A strain of Bifidobacterium longum subsp. longum LF03 and its application in the preparation of antidepressant drugs

Abstract

The present invention discloses a strain of Bifidobacterium longum subspecies LF03 and its application in the preparation of antidepressant drugs, belonging to the field of microbial technology. The present invention discloses a strain of Bifidobacterium longum subspecies LF03, and the deposit number is CGMCC No. 23315. Bifidobacterium longum subspecies LF03 can significantly increase the movement distance, mania time, active time of zebrafish in an in vivo insomnia model, and significantly reduce the static time, and significantly inhibit the activity of monoamine oxidase in zebrafish, and has the potential to be used in vivo to prevent or treat depression. The Bifidobacterium longum subspecies LF03 described in the present invention has huge potential application prospects in the preparation of drugs for the treatment and / or prevention of depression.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, and more specifically to a strain of Bifidobacterium longum subspecies longum LF03 and application thereof in preparing antidepressant drugs. Background Art

[0002] Depression is a mental illness characterized by persistent depression, pessimism, frequent insomnia, mental distress, and physical symptoms in severe cases. Currently, antidepressants are the main treatment for depression. However, antidepressants have poor therapeutic effects on 30-40% of patients, and most drugs are designed to interfere with the activity of neurotransmitters in the brain, causing side effects such as headaches, nausea, vomiting, and diarrhea.

[0003] Therefore, finding a method to treat depression with reliable efficacy and few toxic side effects has become a major research direction. Compared with traditional drug treatments, probiotics with antidepressant effects have attracted widespread attention due to their few side effects and other probiotic effects.

[0004] An adult's intestine contains 10 13 –10 14 microorganisms, at least 10 times the number of human cells. In total, the gut microbiome contains about 3 million genes, 150 times the number of genes found in the human genome. Diet, infection, disease conditions, and other factors affect the composition of the gut microbiome, and abnormal microbiota can alter behavior, immunity, and endocrine function. The intestine contains more than 100 million neurons and is a gathering place for neural, microbial, and immune cells. The gut microbiota regulates central nervous system and peripheral nerve function through its effects on core nervous system processes such as neurogenesis, synaptic plasticity, neurotransmitter signaling, neurodevelopment, and neuroinflammation. In addition, abnormal gut microbiota have been associated with autism spectrum disorders, depression, and Parkinson's disease. Bacteria are essential for normal brain development and adult brain function. Evidence from previous animal and clinical studies supports a key role for the gut microbiota in mood regulation, and when the microbiota is transplanted from patients with major depressive disorder into normal mice, it can cause depressive behavior in mice.

[0005] At present, international probiotics patent applications are concentrated in the United States, Japan, Russia and other countries, but foreign strains may not be suitable for the gastrointestinal physiological conditions of Chinese residents. In addition, the lack of strong scientific research evidence on the functions of probiotics has seriously affected the promotion of probiotics and their products.

[0006] Based on this, it is an urgent problem for technical personnel in this field to deeply explore the functions of bacterial resources and screen out new probiotic strains with independent intellectual property rights, specific functional properties, and suitable for the physiological characteristics of the Chinese population. Summary of the invention

[0007] In view of this, the present invention provides a strain of Bifidobacterium longum subspecies longum LF03, and use thereof in preparing an antidepressant drug.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A strain of Bifidobacterium longum subsp. longum LF03, wherein the Bifidobacterium longum subsp. longum LF03 was deposited in the General Microbiology Center of China Culture Collection Administration on August 30, 2021, with the deposit number CGMCC No. 23315, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Bifidobacterium longum subsp. longum.

[0010] As an inventive concept identical to the above technical solution, the present invention also seeks to protect an engineered strain constructed using the above Bifidobacterium longum subspecies longum LF03 as a starting strain.

[0011] As the same inventive concept as the above technical solution, the present invention also requests protection for the use of Bifidobacterium longum subspecies longum LF03 in the preparation of antidepressant drugs.

[0012] Preferably, the Bifidobacterium longum subspecies longum LF03 can significantly increase the movement distance, manic time, and active time of zebrafish in a reserpine-induced zebrafish depression model, and significantly reduce the immobility time, showing a good antidepressant prebiotic effect.

[0013] As an inventive concept identical to the above technical solution, the present invention also seeks to protect the use of Bifidobacterium longum subspecies longum LF03 in the preparation of a drug for inhibiting the activity of monoamine oxidase.

[0014] Preferably, the Bifidobacterium longum subspecies longum LF03 can significantly inhibit the activity of monoamine oxidase in zebrafish.

[0015] As an inventive concept identical to the above technical solution, the present invention also seeks to protect a drug for treating and / or preventing depression, comprising the above Bifidobacterium longum subspecies longum LF03 or the above engineered strain.

[0016] Preferably, the pharmaceutical dosage form is a bacterial suspension, powder or tablet.

[0017] As an inventive concept identical to the above technical solution, the present invention further seeks to protect a drug for inhibiting the activity of monoamine oxidase, comprising the above Bifidobacterium longum subspecies longum LF03 or the above engineered strain.

[0018] Preferably, the pharmaceutical dosage form is a bacterial suspension, powder or tablet.

[0019] Through the above technical scheme, it can be known that compared with the prior art, the present invention discloses a strain of Bifidobacterium longum subspecies long LF03, and provides the application of Bifidobacterium longum subspecies long LF03 in the preparation of antidepressant drugs. Bifidobacterium longum subspecies long LF03 is isolated and screened from the feces of centenarians in Jiaoling County, Meizhou City, Guangdong Province. In the reserpine-induced zebrafish depression model, it can significantly increase the movement distance, manic time, and active time of zebrafish, and significantly reduce the immobility time; at the same time, it can significantly inhibit the activity of monoamine oxidase in zebrafish, and has the potential to be used in the prevention or treatment of depression. This provides a theoretical reference and guiding basis for the development of probiotic preparations for the prevention or treatment of depression using Bifidobacterium longum subspecies long LF03. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 The accompanying drawing shows the colony morphology of Bifidobacterium longum subspecies longum LF03 of the present invention on an MRS agar plate;

[0022] Figure 2 The accompanying drawing shows the effect of Bifidobacterium longum subspecies longum LF03 of the present invention on the movement trajectory of a zebrafish depression model induced by reserpine;

[0023] Figure 3 The accompanying drawing shows the effect of Bifidobacterium longum subspecies longum LF03 of the present invention on the movement distance of the reserpine-induced zebrafish depression model;

[0024] Figure 4 The accompanying drawing shows the effect of Bifidobacterium longum subspecies longum LF03 of the present invention on the manic time of the reserpine-induced zebrafish depression model;

[0025] Figure 5 The accompanying drawing shows the effect of Bifidobacterium longum subspecies longum LF03 of the present invention on the active time of the reserpine-induced zebrafish depression model;

[0026] Figure 6 The accompanying drawing shows the effect of Bifidobacterium longum subspecies longum LF03 of the present invention on the immobility time of the reserpine-induced zebrafish depression model;

[0027] Figure 7 The accompanying drawing shows the effect of Bifidobacterium longum subspecies longum LF03 of the present invention on monoamine oxidase activity in zebrafish. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1 Isolation, identification and preservation of Bifidobacterium longum subspecies longum LF03

[0030] (1) Separation:

[0031] 1) Dissolve the feces of the centenarian (about 0.1 g) in a 1.5 mL centrifuge tube containing 1 mL of sterile saline, and mix thoroughly by pipetting with a 1 mL sterile pipette tip for later use.

[0032] 2) Add 900uL of sterile saline to each of 6 sterile 1.5mL centrifuge tubes.

[0033] 3) From the first one with 10 -1 Pipette 100uL of the sample diluent into the centrifuge tube (10 -2 ), diluted to 10 -2 ;

[0034] 4) From the second one with 10 -2 Pipette 100uL of the sample diluent into the centrifuge tube (10 -3 ), diluted to 10 -3 ;

[0035] 5) Repeat the previous step until the dilution reaches 10 -4 , 10 -5 , 10 -6 , 10 -7 .

[0036] 6) From the installed 10 -4 Pipette 100uL of sample dilution from the centrifuge tube of the sample dilution and apply it on the BS solid culture medium and MRS solid culture medium respectively. Spread 100uL of bacterial solution flat and apply it dry. Note that the application technique should be gentle and quick, and must be done near the flame of the alcohol lamp. After the application is completed, mark the side of the culture dish, including the name, sample number, culture medium name, culture time, dilution gradient, culture conditions (anaerobic / aerobic), etc.

[0037] 7) Repeat the previous step to complete 10 -5 , 10 -6 , 10 -7 Dilution spread of the dilution gradient.

[0038] 8) After coating, place the culture dishes at 37°C under anaerobic conditions for culture, and observe and record after 48 hours.

[0039] 9) Use an inoculation loop to pick up a single colony on the plate and streak it onto BS solid culture medium. Incubate anaerobically at 37°C for 48 hours to isolate pure colonies.

[0040] 10) Inoculate the pure colonies on the plate into BS liquid culture medium, culture anaerobically at 37°C for 12-16 hours, add 20% glycerol, and store in a -80°C refrigerator.

[0041] (2) Molecular biological identification of strains: Genomic DNA was extracted from the obtained strains, and the 16S rDNA full-length fragment was amplified by PCR using 16S rDNA universal primers 27F and 1492R, followed by sequencing to identify the species of the strains.

[0042] Among them, the primer sequences of universal primers 27F and 1492R are as follows:

[0043] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO.1;

[0044] 1492R: 5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO. 2.

[0045] Experimental results: The strains screened from the feces of centenarians in Jiaoling County, Meizhou City, Guangdong Province were identified through morphological observation and 16SrDNA identification. Among them, strain LF03 was identified as Bifidobacterium longum subsp. longum, and its 16SrDNA sequence is shown in SEQ ID NO.3.

[0046]

[0047] A single colony of strain LF03 was inoculated on BS solid medium and grew well under anaerobic conditions at 37°C. The colonies were milky white, spherical, with neat edges and a smooth surface (see Appendix). Figure 1 ). The strain LF03 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, referred to as CGMCC, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on August 30, 2021. It is classified and named Bifidobacterium longum subsp. longum, and the deposit number is CGMCC No. 23315.

[0048] Example 2 Preparation of Bifidobacterium longum subspecies longum LF03 bacterial suspension (bacteria)

[0049] Bifidobacterium longum subsp. longum LF03 was activated and cultured and inoculated into BS liquid medium. After culturing at 37°C for 24 h, the culture was centrifuged at 4°C and 6000 r / min for 10 min to obtain bacterial pellets. The bacterial pellets were washed twice with PBS and then resuspended in PBS to adjust the cell concentration to 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL to obtain bacterial suspension (bacteria).

[0050] Example 3 Effect of Bifidobacterium longum subspecies longum LF03 on zebrafish depression model

[0051] Reserpine was purchased from Sigma; clorgyline hydrochloride was purchased from MedChemExpress.

[0052] Healthy wild-type AB zebrafish that developed to 3 dpf (days post fertilization) were selected and placed in a 96-well cell culture plate. The experiment set up a normal group, a model group, a positive control group, and a strain intervention group (1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL Bifidobacterium longum subspecies longum LF03), 20 replicate wells were set up in each group, and 1 fish was placed in each well. PBS was added to the normal group and the model group, and clorgyline hydrochloride solution (5 μmol / L) was added to the positive control group, 1×10 4 CFU / mL intervention group added 1×10 4 CFU / mL Bifidobacterium longum subsp. longum LF03, 1×10 5 CFU / mL intervention group added 1×10 5CFU / mL Bifidobacterium longum subsp. longum LF03, 1×10 6 CFU / mL intervention group added 1×10 6 CFU / mL Bifidobacterium longum subspecies longum LF03, 200 μL per well, incubated at 28°C, replaced with new solution after 24 h; after incubation for 48 h, discarded the above solution, PBS was added to the normal group, and 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL long Bifidobacterium longum subspecies longum LF03) were added with reserpine solution (2mg / L), 200μL per well, incubated at 28℃ for 48h, and then the 96-well cell culture plate was placed in a zebrafish behavior analysis system (EthoVisionXT10.4, Nordus Information Technology Co., Ltd.) for trajectory tracking. The light source was turned off to allow the zebrafish to adapt to the environment for 5min, and then the behavior trajectory analysis software was turned on to collect the behavior trajectory of the zebrafish in the dark environment within 5min. The movement distance of the zebrafish was counted; at the same time, the zebrafish was divided into three movement states according to the swimming speed of the zebrafish: static (<4mm / s), active (4-20mm / s), and manic (>20mm / s), and the manic time, active time, and static time of the zebrafish were counted.

[0053] SPSS19.0 software was used to statistically process the data. The experimental data were expressed as x±SEM data and analyzed by T-test. Compared with the normal group: #### p<0.001, compared with the model group: &&&& p<0.001; one-way ANOVA, compared with the model group: *P<0.05, ***P<0.005, ****P<0.001.

[0054] See attached for the results Figure 2-6 ; By the attached Figure 2 and attached Figure 3 It can be seen that the red area is the area covered by the path of zebrafish movement; compared with the normal group, the movement trajectory of zebrafish in the model group was reduced, indicating that the activity level of zebrafish in the model group was reduced; at the same time, compared with the normal group (75.88±2.08cm), the movement distance of zebrafish in the model group (37.37±1.79cm) was significantly shortened (p<0.001), indicating that the zebrafish depression model was successfully established.

[0055] Compared with the model group, the movement trajectory of zebrafish in the positive control group (clogyline hydrochloride) increased, indicating that clogyline hydrochloride can increase the activity of zebrafish in the reserpine-induced zebrafish depression model; at the same time, the movement distance of zebrafish in the positive control group was 68.18±2.62cm, which was significantly different from that in the model group (37.37±1.79cm) (P<0.001). Therefore, clogyline hydrochloride has an antidepressant effect, which is consistent with the clinical results. Compared with the model group, the movement trajectory of zebrafish in the Bifidobacterium longum subspecies LF03 group increased, indicating that Bifidobacterium longum subspecies LF03 can increase the activity of zebrafish in the reserpine-induced zebrafish depression model; at the same time, the concentration of Bifidobacterium longum subspecies LF03 was 1×10 5 CFU / mL and 1×10 6 CFU / mL, the movement distance of zebrafish was 53.09±3.02cm and 60.66±1.85cm, respectively, which was significantly different from the model group (37.37±1.79cm) (P<0.01). Therefore, the above results show that Bifidobacterium longum subspecies longum LF03 can significantly increase the movement distance of zebrafish in the in vivo depression model, showing the effect of alleviating depression.

[0056] By the attached Figure 4 , Attachment Figure 5 and attached Figure 6 It can be seen that compared with the normal group (manic time: 21.28±0.76s, active time: 63.43±1.91s, and immobile time: 215.28±1.88s), the manic time (6.51±0.40s) and active time (35.63±1.60s) of the zebrafish in the model group were significantly reduced (p<0.001), and the immobile time (257.87±1.65s) was significantly increased (p<0.001), indicating that the zebrafish depression model was successfully established.

[0057] Compared with the model group (manic time: 6.51±0.40s, active time: 35.63±1.60s, and immobility time: 257.87±1.65s), the manic time (18.16±0.76s) and active time (58.15±3.83s) of zebrafish in the positive control group were significantly increased (p<0.001), and the immobility time (223.69±3.96s) was significantly reduced (p<0.005), indicating that clorgyline hydrochloride has an antidepressant effect, which is consistent with the clinical results. Compared with the model group (manic time: 6.51±0.40s, active time: 35.63±1.60s, and immobility time: 257.87±1.65s), 1×10 5 CFU / mL Bifidobacterium longum subsp. longum LF03 and 1×10 6CFU / mL Bifidobacterium longum subsp. longum LF03 group zebrafish frenzy time (1×10 5 CFU / mL group: 13.14±0.55s, 1×10 6 CFU / mL group: 15.28±0.72s), active time (1×10 5 CFU / mL group: 45.17±3.10s, 1×10 6 CFU / mL group: 52.02±3.27s) were significantly increased (p<0.05), and the immobility time (1×10 5 CFU / mL group: 241.69±3.11s, 1×10 6 CFU / mL group: 232.70±3.09s) were significantly reduced (p<0.005).

[0058] Therefore, the above results indicate that Bifidobacterium longum subspecies longum LF03 can significantly increase the manic time and active time of zebrafish, and significantly reduce the immobility time in the in vivo depression model, showing an effect of alleviating depression.

[0059] Example 4 Effect of Bifidobacterium longum subsp. longum LF03 on monoamine oxidase activity in zebrafish

[0060] Clogyline hydrochloride was purchased from MedChemExpress; monoamine oxidase assay kit was purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0061] Wild-type AB zebrafish (5 dpf) were placed in a six-well plate. The experiment set up a normal group, a positive control group (clogyline hydrochloride), a strain intervention group (1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL Bifidobacterium longum subspecies longum LF03), 6 replicate wells were set up in each group, and 30 fish were placed in each well. 5 mL PBS was added to each well of the normal group; 5 mL 5 μmol / L clorgyline hydrochloride solution was added to each well of the positive control group; 1×10 4 CFU / mL intervention group added 1×10 4 CFU / mL Bifidobacterium longum subsp. longum LF03, 1×10 5 CFU / mL intervention group added 1×10 5 CFU / mL Bifidobacterium longum subsp. longum LF03, 1×10 6 CFU / mL intervention group added 1×10 6CFU / mL Bifidobacterium longum subsp. longum LF03, 5mL per well, incubated at 28℃, replaced with new solution after 24h; after incubation for 72h, collect zebrafish into 1.5mL centrifuge tubes, 20 zebrafish per tube, and collect 6 tubes for each experimental group; after absorbing the water in the centrifuge tubes, add 100μL of cold PBS, use S-18KS handheld micro electric tissue homogenizer to homogenize the zebrafish until there are no obvious tissue fragments, centrifuge at 12000×g, 4℃ for 10min, collect the supernatant in a centrifuge tube, and use the monoamine oxidase assay kit to detect the monoamine oxidase activity in the supernatant.

[0062] SPSS19.0 software was used to statistically process the data. The experimental data were expressed as x±SD data and analyzed by T-test. Compared with the normal group: #### p<0.001; compared with the normal group by one-way analysis of variance: ****P<0.001 (see Appendix Figure 7 ).

[0063] By the attached Figure 7 It can be seen that the monoamine oxidase activity in zebrafish in the clorgyline hydrochloride group was 0.14±0.01U / min / mL, which was significantly different from that in the normal group (0.50±0.02U / min / mL) (P<0.001), indicating that the detection experiment of monoamine oxidase activity in zebrafish is effective.

[0064] The concentration of Bifidobacterium longum subsp. longum LF03 was 1×10 5 CFU / mL and 1×10 6 CFU / mL, the monoamine oxidase activities in zebrafish were 0.31±0.02U / min / mL and 0.23±0.02U / min / mL, respectively, which were significantly different from those in the normal group (0.50±0.02U / min / mL) (P<0.001).

[0065] There are many free biologically active monoamines in the human body, such as catecholamines adrenaline, norepinephrine, dopamine, etc. These monoamines are substances that have special effects on the human body. As neurotransmitters of neuronal synapses, these substances are usually stored in cells. Nerve impulses are released into the synaptic cleft, bind to receptors, and play a role in impulse conduction. The monoamines in the synaptic cleft are oxidized and deaminated by monoamine oxidase and decomposed and eliminated. Therefore, monoamine oxidase plays a role in regulating the concentration of monoamines in nerve cells and synaptic clefts. The role of monoamine oxidase inhibitors is to inhibit monoamine oxidase, reduce the degradation of neurotransmitters such as serotonin, norepinephrine, and dopamine, and increase their content in brain tissue. Because the reduction of monoamine concentration in the brain can cause people to suffer from depression, monoamine oxidase inhibitors can play a role in treating depression.

[0066] The above results show that the Bifidobacterium longum subspecies longum LF03 of the present invention has an inhibitory effect on monoamine oxidase in zebrafish and has the potential to prevent or treat depression.

[0067] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Bifidobacterium longum subspecies longum LF03, characterized in that: The Bifidobacterium longum subspecies longum LF03 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on August 30, 2021, with a deposit number of CGMCC No. 23315, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named Bifidobacterium longum subspecies longum Bifidobacterium longum subsp. longum .

2. Use of Bifidobacterium longum subspecies longum LF03 according to claim 1 in the preparation of antidepressant drugs.

3. A drug for treating and / or preventing depression, characterized in that: It comprises the Bifidobacterium longum subspecies longum LF03 described in claim 1.

4. The drug for treating and / or preventing depression according to claim 3, characterized in that The drug dosage form is bacterial suspension, powder or tablet.

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