Application of Lactobacillus reuteri CCFM1289 with high yield of indoleacetic acid in relieving anxiety and depression

Abstract

The invention discloses application of Lactobacillus reuteri CCFM1289 with high yield of indoleacetic acid in relieving anxiety and depression, and belongs to the technical field of microorganisms. The Lactobacillus reuteri CCFM1289 can produce heteroauxin at high yield in vivo and in vitro, promote the yield of heteroauxin in fecal flora, relieve anxiety-like and depression-like behaviors of depression mice, relieve HPA hyperfunction, improve the level of brain-derived neurotrophic factor BDNF in hippocampus, promote secretion of 5-HTP and 5-HT in brain, improve the content of heteroauxin in colon, serum and brain, and improve the content of heteroauxin in colon, serum and brain. The compound can be used for improving intestinal flora metabolism imbalance, improving mRNA levels of hippocampal neurogenin-2, synapsin and post-synaptic dense protein 95, maintaining neuronal synaptic function stability, activating AHR signal channels, improving bicortin DCX expression and promoting neurogenesis so as to relieve depression mood, and can be used for drugs, health care products or healthy food for preventing and treating depression.

Description

Technical Field

[0001] The invention relates to application of Lactobacillus reuteri CCFM1289 with high indoleacetic acid production in relieving anxiety and depression, and belongs to the technical field of microorganisms. Background Art

[0002] Depression is a complex mental illness with long-term and continuous low mood as the main symptom. Common symptoms include anhedonia, slow thinking, decreased volitional activity, cognitive impairment, and somatic symptoms. In recent years, the incidence of depression has continued to rise, and the age of onset has gradually decreased. Currently, antidepressant drugs widely used in clinical treatment, such as selective serotonin reuptake inhibitors and monoamine oxidase inhibitors, generally have the disadvantages of slow onset, inconsistent therapeutic effects, large side effects, and drug dependence, which makes the treatment of depression have serious limitations. Therefore, it is urgent to develop safe and effective interventions or treatments for depression.

[0003] Patients with depression are usually accompanied by multi-system dysfunction, such as the serotonergic nervous system, dopaminergic nervous system, noradrenergic system, cholinergic nervous system, glutamatergic nervous system, GABAergic nervous system, hypothalamus-pituitary-adrenal axis and immune system. In recent years, more and more studies have shown that the "microbe-gut-brain" axis plays an important role in the occurrence and development of depression. Depressed people have disorders of intestinal flora and its metabolites. Experiments on humanized mice have shown that intestinal flora and metabolites can also lead to the occurrence of anxiety-like and depressive-like behaviors. By regulating the composition of intestinal flora or the content of its metabolites, depressive symptoms can be alleviated and the occurrence of depression can be slowed down. Metabolomics testing shows that there is a metabolic disorder of indole substances in the tryptophan metabolic pathway in the urine, feces and cerebrospinal fluid of people with severe depression. Studies have shown that indole substances are involved in the body's immune regulation and neural regulation. Among them, indolepropionic acid can relieve anxiety, indole lactic acid can maintain immune homeostasis, and indoleacetic acid can relieve depressive-like behavior in stressed mice. In addition, as ligands of the AHR signaling pathway, indoles can activate the AHR signaling pathway, regulate downstream gene expression, and promote neurogenesis. Depression is accompanied by decreased neurogenesis, and regulating neurogenesis through indoles may become another therapeutic approach to relieve depression. The human body cannot metabolize indoleacetic acid and other indole substances. These substances are produced by intestinal microorganisms in the human body, and their initial site of action in the human body is in the large intestine. Direct intake of these indoles will be absorbed from the small intestine, which violates the normal site and process of indole substances in the human body, causing potential risks, such as easy formation of drug accumulation, which may increase the metabolic burden of the liver and inhibit liver function. When the drug dose is too high, it will inhibit the development of the nervous system; in addition, the half-life of indoleacetic acid in the body is short, and it is difficult to achieve a sustained effect, and the amount of drug required is also difficult to control. Therefore, it is of great significance to develop a safe and effective depression relief pathway that acts from the human large intestine based on indole substances.

[0004] Probiotics are a type of active microorganisms that are beneficial to the host. They can participate in regulating the host's mucosal and systemic immune functions or maintaining the homeostasis of the internal environment and promote the health of the body. Studies have reported that probiotics can relieve depression. For example, Bifidobacterium breve CCFM1025 can improve the depressive-like behavior of depressed mice and increase the levels of 5-hydroxytryptamine, 5-hydroxytryptophan and brain-derived neurotrophic factor in the brains of depressed mice, thereby achieving an antidepressant effect. Lactobacillus paracasei CCFM1229 can reduce anxiety-like and depressive behaviors in mice and maintain the stability of the structure and function of myelin in the central nervous system. These living organisms that can bring mental health benefits to the host after consumption can be called mental probiotics. The application of such probiotics greatly reduces drug side effects and drug dependence, and provides a safe and stable relief method for the clinical treatment of depression. Studies have shown that some probiotics have the ability to metabolize tryptophan to produce indole substances, such as Lactobacillus casei, Lactobacillus plantarum and Lactobacillus mucosa reuteri. It has been reported that Lactobacillus plantarum L168 and Lactobacillus reuteri activate AHR through ICA and ILA, and develop intraepithelial CD4+T helper cells into immunoregulatory T cells to participate in immune regulation. Fecal metagenomic sequencing found that Lactobacillus gallinarum is missing in patients with colorectal cancer. Lactobacillus gallinarum can promote apoptosis of colorectal cancer cells by secreting ILA and regulate intestinal microbiota to prevent the occurrence of colorectal tumors. There is no application of indole-producing probiotics in depression relief. Patent CN114990023A reports a strain of Lactobacillus reuteri that produces high indole derivatives, but the indoleacetic acid production is only 109.66ng / mL, which is a low dose and cannot meet the role of antidepressant effect.

[0005] At present, there has been no report on Lactobacillus reuteri that can both produce high amounts of indoleacetic acid and relieve anxiety and depression. Summary of the invention

[0006] The invention aims to provide screening of Lactobacillus reuteri CCFM1289 with high indoleacetic acid production and application thereof in alleviating depression.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The Lactobacillus reuteri CCFM1289 provided by the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 13, 2022, with a deposit number of GDMCC No: 62969.

[0009] In one embodiment of the present invention, the Lactobacillus reuteri comes from human feces, and the extracted whole genome is sent to a professional sequencing company, and the whole genome of the bacteria is sequenced using a second-generation sequencer and identified as Lactobacillus reuteri, named Limosilactobacillus reuteri CCFM1289.

[0010] In one embodiment of the present invention, a carbon starvation medium is used to screen lactobacillus with high indoleacetic acid production, and the medium components are peptone, 10 g; NaCl, 5 g; and tryptophan, 6 mM.

[0011] In one embodiment of the present invention, the screened high-indoleacetic acid producing Lactobacillus reuteri CCFM1289 can increase the indoleacetic acid production in fecal flora in vitro and in germ-free mice.

[0012] The invention also provides a microbial preparation containing the Lactobacillus reuteri CCFM1289.

[0013] In one embodiment of the present invention, the microbial preparation contains no less than 1.8×10 10 CFU / g or 1.8×10 10 CFU / mL.

[0014] The present invention also provides a product containing the Lactobacillus reuteri CCFM1289 or the microbial preparation, and the product includes food, medicine or health care product.

[0015] In one embodiment of the present invention, the product comprises a solid, semi-solid or liquid product.

[0016] In one embodiment of the present invention, the food includes fruit and vegetable products, bean products or dairy products; preferably, the food includes fermented fruits and vegetables, fermented milk, cheese, milk-containing beverages, and milk powder.

[0017] In one embodiment of the present invention, the medicine further contains a drug carrier and / or a pharmaceutical excipient.

[0018] In one embodiment of the present invention, the dosage form of the drug includes capsules, powders, tablets or liquid preparations.

[0019] The present invention also provides the use of the Lactobacillus reuteri CCFM1289 in the preparation of antidepressant medicines.

[0020] In one embodiment of the present invention, the antidepressant comprises at least one of the functions (a) to (g):

[0021] (a) Reduce anxiety-like and depression-like behaviors;

[0022] (b) Reduce serum corticosterone and adrenocorticotropic hormone concentrations and alleviate HPA axis hyperactivity;

[0023] (c) Increase the level of brain-derived neurotrophic factor (BDNF) in the hippocampus;

[0024] (d) Enhance the transcription level of tryptophan hydroxylase TPH2 and promote the secretion of 5-HTP and 5-HT in the brain;

[0025] (e) Increase the content of indoleacetic acid in the colon, serum and brain, and improve the metabolic imbalance of intestinal flora;

[0026] (f) Increase the mRNA levels of neurogenin 2, synaptophysin, and postsynaptic density protein 95 in the hippocampus to maintain the stability of neuronal synaptic function;

[0027] (g) Activate the AHR signaling pathway, increase the expression of doublecortin DCX, and promote the occurrence of neurogenesis.

[0028] Beneficial effects:

[0029] The Lactobacillus reuteri CCFM1289 provided by the present invention has a high in vitro indoleacetic acid content, which can significantly reduce anxiety-like and depression-like behaviors in mice, relieve hypothalamic-pituitary-adrenal axis hyperfunction, increase the level of brain-derived neurotrophic factor in the brain, improve the function of the serotonin neurotransmitter-energy nervous system, and promote the release of neurotransmitters, thereby relieving the depressive state of anhedonia. Lactobacillus reuteri CCFM1289 can promote the recovery of indoleacetic acid in the gut-brain axis to normal levels by regulating intestinal flora, and activate the AHR signaling pathway to promote the generation of new neurons, thereby playing an antidepressant effect. Lactobacillus reuteri CCFM1289 can be used as a functional probiotic to assist in the treatment of depression, provide new ideas for clinical intervention and control of mental illnesses related to depression, and have good industrial prospects.

[0030] Biomaterial Deposit

[0031] A strain of Limosilactobacillus reuteri CCFM1289, taxonomically named Limosilactobacillus reuteri, was deposited in the Guangdong Provincial Microbiological Culture Collection on November 13, 2022, with the deposit number GDMCC No: 62969, and the deposit address is Guangdong Provincial Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1:Comparison of the ability of lactobacilli to produce indoleacetic acid and in vitro and in vivo fermentation, (A) the production of indoleacetic acid secreted by different lactobacilli; (B) the production of indoleacetic acid corresponding to the fecal fermentation of Lactobacillus reuteri strains; (C) the content of indoleacetic acid in mouse tissues corresponding to the in vivo fermentation of Lactobacillus reuteri strains. Single-factor ANOVA and Tukey post hoc test were used for analysis, and the significance was compared with the model group. The significance was expressed as * P<0.05, ** P < 0.01 and **** P<0.001.

[0033] Figure 2 :Effects of Lactobacillus reuteri with different indoleacetic acid metabolism abilities on behavioral outcomes of depressed mice, (A) buried bead test; (B) open field test; (C) elevated plus maze test; (D) forced swimming test; (E) sugar water preference test; (F) light-dark box test. Single-factor ANOVA and Tukey post hoc test were used for analysis, and significance analysis was performed compared with the model group. Significance is expressed as * P<0.05, ** P<0.01, *** P < 0.001 and **** P<0.001.

[0034] Figure 3 :Detection of corticosterone (A) and adrenocorticotropic hormone (B) levels in mouse serum. One-way ANOVA and Tukey post hoc test were used for analysis, and the significance was compared with the model group. The significance was expressed as * P<0.05, ** P<0.01, *** P < 0.001 and **** P<0.001.

[0035] Figure 4 :Effects of Lactobacillus reuteri CCFM1289 on BDNF levels in the hippocampus. One-way ANOVA and Tukey post hoc test were used to analyze the significance of the model group. The significance is expressed as * P<0.05, ** P < 0.01 and *** P<0.001.

[0036] Figure 5 :Effects of Lactobacillus reuteri CCFM1289 on the serotonin neurotransmitter system in the hippocampus of depressed mice, (A) 5-HTP content in the hippocampus; (B) 5-HT content in the hippocampus; (C) Changes in the mRNA level of Tph2 in the hippocampus. Single-factor ANOVA and Tukey post hoc test were used for analysis, and the significance was compared with the model group. The significance is expressed as *P<0.05, ** P < 0.01 and *** P<0.001.

[0037] Figure 6 :Changes in indoleacetic acid content in different tissues, (A) Changes in indoleacetic acid metabolism in colonic contents; (B) Indoleacetic acid content in serum; (C) Indoleacetic acid detection in hippocampal tissue. Single-factor ANOVA and Tukey post hoc test were used for analysis, and significance analysis was performed compared with the model group. Significance is expressed as * P<0.05, ** P<0.01, *** P < 0.001 and **** P<0.001.

[0038] Figure 7 :Effects of Lactobacillus reuteri CCFM1289 on the transcriptional levels of neuronal synaptic-related proteins in hippocampal tissue, (A) mRNA level of neurogenin 2 (Neurog2); (B) mRNA level of postsynaptic density protein 95 (PSD-95); (C) mRNA level of synaptophysin (SYP). Single-factor ANOVA and Tukey post hoc test were used for analysis, and significance analysis was performed compared with the model group. Significance is expressed as * P<0.05, ** P < 0.01 and **** P<0.001.

[0039] Figure 8 :Effects of Lactobacillus reuteri CCFM1289 on the AHR signaling pathway and new neuron generation, (A) Transcription level of the aryl hydrocarbon receptor encoding gene Ahr; (B) mRNA level of the cytochrome P450 enzyme (CYPlA1) gene; (C) DCX positive cell intensity; (D) DCX immunohistochemistry results in hippocampal tissue. One-way ANOVA and Tukey post hoc test were used for analysis, and significance analysis was performed compared with the model group. Significance is expressed as * P < 0.05 and ** P<0.01. DETAILED DESCRIPTION

[0040] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0041] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0042] The 84 Lactobacillus strains involved in the following embodiments are all from the strain bank of the Food Biotechnology Center of Jiangnan University, and are all identified by genome draft testing (Meiji Biotechnology), including 34 strains of Lactobacillus reuteri, 37 strains of Lactobacillus casei and 14 strains of Lactobacillus rhamnosus, which are renumbered as R1-R34, C1-C37 and Rh1-Rh14 for convenience of description.

[0043] The culture medium involved in the following examples is as follows:

[0044] MRS liquid medium (1L): peptone 10g, beef extract 10g, glucose 20g, sodium acetate 5g, yeast extract 5g, diammonium hydrogen citrate 2g, K2HPO4 2g, MgSO4·7H2O 0.1g, MnSO4·H2O 0.05g, Tween 80 1mL.

[0045] MRS solid culture medium: Add 15-20 g of agar to the MRS liquid culture medium.

[0046] Carbon starvation medium (1 L): peptone 10 g, NaCl 5 g.

[0047] The 5-week-old male C57BL / 6J mice involved in the following examples were purchased from Vitallife.

[0048] Detection of indole substances in bacterial solution:

[0049] 300 μL of bacterial suspension was taken, centrifuged at 6000×g for 10 min, 200 μL of supernatant was taken, 800 μL of pre-cooled methanol was added, oscillated and mixed, and allowed to stand at -20°C for 30 min, centrifuged at 10000×g for 10 min, 4°C, and the supernatant was taken. The supernatant was placed in a vacuum concentrator at 45°C for 3 h to evaporate the liquid, and resuspended in 100 μL of 0.1% formic acid water, centrifuged at 10000×g for 10 min, 4°C, and 50 μL of supernatant was placed in a liquid phase vial. The sample was used to precipitate the sample using an ACQUITY The ultra-high performance liquid phase (DIONEX UltiMate 3000HPLC) with BEH C18 column (1.7μm, 100mm×2.10mm) was coupled with a mass spectrometer (Q-Exactive mass spectrometer) for detection. The injection volume was 2μL, the mobile phase A was 0.1% (v / v) formic acid water, the mobile phase B was acetonitrile, the gradient elution program was: 5% B, 0–3min; 30% B, 3–9min; 100% B, 9–15min; 100% B, 15–16.5min; 5% B, 16.5–20min, the column temperature was 37°C, and the positive ion mode was used for scanning detection.

[0050] The behavioral testing methods involved in the following embodiments are as follows:

[0051] Open Field Test (OFT): The experiment was conducted in a quiet environment. After placing the animal in the center of the open field, the timing and recording were started immediately, and the recording was stopped after 10 minutes of observation. When replacing the animal, in order to avoid affecting the results of the next test, the inner wall and bottom of the open field must be cleaned first to remove the remaining information of the last animal (such as urine, feces, odor, etc.). The video was digitally analyzed using EthoVision software to calculate the percentage of time the mouse stayed in the center area of ​​the open field: (stay time in the center area of ​​the open field / total monitoring time) × 100%.

[0052] Marble Burying Test (MBT): Place 5 cm thick corn cob bedding in the mouse cage, and put 20 glass beads (15 mm in diameter) in it, divided into 5 rows, 4 beads in each row, and 2 cm apart. Record after 30 minutes, and record the total number of glass beads that cover more than 1 / 2.

[0053] Elevated Plus Maze (EPM): At the beginning of the experiment, the mouse was placed in the maze from the central grid to the open arm, and the timing and video recording for 5 minutes were immediately started. After the experiment, the mouse was taken out, the two arms were cleaned, and alcohol was sprayed to remove the odor before the next experiment. The video was digitally analyzed using EthoVision software to calculate the percentage of the open arm stay time (and the number of open arm entries) to the total activity time (and the total number of times).

[0054] Forced Swim Test (FST): The experimental bucket was filled with clean water about 30 cm high, with a water temperature of 24±1°C. 24 hours before the formal experiment, each mouse was subjected to 10 minutes of adaptive swimming training. During the formal experiment, each mouse was subjected to a 6-minute swimming test. The EthoVision real-time monitoring system was used for video recording and analysis, and the immobility time of the mouse (Immobility, the judgment standard was immobility of the limbs or only slight movement of the hind limbs) was calculated.

[0055] Sucrose Preference Test (SPT): Before the test, the mice were first trained to drink sucrose for 3 days, that is, two bottles of 1% sugar water and regular drinking water were placed in the cage, and the positions of the two bottles were swapped from time to time. After 3 days, the mice were fasted and deprived of water for 24 hours, and then a 1-hour sugar water test was started: 2 bottles of regular drinking pure water and 1% sucrose water of equal volume were prepared and placed in the mouse cage. After 1 hour, the experiment was terminated, and the drinking water and sucrose water consumption (g) of each group of mice were recorded respectively, and the sugar water preference value was calculated: (sugar water consumption mass / total mass of liquid consumed) × 100%.

[0056] Light Dark Box Test (LDBT): Place the mouse facing the light box in the middle grid of the shuttle box. Use the EthoVision real-time monitoring system to record the mouse's activities for 5 minutes, and calculate the percentage of the number of times the mouse enters the light box to the total number of shuttles.

[0057] Detection of tryptophan metabolites in different tissues of mice:

[0058] Intestinal contents: The collected intestinal contents were freeze-dried at -107°C and 0.2 mbar. 30 mg of freeze-dried sample was weighed and added to 900 μL of 50% methanol water (v / v). Vortexed for 15 seconds, homogenized, and centrifuged at 15,000 × g for 10 minutes. The supernatant was taken and concentrated in 45°C vacuum for 3 hours. The evaporated sample was resuspended in 100 μL of 10% methanol water, centrifuged at 15,000 × g for 10 minutes, filtered through a 0.2 μm filter membrane, and placed in a liquid phase vial. Quantitative detection was performed using the above-mentioned LC-MS.

[0059] Serum: Take 100 μL serum sample, add 800 μL methanol, vortex for 15 seconds, homogenize the sample, centrifuge at 15000 × g for 10 minutes, take the supernatant, concentrate at 45 ° C vacuum for 3 hours, resuspend in 100 μL 10% methanol water, centrifuge at 15000 × g for 10 minutes, take the supernatant and perform quantitative detection using the above-mentioned LC-MS.

[0060] Tissue: Take a certain amount of sample, add 900μL 50% methanol water, vortex for 15s, homogenize, stand at -20℃ for 1h, centrifuge at 4℃, 15000×g for 10min. Take the supernatant, vacuum concentrate at 45℃ for 3h. Resuspend in 100μL 10% methanol water, centrifuge at 15000×g for 10min, filter the supernatant through a 0.2μm filter membrane, place in a liquid phase vial with an inner liner, and perform quantitative detection using LC-MS and external standard method.

[0061] Detection of corticosterone, adrenocorticotropic hormone in mouse serum and BDNF in hippocampal tissue:

[0062] ELISA kits were used for detection and were purchased from Nanjing Senbeijia Biotechnology Co., Ltd.

[0063] Determination of transcription levels of target protein-coding genes in tissues:

[0064] Total RNA was extracted from mouse tissues using the Trizol method, and its concentration was detected by an ultra-micro spectrophotometer (NanoDrop 2000C). 1 μg of RNA was taken for gDNA removal and cDNA synthesis (HiScriptIII RT SuperMix for qPCR, Vazyme) according to the instructions of the reverse transcription kit. The qPCR system was 5 μL SYBR Green super mix, 1 μL cDNA, 1 μL forward and reverse primers, and 4 μL double distilled water. The real-time fluorescence quantitative gene amplification instrument CFX384TM Real-Time System (Bio-Rad, USA) was used to detect gene transcription levels. Three parallel wells were set up for each sample, and Gapdh was used as the internal reference gene. The results were used for 2 -ΔΔCq The primer sequences used are shown in Table 1.

[0065] Table 1 Primers used for fluorescence quantitative PCR

[0066]

[0067] Mouse hippocampal immunohistochemistry detection:

[0068] The whole brain of the mouse was paraffin-embedded, and the coronal sections of the hippocampus were prepared. The immunohistochemical staining experimental steps were carried out according to the instructions of the immunohistochemical kit (Shanghai Sangon Biotechnology Co., Ltd.), and the hippocampal neurons were detected for antigen-antibody reaction. After sealing and drying for two weeks, the sections were scanned using a pathological section scanner (Pannoramic MIDI) 20 times to obtain the expression images of doublecortin on neurons, and quantitative analysis was performed using Image J.

[0069] Example 1: Screening and identification of Lactobacillus reuteri CCFM1289 with high indoleacetic acid production

[0070] The strain originated from the strain bank of the Biotechnology Center of Jiangnan University and was isolated from human feces. Feces of healthy volunteers were collected, 0.1g of fecal samples were weighed and dissolved in 1mL of normal saline. After gradient dilution, samples of different dilutions were spread on MRS solid plates and cultured at 37°C. After 2 days of culture, single colonies were picked for streaking purification, and purified single colonies were selected and inoculated into fresh MRS culture medium, cultured at 37°C, and 16s rDNA bacterial solution was amplified by PCR after 18 hours. The PCR products were sent to Jinweizhi Biotechnology Company for sequencing, and the strains were identified by NCBI Blast.

[0071] ArAT and TMO amino acid sequences (GenBank: KEK16881.1 and POD81577.1) were used to compare with the central bacterial species gene draft library in NCBI Blast, and 84 strains of different affinities and strains without metabolic enzyme genes were selected for in vitro metabolic testing. 84 lactobacilli stored at -80°C were activated on MRS solid medium, inverted and cultured at 37°C for 24 hours, and single colonies were picked for purification again. After purification, the single colonies were transferred to 2mL MRS liquid medium and cultured at 37°C for 12 hours. 1% inoculation was transferred to 2mL fresh MRS medium, cultured for 12 hours, and after oscillation and mixing, 1mL of bacterial solution was taken in a 1.5mL EP tube, centrifuged at 8000×g for 5min, the supernatant was discarded, and the culture medium was resuspended by adding 0.3mL of carbon starvation medium containing 0.6mM tryptophan. 50μL was taken from the bacterial suspension and diluted to 10 with physiological saline. -7 , 10 -8 and 10 -9 , and set up two parallels, spread on MRS solid medium, and incubate at 37℃ for 24h before counting the viable bacteria. The remaining 250μL of bacterial suspension was placed in a 37℃ incubator for 24h, shaken and mixed, and 200μL was taken for indole metabolite detection using the above method. The indoleacetic acid metabolic capacity of the strain was defined as 10 8 The amount of indoleacetic acid produced by CFU live bacteria was calculated after three biological replicates, and the strain with the highest indoleacetic acid production was obtained by calculating the indoleacetic acid production of each lactobacillus strain and comparing the significance. The strains of different strains were also identified.

[0072] Figure 1 A shows that most Lactobacillus reuteri have higher indoleacetic acid metabolism ability than Lactobacillus casei and Lactobacillus rhamnosus, and the strain with the highest indoleacetic acid metabolism ability of Lactobacillus reuteri is named Lactobacillus reuteri CCFM1289. The Lactobacillus reuteri CCFM1289 has been deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC No: 62969.

[0073] Example 2 In vitro and in vivo fermentation of Lactobacillus reuteri CCFM1289 to produce indoleacetic acid

[0074] 1. In vitro fecal fermentation of high-yield IAA strains

[0075] 1.0 g of fresh feces from three healthy volunteers was collected and soaked in 10 mL of PBS with a pH of 7.2. After thorough mixing, centrifugation at 600 × g and 4 ° C for 5 min, the solids were removed, the supernatant was resuspended in PBS, and immediately mixed with an equal volume of 40% sterile glycerol to a final concentration of 20%. It should be stored at -80 ° C for no more than 8 weeks. The feces were immediately transferred to an anaerobic glove box, diluted with PBS buffer at a solid-liquid ratio of 1:7, vortexed and mixed, centrifuged at 500 × g for 5 min, and the supernatant was passed through 3 layers of sterile gauze to obtain fecal filtrate. Lactobacillus mucosus R11 (non-producing), R33 and R27 (medium producing) with different indoleacetic acid production and CCFM1289 were activated on MRS solid medium. After single colony culture, the culture was expanded in MRS liquid medium at a 1% (v / v) inoculation amount. The obtained culture solution was centrifuged at 4 ° C and 8000 × g for 5 min, and the bacteria were collected. 10 8 CFU lactobacilli were resuspended in 1 mL of in vitro fecal filtrate, anaerobically fermented at 37 °C for 24 h, and the supernatant was collected for analysis after centrifugation at 8000 × g for 15 min. Figure 1 As shown in B, the indoleacetic acid production in the culture medium of Lactobacillus reuteri CCFM1289 reached 2.03±1.62μg / mL (33.08ng / 10 8 CFU), which is higher than the strain in patent CN114990023A.

[0076] 2. Determination of indoleacetic acid secretion in vivo by Lactobacillus reuteri

[0077] Fifteen 7-week-old C57BL / 6J germ-free mice (18-20 g) were randomly divided into five groups, namely Control, R11, R27, R33 and CCFM1289, with three mice in each group. The mice were kept in a sterile experimental animal environment, and each mouse was given 200 μL of Lactobacillus suspension, with the same initial inoculation number of 5×10 9 CFU. After 24 hours, the mice were euthanized and samples were obtained to obtain the small intestinal contents, cecal contents, colonic contents, colonic tissues and serum, and pre-processed and indoleacetic acid tested according to the above methods. At the same time, a certain amount of colonic contents was taken for colony counting.

[0078] The results are as follows Figure 1As shown in C, the level of indoleacetic acid in germ-free mice is at a low level. The intake of Lactobacillus reuteri CCFM1289 can promote the distribution and accumulation of indoleacetic acid in germ-free mice. The IAA contents in small intestine contents, colon, colon contents, cecum contents, hippocampus and serum are 0.02±0.01, 0.32±0.06, 0.67±0.06, 2.08±0.26, 0.04±0.01 and 0.24±0.03 mg / kg, respectively, which are 1.41, 4.12, 3.69, 4.79, 3.48 and 2.34 times that of blank mice, respectively, which can contribute to immune balance.

[0079] Example 3: Lactobacillus reuteri CCFM1289 with high indoleacetic acid production improves anxiety-depression-like behaviors in chronically stressed mice

[0080] The specific steps are as follows:

[0081] 1. Preparation of oral gavage:

[0082] Lactobacillus reuteri R11, R33 and R27 with different indoleacetic acid production and CCFM1289 were activated on MRS solid medium. After purification, single colonies were picked and placed in fresh MRS liquid medium and cultured at 37°C for 24 h. The culture was expanded at a 1% inoculum. After culturing for 12 h, the culture solution was centrifuged at 4°C and 8000×g for 5 min, the cells were collected, the supernatant was discarded, and the cells were resuspended in 0.85% saline to prepare 5×10-10 viable cells. 9 CFU / mL bacterial suspension. Indoleacetic acid was dissolved in 0.85% saline to a final concentration of 5 mg / mL. Fluoxetine, a drug control, was dissolved in saline to a final concentration of 1 mg / mL.

[0083] The intragastric volume of the above-mentioned gavage agent was 200 μL / mouse / day.

[0084] 2. Animal experiments:

[0085] 48 5-week-old male C57BL / 6J mice were randomly divided into 8 groups after one week of adaptive feeding: normal control group, depression model group, fluoxetine group, indoleacetic acid positive control group, Lactobacillus reuteri R11 intervention group, Lactobacillus reuteri R27 intervention group, Lactobacillus reuteri R33 intervention group and Lactobacillus reuteri CCFM1289 intervention group, each group contained 6 mice, and the intervention was carried out at the same time as the modeling, and the intervention lasted for 5 weeks. The animal grouping is shown in Table 2, where the control solvent is 0.85% saline.

[0086] Table 2 Experimental animal groups

[0087]

[0088] The method of establishing the mouse chronic unpredictable stress model is as follows:

[0089] 1-2 stimuli were randomly applied every day, and the time of stimulation was random every day to avoid circadian rhythm for 5 weeks. To prevent mice from adapting to stress, the same stimulation was not repeated within two days. Stimuli include: fasting for 24 hours; moist bedding for 24 hours (add about 100 mL of sterile water to corn cob bedding, mix well, and form moist bedding); braking for 2 hours (place the experimental mouse in a 50 mL centrifuge tube with vents at the bottom, top, and around the tube wall for 2 hours. The operation process should ensure that the whole body enters the bottle intact, and the nostrils of the experimental mouse are located at the vent at the bottom to prevent suffocation); tail clamping for 2 minutes (the clamp is 1 cm away from the tip of the mouse's tail, and the length of the clamp should not be too long to prevent serious damage to the tail); continuous light for 24 hours; no bedding 24h (the corn cob bedding was removed and the experimental mice were kept in the cages for 24h); forced swimming for 5 minutes (each mouse was placed in a 5L beaker containing 3L of water, the water temperature was 25±2℃, and forced to swim for 5min. This experiment was carried out in winter. Due to the large temperature difference between indoor and outdoor, the water temperature must be adjusted, and after swimming, the water on the experimental mice was wiped off with a towel and blown dry with a hair dryer); water deprivation for 24h; crowding stimulation for 24h (fences were used to reduce the activity space of each cage of experimental mice to create a crowded environment); solitary housing for 24h (the mice were kept separately in individual cages).

[0090] Five weeks after the mice were stimulated, behavioral tests were performed, including the buried bead test, open field test, elevated plus maze test, forced swimming test, sugar water preference test, and light-dark box test.

[0091] Behavioral results such as Figure 2 As shown in the figure, compared with normal mice, the model group mice buried more beads, stayed in the central area of ​​the open field less, spent less time in the open arms, spent more time immobile, had a lower preference for sugar water, and entered the light box less frequently, showing obvious anhedonia, anxiety, and depression-like behaviors. Consistent with the results of the positive control IAA group, the high-indoleacetic acid-producing Lactobacillus mucosa CCFM1289 intervention group significantly improved the anxiety and depression behaviors of mice, the medium-producing strain intervention group significantly increased the time spent in the open arms in the elevated plus maze test, and alleviated the anxiety-like behaviors, while the non-indoleacetic acid-producing lactobacillus intervention group had no significant relief effect. The fluoxetine group significantly improved the anxiety-like behaviors of mice and increased the sugar water preference values ​​of mice, but did not significantly improve other depression indicators such as despair time and light box shuttle rate. In contrast, CCFM1289 intervention maintained the antidepressant effect.

[0092] Example 4: Lactobacillus reuteri CCFM1289 significantly alleviates HPA axis hyperactivity

[0093] After the behavioral test, the mice were euthanized, and the blood was collected. After standing for 2 hours, the blood was centrifuged at 3000×g for 15 minutes to obtain serum. The levels of corticosterone (CORT) and adrenocorticotropic hormone (ACTH) in the serum were detected using ELISA kits.

[0094] Test results such as Figure 3 As shown in the results, the depression model mice had hyperfunction of the hypothalamus-pituitary-adrenal axis (HPA) due to continuous chronic stress, and the serum corticosterone and adrenocorticotropic hormone increased significantly. Similar to the positive IAA control group, the intervention of indoleacetic acid-producing Lactobacillus reuteri R27, R33 and CCFM1289 significantly reduced the CORT and ACTH levels and alleviated the overactivity of the HPA axis. Although the drug control group could reduce the CORT level, it had no effect on ACTH. In addition, the high-yield strain CCFM1289 could adjust the ACTH level (23.68±3.11ng / L) back to the normal group level (24.34±2.34ng / L), and the relief effect was better in comparison.

[0095] Example 5: Lactobacillus reuteri CCFM1289 significantly increases BDNF levels in depressed mice

[0096] A certain mass of fresh hippocampal tissue was taken, and 9 volumes of normal saline were added (equivalent to 1 g of tissue plus 9 mL of homogenate normal saline), and homogenized with a tissue homogenizer, centrifuged at low speed, and the supernatant was used to detect the content of hippocampal BDNF using an ELISA kit.

[0097] The experimental results are as follows Figure 4 As shown, the BDNF contents in the hippocampus of mice in the normal control group, depression model group, fluoxetine group, IAA positive control group, R11 intervention group, R27 intervention group, R33 intervention group and CCFM1289 intervention group were 227.29±56.59, 159.08±19.44, 180.10±24.28, 239.69±34.66, 185.89±24.94, 206.71±18.51, 226.38±50.61, and 259.84±22.94 ng / mL, respectively. The BDNF level in the hippocampus of depressed mice was significantly lower than that in normal mice. Compared with the model group, the drug group had no effect on the BDNF level. However, IAA, Lactobacillus reuteri R33 and Lactobacillus reuteri CCFM1289 significantly improved this situation after intervention in depressed mice, and Lactobacillus reuteri CCFM1289 had the best improvement effect.

[0098] Example 6: Lactobacillus reuteri CCFM1289 can significantly improve the serotonin neurotransmitter energy system in the brain of depressed mice

[0099] A certain amount of hippocampal tissue was taken, and the changes in neurotransmitter metabolism in the tryptophan metabolic pathway were detected using LC-MS according to the above method, and quantified by the external standard method.

[0100] Depression patients are accompanied by decreased neurotransmitter function, and the serotonin content in their bodies is significantly lower than that of healthy people. Figure 5 As shown, compared with normal mice, the neurotransmitter 5-HT (0.10±0.08 mg / kg) and its precursor 5-HTP (101.7±69.89 μg / kg) in the hippocampus of chronic unpredictable stress-induced depressed mice were significantly reduced. Fluoxetine could significantly increase the level of 5-HT (0.22±0.06 mg / kg) but had little effect on its precursor level. In contrast, supplementation with Lactobacillus reuteri CCFM1289 increased Tph2 transcription expression, promoted 5-HTP secretion (194.00±68.51 μg / kg) and downstream metabolism (0.32±0.19 mg / kg), and restored the serotonin level in the body to a healthy state.

[0101] Example 7: Lactobacillus reuteri CCFM1289 can significantly increase the content of indoleacetic acid in the tissues of depressed mice

[0102] Mouse serum, colon contents, and hippocampal tissues were collected for the detection of indoleacetic acid content as described above.

[0103] The experimental results are as follows Figure 6 As shown in the results, the intestinal flora of depression model mice secreted an imbalance of indoleacetic acid, accompanied by a decrease in the amount of indoleacetic acid circulating in the serum and brain. The drug control group could not increase the level of indoleacetic acid in the body, while supplementing indoleacetic acid could make up for the lack of material metabolism and improve depressive traits. Similarly, Lactobacillus mucosa CCFM1289, which produces high indoleacetic acid, can significantly increase the concentration of IAA in the brain-gut axis and improve depression.

[0104] Example 8: Lactobacillus reuteri CCFM1289 can regulate the transcription levels of neurokines and neuronal synaptic proteins

[0105] Mouse hippocampal tissue was isolated and total RNA was extracted by Trizol method. Reverse transcription and RT-PCR were performed according to the above method to detect the mRNA levels of neurog2, synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95) in hippocampus.

[0106] The results are as follows Figure 7As shown, the depression model will cause a decrease in the transcription levels of Neurog2, SYP and PSD-95 in the hippocampus, thereby affecting the synaptic function of neurons. Supplementation with Lactobacillus reuteri CCFM1289 can significantly increase the mRNA levels of Neurog2, SYP and PSD-95, maintain the stability of neuronal synaptic function, and show a good antidepressant effect. Fluoxetine intervention has no obvious effect on synaptic function.

[0107] Example 9: Effects of Lactobacillus reuteri CCFM1289 on AHR signaling pathway and neurogenesis

[0108] The cDNA obtained by reverse transcription of the hippocampal tissue was used to perform RT-PCR reaction using Ahr and Cypla1 gene primers to detect its transcription level. After the brain tissue was fixed, coronal sections of the hippocampus were taken, and the newborn neurons in the DG region were detected using doublecortin (DCX) antibody combined with immunohistochemistry kit instructions. After scanning the slices according to the above method, semi-quantitative processing was performed using Image J software.

[0109] Studies have shown that activation of the AHR signaling pathway can promote the generation of new neurons, and DCX is a marker of neurogenesis. Figure 8 As shown in the results, the generation of new neurons in the hippocampal DG region of depressed mice was significantly reduced compared with normal mice. The AHR signal was not enhanced and the generation of neurons did not change significantly in the fluoxetine group, indicating that the relief of anxiety-like depression-like behaviors by fluoxetine is mainly related to the generation of 5-HT, rather than the AHR-AHN pathway. Lactobacillus reuteri CCFM1289 can activate the AHR signaling pathway by increasing the level of Cyp1a1, thereby regulating neurogenesis and achieving an antidepressant effect.

[0110] Example 10 Preparation of Lactobacillus reuteri CCFM1289 probiotic preparation

[0111] A single colony of Lactobacillus reuteri CCFM1289 obtained in Example 1 was selected and inoculated into MRS liquid culture medium, cultured at 37°C for 24 h, transferred into fresh MRS liquid culture medium at an inoculum volume of 1% (v / v), cultured at 37°C for 24 h, centrifuged the bacterial solution at 6000×g for 15 min, collected the precipitate, washed twice with 0.85% saline, and then centrifuged to collect the bacteria. The Lactobacillus reuteri CCFM1289 bacteria were resuspended to a cell concentration of 1×10 11 CFU / mL, the bacterial liquid of Lactobacillus mucilaginosus CCFM1289 was freeze-dried to obtain bacterial powder, and the number of viable bacteria was 1.8×10 10 CFU / g.

[0112] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Limosilactobacillus reuteri CCFM1289 was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 13, 2022, with the deposit number GDMCC No: 62969.

2. A microbial preparation containing the Lactobacillus reuteri CCFM1289 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the content of Lactobacillus reuteri CCFM1289 is not less than 1.8×10 10 CFU / g or 1.8×10 10 CFU / mL.

4. A product containing Lactobacillus reuteri CCFM1289 according to claim 1, or containing the microbial preparation according to claim 2 or 3, characterized in that: The products include food, medicine or health products.

5. The product according to claim 4, characterized in that The product includes a solid, semi-solid or liquid product.

6. The product according to claim 5, characterized in that The food includes fruit and vegetable products, bean products or dairy products; preferably, the food includes fermented fruits and vegetables, fermented milk, cheese, milk-containing beverages and milk powder.

7. The product according to claim 5, characterized in that The medicine also contains a drug carrier and / or a pharmaceutical excipient.

8. The product according to claim 7, characterized in that The dosage form of the drug includes capsules, powders, tablets or liquid preparations.

9. Use of the Lactobacillus reuteri CCFM1289 according to claim 1 in the preparation of antidepressant medicines.

10. The use according to claim 9, characterized in that: The antidepressant includes at least one of the following functions: (a) Reduce anxiety-like and depression-like behaviors; (b) Reduce serum corticosterone and adrenocorticotropic hormone concentrations and alleviate HPA axis hyperactivity; (c) Increase the level of brain-derived neurotrophic factor (BDNF) in the hippocampus; (d) Enhance the transcription level of tryptophan hydroxylase TPH2 and promote the secretion of 5-HTP and 5-HT in the brain; (e) Increase the content of indoleacetic acid in the colon, serum and brain, and improve the metabolic imbalance of intestinal flora; (f) Increase the mRNA levels of neurogenin 2, synaptophysin, and postsynaptic density protein 95 in the hippocampus to maintain the stability of neuronal synaptic function; (g) Activate the AHR signaling pathway, increase the expression of doublecortin DCX, and promote the occurrence of neurogenesis.

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