Methods and applications for producing N-acetylserotonin and short-chain fatty acids using *Osmotherium visceratum* DSM 20712

Abstract

This invention discloses a method for producing N-acetylserotonin and short-chain fatty acids using *Osmobacterium visceratum* DSM 20712 and its applications, belonging to the field of microbiology and biomedicine. The strain's accession number is DSM 20712. Testing showed that this strain can metabolize and produce N-acetylserotonin under pure in vitro culture conditions; the addition of melatonin to the fermentation system resulted in a significant synergistic effect. Simultaneously, this strain can independently produce six short-chain fatty acids under conditions detached from the host metabolic environment, with propionic acid accounting for the highest proportion (68.5%). This invention also provides a method for producing N-acetylserotonin and / or short-chain fatty acids using this strain, as well as a probiotic composition containing this strain. This strain possesses the dual ability to metabolize and produce both N-acetylserotonin and short-chain fatty acids, providing a safe and efficient new route for their bioprocessing.

Description

Technical Field

[0001] This invention belongs to the field of microbial and biomedical technology, specifically relating to a method for producing N-acetylserotonin and short-chain fatty acids using *Ostomyces visceratus* DSM 20712 and its applications. Background Technology

[0002] N-acetylserotonin (NAS) is a precursor to melatonin and possesses various physiological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. Short-chain fatty acids (SCFAs) are important metabolites of dietary fiber fermented by gut microbiota and play a crucial role in maintaining intestinal barrier function and regulating immunity and metabolic homeostasis. The applicant's prior patent (ZL 202411711276.0) discloses a novel use of NAS in the preparation of drugs for the prevention and treatment of diabetes, providing an important direction for the application of NAS.

[0003] Currently, NAS production mainly relies on chemical synthesis or genetically engineered microbial fermentation. Chemical synthesis typically requires harsh conditions, is prone to organic solvent contamination, and is costly. While genetically engineered bacteria (such as recombinant Escherichia coli and Streptomyces) can achieve heterologous NAS production, they suffer from drawbacks such as complex genetic manipulation, easy plasmid loss, and the need for additional verification of the safety of environmental release from engineered strains. Therefore, finding new microbial resources that can naturally and safely produce NAS has significant application value.

[0004] Odoribacter splanchnicus is a commensal bacterium found in the human gut. Existing research indicates that colonization of this strain in the gut of germ-free mice results in the production of short-chain fatty acids, which exert immunomodulatory effects (Svetlana Lima et al., Gastroenterology, 2022). However, these results are from in vivo experiments and are subject to the complex influences of the host's physiological environment, thus failing to reflect the strain's independent metabolic capacity under host-free conditions. Furthermore, existing literature lacks a systematic identification and proportional analysis of the produced short-chain fatty acids. Similarly, the current technology lacks research and understanding regarding whether this strain possesses the ability to produce other important bioactive substances (such as N-acetylserotonin).

[0005] Therefore, finding a new microbial resource that is safer, requires no genetic modification, and has diverse metabolic functions to achieve the safe and efficient production of N-acetylserotonin and / or short-chain fatty acids remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] In view of the above, it is necessary to provide a method and its application for producing N-acetylserotonin and short-chain fatty acids using *Osmotherium visceratum* DSM 20712. This strain is a natural human symbiotic bacterium that has not been genetically modified and has high safety. By adding melatonin, it can produce a significant synergistic effect and can simultaneously produce high yields of short-chain fatty acids, mainly propionic acid, providing a safe and efficient new route for related bioprocessing.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows.

[0008] This invention proposes a method for producing N-acetylserotonin, comprising the following steps:

[0009] (a) The Odoribacter splanchnicus strain with accession number DSM 20712 was inoculated into the fermentation medium;

[0010] (b) Fermentation culture under anaerobic conditions;

[0011] (c) Collect N-acetylserotonin from the fermentation products.

[0012] In this invention, melatonin is further added to the fermentation culture medium, and the final concentration of melatonin in the fermentation culture medium is 0.1-0.3 mg / mL.

[0013] Furthermore, in this invention, the *Odoribacter splanchnicus* strain simultaneously produces short-chain fatty acids during fermentation, including acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid.

[0014] Furthermore, in this invention, propionic acid accounts for the highest proportion of the short-chain fatty acids produced.

[0015] This invention also proposes a method for producing short-chain fatty acids, comprising the following steps:

[0016] (a) The Odoribacter splanchnicus strain with accession number DSM 20712 was inoculated into the fermentation medium;

[0017] (b) Fermentation culture under anaerobic conditions;

[0018] (c) Collecting short-chain fatty acids from fermentation products;

[0019] The short-chain fatty acids include acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid, and the production of propionic acid accounts for no less than 60% of the total production of all short-chain fatty acids.

[0020] The present invention also proposes a probiotic composition comprising Odoribacter splanchnicus strain with accession number DSM 20712 as an active ingredient, and pharmaceutically or food-grade excipients.

[0021] Furthermore, in this invention, the composition further comprises melatonin.

[0022] The present invention also proposes the use of Odoribactersplanchnicus, accession number DSM 20712, in the preparation of formulations for increasing N-acetylserotonin production.

[0023] In this invention, the application further includes the combined use of Odoribacterplanchnicus with melatonin.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects.

[0025] 1. This invention provides a novel pathway for the production of N-acetylserotonin using the natural gut symbiotic bacterium *Odoribacterplanchnicus* DSM 20712. Unlike existing chemical synthesis or genetically engineered bacterial fermentation routes, this strain is a wild-type gut symbiotic that has not been genetically modified, resulting in higher safety and milder culture conditions. Experiments have shown that this strain can metabolize and produce N-acetylserotonin under pure in vitro culture conditions, with an intracellular concentration of 1.22 ng / mL and an extracellular concentration of 0.26 ng / mL. In particular, the addition of melatonin to the fermentation medium significantly increased the N-acetylserotonin yield to 2.55 ng / mL, approximately 10 times higher than the untreated group, demonstrating a significant synergistic effect and providing a new technical solution for the efficient bioproduction of N-acetylserotonin.

[0026] 2. This invention systematically identified the short-chain fatty acid production profile of *Odoribactersplanchnicus* DSM 20712 in an in vitro pure culture system. Under conditions removed from the host metabolic environment, this strain can independently and stably produce six short-chain fatty acids, with the following composition: acetic acid 6.6% : propionic acid 68.5% : butyric acid 7.4% : valeric acid 3.1% : isobutyric acid 10.4% : isovaleric acid 4.0%, with propionic acid accounting for the largest proportion. This finding clarifies that this strain has a propionic acid-dominant metabolic characteristic, providing a clear technical basis for the targeted production of beneficial short-chain fatty acids such as propionic acid.

[0027] 3. This invention is the first to discover that *Odoribacter splanchnicus* DSM 20712 possesses dual metabolic functions, producing both N-acetylserotonin and short-chain fatty acids, thus achieving "multi-purpose use of a single strain." The N-acetylserotonin and propionic acid produced by this strain both have known physiological activities, indicating that this strain has application potential in the field of functional foods. Attached Figure Description

[0028] Figure 1 The growth curves of Odoribacter splanchnicus DSM 20712 in different experimental groups during fermentation are shown in the figure. In the figure, A is the growth curve of the live bacteria group and B is the growth curve of the live bacteria + melatonin group.

[0029] Figure 2 LC-MS / MS peak area plots of N-acetylserotonin (NAS) production in different experimental groups.

[0030] Figure 3 This is a standard curve of N-acetylserotonin (NAS).

[0031] Figure 4 A comparative graph showing the detection results of N-acetylserotonin (NAS) production in different experimental groups.

[0032] Figure 5 Standard curves for six short-chain fatty acids (SCFAs).

[0033] Figure 6 This is a graph showing the yield and composition ratio of short-chain fatty acids produced by Odoribacter splanchnicus DSM 20712 under in vitro pure culture conditions (propionic acid accounts for the highest proportion). Detailed Implementation

[0034] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0035] Example 1: Strain source, activation and establishment of fermentation system.

[0036] 1.1 Source and activation of strains.

[0037] The strain used in this invention is *Odoribacter splanchnicus*, accession number DSM20712, sourced from the German Microbiological Culture Collection Center and purchased from Ningbo Mingzhou Biotechnology Co., Ltd., and preserved under strictly anaerobic conditions. This strain was isolated from the human intestine, belongs to the human intestinal symbiotic flora, and is a naturally occurring wild-type strain without any genetic engineering modifications.

[0038] Take a quantitative bacterial suspension of Odoribacter splanchnicus DSM 20712 (1×10⁻⁶). 9 The bacterial culture (CFU / mL) was inoculated into CMC liquid medium under strictly anaerobic conditions at an inoculation ratio of 1:10 (bacterial culture: medium). It was then incubated statically at 37°C for 24 hours to obtain activated seed culture. During subculturing, OD600 values ​​were measured at 0h, 6h, 9h, 12h, 15h, 18h, and 24h to plot growth curves. The bacterial culture at 33h after the second subculturing was used for subsequent fermentation experiments. The remaining bacterial culture was mixed with 40% glycerol at a 1:1 volume ratio and stored at -80°C for later use.

[0039] 1.2 Establishment of the fermentation system.

[0040] (1) Experimental groups.

[0041] The experiment set up the following 4 treatment groups, as shown in Table 1.

[0042]

[0043] Note: The final concentration of the live bacteria group was approximately 1×10⁻⁶. 8 CFU / mL; the final concentration of melatonin in the live bacteria + melatonin group was 150 μg / mL (i.e. 0.15 mg / mL).

[0044] (2) Preparation of melatonin solution.

[0045] Weigh 150 mg of melatonin powder using an analytical balance and place it in a 1.5 mL EP tube. Add 1500 μL of DMSO solution, shake well, and transfer to a 10 mL volumetric flask. Rinse the EP tube repeatedly and add PBS to bring the volume to 10 mL to obtain a 15 mg / mL melatonin solution (containing 15% DMSO). Filter the solution through a 0.22 μm filter membrane for sterilization before use.

[0046] (3) Fermentation conditions.

[0047] Total reaction volume: 4200 μL; Final concentration of viable bacteria: 1 × 10⁻⁶ 8CFU / mL; Culture conditions: strictly anaerobic (anaerobic incubator, 37℃); Fermentation time: 33 hours; Initial pH range of culture medium: 6.8-7.0.

[0048] (4) Growth of the strain during fermentation.

[0049] During the formal fermentation experiment, the growth of each group of strains was as follows: Figure 1 As shown in the figure, A represents the growth curve of the live bacteria group, and B represents the growth curve of the live bacteria + melatonin group. These results indicate that the live bacteria group and the live bacteria + melatonin group grew well during fermentation. The period from 0-12 hours was the logarithmic growth phase, 12-33 hours was the stationary phase, and after 33 hours, the bacteria began to decline. Therefore, 33 hours was chosen as the fermentation endpoint, at which point the accumulation of metabolic products was most abundant.

[0050] Example 2: Synergistic effect of N-acetylserotonin production detection and melatonin.

[0051] 2.1 Sample collection.

[0052] After fermentation for 33 hours, 1.2 mL of bacterial culture was taken from each test tube and placed into a 2 mL centrifuge tube. 600 μL of methanol was added to terminate the reaction. The mixture was then thoroughly vortexed and stored at -20°C for later use.

[0053] 2.2 Sample pretreatment.

[0054] After fermentation in each group was terminated, the samples stored at -20°C were removed and vigorously vortexed to mix. Each fermentation sample was 600 μL, and the samples were extracted and analyzed for NAS according to the following procedure.

[0055] (1) Sample extraction of fermentation broth (including extracellular metabolites of live bacteria): Take 600 μL of fermentation broth, centrifuge at 12000 r / min for 15 minutes at 4℃, and collect the supernatant. Take 0.10 mL of sample and place it in a 2 mL centrifuge tube, add 0.30 mL of methanol, vortex mix for 1 min, sonicate at 4℃ for 30 min, let stand at 4℃ for 30 min, centrifuge at 12000 rpm at 4℃ for 15 min, and collect the supernatant for instrumental analysis.

[0056] (2) Extraction of intracellular metabolites from live bacteria: Take 600 μL of fermentation broth, centrifuge at 12000 r / min for 15 minutes at 4℃, discard the supernatant, and collect the bacterial pellet. Add 1 mL of pre-cooled PBS buffer to the pellet, gently resuspend the bacterial cells by pipetting, centrifuge at 12000 r / min for 5 minutes at 4℃, discard the supernatant, and repeat the washing once. Weigh the appropriate weight of the sample into a 2 mL centrifuge tube, add 0.80 mL of methanol, vortex for 1 min, grind for 5 min, sonicate at 4℃ for 30 min, let stand at 4℃ for 30 min, centrifuge at 12000 rpm at 4℃ for 15 min, and collect the supernatant for analysis.

[0057] 2.3 Instrument parameters.

[0058] Instruments: Waters Acquity UPLC liquid chromatography, AB SCIEX 5500 Qtrap-MS mass spectrometry.

[0059] Column: Acquity UPLC HSS T3 (1.8 µm, 2.1 mm * 100 mm).

[0060] UPLC-Qtrap-MS method.

[0061] Chromatographic separation conditions: column temperature: 40℃, flow rate: 0.30 mL / min.

[0062] Mobile phase composition: A-water (0.1% formic acid), B-acetonitrile (0.1% formic acid).

[0063] Run time: 5 min, injection volume: 6 μL, sample gradient elution program is shown in Table 2.

[0064]

[0065] Mass spectrometry conditions.

[0066] Ion source: ESI ion source.

[0067] Curtain Gas: 35 arb.

[0068] Collision gas (GAS): 9 arb.

[0069] IonSpray voltage: 4500V.

[0070] Ion source temperature: 450 ℃.

[0071] Ion Source Gas (IonSource Gas1): 55 arb.

[0072] Ion Source Gas 2: 55 arb.

[0073] MRM acquisition parameters: According to the above chromatographic and mass spectrometric conditions, add the prepared standard solution to the injection bottle and inject the sample; the retention times of the determined substance peaks are shown in Table 3.

[0074]

[0075] 2.4 Data Analysis and Standard Curve.

[0076] MultiQuant software was used to integrate the chromatographic peaks, and the content of N-acetylserotonin in the sample was calculated using the standard curve method.

[0077] Accurately measure 1 mL of N-acetylserotonin standard solution (100.0 ng / mL) and serially dilute it with methanol to prepare a series of standard working solutions with concentrations of 0.49 ng / mL, 0.98 ng / mL, 1.95 ng / mL, 3.91 ng / mL, 7.81 ng / mL, 15.63 ng / mL, 31.25 ng / mL, and 62.50 ng / mL. Prepare the standard working solutions fresh before use. Inject the above series of standard working solutions according to the instrument conditions described in Section 2.3 and record the peak area corresponding to each concentration. Plot the standard curve of N-acetylserotonin with concentration on the x-axis and peak area on the y-axis. The R-squared value of the obtained linear regression equation is... 2 A value greater than 0.99 indicates a good linear relationship within this concentration range, suitable for quantitative calculation of N-acetylserotonin levels in samples. The standard curve is shown below. Figure 3 As shown.

[0078] 2.5 Detection Results and Analysis.

[0079] The quantitative detection results of N-acetylserotonin (NAS) are as follows: Figure 4 As shown, the raw data comes from the characteristic ion peak area integral values ​​recorded by LC-MS / MS targeted detection NAS. See the attached data for details. Figure 2 . Figure 2 The values ​​shown are peak area response values ​​obtained by monitoring the mass-to-charge ratio of NAS characteristic ion pairs (mother ion mass-to-charge ratio 219.2 Da → daughter ion mass-to-charge ratio 160.5 Da, mother ion mass-to-charge ratio 219.2 Da → daughter ion mass-to-charge ratio 115.7 Da) in MRM mode. Multiple replicate samples were set for each group. This peak area data was obtained by... Figure 3 The standard curve shown (R) 2 >0.99) Regression calculation, finally converted to Figure 4 The NAS concentrations for each group are shown. It should be noted that the intracellular samples of the live bacteria group are cell precipitates, and the original quantitative results are in ng / g. To maintain consistency with the concentration units (ng / mL) of the extracellular samples of each group for comparison, the concentrations have been converted to equivalent ng / mL based on the wet weight of the cell precipitate and the corresponding original fermentation broth volume.

[0080] Based on the above quantitative results, the analysis is as follows.

[0081] (1) Odoribacter splanchnicus DSM 20712 possesses the ability to produce NAS through metabolism. NAS was detected both intracellularly and extracellularly in the viable bacterial group, with an intracellular NAS content of 1.22 ng / mL and an extracellular NAS content of 0.26 ng / mL, indicating that this strain can autonomously metabolize and produce NAS and secrete it extracellularly. KEGG pathway annotation confirmed that the genome of Odoribacter splanchnicus DSM 20712 encodes enzyme systems related to de novo tryptophan synthesis and catabolism, providing a genetic basis for its NAS production.

[0082] (2) There was a significant synergistic effect between melatonin and Odoribacter splanchnicus DSM 20712. A small amount of NAS (0.86 ng / mL) was detected in the PBS + melatonin group (melatonin only, under sterile conditions), indicating that melatonin can spontaneously transform, but the transformation amount is low. When Odoribacter splanchnicus DSM 20712 was co-cultured with melatonin (live bacteria + melatonin group), the NAS production jumped to 2.55 ng / mL, which was 9.8 times that of the extracellular production of the live bacteria group without melatonin (0.26 ng / mL), and was also significantly higher than the sum of the contributions of Odoribacter splanchnicus DSM 20712 and melatonin alone (1.12 ng / mL), with the excess reaching 1.43 ng / mL. This indicates that Odoribacter splanchnicus DSM 20712 can efficiently utilize exogenous melatonin to catalyze the generation of NAS, and the two exhibit a synergistic effect.

[0083] At the qualitative confirmation level, the MRM acquisition parameters set in Table 3 (mother ion mass-to-charge ratio 219.2 Da, daughter ion mass-to-charge ratios 160.5 Da and 115.7 Da) are characteristic ion pairs of NAS substances. Under the above mass spectrometry conditions, the live bacteria group, the live bacteria + melatonin group, and the NAS standard all showed corresponding signal peaks at the same retention time, confirming the specificity of the quantified substance.

[0084] Example 3: Short-chain fatty acid yield and spectral identification under in vitro pure culture conditions.

[0085] 3.1 Sample collection and processing.

[0086] The collection method is the same as in Example 2, and the sample detection process is as follows.

[0087] (1) The sample was thawed in a 4 ℃ refrigerator, and 0.50 mL of the sample was placed in a 2 mL centrifuge tube for freeze-drying.

[0088] (2) Add 0.40 mL of 50% acetonitrile water to the freeze-dried sample, vortex mix for 1 min, and sonicate at 4 ℃ for 30 min.

[0089] (3) Centrifuge at 12,000 rpm for 15 min at 4 ℃.

[0090] (4) Add 3-NPH (200 mM) and EDC (120 mM; containing 6% pyridine) solution (2:1:1 v / v / v) and vortex for 1 min to mix (total volume 0.80 mL). React at 40 °C for 1 hour, shaking once every 5 minutes during the reaction.

[0091] (5) After the reaction is complete, centrifuge at 12000 rpm and 4 ℃ for 15 min, take the supernatant and filter it through a 0.22 μm filter membrane and then perform LC-MS / MS analysis.

[0092] 3.2 Instrument parameters.

[0093] Instruments: Waters Acquity UPLC liquid chromatography, AB SCIEX 5500 Qtrap-MS mass spectrometry.

[0094] Column: Acquity UPLC HSS T3 (1.8 μm, 2.1 mm * 100 mm).

[0095] UPLC-Qtrap-MS method.

[0096] Chromatographic separation conditions: column temperature: 40℃, flow rate: 0.300 mL / min.

[0097] Mobile phase composition: A-water (0.05% formic acid), B-acetonitrile (0.05% formic acid).

[0098] Run time: 8 min, injection volume: 3 μL, sample gradient elution program is shown in Table 4.

[0099]

[0100] Mass spectrometry conditions.

[0101] Ion source: ESI ion source.

[0102] Curtain Gas: 35 arb.

[0103] Collision gas (GAS): 9 arb.

[0104] IonSpray voltage: 4500V.

[0105] Ion source temperature: 450℃.

[0106] Ion Source Gas (IonSource Gas1): 55 arb.

[0107] Ion Source Gas 2: 55 arb.

[0108] MRM acquisition parameters: According to the above chromatographic and mass spectrometric conditions, add the prepared standard solution to the injection bottle and inject the sample; the retention times of the determined substance peaks are shown in Table 5.

[0109]

[0110] 3.3 Data Analysis and Standard Curve.

[0111] MultiQuant software was used for integration, and a standard curve was used for content calculation.

[0112] Accurately weigh 5 mg of the standard and dissolve it in 1 mL of 50% acetonitrile solution; then, separately transfer the standard and the prepared 3-NPH (200 mM) and EDC (120 mM; containing 6% pyridine) solutions (2:1:1 v / v / v) and vortex for 1 min to mix (total volume 1 mL); 40 The reaction was carried out at ℃ for 1 hour, with shaking every 5 minutes. First, derivatization was performed using a single standard. After derivatization, a mixed standard was prepared using 50% acetonitrile. Finally, the solutions were diluted with 50% acetonitrile to prepare a series of standard solutions with appropriate concentrations: 0.49 ng / mL, 0.98 ng / mL, 1.95 ng / mL, 3.91 ng / mL, 7.81 ng / mL, 15.63 ng / mL, 31.25 ng / mL, 62.50 ng / mL, 125.00 ng / mL, 250.00 ng / mL, 500.00 ng / mL, and 1000.00 ng / mL. The standard solutions were prepared fresh before use. A standard curve was plotted based on the peak areas of the standards at different concentrations. R0 2All values ​​greater than 0.99 indicate a good linear relationship in the standard curve, making it suitable for calculating the concentration of substances in the sample. The standard curve is shown below. Figure 5 As shown.

[0113] 3.4 Detection results and analysis.

[0114] Based on the above LC-MS / MS method, short-chain fatty acids in the fermentation supernatant of the viable bacteria were quantitatively detected. The results are shown in [Figure 1]. Figure 6 As shown in the figure, under the experimental conditions, the Odoribacter splanchnicus DSM20712 strain can stably produce six short-chain fatty acids in an in vitro pure culture system, and their yield and composition ratio are clearly defined.

[0115] The following conclusions were obtained under the experimental conditions.

[0116] (1) In vitro pure culture confirmed the short-chain fatty acid production capacity of this strain. Using an in vitro pure culture system, the interference of host factors was eliminated, proving that Odoribacter splanchnicus DSM 20712 has the ability to produce short-chain fatty acids under conditions without host interference.

[0117] (2) The short-chain fatty acid profile was clarified. This invention quantified the six short-chain fatty acids produced by this strain under in vitro pure culture conditions, with proportions of 6.6%:68.5%:7.4%:3.1%:10.4%:4.0%. Propionic acid had the highest yield (68.5%), followed by isobutyric acid and butyric acid (e.g., ...). Figure 6 (As shown in the figure). This result directly supports the characteristic that the strain described in this invention has a specific SCFA production spectrum and is dominated by propionic acid.

[0118] Example 4: Probiotic composition.

[0119] This embodiment provides a probiotic composition comprising an effective dose of live Odoribacter splanchnicus cells with accession number DSM 20712, and pharmaceutically or food-grade excipients. The excipients may be selected from fillers (such as starch, dextrin), stabilizers (such as trehalose, skim milk powder), etc.

[0120] In a preferred dosage form, the composition further comprises melatonin as a synergistic prebiotic. Specifically, a probiotic capsule is provided, each capsule containing: 1 × 10⁶ live cells of *Odoribacter splanchnicus* DSM 20712. 9CFU, melatonin 0.5 mg, trehalose 50 mg, and an appropriate amount of microcrystalline cellulose were used to prepare capsule contents with a total weight of 500 mg. Upon release, this composition, under the conditions of Examples 2 and 3, effectively produces N-acetylserotonin and short-chain fatty acids, primarily propionic acid, thereby exerting a dual beneficial function in situ in the intestine. This composition can also be formulated into oral liquids, tablets, or powders using conventional methods.

[0121] The above embodiments only illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention.

Claims

1. A method for producing N-acetylserotonin, characterized in that, Includes the following steps: (a) Visceral odorifera with accession number DSM 20712 ( Odoribacter splanchnicus The strain was inoculated into a fermentation medium containing melatonin, with a final concentration of 0.1-0.3 mg / mL. The fermentation medium was CMC liquid medium. (b) Fermentation culture under anaerobic conditions; (c) Collect N-acetylserotonin from the fermentation products.

2. The method according to claim 1, characterized in that, The visceral odor bacillus ( Odoribacter splanchnicus During fermentation, the strain simultaneously produces short-chain fatty acids, including acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, and isovaleric acid; among the short-chain fatty acids produced, propionic acid accounts for the highest proportion of the yield.

3. Visceral odor bacillus with accession number DSM 20712 ( Odoribacter splanchnicus The application of visceral odor bacterium ( ) in the preparation of formulations for increasing N-acetylserotonin production, said application including the use of visceral odor bacterium ( ) Odoribacter splanchnicus It can be used in combination with melatonin.

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