacillus mucosae strain DSM 33293, such as the L. mucosae strain DSM 33293 in lyophilized or otherwise dried form, to the L. mucosae strain DSM 33293 for use as a medicament, and to the L. mucosae strain DSM 33293 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency, and to the use of the L. mucosae strain DSM 33293 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0110] Embodiments of the present invention also relate to the Lactobacillus mucosae strain DSM 33661, for example the L. mucosae strain DSM 33661 in lyophilized or otherwise dried form, the L. mucosae strain DSM 33661 for use as a medicament, and the L. mucosae strain DSM 33661 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency, and the use of the L. mucosae strain DSM 33661 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0111] Further embodiments of the present invention relate to the Lactobacillus plantarum strain DSM 33295, such as the L. plantarum strain DSM 33295 in lyophilized or otherwise dried form, the L. plantarum strain DSM 33295 for use as a medicament, and the L. plantarum strain DSM 33295 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency, and the use of the L. plantarum strain DSM 33295 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0112] Embodiments of the present invention further relate to the Lactobacillus plantarum strain DSM 33662, such as the L. plantarum strain DSM 33662 in lyophilized or otherwise dried form, to the L. plantarum strain DSM 33662 for use as a medicament, and to the L. plantarum strain DSM 33662 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency, and to the use of the L. plantarum strain DSM 33662 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0113] Further embodiments of the present invention relate to the Lactobacillus reuteri strain DSM 33509, such as the L. reuteri strain DSM 33509 in lyophilized or otherwise dried form, the L. reuteri strain DSM 33509 for use as a medicament, the L. reuteri strain DSM 33509 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency and the use of the L. reuteri strain DSM 33509 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0114] Embodiments of the present invention relate to the Lactobacillus reuteri strain DSM 33632, such as the L. reuteri strain DSM 33632 in lyophilized or otherwise dried form, the L. reuteri strain DSM 33632 for use as a medicament, the L. reuteri strain DSM 33632 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency and the use of the L. reuteri strain DSM 33632 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0115] Embodiments of the present invention also relate to the Lactobacillus reuteri strain DSM 33633, such as the L. reuteri strain DSM 33633 in lyophilized or otherwise dried form, the L. reuteri strain DSM 33633 for use as a medicament, the L. reuteri strain DSM 33633 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency and the use of the L. reuteri strain DSM 33633 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0116] Further embodiments of the present invention relate to the Lactobacillus reuteri strain DSM 33634, such as the L. reuteri strain DSM 33634 in lyophilized or otherwise dried form, the L. reuteri strain DSM 33634 for use as a medicament, the L. reuteri strain DSM 33634 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency and the use of the L. reuteri strain DSM 33634 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0117] Further embodiments of the present invention relate to the Lactobacillus reuteri strain DSM 33635, such as the L. reuteri strain DSM 33635 in lyophilized or otherwise dried form, the L. reuteri strain DSM 33635 for use as a medicament, the L. reuteri strain DSM 33635 for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency and the use of the L. reuteri strain DSM 33635 for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0118] Thus, the present invention relates to Lactobacillus mucosae DSM 33291, L. mucosae DSM 33292, L. mucosae DSM 33293, L. mucosae DSM 33661, L. plantarum DSM 33295, L. plantarum DSM 33662, L. reuteri DSM 33509, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634 and L. reuteri DSM The present invention relates to a lactic acid producing bacterial strain, e.g. in lyophilized or another dried form, selected from the group consisting of 33635. The present invention also relates to a lactic acid producing bacteria selected from the above group for use as a medicament for use in the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency, and to its use for the manufacture of a medicament for the treatment of serotonin deficiency and / or diseases associated with serotonin deficiency.

[0119] One aspect of the present invention relates to a bacterial strain that produces and releases serotonin extracellularly under aerobic conditions. In one embodiment, the bacterial strain is selected from the group consisting of Lactobacillus mucosae DSM 33291, L. mucosae DSM 33292, L. mucosae DSM 33293, L. mucosae DSM 33661, L. plantarum DSM 33295, L. plantarum DSM 33662, L. reuteri DSM 33509, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, and L. reuteri DSM 33635.

[0120] In a particular embodiment, the bacterial strain is a Lactobacillus mucosae strain selected from the group consisting of L. mucosae DSM 33291, L. mucosae DSM 33292, L. mucosae DSM 33293 and L. mucosae DSM 33661.

[0121] In another particular embodiment, the bacterial strain is a Lactobacillus plantarum strain selected from the group consisting of L. plantarum DSM 33295 and L. plantarum DSM 33662.

[0122] In a further specific embodiment, the bacterial strain is a Lactobacillus reuteri strain selected from the group consisting of L. reuteri DSM 33509, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634 and L. reuteri DSM 33635.

[0123] Another aspect of the present invention relates to a bacterial strain capable of producing serotonin under aerobic conditions, releasing it extracellularly, and adhering to mucus. In one embodiment, the bacterial strain is selected from the group consisting of Lactobacillus mucosae DSM 33291, L. mucosae DSM 33292, L. mucosae DSM 33293, L. mucosae DSM 33661, L. plantarum DSM 33662, L. reuteri DSM 33509, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634, and L. reuteri DSM 33635.

[0124] In a particular embodiment, the bacterial strain is a Lactobacillus mucosae strain selected from the group consisting of L. mucosae DSM 33291, L. mucosae DSM 33292, L. mucosae DSM 33293 and L. mucosae DSM 33661.

[0125] In another particular embodiment, the bacterial strain is Lactobacillus plantarum DSM 33662.

[0126] In a further specific embodiment, the bacterial strain is a Lactobacillus reuteri strain selected from the group consisting of L. reuteri DSM 33509, L. reuteri DSM 33632, L. reuteri DSM 33633, L. reuteri DSM 33634 and L. reuteri DSM 33635.

[0127] The preferred mode of administration of the lactic acid producing bacterial strains is oral. Other modes of administration include nasal, intraocular, topical, or any other form of local administration to the skin, rectum, nose, eyes, vagina or gums.

[0128] The appropriate dose of the lactic acid-producing bacteria strain defined herein can be easily selected depending on the disease associated with the serotonin deficiency to be treated, the mode of administration, the subject and the relevant formulation. For example, the dosage and administration regimen are selected so that the lactic acid-producing bacteria administered to the subject according to the present invention can provide the desired therapeutic effect, preventive effect or health benefit. Thus, preferably, the dosage is a therapeutically or prophylactically effective dosage appropriate for the type of mammal and the serotonin deficiency to be treated. For example, the total colony forming units (CFU) of the bacteria is 10 4 ~10 11 , e.g. 10 5 ~10 9 , or 106 ~10 8 , or 10 8 ~10 10 A daily dose of about 10 8 Total CFU, e.g. 10 7 ~10 9 or 10 8 ~10 9 It is a CFU.

[0129] The lactic acid-producing bacteria strain is preferably administered in pure, isolated, dried, lyophilized or freeze-dried form. The lactic acid-producing bacteria can also be administered in a frozen or active formulation (e.g., fermented food). Thus, the lactic acid-producing bacteria strain is preferably produced or prepared in a lyophilized or freeze-dried form.

[0130] The lactic acid-producing bacterial strain may be included in a composition, preferably in lyophilized form, which comprises, in addition to the lactic acid-producing bacterial strain, at least one excipient and / or other active agent, non-limiting but illustrative examples of such excipients include fillers, anti-adherents, binders, coatings, colorants, disintegrants, flavors, glidants, lubricants, preservatives, adsorbents, sweeteners and vehicles.

[0131] Other active agents that may be included in the composition include those active agents used in the treatment of any of the above examples of serotonin deficiency diseases or disorders.

[0132] A related aspect of the invention defines a method for preventing, inhibiting or treating serotonin deficiency and / or a disease associated with serotonin deficiency in a subject, the method comprising administering to the subject a bacterium of a lactic acid-producing bacterial strain capable of producing and releasing serotonin extracellularly under aerobic conditions.

[0133] Any of the routes of administration described above can be used. The lactic acid producing bacterial strains can advantageously be administered in the form of a composition as described above.

[0134] The subject treated with the lactic acid producing bacteria strain or composition is a mammal or bird, preferably a human.However, the lactic acid producing bacteria strain or composition can also or alternatively be used for veterinary purposes.In such cases, the subject can be selected from, for example, cats, dogs, sheep, goats, cows, horses, as illustrative but non-limiting examples.

[0135] example The gut microbiota produces a wide range of metabolites that affect host physiology, such as secondary bile acids and short-chain fatty acids. However, the microbiota has a vast and relatively unexplored potential for the transformation of aromatic amino acids. This example demonstrates that serotonin produced in the lumen by gut microbes such as Lactobacillus is absorbed and has effects both locally and peripherally. Microbially produced serotonin is bioactive and can rescue the developmental phenotype of the enteric nervous system. This example also shows that specific good serotonin-producing bacteria can be isolated and selected for their ability to produce serotonin under aerobic conditions. It has also been shown that such serotonin-producing bacteria can produce serotonin in vivo.

[0136] Example 1 - In vitro and in vivo production of serotonin and evaluation of suitable bacterial strains material and method animal Eight to 12 week old Tph1+ / + (as littermate controls) and Tph1- / - (C57Bl / 6 background) mice were housed in a climate controlled room (22 ± 2 °C) subjected to a 12-h light / dark cycle (7:00 AM to 7:00 PM) and allowed free access to water and food. Tph1- / - mice have been described previously (Cell 135:825-837 (2008)). All animal procedures were approved by the Ethics Committee on Animal Care and Use, Gothenburg, Sweden. Animals were obtained from heterozygous breeding and genotyped before the experiments. Proximal colon, serum from the vena cava, material from the intestinal lumen and cecum were obtained from each animal after the experiments. TPH1 is the rate-limiting enzyme involved in endogenous serotonin synthesis in the periphery, including the gastrointestinal tract. Thus, Tph1 knockout (Tph1- / -) mice are deficient in endogenous production of serotonin outside the brain. Knockout of Tph1 expression in the gastrointestinal tract of Tph1- / - mice was confirmed by quantitative polymerase chain reaction (qPCR). Some very low levels of serotonin in these knockout animals remained even after re-induction of the animals as germ-free (GF). These low levels could have been produced by compensatory mechanisms common in knockout animals, for example, by nonspecific hydroxylation of tryptophan by phenylalanine or tyrosine hydroxylase in the intestine.

[0137] Bacterial culture, Lactobacillus For in vitro experiments, Lactobacillus reuteri (DSM 17938, DSM 32846, DSM 32848, DSM 32849, DSM 27131 and ATCC PTA 6475), L. plantarum 36E (obtained from the Culture Collection University of Gothenburg as “CCUG 61730”), L. casei LMG 6904 (obtained from the Belgian Coordinated Collections of Microorganisms) and L. acidophilus ATCC 4356 (obtained from the American Type Culture Collection) were cultivated in tryptophan-modified decarboxylase (DECT) broth under anaerobic conditions, i.e. in a Coy chamber. In parallel experiments, Lactobacillus reuteri strains (DSM 17938, DSM 32846, DSM 32848, DSM 32849, DSM 27131 and ATCC PTA 6475) were cultivated in tryptophan modified DECT broth under ambient aerobic conditions. Each culture was sampled at 24 h and the samples were centrifuged (10,000 × g, 4 °C, 2 min). The pellets were collected, stored in 20% glycerol (cryoprotectant) and frozen at -80 °C until use.

[0138] Tryptophan modified DECT broth was designed to promote tryptophan decarboxylation and production of serotonin by bacteria. DECT contained 0.5% beef extract (BD Biosciences #212303), 0.1% yeast extract (Oxoid Fisher-Scientific #LP0021), 0.05% d-glucose (Millipore #346351), 1% L-tryptophan (Sigma-Aldrich #93659) in distilled water, and 0.05% cysteine ​​for anaerobic conditions. To obtain anoxic variants of DECT, 0.05% cysteine ​​was added as a reducing agent. DECT was autoclaved at 121 °C and 15 PSI for 30 min.

[0139] Mouse inoculation experiment, Lactobacillus For these experiments, the bacterial strains L. reuteri (DSM 17938, DSM 27131) and L. casei (LMG 6904) were cultivated under anaerobic conditions as described above in De Man, Rogosa and Sharpe (MRS) medium instead of DECT broth. Culture samples were collected at 24 h and centrifuged (4500 × g, 20 min, 4 °C), after which the supernatant was removed. The bacterial pellet was resuspended in 5 ml of sterile reducing buffer (NaHCO 3 In the presence of 4 g / L Na 2 S 9H 2 The bacteria were resuspended in Hungate tubes containing reduced sterile PBS mixed with 0.24 g / L O and 0.5 g / L cysteine. Adult Tph1- / - mice were transferred to an experimental isolator and fasted for 4 hours. The mice were then gavaged with 200 μL of a solution containing one of the bacterial strains (feeding, inoculation). 14 days after inoculation, serum from the proximal colon, longitudinal muscle-myenteric plexus (LMMP), and vena cava were collected from these animals and processed for further analysis.

[0140] Serum serotonin measurement by ELISA Serum was prepared from blood samples taken from the vena cava using Microvette® 500 Z-Gel (Sarstedt) tubes after centrifugation (5 min; 10,000 × g; room temperature (RT), 20-25 °C). Serotonin concentrations in serum samples were assessed using the ELISA kit ADI-900-175 (Enzo Life Sciences) according to the manufacturer's instructions.

[0141] Determination of tryptophan metabolites in broth by mass spectrometry (MS) - A comprehensive MS method Broth supernatants (50 μL) were extracted with 500 μL of methanol / acetic acid [99 / 1; v / v] containing d6-kynurenine 500 nM, d5-5-HIAA 1.25 μM and d4-serotonin 250 nM as internal standards (Sigma-Aldrich, Stockholm, Sweden). After vortexing and centrifugation, samples were purified by transferring the supernatants through an OSTROSPE 96 plate (Waters, Milford, MA) using a positive pressure manifold (Biotage AB, Uppsala, Sweden). After evaporation of the eluate under a stream of nitrogen at 40 °C, samples were reconstituted in 150 μL of injection solvent (methanol:water [10:90] + 0.1% hydrochloric acid (v / v)). Standard curves including serotonin, tryptophan, 5-HIAA, kynurenine, indole, tryptamine and 5-hydroxytryptophan (Sigma-Aldrich, Stockholm, Sweden) were prepared in methanol / acetic acid [99 / 1; v / v] and treated in the same way as the samples. Samples and calibration curve samples were injected into a Waters Acquity UPLC system equipped with a Waters BEH C18 column (2.1 × 100 mm; 1.7 μm particle size). The column temperature was maintained at 60 °C and the flow rate was 0.4 ml / min. The autosampler temperature was maintained at 10 °C. The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). Gradient elution started with an isocratic elution at 1% B over 1 min. The gradient was then increased linearly from 1 to 15% B over 2 min, followed by 15 to 99% B over 1 min. After a 1 min isocratic elution at 99% B, the gradient returned to 1% B and held for 2 min, for a total run time of 7 min. Tryptophan metabolites were detected using a Xevo TQ-XS (Waters, Milford, MA) using positive electrospray.After optimization, the ion source parameters were: capillary: 1.00 kV, desolvation temperature 500 °C, desolvation gas flow 1000 L / h, cone gas flow 150 L / h, nebulizer gas pressure 7.0 bar, cone voltages (V) and collision energies (eV): serotonin 30 V and 10 eV; tryptophan 25 V and 25 eV; 5-HIAA 20 V and 20 eV; kynurenine 20 V and 18 eV; indole 50 V and 23 eV; tryptamine 20 V and 18 eV; 5-hydroxytryptophan 25 V and 18 eV. The most intense precursor / product ion transitions were selected. The resulting MRM transitions used were as follows: serotonin m / z 177.1>160.3; tryptophan 205.2>118.25; 5-HIAA 192.4>146.2; kynurenine 209.1>146.1; indole 118.2>91.2; tryptamine 161.3>144.3; 5-hydroxytryptophan 221.2>162.2; d6-kynurenine 215.1>152.1; d4-serotonin 181.2>164.2; d5-5-HIAA 197.3>151.2. The retention time was 24 ms.

[0142] Serotonin measurement in broth by mass spectrometry (MS) - Serotonin-specific, highly sensitive, MS Broth supernatants (50 μL) were extracted with 250 μL of methanol / acetic acid [99 / 1; v / v] containing serotonin (unlabeled) as a calibrator and d6-kynurenine 500 nM (Sigma-Aldrich, Stockholm, Sweden) as an internal standard. After vortexing and centrifugation, samples were purified by transferring the supernatant through an OSTROSPE 96 plate (Waters, Milford, MA) using a positive pressure manifold (Biotage AB, Uppsala, Sweden). After evaporation of the eluate under a stream of nitrogen at 40 °C, samples were reconstituted in 150 μL of injection solvent (methanol:water [10:90] + 0.1% hydrochloric acid (v / v)). A standard curve containing serotonin (Sigma-Aldrich, Stockholm, Sweden) was prepared in methanol / acetic acid [99 / 1; v / v] and treated in the same way as the samples. Samples and calibration curve samples were injected into a Waters Acquity UPLC system equipped with a Waters BEH C18 column (2.1 × 100 mm; 1.7 μm particle size). The column temperature was maintained at 60 °C and the flow rate was 0.4 ml / min. The autosampler temperature was maintained at 10 °C. The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). Gradient elution started with an isocratic elution at 1% B over 1 min. The gradient was then increased linearly from 1 to 15% B over 2 min, followed by 15 to 99% B over 1 min. After the 1 min isocratic elution at 99% B, the gradient returned to 1% B and held for 2 min, with a total run time of 7 min. Serotonin was detected using a Xevo TQ-XS (Waters, Milford, MA) using positive electrospray. After optimization, the ion source parameters were: capillary: 1.00 kV, desolvation temperature 500 °C, desolvation gas flow 1000 L / h, cone gas flow 150 L / h, nebulizer gas pressure 7.0 bar, cone voltage (V) and collision energy (eV): serotonin 30 V and 10 eV. The most intense precursor / product ion transition was selected. As a result, the MRM transition used was serotonin m / z 177.1>160.3. The dwell time was 27 ms.

[0143] result Lactic acid-producing bacterial strains are commonly present in the human intestine, and many lactic acid-producing bacterial strains are also well-studied probiotic bacteria. Therefore, the serotonin-producing ability of many Lactobacillus strains, including the strain species L. reuteri, L. plantarum, L. casei and L. acidophilus, was tested with an extensive mass spectrometry (MS) assay optimized for specific tryptophan metabolites, including serotonin, to identify bacterial strains that have the ability to produce serotonin under anaerobic and aerobic conditions. In this way, it was found that all tested L. reuteri strains produced serotonin when cultured anaerobically in DECT broth for 24 hours (Figure 1A and Table 1). Surprisingly, it was also found that L. reuteri bacterial strains produced serotonin when cultured aerobically in DECT broth for 24 hours (Figure 1A and Table 1). Importantly, and even more surprisingly, the serotonin levels produced by the L. reuteri strain under aerobic conditions were significantly higher under aerobic conditions compared to anaerobic conditions, resulting in a ratio of aerobic / anaerobic serotonin production of >1.0. Conversely, no serotonin production was observed in the other bacterial strains tested, namely L. casei LMG 6904, L. plantarum 36 E or L. acidophilus ATCC 4356, when they were cultivated under anaerobic conditions (Table 1). [Table 1]

[0144] Three strains, L. casei LMG 6904, L. plantarum 36E and L. acidophilus ATCC 4356, were also tested for serotonin production using another, more sensitive, serotonin-specific mass spectrometry method. Using this method, low or very low levels of serotonin were detected in the medium obtained from all these bacterial strains after cultivation under anaerobic conditions (Table 2). However, no serotonin was detected by this sensitive serotonin-specific method after cultivation of these bacterial strains under aerobic conditions (Table 2). [Table 2]

[0145] These results indicate that some, but not all, lactate-producing bacterial strains can produce serotonin under anaerobic and / or aerobic conditions. Also, some lactate-producing bacterial strains can produce serotonin under anaerobic conditions but cannot produce serotonin under aerobic conditions. The results also demonstrate that various lactate-producing bacterial strains produce serotonin better than other lactate-producing bacterial strains under in vitro conditions that mimic the in vivo conditions of the gastrointestinal tract.

[0146] Further experiments will investigate whether the serotonin produced by the L. reuteri bacterial strain is bioactive and can be transported to and contribute to the circulating serotonin pool in vivo in Tph1- / -GF mice. These mice, which are deficient in endogenous serotonin production, were inoculated with L. reuteri DSM 17938 or L. reuteri DSM 27131. After inoculation with either L. reuteri strain, an increase in serum serotonin levels in Tph1- / -GF mice was observed using ELISA (Figure 1B), and the increase was more pronounced in DSM 27131 compared to DSM 17938. In contrast, when GF Tph1- / - mice were inoculated with L. casei LMG 6904, which did not produce serotonin in vitro (Figure 1A, Table 1, Table 2), serum levels of serotonin did not increase (Figure 1B). The results of these experiments clearly demonstrate that certain Lactobacillus species and certain lactic acid-producing bacterial strains have the ability to produce serotonin locally in the gastrointestinal tract that is bioavailable to the host and can be transported from the gastrointestinal tract to the periphery, thus contributing to both the gastrointestinal and systemic serotonin pools.

[0147] Thus, the results show that L. reuteri DSM 17938 and L. reuteri DSM 27131 are examples of lactic acid-producing bacterial strains that can be selected based on their aerobic serotonin production, high ratio of aerobic / anaerobic serotonin production, and can be used to increase serotonin levels in vivo. Therefore, these two bacterial strains, and other lactic acid-producing bacterial strains that have the same or similar ability to produce serotonin under aerobic conditions, such as the L. reuteri bacterial strains in Table 1, can be considered to be effective in treating serotonin deficiency and diseases related to serotonin deficiency. On the other hand, despite being able to produce low levels of serotonin under anaerobic conditions, bacterial strains such as L. plantarum 36E, L. casei LMG 6904 or L. acidophilus ATCC 4356 are not selected or considered effective in treating serotonin deficiency based on their inability to produce serotonin under aerobic conditions in vitro.

[0148] Example 2 - In vitro and in vivo production of serotonin and evaluation of additional suitable bacterial strains Since the Lactobacillus reuteri bacterial strain of Example 1 was found to produce serotonin well under both anaerobic and aerobic conditions, several additional L. reuteri bacterial strains (Table 3) were evaluated for their serotonin production.

[0149] material and method All bacterial cultures and serotonin measurements were performed as described in Example 1 using a serotonin-specific and sensitive MS method. The lactic acid-producing bacterial strains tested in this example and the levels of serotonin detected are listed in Table 3 below.

[0150] result The additional L. reuteri bacterial strains were cultured in DECT both anaerobically and aerobically for 24 hours, and then the concentration of serotonin in each medium was evaluated (Table 3). It was found that all of these additional L. reuteri bacterial strains were able to produce low but detectable amounts of serotonin under anaerobic conditions. It was further found that all of these L. reuteri bacterial strains produced more serotonin under aerobic conditions compared to anaerobic conditions (Table 3). Therefore, all of these L. reuteri strains could be selected as suitable strains according to the present invention for use in treating serotonin deficiency and diseases related to serotonin deficiency. [Table 3]

[0151] Example 3 - Bacterially produced serotonin has in vivo effects on ENS development and is important for crypt innervation of the colon material and method Bacterial culture, mouse cecum Cecums were harvested from conventional Tph1+ / + mice and cultured in DECT for 48 hours under anaerobic conditions at 37°C. At this point, samples were centrifuged (10,000 x g, 4°C, 2 min) and pellets were collected. Pellets were stored in 20% glycerol (cryoprotectant) and frozen at -80°C until use.

[0152] Bacterial culture, Lactobacillus For in vivo experiments, L. reuteri DSM 27131 and L. casei LMG 6904 were cultivated in MRS under anaerobic conditions. Each culture was sampled at 24 hours and the samples were centrifuged (10,000×g, 4° C., 2 min). The pellets were stored in 20% glycerol (cryoprotectant) and frozen at −80° C. until use.

[0153] Mouse inoculation experiment, mouse cecum and Lactobacillus For these experiments, mouse cecum, 48-h cultures of mouse cecal contents in DECT broth, or 24-h cultures of L. reuteri DSM 27131 and L. casei LMG 6904 in MRS were used. Pellets obtained from mouse cecum and Lactobacillus cultures were dissolved in 5 ml of sterile reducing buffer (NaHCO 3 In the presence of 4 g / L Na 2 S 9H 2 The cells were resuspended in Hungate tubes containing reduced sterile PBS mixed with 0.24 g / L O and 0.5 g / L cysteine. Adult Tph1- / - mice were transferred to an experimental isolator and fasted for 4 hours. Mice were gavaged with 200 μL of either a solution containing bacteria from mouse cecum or Lactobacillus. 14 days after inoculation, proximal colon, LMMP, serum from vena cava, luminal contents of colon and cecum were collected from these animals.

[0154] immunohistochemistry Proximal colon samples were fixed overnight in PFA 4% at 4°C and kept in 70% EtOH at 4°C before sectioning. Tissues were embedded in paraffin and sectioned at 10 μm thickness using a Leica System, deparaffinized by successive steps (TissuClear 2×12 min; 99% EtOH 2×2 min; 95% EtOH 2×2 min). For antigen retrieval, slides were incubated with 10 mM sodium citrate / 0.05% TWEEN® 20 solution (pH 6.0), warmed in a water bath (95°C) for 20 min, incubated at room temperature for 20 min, and rinsed twice with PBS-0.05% TWEEN® 20. Slides were then incubated with blocking solution (PBS / 4% BSA / 4% donkey serum) for 1 h at room temperature; primary antibodies diluted in blocking solution (Table 4) overnight at 4° C.; secondary antibodies diluted in blocking solution (Table 4) for 1 h at room temperature; and Hoechst solution for 5 min at room temperature. From the incubation with primary antibodies, two washing steps in PBS were performed after each incubation step. Slides were mounted with mounting medium (Dako). [Table 4]

[0155] After dissection, LMMP samples were fixed in PFA 4% overnight at 4 °C and rinsed three times for 10 min with cold PBS. LMMPs were then stored in NaN at 4 °C until immunostaining. 3 Solution (0.1% NaN diluted in PBS) 3 LMMP samples were kept in 300 μl of blocking solution (NaN 3 The tissues were incubated for 1 h in Triton X 100 0.5%, BSA 4% and donkey serum 4% in a 0.1% solution. The tissues were then incubated overnight with primary antibodies (Table 4) diluted in blocking solution, followed by 3 washes for 10 min in PBS and incubation for 1.5 h with secondary antibodies (Table 4). The tissues were washed 3 times for 10 min in PBS, incubated for 5 min in Hoechst solution and washed again 3 times for 10 min in PBS. The LMMP samples were mounted with fluorescent mounting solution.

[0156] Immunostained tissues were imaged by confocal microscopy using a Zeiss Laser Scanning Inverted Microscope LSM-700 equipped with a 20× / 0.8 NA objective and Black Zen software (Carl Zeiss).

[0157] PGP9.5 and TuJ1 are well-established markers for mature enteric neurons. Therefore, for the proximal colon, we quantified the percentage of immunopositive area for PGP9.5 or TUJ1 in the colonic tissue, and the number of epithelial cells positive for serotonin was counted and divided by the mucosal area. Two to three images were analyzed per tissue, and three tissue samples were analyzed per animal.

[0158] For ENS immunostaining, the area of ​​fluorescence positive for serotonin was quantified. Two to three images were analyzed per tissue sample.

[0159] result As serotonin is essential for normal development and maturation of the ENS, we investigated the effect of microbially produced serotonin on mouse ENS development (Figure 2A-F). Immunostaining revealed that colonization of GF Tph1- / - mice with mouse cecal microbiota increased serotonin levels within enteric neurons in the proximal colon (Figure 2A). The presence of cecal microbiota also increased well-established markers of mature enteric neurons, PGP9.5 and TUJ1, within colonic tissue (Figure 2B and 2C), demonstrating that bacterially produced serotonin plays a role in ENS development and maturation. GF Tph1- / - mice were also inoculated with L. reuteri DSM 27131 or L. casei LMG 6904 for 14 days and assessed for serotonin supplementation of the ENS. We found that inoculation with L. reuteri DSM 27131, which has previously been shown to produce serotonin under aerobic conditions in vitro as well as in vivo, increased neuronal serotonin levels (Figure 2D), and that DSM 27131 also increased markers of mature neurons, PGP9.5 and Tuj1, in the proximal colon, whereas the non-serotonin-producing L. casei LMG 6904 had no effect on either neuronal serotonin levels or markers of neuronal maturation in the proximal colon.

[0160] Example 4 - Isolation and selection of serotonin-producing human bacterial strains material and method Human fecal collection Human fecal sample collection was performed from three healthy donors aged 30–50 years. They were treatment-naive volunteers and had not taken any antibiotics. Human fecal sample collection and subsequent use of the samples were performed in accordance with Swedish ethical policies and regulations.

[0161] Bacterial Culture – Human Feces Freshly voided human feces (1% (w / v) inoculum) was inoculated into DECT broth under anaerobic conditions, i.e., in a Coy chamber, or under ambient aerobic conditions. Each culture was sampled at different time points (0 h, i.e., before inoculation, 12 h, 24 h, 36 h, 48 h, 60 h) and the samples were centrifuged (10,000×g, 4° C., 2 min). The supernatants and pellets were frozen at −80° C. The supernatants were used for serotonin measurements and the pellets were used to analyze the bacterial communities within the samples by utilizing 16S rRNA gene analysis. One glycerol stock of each sample was also retained from each of the above time points.

[0162] Isolation of bacterial serotonin-producing strains DECT broth was made according to Example 1. LYBHI medium contained Brain Heart Infusion (BHI) 3.7% (Oxoid Fisher-Scientific # CM 1135), L-cysteine ​​0.05% (Sigma-Aldrich #W 326305), D-(+)-cellobiose 0.1% (Sigma-Aldrich #22150), maltose 0.1% and hemin 0.0006% (Sigma-Aldrich #51280). To obtain anoxic variants of DECT and LYBHI, cysteine ​​0.05% was added as a reducing agent. Growth media were autoclaved at 121°C and 15 PSI for 30 min. 36-h glycerol stocks obtained from human feces cultured in DECT broth were plated under anaerobic conditions on LYBHI (LYBHI broth with 2% agar; Nordic Biolabs #214010), DECT (DECT broth with 2% agar) and MRS (50 g of MRS powder from Sigma-Aldrich (Sigma-Aldrich #69966) and 1 ml of TWEEN® 80 (Sigma-Aldrich #P8074) in 1 L of distilled water with 2% agar) plates. Plating was done in triplicate, meaning that a second culture plating was done from sampling from the most abundant area in the first plate onto a second plate and from the second plate onto a third plate. Each isolated colony was then cultured in LYBHI, DECT and MRS, respectively, to make glycerol stocks and DNA was recovered after centrifugation (10,000×g, 4° C., 2 min). DNA was collected for identification of each bacterial strain by sequencing the 16S rRNA gene. The purity of each isolate was confirmed by Gram staining. For serotonin production kinetics, each isolate was cultured in DECT broth under aerobic and anaerobic conditions for 24 h. Samples from the cultures were then collected and centrifuged (10,000 × g, 4 °C, 2 min) to collect the supernatant and analyze the levels of serotonin.

[0163] Extraction of total fecal genomic DNA genome DNA was isolated from pellets obtained from 1.5 ml culture samples using the NucleoSpin Soil kit (MACHEREY-NAGEL, Germany) according to the manufacturer's instructions, but with a modified cell lysis step. Samples were suspended in SL2 buffer supplemented with SX and then mixed at maximum speed for 2 min on a vortex with a horizontal adapter. Cells were lysed by heating at 90°C for 10 min followed by three rounds of 60 s bead-beating at 5.5 m / s using a FastPrep-24 instrument (MP Biomedicals). The heating and bead-beating steps were repeated one more time and the supernatants from the two extractions were pooled before purification using the columns in the kit. DNA was eluted using elution buffer and DNA concentration and quality were confirmed using a NanoDrop spectrophotometer.

[0164] 16S rRNA gene sequencing and identification of isolates The full length of the 16S rRNA gene was amplified using 27F and 1492R primers designed for dual indexing (Appl Environ Microbiol 79:5112-5120(2013)). Each sample was amplified in a 50 μL reaction containing HotStarTaq Master Mix 2X (QIAGEN), 10 μmol / L of each primer, and 250 ng of genomic DNA. PCR was performed under the following conditions: initial denaturation at 94 °C for 5 min, followed by 26 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 40 s and extension at 72 °C for 90 s, and a final extension step at 72 °C for 7 min. Samples were purified with NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and quantified using a NanoDrop spectrophotometer. Dilutions of 10 ng / μl in a volume of 40 μL were prepared and sent to Eurofins for sequencing. Forward and reverse amplicon sequences were obtained for each isolate and aligned to find a consensus sequence, which was then blasted using the Basic Local Alignment Search Tool from NCBI to identify the bacteria.

[0165] Serotonin measurement in broth using a highly sensitive serotonin-specific MS method The serotonin produced by these bacterial strains was quantified using a serotonin-specific and sensitive mass spectrometry method as described in Example 1.

[0166] result Serotonin production To identify serotonin-producing microorganisms from the human gut, fecal samples from the two donors with the highest total serotonin production, i.e., donor #1 and donor #2, were cultured in DECT under anaerobic conditions. Using LYBHI, DECT and MRS agar plates, 122 bacterial isolates from donor #1 and 77 from donor #2 were obtained, totaling 199 isolates. The levels of serotonin production by these isolates were then determined by growing the resulting isolates as monocultures under anaerobic or aerobic conditions. Of the 199 isolates, only 6 isolates from donor #1 and 11 isolates from donor #2 produced detectable levels of serotonin in DECT broth under anaerobic conditions (Table 5; Figure 3). Of the 199 isolates, a total of 24 isolates were found to produce serotonin under aerobic conditions, including isolates that also produced serotonin under anaerobic conditions. For all these 24 isolates, serotonin production was improved under aerobic conditions compared to anaerobic conditions. Full-length 16S rRNA sequencing analysis identified the 24 isolates as Lactobacillus mucosae (n = 13), Lactobacillus plantarum (n = 6), and Enterococcus spp. (n = 1) (Table 5). Finally, four isolates from donor #2 were not clearly identified, but these isolates aligned closely with Escherichia, Shigella, or Brenneria bacterial strains (Table 5). Nevertheless, these results suggest an important role for Lactobacillus genus, especially Lactobacillus mucosae and Lactobacillus plantarum bacterial strains, in serotonin production under both anaerobic and aerobic conditions.

[0167] Lactic acid producing bacterial strains belonging to either L. mucosae or L. plantarum were selected, which produce serotonin well under aerobic conditions (Table 5). More specifically, bacterial strains L. mucosae h1D22g, h1M31d, h1M31g, and h1M32d, and L. plantarum h2M3E and h2M3i were selected and deposited in DSMZ based on their serotonin production under aerobic conditions (the corresponding deposit numbers are DSM 33291, DSM 33292, DSM 33293, DSM 33661, DSM 33662, and DSM 33295, respectively). [Table 5-1] [Table 5-2]

[0168] Example 5 - Mouse inoculation experiment with human serotonin-producing bacterial strains material and method Inoculation of mice with human serotonin-producing bacterial strains Mouse inoculations were performed using germ-free Tph1- / - mice. Animals were inoculated with either Lactobacillus mucosae DSM 33661 or Lactobacillus plantarum DSM 33662 using the same method as described in Example 3. Serum and intestinal luminal samples, as well as tissue from the proximal colon, were collected 14 days after inoculation and analyzed for serotonin.

[0169] Serum and intestinal lumen serotonin measurement by ELISA Serum was prepared from blood samples taken from the vena cava using Microvette® 500 Z-Gel (Sarstedt) tubes after centrifugation (5 min; 10,000 × g; room temperature (RT), 20-25 °C). The luminal contents of the gastrointestinal tract were sampled from the proximal colon or cecum. The luminal contents were weighed and flash frozen. Serotonin from the luminal contents was then extracted as follows: A fixed amount of Tris-HCl 25 mM / EDTA 1 mM / EGTA 1 mM solution was added to each sample (approximately 1 mg / 2 μL w / v), homogenized with 5 mm steel beads and a TissueLyser (Qiagen, 25 Hz, 2 min), incubated overnight at 4 °C, and centrifuged at 10,000 × g for 20 min at 4 °C. The supernatant of each sample was collected and kept at -80 °C until analysis. Broth supernatants from bacterial cultures were obtained to measure serotonin, and non-cultured broth was used as a blank. Serotonin from all samples was assessed using ELISA kit ADI-900-175 (Enzo Life Sciences) according to the manufacturer's instructions.

[0170] Serotonin immunopositive area in the proximal colon Serotonin immunopositive areas in tissue samples from the proximal colon were analyzed and quantified as described in Example 3.

[0171] result Two isolated human bacterial strains, L. mucosae DSM 33661 and L. plantarum DSM 33662, were selected based on their serotonin production under anaerobic and aerobic conditions in vitro. These strains were found to produce functional serotonin in vivo as well (Figure 4). The graph shows that both these bacterial strains were able to increase serotonin levels locally in the gut lumen (Figure 4A) and in the systemic circulation (serum samples, Figure 4B) compared to the levels found in corresponding germ-free mice. Furthermore, both these human strains were found to increase the serotonin-positive area of ​​the ENS of the proximal colon (Figure 4C). Thus, these human bacterial strains are able to increase serotonin levels locally in the gut lumen (cecum) in vivo, and this locally produced serotonin is bioactive and can be transported to the systemic circulation (serum) and to neurons of the enteric nervous system of the proximal colon. The results demonstrate that serotonin-producing human commensal bacterial strains can be isolated, identified, and selected to increase serotonin levels in vivo.

[0172] Example 6 - Adhesion of serotonin-producing bacterial strains to mucus material and method Mucus preparation and plate coating Mucus was prepared from porcine small intestine. The inside of 5 cm of intestine was scraped with a spatula and the material was removed and collected in 5 ml of ice-cold PBS. To remove cells and particulate matter, the resulting suspension was first centrifuged at 11,000 × g for 10 min and then at 26,000 × g for 15 min. Mucus preparations were stored at -20 °C until use. The mucus material was diluted in PBS pH 6.0 with A 280 = 0.1 (absorbance at 280 nM = 0.1) and incubated in microtiter wells (Nunc MaxiSorb) (150 μl / well) with gentle agitation overnight at 4° C. The wells were then washed with PBS (pH 6.0) containing 0.05% TWEEN® 20 (PBST), then blocked with 0.2 ml PBS pH 6.0 containing 1% TWEEN® 20 for 1 hour and finally washed twice with PBST.

[0173] Preparation of bacteria Mucus adhesion experiments were performed with the following bacterial strains: Lactobacillus reuteri DSM 27131, DSM 32465, DSM 33632, DSM 33633, DSM 33634, DSM 33635, and DSM 33509; Lactobacillus mucosae DSM 33291, DSM 33292, DSM 33293, and DSM 33661; and Lactobacillus plantarum DSM 33662. Each Lactobacillus strain was inoculated from a frozen stock at -70 °C and grown in 10 ml of MRS broth (Merck) at 37 °C for 16 h. The OD was measured at 600 nM and the samples were diluted to OD 0.5 (total volume is 1 ml). The diluted bacterial solution was then washed twice with PBST pH 7.4 and resuspended in PBST pH 6.0. After a final wash and resuspension in PBST pH 6.0, the bacteria were diluted and plated on MRS agar to analyze the starting values ​​(colony forming units (cfu) / ml). The same bacterial suspension was used for the binding experiments.

[0174] Mucus binding, trypsin release and quantification Bacterial suspension (150 μl) was added to each well and incubated at 37 °C for 4 h with gentle agitation. The wells were washed four times with PBST (pH 6.0) to remove unbound bacteria. After washing, 150 μL of trypsin-EDTA solution (0.25% porcine trypsin and 1 mM EDTA·4Na in Hank's balanced salt solution) was added to the wells and incubated at 37 °C for 30 min. The released bacteria were then mixed thoroughly, serially diluted, and plated on MRS agar plates. The plates were incubated in anaerobic conditions at 37 °C for 24 h, after which colonies were counted (colony forming units (cfu)) and the number of attached bacteria per well was calculated.

[0175] result We investigated whether serotonin-producing lactobacilli can also adhere to mucus in vitro and show that they can also adhere to the intestinal mucosa in vivo, thereby allowing the uptake of bacterially produced and secreted serotonin by the intestinal mucosa, and in particular by the intestinal epithelium. The bacterial strains tested for adhesion were selected based on their ability to produce serotonin under aerobic conditions. L. reuteri DSM 27131, which can produce serotonin under aerobic conditions in vitro and can also contribute to the systemic pool of serotonin in vivo, was found to be able to adhere to mucus (Figure 10A). This ability was also observed and further increased in all other L. reuteri strains tested (DSM 32465, DSM 33632, DSM 33633, DSM 33634, DSM 33635, DSM 33509), and in all other L. mucosae strains tested (DSM 33291, DSM 33292, DSM 33293, DSM 33661) and the L. plantarum strain DSM 33662 (Figures 10A and 10B). The adhesion ability of each of the L. reuteri bacterial strains DSM 33632, DSM 33633, DSM 33634, DSM 33635, and DSM 33509 was found to be improved compared to that of their corresponding parent strains (data not shown). These results indicate that certain lactic acid bacteria strains of the species Lactobacillus reuteri, Lactobacillus mucosae, and Lactobacillus plantarum are able to adhere to the intestinal mucosa, where metabolic products such as serotonin can be produced and secreted in the vicinity of the intestinal epithelium lining the gastrointestinal tract.

[0176] Example 7 - Production of an oil-based formulated probiotic product for use in serotonin deficiency In this example, a probiotic product is produced for use in serotonin deficiency. A lactic acid producing bacterial strain capable of producing serotonin under aerobic conditions, for example any of the Lactobacillus bacterial strains of Examples 1, 2, 4 or 6, is selected as disclosed herein. These strains are then grown separately as pure cultures and freeze-dried using standard industry methods for growing Lactobacillus bacterial strains. The product is an oil-based formulation with a single (pure) bacterial strain, made for good stability and shelf life. A unique feature of the manufacturing process is the step of drying the oil by placing it under vacuum to remove most of the water in the oil and increase the stability of the formulation. The oil used herein is a pure edible vegetable oil, preferably sunflower oil and medium chain triglyceride.

[0177] The ingredients are mixed. 1. Medium chain triglyceride, e.g. Akomed R (Karlshamns AB, Karlshamn Sweden) and sunflower oil, e.g. Akosun (Karlshamns AB, Karlshamn Sweden) are mixed with silicon dioxide (Cab-o-sil M5P, M5P, Cabot) in a Bolz mixer / tank (Alfred BOLZ Apparatebau GmbH, Wangen im Allgau, Germany). 2. A homogenization Sine pump and dispax (Sine Pump, Arvada, Colorado) is connected to the Bolz mixer and the mixture is homogenized. 3. Vacuum dry: The mixture is dried in a Bolz tank under 10 mBar vacuum for 12 hours. 4. Add Lactobacillus bacteria. Transfer approximately 20 kg of the dry oil mixture into a 50 liter stainless steel container. The Lactobacillus bacteria powder is preferably freeze-dried. The amount of Lactobacillus bacteria used will vary depending on the amount required in the oil, but one example is 10 11 0.2 kg of culture with CFU / g will be added. Mix slowly until homogenous. 5. The premix containing the mixed Lactobacillus bacteria is returned to the Bolz mixer. 6. The release suspension is released into a 200 liter glass container and blanketed with nitrogen. The suspension is retained in the container until it is filled into spray bottles for oral administration to humans for the prevention or treatment of serotonin deficiency.

[0178] Example 8 - Manufacturing of a probiotic product in tablet form for use in serotonin deficiency In this example, a Lactobacillus strain capable of producing serotonin under aerobic conditions is used to manufacture tablets for use in preventing, inhibiting and / or treating serotonin deficiency in humans.The Lactobacillus strain is grown and lyophilized using standard methods for growing Lactobacillus in the industry.

[0179] The following steps provide an example of a manufacturing process for tablets containing selected bacterial strains, including glucose encapsulation: It is understood that excipients, fillers, flavorings, encapsulants, lubricants, anti-caking agents, sweeteners and other ingredients of tablet products may be used as known in the art without affecting the efficacy of the product.

[0180] 1. Molten SOFTISAN™ 154 (SASOL GMBH, Bad Homburg, Germany) is melted in a container and heated to 70° C. to ensure complete destruction of the crystalline structure. It is then cooled to 52-55° C. (just above the hardening point). 2. Transfer the granulated Lactobacillus bacteria freeze-dried powder to a Diosna high shear mixer / granulator or equivalent. Slowly add the molten SOFTISAN™ 154 to the Lactobacillus bacteria powder for approximately 1 minute. Use the chopper during addition. 3. Wet sieving: Immediately after granulation, the granules are passed through a 1 mm sieve mesh using a tornado mill. The sieved granules are packed into aluminum pouches made from PVC-coated aluminum foil, sealed with a heat sealer to form a pouch with a desiccant pouch, and stored refrigerated until blending. The granulation batch is divided into two tablet batches. 4. Add encapsulated D-glucose (G 8270, >99.5% glucose, Sigma) encapsulated using standard microencapsulation methods known in the art. The amount of sugar depends on the total CFU of the dried Lactobacillus bacteria powder added, with standard levels ranging from 10 8 The preferred range may be 1 gram of sugar per total CFU of bacteria, but this may vary from 0.1 grams, or from 0.01 grams to 10 grams or even 100 grams of sugar. 5. Mixing Mix all ingredients in a mixer to a homogenous blend. 6. Compression The final blend is transferred to the hopper of a rotary tablet press and compressed into tablets of 765 mg total weight in a Kilian compression machine. 7. Bulk Packaging The tablets are packed in aluminum pouches along with a desiccant pouch of molecular sieves. The aluminum pouches are stored in plastic buckets in a cool place for at least one week prior to final packaging. The use of SOFTISAN™, a hydrogenated palm oil, allows the Lactobacillus cells to be encapsulated in fat and environmentally protected.

[0181] As noted above, the products of the embodiments may be in forms other than tablets, and standard methods for preparing basic products known in the art are advantageously used to prepare the products of the invention containing the selected Lactobacillus cultures.

[0182] The above embodiments should be understood as some illustrative examples of the present invention. Those skilled in the art will understand that various modifications, combinations and changes can be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in different embodiments can be combined in other configurations, if technically possible. However, the scope of the present invention is defined by the appended claims.

Claims

1. 1. A method for selecting a bacterial strain for use in treating a disease associated with serotonin deficiency, comprising: Cultivating bacteria of a Lactobacillus bacterial strain selected from the group consisting of L. reuteri and L. mucosae bacterial strains in a medium containing tryptophan under aerobic conditions (S1); Detecting any serotonin produced by the bacteria of the Lactobacillus bacterial strain in the medium or a sample thereof (S2); Determining whether the bacteria of said Lactobacillus bacterial strain are capable of adhering to intestinal mucus and / or to the gastrointestinal mucosa (S15); and selecting said Lactobacillus bacterial strain as being effective for treating a disease associated with serotonin deficiency in a subject if serotonin is detected in said medium or a sample thereof and if said bacteria of said Lactobacillus bacterial strain are capable of adhering to intestinal mucus and / or to gastrointestinal mucosa (S3); Including, The above method, wherein the disease associated with serotonin deficiency is selected from the group consisting of anxiety, depression, aggression, memory problems, eating disorders, obsessive-compulsive disorder, panic disorder, post-traumatic stress disorder, social anxiety disorder, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), cardiovascular disease, osteoporosis, gastrointestinal motility disorders, autoimmune polyendocrine deficiency-candidiasis-ectodermal dystrophy (APECED), osteopenia and bone loss conditions.

2. Cultivating the bacteria of the Lactobacillus bacterial strain in a medium containing tryptophan under anaerobic conditions (S10); Detecting any serotonin produced by the bacteria of the Lactobacillus bacterial strain in the medium or a sample thereof when cultured under anaerobic conditions (S11); and Calculating the ratio between the concentration of serotonin detected in the medium or a sample thereof when cultured under aerobic conditions and the concentration of serotonin detected in the medium or a sample thereof when cultured under anaerobic conditions (S12). Further comprising: The method of claim 1, wherein selecting (S3) the Lactobacillus bacterial strain comprises selecting (S3) the Lactobacillus bacterial strain as being effective for treating a disease associated with serotonin deficiency in a subject if the ratio is greater than or equal to 1.0 and if the bacteria of the Lactobacillus bacterial strain are capable of adhering to intestinal mucus and / or gastrointestinal mucosa.

3. The method of claim 2, wherein selecting (S3) the Lactobacillus bacterial strain comprises selecting (S3) the Lactobacillus bacterial strain as being effective for treating a disease associated with serotonin deficiency in a subject if the ratio is greater than 1.0 and if the bacteria of the Lactobacillus bacterial strain are capable of adhering to intestinal mucus and / or gastrointestinal mucosa.

4. The method according to any one of claims 1 to 3, wherein the step of selecting (S3) the Lactobacillus bacterial strain comprises: selecting (S3) the Lactobacillus bacterial strain as being effective in treating a disease associated with serotonin deficiency in a subject if serotonin is detected in the medium or a sample thereof, and if the bacteria of the Lactobacillus bacterial strain have a mucoadhesive ability higher than that of L. reuteri DSM 27131.

5. Lactobacillus reuteri strain DSM 33509.

6. Lactobacillus reuteri strain DSM 33632.

7. Lactobacillus reuteri strain DSM 33633.

8. Lactobacillus reuteri strain DSM 33634.

9. Lactobacillus reuteri strain DSM 33635.

10. Lactobacillus mucosae strain DSM 33291.

11. Lactobacillus mucosae strain DSM 33292.

12. Lactobacillus mucosae strain DSM 33293.

13. Lactobacillus mucosae strain DSM 33661.

14. A pharmaceutical composition for use as a medicament, comprising a Lactobacillus reuteri strain according to any one of claims 5 to 9 or a Lactobacillus mucosae strain according to any one of claims 10 to 13.

15. 14. A pharmaceutical composition for use in the treatment of a disease associated with serotonin deficiency, said pharmaceutical composition comprising a Lactobacillus reuteri strain according to any one of claims 5 to 9 or a Lactobacillus mucosae strain according to any one of claims 10 to 13, and wherein said disease associated with serotonin deficiency is selected from the group consisting of anxiety, depression, aggression, poor memory, eating disorders, obsessive-compulsive disorder, panic disorder, post-traumatic stress disorder, social anxiety disorder, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), cardiovascular disease, osteoporosis, gastrointestinal motility disorders, autoimmune polyendocrine deficiency-candidiasis-ectodermal dystrophy (APECED), osteopenia and osteopenic conditions.

16. 16. The pharmaceutical composition of claim 15, wherein the disease associated with serotonin deficiency is selected from the group consisting of IBS, IBD, cardiovascular disease, osteoporosis, gastrointestinal motility disorders, APECED, osteopenia and bone loss conditions.

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