Lactobacillus reuteri CCFM1288 for relieving depressive mood and application of lactobacillus reuteri CCFM1288

Abstract

The invention discloses lactobacillus reuteri CCFM1288 for relieving depressive mood and application of the lactobacillus reuteri CCFM1288, and belongs to the technical field of microorganisms. The lactobacillus reuteri CCFM1288 has the tryptophan metabolism capability, and can survive for 7 days in the intestinal tract; after the lactobacillus reuteri CCFM1288 intervenes a depressive mouse, anxiety-like and depression-like behaviors of the mouse can be obviously relieved, flora alpha-diversity is improved, intestinal symbiotic bacteria are promoted to secrete indole substances, intestinal homeostasis is maintained, hypothalamic-pituitary-adrenal axis hyperfunction is relieved, dopamine and serotonin neurotransmitter nervous system functions are improved, and the effect of preventing and treating depression is achieved. The neurotransmitter release is promoted, and the anti-depression effect is achieved. In clinical tests, intervention of Lactobacillus reuteri CCFM1288 can significantly improve the depressive symptom of MDD patients, and a new safe alleviation approach is provided for depression treatment.

Description

Technical Field

[0001] The invention relates to Lactobacillus reuteri CCFM1288 for relieving depression and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Major Depressive Disorder (MDD) is a mental illness characterized by long-term low mood, lack of pleasure, pessimism, lack of initiative and autonomic nervous system dysfunction. Depressive mood seriously disturbs the patient's life and work, brings a heavy burden to the family and society, and is a major risk factor in the disease burden. Factors that cause depression include: genetic factors, physical factors, abnormal functions and metabolism of central nervous mediators, mental factors, etc. Due to the low effectiveness, delayed onset and side effects of traditional drugs (such as serotonin reuptake inhibitors, etc.), the treatment of depression has also become a medical problem.

[0003] Clinical studies have shown that patients with depression have disordered intestinal flora, such as reduced diversity and reduced abundance of beneficial bacterial groups. Gut flora plays a vital role in the occurrence and development of depression, and reports have shown that Tyzzerella and Jeotgalicoccus are associated with the severity of depressive symptoms. By adjusting the proportion of species, depressive mood may be improved, which may become a potential therapeutic target. Gut flora and its metabolites are also involved in regulating central nervous system and peripheral nerve function, including neurogenesis, synaptic plasticity, neurotransmitter signaling, neurodevelopment and neuroinflammation. In the absence of intestinal bacteria, changes in the permeability of the blood-brain barrier, impaired immune response of microglia, hyperactivity of the hypothalamic-pituitary-adrenal axis, changes in brain neurochemistry, and reduced anxiety and social behavior can occur.

[0004] Probiotics are a class of active microorganisms that have beneficial effects on the host after sufficient intake. Some probiotics can colonize in the human intestine and play a variety of functions such as improving the balance of the host's intestinal flora, regulating energy metabolism, and enhancing immunity. Probiotic active preparations have been widely used in the food, health care products and pharmaceutical industries, including lactobacillus, bifidobacteria and yeast. A large number of clinical studies have shown that probiotics can promote the secretion of neurotransmitters, effectively relieve depression, and slow down the process of depression. Daily intake of specific probiotics can help regulate the balance of intestinal microecology and thus improve physiological and mental health. Limosilactobacillus reuteri is a probiotic that has been reported to exist naturally in the intestines of almost all vertebrates and mammals. Limosilactobacillus reuteri has a strong adhesion ability to the intestinal mucosa, which can improve the distribution of intestinal flora, antagonize the colonization of harmful bacteria, and improve the body's immunity.

[0005] At present, there is no report on the use of Lactobacillus reuteri with tryptophan metabolism function to relieve depression-like behavior and symptoms by regulating the metabolic balance of indole in the intestinal flora, and there is no report on the abundance of Tyzzerella and Jeotgalicoccus to relieve depression. And according to the list of edible probiotics, it has not yet been approved to increase the abundance in the body by taking Tyzzerella bacteria to achieve depression relief. In addition, there are some patent applications related to probiotics that relieve depression, such as CN111110705A discloses a plant lactobacillus in relieving anxiety and depression, CN113430135A discloses a helveticus lactobacillus that can improve depression-like behavior, by increasing the level of 5-hydroxytryptamine and brain-derived neurotrophic factor in the hippocampus and reducing the level of serum corticosterone to achieve the purpose of depression relief, and CN114717148A discloses a paracasei lactobacillus that reduces the activity of xanthine oxidase in the cerebral cortex, increases the level of Mbp mRNA in the prefrontal lobe, maintains the stability of the structure and function of the CNS myelin sheath, and then relieves anxiety and depression-like behavior. However, the above patents all focus on the effects of strains on the body, and do not analyze the important role of intestinal flora and metabolic molecules. No probiotics have been found that can effectively relieve anxiety and depression by regulating intestinal flora and its metabolites. Therefore, it is of great significance to screen edible strains that can target and regulate intestinal flora and intestinal metabolites. Summary of the invention

[0006] The purpose of the present invention is to provide a Lactobacillus reuteri CCFM1288 for improving anxiety and depression and its application. The present invention proposes a Lactobacillus reuteri with tryptophan metabolism function, which can relieve depression by regulating the intestinal flora structure and flora tryptophan metabolism, and can survive in the body for a long time. At the same time, it shows great potential for relieving depression in clinical trials, and provides a new method for auxiliary treatment of depression.

[0007] In one embodiment of the present invention, the Lactobacillus reuteri is isolated from fecal samples of healthy people. The Lactobacillus reuteri (Limosilactobacillus reuteri) CCFM1288 provided by the present invention was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 13, 2022, with a collection number of GDMCC No: 62968.

[0008] In one embodiment of the present invention, Lactobacillus reuteri CCFM1288 has the ability to metabolize tryptophan.

[0009] In one embodiment of the present invention, Lactobacillus reuteri CCFM1288 can survive in the intestine for 7 days.

[0010] The invention also provides a microbial preparation containing the Lactobacillus reuteri CCFM1288.

[0011] In one embodiment of the present invention, the viable count of Lactobacillus reuteri CCFM1288 in the microbial preparation is not less than 10 9 CFU / mL.

[0012] In one embodiment of the present invention, the microbial preparation further contains a protective agent; the protective agent includes one or more of skimmed milk powder, sucrose, fructooligosaccharides, galacto-oligosaccharides, and xylo-oligosaccharides.

[0013] The invention also provides a product containing the Lactobacillus reuteri CCFM1288 or the microbial preparation.

[0014] In one embodiment of the present invention, the product includes food, medicine or health care product.

[0015] In one embodiment of the present invention, the food includes fermented fruits and vegetables, fermented bean products, fermented dairy products or other foods containing Lactobacillus reuteri CCFM1288.

[0016] In one embodiment of the present invention, the medicine includes biological medicine, traditional Chinese medicine or chemical medicine.

[0017] In one embodiment of the present invention, the product is in a solid, liquid or semi-solid state.

[0018] The present invention also provides the Lactobacillus reuteri CCFM1288 or the use of the microorganism in preparing medicines for relieving depression.

[0019] In one embodiment of the present invention, the depression relief comprises at least one of the following functions:

[0020] (a) Reduce anxiety-like and depressive-like behaviors;

[0021] (b) Improve the α-diversity of intestinal flora, increase species abundance and diversity, increase the abundance of beneficial bacteria Faecalibaculum, Bacteroides, Ruminococcaceae UCG-004, Ruminiclostridium 5, Tyzzerella and Lachnospiraceae FCS020 group, and reduce the abundance of harmful bacteria Jeotgalicoccus;

[0022] (c) Promote the metabolism of indole substances in the intestinal flora and improve the metabolic imbalance of the intestinal flora;

[0023] (d) Reduce the levels of corticotropin-releasing factor in the hypothalamus and corticosterone and adrenocorticotropic hormone in serum, alleviating the hyperactivity of the HPA axis;

[0024] (e) Increases the levels of dopamine and 5-hydroxytryptamine in the brain and promotes 5-HT secretion in the intestine;

[0025] (f) Reduce the mRNA levels of IL-6 and TNF-α and increase IL-22 and to maintain immune regulation homeostasis.

[0026] In one embodiment of the present invention, Lactobacillus reuteri CCFM1288 can significantly improve the depression of MDD patients and significantly reduce the HAMD, MADRS and SDS scores.

[0027] In one embodiment of the present invention, the drug is a drug for alleviating depression and anxiety in mammals.

[0028] In one embodiment of the present invention, the mammal includes but is not limited to humans.

[0029] The present invention also provides the Lactobacillus mucilaginosus CCFM1288 or the use of the microorganism in preparing products for regulating intestinal flora.

[0030] In one embodiment of the present invention, the targeted regulation of intestinal flora includes increasing the abundance of beneficial bacteria Faecalibaculum, Ruminiclostridium 5 and Tyzzerella, and reducing the abundance of harmful bacteria Jeotgalicoccus; the product includes a health product or a medicine.

[0031] Beneficial effects:

[0032] The Lactobacillus reuteri CCFM1288 provided by the present invention has the following beneficial effects:

[0033] 1. It has the ability to metabolize tryptophan. The in vitro ILA production is 79.86±4.74μg / mL, the IAA production is 15.75±1.49μg / mL, and the ICA production is 21.82±2.14μg / mL.

[0034] 2. It can stay in the intestine for 7 days.

[0035] 3. CCFM1288 intervention in depressed mice can significantly reduce their anxiety-like and depression-like behaviors, improve the α-diversity of intestinal flora, increase species abundance and diversity, increase the abundance of beneficial bacteria Faecalibaculum, Bacteroides, Tyzzerella and Lachnospiraceae FCS020 group, reduce the abundance of harmful bacteria Jeotgalicoccus, promote the secretion of indole substances by intestinal symbiotic bacteria, maintain intestinal homeostasis, and thereby relieve the hyperfunction of the hypothalamic-pituitary-adrenal axis, improve the function of the dopamine and serotonin neurotransmitter-energy nervous system, promote the release of neurotransmitters, and relieve the depressive state of lack of pleasure.

[0036] In clinical trials, intervention with Lactobacillus reuteri CCFM1288 can significantly improve depressive symptoms in patients with MDD, providing a new and safe way to relieve depression.

[0037] Biomaterial Deposit

[0038] A strain of Limosilactobacillus reuteri CCFM1288, taxonomically named Limosilactobacillus reuteri, was deposited in the Guangdong Provincial Microbiological Culture Collection on November 13, 2022, with the deposit number GDMCC No: 62968, 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

[0039] Figure 1 : In vivo survival ability of Lactobacillus reuteri CCFM1288.

[0040] Figure 2 :Behavioral test results of experimental mice, (A) open field test; (B) elevated plus maze test; (C) forced swimming test; (D) sugar water preference test. 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.

[0041] Figure 3 :Effects of Lactobacillus reuteri CCFM1288 on the abundance and diversity of intestinal flora, (A) Chao1 index; (B) Shannon index; (C) Simpson diversity index. 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 and ***P<0.001.

[0042] Figure 4 :Differential analysis of the effects of Lactobacillus reuteri CCFM1288 intervention on intestinal flora structure.

[0043] Figure 5 :Analysis of differential bacterial genera significantly regulated by Lactobacillus reuteri CCFM1288 intervention, using one-way ANOVA and Tukey post hoc test analysis, compared with the model group for significance analysis, significance is expressed as * P<0.05, ** P<0.01, *** P < 0.001 and **** P<0.0001.

[0044] Figure 6 :Changes in tryptophan metabolic pathways and cytokine transcription levels of intestinal flora in the colon, (A) tryptamine TA content; (B) indole lactic acid ILA content; (C) indole formaldehyde ICA content; (D) indole acetic acid IAA content; (E) indole acrylic acid IA content; (F) indole propionic acid IPA content; (G) interleukin IL-22 mRNA level; (H) α tumor necrosis factor TNF-α mRNA level; (I) interleukin IL-6 mRNA level. Single-factor ANOVA and Tukey post hoc test were used for analysis, and significance analysis was performed compared with the model group, and significance was expressed as * P < 0.05 and *** P<0.001.

[0045] Figure 7 :Effects of Lactobacillus reuteri CCFM1288 on the HPA axis, (A) serum corticosterone CORT content; (B) serum adrenocorticotropic hormone ACTH content; (C) hypothalamic adrenocorticotropic hormone releasing factor CRF content. 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, *** P < 0.001 and **** P<0.0001.

[0046] Figure 8 :Changes in neurotransmitter levels in different tissues of mice, (A) dopamine DA content in hippocampus; (B) 5-HT concentration in hippocampus; (C) 5-HT concentration in serum; (D) 5-HT content in colon contents. 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<0.0001.

[0047] Fig. 9 :The effect of Lactobacillus reuteri CCFM1288 on the psychological scale scores of patients with MDD, (A) Hamilton Depression Rating Scale HAMD score; (B) Montgomery Depression Rating Scale MADRS score; (C) Self-rating Depression Scale SDS score. Statistical analysis used student's t test, and significance was expressed as *** P < 0.05 and **** P<0.001. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0049] The experimental methods in the following examples are all conventional methods unless otherwise specified; the biological and chemical reagents used are all conventional reagents in the art and can be obtained commercially.

[0050] The culture medium involved in the following examples is as follows:

[0051] 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.

[0052] MRS solid culture medium: Add 15-20 g of agar to the MRS liquid culture medium.

[0053] Carbon starvation medium (1 L): peptone 10 g, NaCl 5 g.

[0054] The 5-week-old male C57BL / 6J mice involved in the following examples were purchased from Vitallife.

[0055] Example 1: Screening and identification of Lactobacillus reuteri CCFM1288 and in vitro tryptophan metabolism

[0056] (1) Screening and molecular biological identification of Lactobacillus reuteri CCFM1288

[0057] 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.

[0058] The strain was taken out from -80 ° C, streaked and activated on MRS solid medium, inverted and cultured at 37 ° C for 24 hours, and a single colony was picked for purification again. After purification, the single colony was transferred to 2mL MRS liquid medium and cultured at 37 ° C for 12 hours. After mixing, it was transferred to 20mL fresh MRS medium at a 1% inoculation amount. After culturing for 16 hours, the bacteria were collected by centrifugation at 8000×g for 5 minutes. Weigh 5g of precipitation and send it to Shanghai Meiji Biomedical Technology Co., Ltd. for whole genome draft testing and strain identification. The strain was identified as Lactobacillus reuteri by whole genome draft testing and NCBI database comparison, named Lactobacillus reuteri CCFM1288, and has been deposited in Guangdong Institute of Microbiology, Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou, with a deposit number of GDMCC No: 62968.

[0059] (2) In vitro tryptophan fermentation

[0060] Take 1 mL of bacterial suspension in a 1.5 mL EP tube, centrifuge at 8000 × g for 5 min, discard the supernatant, wash twice with saline, centrifuge to remove the supernatant, add 0.3 mL of carbon starvation medium containing 0.6 mM tryptophan to resuspend the medium, take 50 μL of the bacterial suspension and dilute it to 10 with 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 300μL was taken for indole metabolite detection. The metabolic capacity of the strain was defined as 10 8 CFU refers to the amount of indole substances produced by live bacteria.

[0061] Determination of indole substances in bacterial suspension: 300 μL of bacterial suspension was 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 assembled using ACQUITY Ultra-high performance liquid chromatography (DIONEX UltiMate 3000HPLC) with a BEH C18 column (1.7 μm, 100 mm × 2.10 mm) was coupled to a mass spectrometer (Q-Exactive massspectrometer) for detection. The injection volume was 2 μL, 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, 0–3 min; 30% B, 3–9 min; 100% B, 9–15 min; 100% B, 15–16.5 min; 5% B, 16.5–20 min, the column temperature was 37 °C, and the positive ion mode was used for scanning detection. The detection standards included tryptamine (TA), indole lactic acid (ILA), indole acetic acid (IAA), indole propionic acid (IPA), indole acrylic acid (IA) and indole carboxaldehyde (ICA), and the external standard method was used for quantification.

[0062] The results of in vitro tryptophan metabolism assay showed that the contents of IPA, TA and IA in the supernatant of CCFM1288 were below the detection limit, while the yield of ILA was 79.86±4.74μg / mL (172.50±10.24ng / 10 8 CFU), IAA production was 15.75±1.49μg / mL (34.01±3.21ng / 10 8 CFU), ICA production was 21.82±2.14μg / mL (47.14±4.62ng / 10 8 The results showed that Lactobacillus reuteri has the ability to metabolize tryptophan and produces high amounts of indole lactic acid, indole formaldehyde and indole acetic acid.

[0063] Example 2: In vivo survival ability of Lactobacillus reuteri CCFM1288

[0064] (1) Screening of rifampicin-resistant mutants of Lactobacillus reuteri CCFM1288

[0065] Lactobacillus reuteri CCFM1288 was activated on MRS, and a single colony was picked and placed in fresh MRS liquid medium, incubated at 37°C for 24 hours, transferred to 10 mL MRS liquid medium at a 1% inoculum, cultured overnight, and collected after centrifugation at 4°C and 8000×g for 5 minutes. The cells were resuspended in 200 μL PBS, spread on MRS solid medium containing 50 μg / mL rifampicin, and cultured at 37°C for 3 days to obtain Rifampicin. r Lactobacillus reuteri CCFM1288.

[0066] (2) Determination of the in vivo survival time of Lactobacillus reuteri CCFM1288 mutants

[0067] The activated Rif r Lactobacillus reuteri CCFM1288 was expanded and cultured for 12 hours. After centrifugation at 4°C and 8000×g for 5 minutes, the bacteria were collected and washed twice with an equal volume of 0.85% saline. The supernatant was discarded, and the bacteria were resuspended in 1 / 5 volume of saline. 100μL of the bacterial suspension was taken for live bacteria counting. The remaining bacterial suspension was given to 3 mice, and the gavage volume was 200μL. After gavage, the feces of the 3 mice on the 1st, 2nd, 3rd, 4th, 6th, and 7th days were collected. After the feces were weighed, 1mL of saline was added, and after shaking, 100μL of the suspension was diluted and applied to Rif r The cells were incubated on MRS solid medium at 37°C for 24 h, and the colonies were counted. r The amount of Lactobacillus reuteri CCFM1288 was calculated as the log value of the number of viable bacteria per gram of feces.

[0068] On day 0, the experimental mice consumed 7.8×10 9 CFU / mL of Lactobacillus reuteri, the in vivo survival test results are as follows Figure 1 As shown in the figure, as time went on, the number of live bacteria of Lactobacillus reuteri CCFM1288 in the mouse feces gradually decreased, and the number of live bacteria was less than 10 CFU on the 7th day, indicating that Lactobacillus reuteri CCFM1288 can survive in the mouse body for 7 days.

[0069] Example 3: Lactobacillus reuteri CCFM1288 alleviates anxiety-like and depression-like behaviors in chronically stressed mice

[0070] (1) Preparation of intragastric lavage agent:

[0071] The activated Lactobacillus reuteri CCFM1288 single colony was placed in a fresh MRS liquid medium and cultured at 37°C for 24 h. The culture was expanded at a 1% inoculum size. After culturing for 12 h, the obtained 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 a viable cell count of 5×10 9 CFU / mL bacterial suspension. Fluoxetine, the drug control, was dissolved in normal saline with a final concentration of 1 mg / mL. The oral volume for mice was 200 μL / mouse / day.

[0072] (2) Animal experiments:

[0073] Twenty-four 5-week-old male C57BL / 6J mice were randomly divided into 4 groups after one week of adaptive feeding: normal control group, depression model group, drug control group and Lactobacillus reuteri CCFM1288 intervention group, each group containing 6 mice, and the intervention was carried out at the same time as the modeling, and the intervention lasted for 5 weeks. The animal groups are shown in Table 1.

[0074] Table 1 Experimental animal groups

[0075]

[0076] The method of establishing the mouse chronic unpredictable stress model is as follows:

[0077] 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).

[0078] Five weeks after the mice were stimulated, behavioral tests were performed, including the open field test, elevated plus maze test, forced swimming test, and sugar water preference test.

[0079] The behavioral testing method is as follows:

[0080] 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%.

[0081] 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).

[0082] 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.

[0083] 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%.

[0084] The open field test, elevated plus maze test, forced swimming test and sugar water preference test can be used to test the differences in anxiety-like and depression-like behaviors of animals and to measure the establishment of depression models in mice. Figure 2 As shown in the results, compared with normal mice, the model mice had significantly reduced time spent in the center of the open field and the time spent in the elevated plus maze, decreased interest in sugar water preference, and increased despair time in forced swimming, indicating that the depression model was successfully established. Supplementation with Lactobacillus reuteri CCFM1288 reversed this condition and significantly alleviated anxiety and depression-like behaviors. The drug control group increased the sugar water preference value of mice, but CCFM1288 intake was more effective in alleviating the despair time and preference value of mice, achieving a more stable therapeutic effect.

[0085] Example 4: Regulatory effect of Lactobacillus reuteri CCFM1288 on intestinal flora of depressed mice

[0086] According to the instructions of MP's fecal kit, total DNA from mouse colon contents was extracted. The whole genome of intestinal flora was used as a template, and the V3-V4 region fragment of 16S rDNA was amplified using the universal primer pair 341F / 806R. The target fragment length was about 465bp. The PCR product was separated by 1.5% agarose gel, electrophoresed at 120V for 40min, and the target band gel block was cut out. The fragment was purified according to the instructions of the gel recovery kit. The sample DNA concentration was detected using the Qubit DNA3.0 kit, and then the library was mixed and constructed according to the DNA concentration. The total volume of each library was 50μL. Sequencing was performed on the Illumina Miseq platform, and the offline data was analyzed by QIIMEⅡ software for quality control, splicing, annotation, etc. The α-diversity and β-diversity of the sample were calculated to evaluate the diversity of the sample's flora. Among them, α-diversity was characterized by Shannon index, Simpson index and Chao1 index.

[0087] The results of intestinal flora α-diversity are as follows Figure 3As shown in the figure, the Chao1 value, Shannon index and Simpson index of the intestinal flora of healthy mice were 113.00±23.85, 6.48±0.22 and 0.98±0.01, respectively, while those of the depression model mice were 86.00±10.77, 6.15±0.26 and 0.96±0.01. In contrast, the species abundance and diversity of the intestinal flora in the model group showed a downward trend. After the intervention of Lactobacillus mucosa CCFM1288, the α-diversity results were 113.50±17.09, 6.53±0.17 and 0.98±0.01, respectively, which significantly improved the diversity of the intestinal flora of depressed mice and maintained the stability of the intestinal flora. Lefse difference analysis found that CCFM1288 intervention could improve the structural composition of the intestinal flora, which was significantly different from the model group ( Figure 4 ). The results of the significant difference analysis are as follows Figure 5 As shown, the model group Faecalibaculum, Bacteroides, Ruminococcaceae UCG-004, Ruminiclostridium 5, Tyzzerella, Lachnospiraceae The relative abundances of FCS020group and Jeotgalicoccus were 0.40±0.36%, 0.17±0.42%, 0.08±0.07%, 0.02±0.01%, 0.05±0.04%, 0% and 0.67±0.29%, respectively, and those of CCFM1288 were 2.96±1.21%, 1.44±0.35%, 0.46±0.27%, 0.21±0.20%, 0.12±0.04%, 0.22±0.22% and 0.10±0.10%, respectively. Compared with the model group, the CCFM1288 group could significantly increase the abundance of beneficial bacteria Faecalibaculum, Bacteroides, Ruminococcaceae UCG-004, Ruminiclostridium 5, Tyzzerella and Lachnospiraceae FCS020group. group, reduced the abundance of the harmful bacteria Jeotgalicoccus, and had unique advantages in regulating Faecalibaculum, Ruminiclostridium 5 and Tyzzerella, while the drug group had little effect on the microbiota. CCFM1288 could reshape the normal microbiota structure to promote the relief of depression.

[0088] Example 5: Effects of Lactobacillus reuteri CCFM1288 on indole metabolites and cytokine transcription levels in intestinal flora

[0089] Detection of neurotransmitters in different tissues of mice:

[0090] Colonic contents: The collected colonic 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.

[0091] 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.

[0092] Hippocampal tissue: Take a certain amount of sample, add 900 μL 50% methanol water, vortex for 15 seconds, homogenize, stand at -20℃ for 1 hour, centrifuge at 4℃, 15000×g for 10 minutes. Take the supernatant, vacuum concentrate at 45℃ for 3 hours. Resuspend in 100 μL 10% methanol water, centrifuge at 15000×g for 10 minutes, filter the supernatant through a 0.2μm filter membrane, place in a liquid phase vial containing an inner liner, and perform quantitative detection using the above-mentioned LC-MS.

[0093] Determination of transcription levels of target protein-coding genes in tissues:

[0094] Total RNA was extracted from mouse colon tissue using the Trizol method, and its concentration was detected by an ultra-micro spectrophotometer (NanoDrop2000C). 1 μg of RNA was taken for gDNA removal and cDNA synthesis (HiScript III RT SuperMix for qPCR, Vazyme) according to the instructions of the reverse transcription kit. The qPCR system was 5 μL SYBR Green supermix, 1 μL cDNA, 1 μL primer pair, 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 2.

[0095] Table 2 Primers used for fluorescence quantitative PCR

[0096]

[0097] Studies have shown that tryptophan metabolites in the intestinal flora of patients with depression are unbalanced. The content of indole substances in the colon contents of depressed mice is as follows Figure 6 As shown, the concentrations of TA, ILA, ICA and IAA in the model group (0.41±0.23, 0.26±0.19, 0.21±0.07 and 0.89±0.31 mg / kg) were significantly lower than those in the normal control group (1.46±0.72, 1.33±0.53, 0.42±0.09 and 1.50±0.35 mg / kg), indicating that the depression of mice is accompanied by metabolic disorders and reduced content of indole substances in the intestinal flora. After CCFM1288 intervention, the concentrations of TA, ILA, ICA and IAA in the intestine increased to 1.94±0.47, 0.81±0.25, 0.47±0.25 and 2.13±0.31 mg / kg, indicating that it can promote the downward metabolism of tryptophan by intestinal flora and help improve depression. Secondly, patients with depression are also accompanied by changes in immune levels. Figure 6 GI showed that compared with the normal group TNF-α, IL-6 and IL-22 transcription levels (0.56±0.10, 0.63±0.20 and 1.12±0.18), the transcription levels of pro-inflammatory factors TNF-α and IL-6 in the colon immune cells of depressed mice were increased (1.01±0.47 and 1.02±0.20), and the transcription level of anti-inflammatory factor IL-22 was decreased (0.94±0.01). Fluoxetine had no significant effect on immunomodulatory effects, while CCFM1288 was 0.52±0.12, 0.51±0.22 and 1.13±0.13, which can significantly alleviate the imbalance of inflammatory factors and relieve depression.

[0098] Example 6: Lactobacillus reuteri CCFM1288 relieves hyperactivity of the HPA axis

[0099] 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 an ELISA kit. The hypothalamic tissue of the mice was taken, weighed, and 9 times the volume of normal saline (equivalent to 1g of tissue plus 9mL of normal saline) was added, homogenized with a tissue homogenizer, centrifuged at low speed, and the supernatant was tested for the content of adrenocorticotropic hormone releasing factor (CRF) using an ELISA kit.

[0100] HPA axis hyperfunction is one of the common neurobiological manifestations of depression and a prominent feature of depression. Figure 7As shown in the figure, the levels of corticosterone and adrenocorticotropic hormone in the serum of depressed mice increased, and the level of adrenocorticotropic hormone releasing factor in the hypothalamus increased significantly, indicating that the HPA axis was overactive. However, the mice in the Lactobacillus reuteri CCFM1288 intervention group did not show abnormal HPA axis function, and the levels of CORT, ACTH and CRF were similar to those of normal mice, indicating that it can alleviate the hyperactivity of the HPA axis and thus relieve depression.

[0101] Example 7: Lactobacillus reuteri CCFM1288 promotes the secretion of dopamine and serotonin neurotransmitters

[0102] The colon content pretreatment method and the indole detection method are the same as those in Example 5;

[0103] Neurotransmitter test results Figure 8 As shown in the figure, compared with normal mice, depressed mice have disordered neurotransmitter function, low levels of dopamine and 5-hydroxytryptamine in the brain, reduced serotonin levels in peripheral blood, and insufficient secretion of 5-HT in the intestine. Fluoxetine intake increased the content of 5-hydroxytryptamine in different tissues of mice, but had no significant effect on dopamine levels. However, dopamine and serotonin levels in depressed mice supplemented with Lactobacillus reuteri CCFM1288 increased significantly, reaching a level comparable to that of the control group, which had an antidepressant effect.

[0104] Example 8: Clinical efficacy evaluation of Lactobacillus reuteri CCFM1288 in relieving symptoms in patients with depression

[0105] (1) Preparation of probiotic preparations

[0106] Freeze-drying protective agent: 13% skim milk, 3% sucrose, 3% fructo-oligosaccharides, 1% galacto-oligosaccharides, 12% xylo-oligosaccharides, sterilized at 65℃ for 30min.

[0107] Food grade culture medium (1L): 20g glucose, 15g yeast extract, 2g sodium acetate, 2g diammonium hydrogen citrate, 2g K2HPO4, 0.1g MgSO4·7H2O, 0.05g MnSO4·H2O, 1mL Tween 80, 1.5% agar added to solid culture medium. All raw materials are food grade standards.

[0108] Lactobacillus reuteri CCFM1288 was activated on a food-grade culture medium, and a single colony was inoculated in a food-grade liquid culture medium and cultured overnight. The culture was transferred to 500 mL of food-grade culture medium at a 2% inoculation rate, cultured at 37°C for 16 hours, and then transferred to 12 L of food-grade liquid culture medium for culture. The culture solution was centrifuged at 4°C and 7000×g for 5 minutes, the bacteria were washed twice with 0.85% saline, resuspended in 300 mL of freeze-drying protective agent, mixed and freeze-dried in a vacuum freeze dryer. After the freeze-drying process is completed, 0.1 of the freeze-dried powder is taken for live bacteria counting. The remaining freeze-dried powder is weighed and sent to Jiangsu Weikang Biotechnology Co., Ltd. for packaging and production. Each package of probiotic preparation contains 2g of powder, and the amount of live bacteria must reach 10 9 Above CFU, maltodextrin was added as a filler. The placebo preparation was filled and packaged with maltodextrin and did not contain any active probiotics.

[0109] (2) A randomized double-blind trial of probiotic intervention in patients with MDD

[0110] With reference to the good clinical practice standards formulated by the International Medical Regulatory Council, this study passed the review of the Wuxi Mental Health Center Ethics Committee and was completed under the supervision of the Ethics Committee, with ethics approval number: WXMHCIRB2023LLky002.

[0111] Inclusion and exclusion criteria:

[0112] Inclusion criteria: 18 to 65 years old; Hamilton Depression Rating Scale score ≥ 14; meet the diagnostic criteria for depressive episodes of the Chinese Classification and Diagnostic Criteria of Mental Disorders (CCMD); understand and voluntarily participate in this trial and sign the informed consent.

[0113] Exclusion criteria: refractory depression; other psychiatric disorders diagnosed according to DSM-IV Axis I; suicidal or self-harming behaviors; patients with schizophrenia, bipolar disorder, or neurodegenerative diseases; severe physical illnesses, such as HIV, history of epilepsy, history of heart disease, hyperthyroidism, and other severe physical illnesses; pregnant or lactating women; men or women preparing for pregnancy; and those who have taken antibiotics or other probiotic preparations in the past month.

[0114] Withdrawal criteria: serious adverse events occur during the study; the subject requests to withdraw informed consent; the researcher considers it necessary for the subject to terminate the study from a medical perspective.

[0115] Eligible subjects signed informed consent and were enrolled and randomly divided into a probiotic group and a placebo group. The trial period was 1 month, and patients took 1 pack of the above preparations daily. Before and after the intervention, the Hamilton Depression Rating Scale (HAMD), Montgomery Depression Rating Scale (MADRS) and Self-rating Depression Scale (SDS) were used to evaluate the degree of depression in patients. The difference in scale scores before and after the intervention was used to evaluate the degree of depression relief.

[0116] This study planned to recruit 60 people, but actually recruited 63 people, and a total of 50 people completed the experiment, including 25 people in the probiotic group and 25 people in the placebo group. The differences in HAMD, MADRS and SDS scores before and after the probiotic group intervention were 10.60±0.76, 8.32±0.59 and 17.00±1.15, respectively, and the differences in the placebo group were 4.68±0.84, 3.56±0.67 and 7.00±0.85, respectively. Fig. 9 As shown, the difference in scale scores before and after the intervention of Lactobacillus reuteri CCFM1288 was significantly higher than that of the placebo group, indicating that it has a higher degree of relief on the depressive mood of MDD patients and has a significant improvement effect on depression.

[0117] 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 CCFM1288 was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 13, 2022, with the deposit number GDMCC No: 62968.

2. A microbial preparation containing the Lactobacillus reuteri CCFM1288 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The viable count of Lactobacillus reuteri CCFM1288 in the microbial preparation is not less than 10 9 CFU / mL or 10 9 CFU / g.

4. The microbial preparation according to claim 2 or 3, characterized in that The microbial preparation also contains a protective agent; the protective agent includes one or more of skimmed milk powder, sucrose, oligofructose, oligogalactose and oligoxylose.

5. A product containing Lactobacillus reuteri CCFM1288 according to claim 1, or a microbial preparation according to any one of claims 2 to 4.

6. The product according to claim 5, characterized in that The products include food, medicine or health products; preferably, the food includes fermented fruits and vegetables, fermented bean products or fermented dairy products; the medicine includes biological medicine, traditional Chinese medicine or chemical medicine.

7. The product according to claim 6, characterized in that The product may be in a solid, liquid or semi-solid state.

8. Use of Lactobacillus reuteri CCFM1288 according to claim 1, or a microorganism containing any one of claims 2 to 4 in the preparation of a medicine for relieving depression.

9. The use according to claim 8, wherein the depression relief comprises at least one of the following functions: (a) Reduce anxiety-like and depressive-like behaviors; (b) Regulate intestinal flora, increase the abundance of beneficial bacteria Faecalibaculum, Ruminiclostridium 5, and Tyzzerella, and reduce the abundance of harmful bacteria Jeotgalicoccus; (c) increasing the content of indole substances in intestinal flora; (d) Increase the secretion of neurotransmitters dopamine and serotonin; (e) Decreased concentrations of corticotropin-releasing factor, corticosterone, and adrenocorticotropic hormone; (f) Reduce the expression of pro-inflammatory factors and increase the expression of anti-inflammatory factors.

10. The use of Lactobacillus reuteri CCFM1288 according to claim 1, or a microorganism containing any one of claims 2 to 4 in the preparation of a product for targeted regulation of intestinal flora, characterized in that: The targeted regulation of intestinal flora includes increasing the abundance of beneficial bacteria Faecalibaculum, Ruminiclostridium 5 and Tyzzerella, and reducing the abundance of harmful bacteria Jeotgalicoccus; the product includes a health product or a medicine.

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