Serotonin-producing bacteria
A serotonin, bacterial technology for use in the treatment of serotonin deficiency and in the treatment of conditions and diseases associated with serotonin deficiency
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Embodiment 1
[0146] Example 1 - In vitro and in vivo production of serotonin and evaluation of suitable bacterial strains
[0147] Materials and methods
[0148] animal
[0149] 8-12 weeks old Tph1++ / + (as littermate controls) and Tph1- / - (C57Bl / 6 background) mice were housed in a climate-controlled room (22±2°C) and subjected to a 12h light / dark cycle (7:00 AM-7:00 PM) with free access to water and food. described earlier Tph1- / - mice ( Cell 135: 825-837 (2008)). All animal procedures were approved by the Ethics Committee on Animal Care and Use in Gothenburg, Sweden. Animals were obtained from heterozygous breeding and genotyped prior to experiments. After the experiment, the proximal colon, serum from the vena cava, material from the intestinal lumen and cecum were obtained from each animal. TPH1 is the rate-limiting enzyme involved in the synthesis of endogenous serotonin in the periphery, including in the gastrointestinal tract. therefore, Tph1 knockout( Tph1- / - ) mi...
Embodiment 2
[0171] Example 2 - In vitro and in vivo production of serotonin and evaluation of additional suitable bacterial strains
[0172] Since the L. reuteri bacterial strain in Example 1 was found to be a good producer of serotonin under both anaerobic and aerobic conditions, a number of additional L. reuteri bacterial strains (Table 3) were evaluated for serotonin production .
[0173] Materials and methods
[0174] All bacterial culture and serotonin measurement methods 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 detected levels of serotonin are listed in Table 3 below.
[0175] result
[0176] Additional Lactobacillus reuteri bacterial strains were incubated anaerobic and aerobic in DECT for 24 hours, and then the serotonin concentration in the respective media was assessed (Table 3). All of these additional Lactobacillus reuteri bacterial strains wer...
Embodiment 3
[0179] Example 3 - Bacterial production of serotonin has in vivo effects on ENS development and is critical for crypt innervation in the colon
[0180] Materials and methods
[0181] Bacterial culture, mouse cecum
[0182] from regular Tph1++ / + Mice were cecum harvested and cultured in DECT for 48 h at 37 °C under anaerobic conditions. At this point the samples were centrifuged (10,000 xg, 4°C, 2 minutes) and the pellet collected. The pellet was stored in 20% glycerol (antifreeze) and frozen at -80°C until use.
[0183] Bacterial culture, Lactobacillus
[0184] For in vivo experiments, Lactobacillus reuteri DSM 27131 and Lactobacillus casei LMG 6904 were grown 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 pellet was stored in 20% glycerol (antifreeze) and frozen at -80°C until use.
[0185] Inoculation experiments in mice, mouse cecum and Lactobacillus
[0186] For these ...
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