Phytobacterium plantarum GOLDGUT-HNU082 and application thereof

Abstract

The invention discloses a strain of lactobacillus plantarum GOLDGUT-HNU082 and application thereof, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.33075, the preservation date is December 16, 2024, and the strain is named as lactobacillus plantarum by taxonomy. The GOLDGUT-HNU082 disclosed by the invention can be used for remarkably relieving depression-like behaviors of mice caused by chronic unpredictable mild stress, including reduction of anxiety and despair behaviors, increase of pleasant sensation and improvement of depression; the levels of key neurotransmitters such as serotonin (5-HT), dopamine (DA), gamma-aminobutyric acid (GABA) and noradrenaline (NE) in colon, serum and brain are remarkably increased, and emotional response is regulated and controlled; the levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS) are obviously reduced, and inflammatory response is relieved; the brain neuron injury is relieved; the intestinal flora disorder phenomenon of mice with depression is improved, harmful bacteria are reduced, and the abundance of beneficial bacteria is increased.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and more specifically to a Lactobacillus plantarum GOLDGUT-HNU082 and an application thereof. Background Art

[0002] Depression is a common and serious mental disorder that affects more than 280 million people worldwide. Depression has become one of the leading causes of disability, and severe cases may even lead to suicide. Although existing antidepressant treatments such as psychotherapy and drug therapy have alleviated patients' symptoms to a certain extent, there are still problems such as limited efficacy and significant side effects. Antidepressant drugs with good efficacy, such as agomelatine, escitalopram, vortioxetine and fluoxetine, still have high treatment failure rates and side effects, including nausea, rash, liver and kidney damage, etc. Therefore, it is particularly important to explore new, safe and effective strategies for the prevention and treatment of depression.

[0003] In recent years, more and more studies have shown that the gut-brain axis (GBA) plays an important role in regulating emotions and behaviors. The gut-brain axis refers to the interaction between the intestinal microbiota, the intestines, the immune system, and the central nervous system. The intestinal microbiota plays an important role in brain function and emotional regulation by affecting the nervous system, immune system, endocrine system, and metabolic pathways. Probiotics, as a method to regulate the intestinal microbiota, can regulate the intestinal microorganisms to produce a variety of neurotransmitters and metabolites, such as serotonin, γ-aminobutyric acid, dopamine, and norepinephrine. These substances enter the brain through the vagus nerve or through the enteric nervous system, affecting emotions and behaviors.

[0004] Therefore, how to alleviate depression by regulating the gut-brain axis through probiotics is an urgent problem that technicians in this field need to solve. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a Lactobacillus plantarum strain GOLDGUT-HNU082 and its application to solve the deficiencies in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A strain of Lactobacillus plantarum GOLDGUT-HNU082, which is deposited in the China General Microbiological Culture Collection Center (CGMCC), the storage address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the storage number is CGMCC NO.33075, the storage date is December 16, 2024, and the taxonomic name is Lactiplantibacillus plantarum.

[0008] The Lactobacillus plantarum GOLDGUT-HNU082 of the present invention is separated from fermented food. The whole genome sequencing results show that GOLDGUT-HNU082 has great potential for development as a probiotic in terms of physiological function.

[0009] The present invention aims to use chronic unpredictable mild stress to induce depression in mice and systematically evaluate the potential role of GOLDGUT-HNU082 in the prevention and treatment of depression. A series of behavioral tests were used to evaluate the depressive-like behavior of mice, and the levels of inflammatory factors and neurotransmitters in the brain and serum were measured. In addition, the fecal shotgun metagenomic technology was used to analyze the intestinal flora and its metabolites of mice to comprehensively explore the effects of GOLDGUT-HNU082 on the physiology and intestinal flora of mice, bringing new insights into the application of probiotics GOLDGUT-HNU082 in anti-depression.

[0010] The present invention also claims to protect a use of the above Lactobacillus plantarum GOLDGUT-HNU082 in preparing a medicine for relieving depression.

[0011] Furthermore, the above depression is caused by unpredictable mild stress (CUMS).

[0012] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] 1. The plant lactobacillus GOLDGUT-HNU082 of the present invention can significantly alleviate the depressive-like behavior of mice caused by chronic unpredictable mild stress, including reducing anxiety, despair behavior and increasing pleasure, and improving depression; significantly increase the levels of key neurotransmitters such as serotonin (5-HT), dopamine (DA), γ-aminobutyric acid (GABA) and norepinephrine (NE) in the colon, serum and brain, and regulate emotional responses; significantly reduce the levels of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and lipopolysaccharide (LPS), and relieve inflammatory reactions; reduce brain neuron damage; improve the intestinal flora disorder of depressed mice, reduce harmful bacteria and increase the abundance of beneficial bacteria.

[0014] 2. Metagenomic results further showed that the present invention clarified the intrinsic mechanism of GOLDGUT-HNU082 in chronic unpredictable mild stress-induced depression, providing a solid scientific basis for the development of probiotic-based microbial products as an alternative treatment strategy for delayed depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the appearance diagram of GOLDGUT-HNU082;

[0016] Figure 2 This is the whole genome comparison result of GOLDGUT-HNU082;

[0017] Figure 3 The effect of GOLDGUT-HNU082 on the depressive-like behavior of mice;

[0018] Figure 4 The effects of GOLDGUT-HNU082 on key neurotransmitter levels in the colon, serum, and brain of mice;

[0019] Figure 5 The effect of GOLDGUT-HNU082 on the inflammatory response of mice;

[0020] Figure 6 The effect of GOLDGUT-HNU082 on brain neurons of mice;

[0021] Figure 7 This is the effect of GOLDGUT-HNU082 on the intestinal flora of mice. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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.

[0023] Example 1

[0024] Isolation, Screening and Identification of Lactobacillus plantarum GOLDGUT-HNU082

[0025] More than 30 fermented food samples were collected from farmers' markets in various counties and cities in Hainan Province, including Wanning, Wenchang, Wuzhishan, Baisha, and Lingshui. The collected fermented food samples were diluted tenfold in sterilized saline, and then a 10-fold dilution method was used to prepare a gradient of 10 -1 , 10 -2 , 10 -3 , 10 -4, 10 -5 Sample diluent, 0.1mL of 10 -3 , 10 -4 , 10 -5 The gradient samples were spread on MRS agar medium and cultured anaerobically at 37°C for 48 hours. After multiple separations, streaking, purification, culture and identification, 5 strains of Lactobacillus plantarum were screened out. The 5 strains were cultured in MRS liquid for 24 hours, and the OD value and viable count were measured. Finally, a strain of Lactobacillus plantarum with relatively good growth activity was screened out and named GOLDGUT-HNU082. After the purified GOLDGUT-HNU082 strain was inoculated in MRS broth medium for activation for 24 hours, it was spread on MRS agar medium. After cultured at 37°C for 24 hours, the colonies were white with a diameter of 0.1-0.2 cm, a smooth, moist and raised surface, and neat edges (such as Figure 1 Observed under an optical microscope, the bacteria are rod-shaped.

[0026] Identification information: The whole genome of GOLDGUT-HNU082 strain was sequenced. The sequence obtained by whole genome sequencing was used for genome alignment. The genome similarity analysis was performed using the online analysis platform Galaxy (https: / / usegalaxy.org / ), and the software used was FastANI (https: / / github.com / ParBLiSS / FastANI). The analysis results showed that the ANI value of the whole genome sequence of GOLDGUT-HNU082 and Lactiplantibacillus plantarum SRCM100442 was 99.0642 (e.g. Figure 2 shown).

[0027] Example 2

[0028] 1 Experimental Materials

[0029] 1.1 Materials and animals

[0030] As in Example 1, GOLDGUT-HNU082 was isolated from fermented food.

[0031] Twenty-four 5-week-old male C57BL / 6J mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The common feed used in the experiment was from Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd. The energy sources of the common feed were protein (22.9%), fat (11.1%) and carbohydrates (66%).

[0032] 1.2 Experimental design

[0033] During the experiment, mice were housed in an SPF-level animal laboratory, maintained with a 12-h light-dark cycle, temperature controlled at 23±2°C, and humidity maintained at 55%±5%. Mice had free access to water and feed. After a week of adaptive feeding, the mice were randomly divided into three groups, with 8 mice in each group, namely the control group, the model group, and the probiotic group. Except for the control group, the mice were subjected to various repeated and unpredictable stressors twice a day for 8 weeks. Stressors included wet litter, no litter, restraint, cage tilt, changed light-dark cycle, flash, and 4°C cold water bath. An average of two stressors were applied every day. During this period, mice in the control and model groups were gavaged with normal saline, and mice in the probiotic group received 1×10 9 cfu GOLDGUT-HNU082 was gavaged. After 8 weeks, all mice were subjected to a series of behavioral tests. After the behavioral test, fresh fecal samples of the mice were collected for metagenomic sequencing. All mice were fasted for 12 hours, and the mice were killed by dislocation after orbital blood collection. After the blood samples were allowed to stand at room temperature for 2 hours, they were centrifuged at 4°C and 3000rpm for 20 minutes to separate the serum, which was then aliquoted and stored at -80°C for subsequent experiments. At the same time, the colon and brain of the mice were collected, one part was fixed in 4% paraformaldehyde solution, and the other part was quickly frozen in liquid nitrogen and then stored at -80°C.

[0034] 1.3 Evaluation of physiological and biochemical indicators

[0035] The levels of serotonin, dopamine, γ-aminobutyric acid, norepinephrine, interleukin-6, tumor necrosis factor α, and lipopolysaccharide in serum, colon, and brain were measured using ELISA kits provided by Shanghai Xinyu Biotechnology Co., Ltd.

[0036] 1.4 Histopathological analysis

[0037] The hippocampus was fixed in 4% paraformaldehyde solution, dehydrated, and embedded in paraffin before sectioning. After dewaxing, antigen retrieval, and nonspecific binding blocking, the sections were incubated overnight with primary antibodies for brain-derived neurotrophic factor (BDNF), postsynaptic density-95 (PSD-95), and glial fibrillary acidic protein (GFAP) labeled with red or green fluorescence, respectively. Subsequently, the sections were reacted with fluorescently labeled secondary antibodies, and the nuclei were stained with DAPI, and finally the sections were sealed with anti-fading agents for observation under a fluorescence microscope.

[0038] 1.5 Fecal metagenomic sequencing and microbial species annotation

[0039] Total DNA was extracted from mouse fecal samples using the QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) according to the instructions, and the purity and integrity of the obtained DNA were evaluated. Sequencing libraries were prepared using the IlluminaNEBNext@Ultra DNA Library Preparation Kit (NEB, USA). DNA fragments of approximately 350 bp were obtained by ultrasonic fragmentation, and shotgun metagenomic sequencing was performed on the Illumina NovaSeq 2500 platform of Novogene (Beijing, China). The obtained metagenomic data were analyzed using HUMAnN3 software to obtain microbial species, and the annotation and abundance information of metabolic pathways were predicted based on the MetaCyc database.

[0040] 1.6 Animal behavior test

[0041] 1.6.1 Sucrose water preference test

[0042] The sucrose preference test consists of two parts, namely the adaptation training part and the test part. During the training, two bottles of 1% (w / v) sucrose aqueous solution are placed on each mouse cage. After 24 hours, one of the bottles will be replaced with pure water. After 12 hours, the position of the water bottle will be swapped, and the training will end after 12 hours. Subsequently, the mice will be deprived of food and water for 24 hours. After 24 hours, a bottle of 1% (w / v) sucrose aqueous solution and a bottle of pure water will be placed on the mouse cage for a 12-hour sucrose water preference test. After 12 hours, the consumption of sucrose aqueous solution and pure water will be recorded. During the test, the mice will be prohibited from obtaining food. Sucrose water preference rate = sucrose aqueous solution consumption / (sucrose aqueous solution consumption + pure water consumption).

[0043] 1.6.2 Open field test

[0044] Place the mice in a soundproof, constant temperature and humidity environment for 1 hour. After 1 hour, place the mice in the center of the bottom of the open field box, use a camera to record the activities of the mice for the next 8 minutes, and use VisuTrack animal behavior software to analyze the activities of the mice. Before each open field test, 75% medical alcohol must be used for disinfection and deodorization.

[0045] 1.6.3 Tail suspension test

[0046] The mice were suspended with tape in an inescapable position for 6 minutes, including 2 minutes of adaptation and 4 minutes of calculation of immobility. The first 2 minutes were used for adaptation, and the last 4 minutes were used for calculation of immobility. The less time spent trying to escape in the last 4 minutes (the longer the immobility time), the higher the degree of depression.

[0047] 1.6.4 Forced swim test

[0048] Add about 18 cm of water (23-25°C) to a plastic cylinder (19 cm in diameter, 25 cm in depth), place mice in the water for 5 minutes, record the behavior of the animals with a camera, and use software to automatically evaluate the immobility time. The longer the immobility time, the higher the degree of depression of the mice.

[0049] 1.7 Statistical analysis

[0050] Statistical analysis and data visualization were performed using GraphPad Prism and R software. Experimental values ​​are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance was used to calculate the significance of the data, followed by Tukey's test for pairwise comparisons. When the data did not meet the normal distribution or homogeneity of variance, the Kruskal-Wallis test was used for difference significance analysis, and Dunn's test was used for post hoc pairwise comparisons. p < 0.05 was considered statistically significant, p < 0.01 was considered strongly statistically significant, and p < 0.001 was considered very strongly statistically significant.

[0051] 2 Results

[0052] 2.1 GOLDGUT-HNU082 alleviates depressive-like behavior in mice

[0053] Long-term chronic unpredictable mild stress induces depressive-like behaviors in mice, including decreased exploratory behavior, increased anhedonia, and despair behavior. Figure 3 As shown, the central area activity time (27.29s) and sucrose water preference rate (48.56%) of the model group mice were significantly lower than those of the control group (49.15s) and sucrose water preference rate (81.27%), and the tail suspension immobility time (177.41s) and forced swimming immobility time (174.41s) of the model group were significantly higher than those of the control group (119.59s) and forced swimming immobility time (123.24s). In contrast, the central area activity time (44..85s) and sucrose water preference rate (85.39%) of the probiotic group mice treated with GOLDGUT-HNU082 were significantly higher than those of the model group, and the tail suspension immobility time (132.23s) and forced swimming immobility time (140.57s) were significantly lower than those of the model group. In summary, GOLDGUT-HNU082 can significantly alleviate the depressive-like behavior of mice induced by chronic unpredictable mild stress.

[0054] GOLDGUT-HNU082 modulates levels of key neurotransmitters in colon, serum, and brain

[0055] The "monoamine hypothesis" is one of the mainstream hypotheses for the onset of depression. The reduction in the levels of monoamine neurotransmitters such as serotonin or norepinephrine has always been considered the main cause or physiological indicator. The levels of neurotransmitters such as serotonin, dopamine, γ-aminobutyric acid or norepinephrine in patients with depression are dysregulated. Therefore, we measured the levels of related neurotransmitters in the colon tissue, serum and brain tissue of mice. The results are as follows Figure 4 As shown, the serotonin (330.49pg / mL), dopamine (69.90nmol / L) and γ-aminobutyric acid (10.60ng / mL) in the colon of the model group mice were significantly lower than those in the colon of the control group mice (446.39pg / mL), dopamine (82.33nmol / L) and γ-aminobutyric acid (13.90ng / mL). The serotonin (198.46pg / mL), dopamine (38.20nmol / L), γ-aminobutyric acid (8.62ng / mL) and norepinephrine (84.44pg / mL) in the serum of the model group mice were significantly lower than those in the serum of the control group mice (225.65pg / mL), dopamine (45.83nmol / L), γ-aminobutyric acid (9.92ng / mL) and norepinephrine (99.16pg / mL). The levels of serotonin (253.41 pg / mL), γ-aminobutyric acid (8.60 ng / mL), and norepinephrine (99.72 pg / mL) in the brains of mice in the model group were significantly lower than those in the brains of mice in the control group (333.32 pg / mL), γ-aminobutyric acid (10.03 ng / mL), and norepinephrine (124.08 pg / mL).

[0056] In contrast, the probiotic group mice treated with GOLDGUT-HNU082 had significantly higher levels of γ-aminobutyric acid (12.64 ng / mL) in the colon, serotonin (232.10 pg / mL), dopamine (44.41 nmol / L), γ-aminobutyric acid (9.77 ng / mL) and norepinephrine (102.65 pg / mL) in the serum, and serotonin (305.59 pg / mL), γ-aminobutyric acid (9.41 ng / mL) and norepinephrine (125.55 pg / mL) in the brain than the control group. The above results show that GOLDGUT-HNU082 can regulate the levels of key neurotransmitters such as serotonin, relieve depression, and provide new perspectives for the development of antidepressant measures.

[0057] 2.3 GOLDGUT-HNU082 reduces inflammatory response

[0058] Neuroinflammation is also considered to be one of the causes of depression. To this end, we measured the levels of interleukin-6, tumor necrosis factor-α, and lipopolysaccharide in the colon tissue, serum, and brain tissue of mice. Figure 5 As shown, the levels of tumor necrosis factor α in the colon (1018.68 ng / L), lipopolysaccharide in serum (446.14 ng / L), and lipopolysaccharide in the brain (381.25 ng / L) of mice in the model group were significantly higher than those in the control group (690.38 ng / L), lipopolysaccharide in serum (419.22 ng / L), and lipopolysaccharide in the brain (379.69 ng / L). The levels of tumor necrosis factor α in the colon (876.61 ng / L), lipopolysaccharide in serum (361.16 ng / L), and lipopolysaccharide in the brain (280.36 ng / L) of mice in the probiotic group were significantly lower than those in the model group. In addition, there was no significant difference in interleukin 6 (214.75 pg / mL) in the colon of the model group and interleukin 6 (225.05 pg / mL) in the colon of the control group, but interleukin 6 (144.27 pg / mL) in the colon of the probiotic group was significantly lower than that of the model group and the control group. The above results show that GOLDGUT-HNU082 can reduce the inflammatory response in depression, which helps to prevent the development of depression.

[0059] 2.4GOLDGUT-HNU082 relieves brain neuron damage

[0060] Due to the imbalance of neurotransmitter levels and the occurrence of neuroinflammation, the brains of patients with depression are often accompanied by neuronal damage. Among them, brain-derived neurotrophic factor is a key mediator of neuronal synaptic plasticity in the brain, which can promote the growth of neurons and also promote the formation and stabilization of synapses of brain nerve cells. Postsynaptic density-95 is a scaffolding protein that plays a key role in synaptic plasticity. Glial fibrillary acidic protein is a marker of astrocyte activation when nerves are damaged. In order to further understand the therapeutic effect of GOLDGUT-HNU082 on depression, we performed immunofluorescence staining in the DG region of the hippocampus of mice to observe neuronal damage. Figure 6 As shown in Figure 2, compared with the control group, the positive area ratios of BDNF and PSD-95 in the model group were significantly reduced, and the positive area ratio of GFAP was significantly increased ( Figure 6 Middle A, Figure 6 Middle B, Figure 6 Middle C, Figure 6 (D). After GOLDGUT-HNU082 intervention, the positive area ratios of BDNF and PSD-95 in Lp082 significantly increased, and the positive area ratio of GFAP significantly decreased compared with the Mod group. The above results indicate that GOLDGUT-HNU082 can significantly alleviate nerve damage and thus relieve depression.

[0061] 2.5GOLDGUT-HNU082 improves intestinal flora disorders in mice with depression

[0062] Metagenomic sequencing was performed on the intestinal contents of different groups of mice. Figure 7 As shown, there was no significant difference in the Shannon index and Simpson index of the intestinal flora of each group of mice, indicating that there was no significant difference in α diversity ( Figure 7 Middle A, Figure 7 The results of principal coordinate analysis (PCoA) based on Bray-Curtis distance showed that there were significant differences in the β diversity of intestinal flora among the Con group, Mod group and Lp082 group ( Figure 7 C, p<0.05), the intestinal flora structure between the groups was significantly separated. The intestinal flora results of the Mod group were far away from the Con group, while the Lp082 group was closer to the Con group. The results of differential bacterial species analysis showed that compared with the Con group, the abundance of some harmful bacteria in the Mod group increased significantly, including Alistipes sp., Lachnospiraceae bacterium, Bacteroides sp.L10_4, Muribaclumintestinale and Duncanielafreteri; the abundance of beneficial bacteria such as Bifidobacterium pseudolongum decreased significantly ( Figure 7 (D) After GOLDGUT-HNU082 intervention, the abundance of the above microorganisms was significantly reversed. In addition, the abundance of another beneficial bacterium, Akkermansia muciniphila, also increased significantly in the Lp082 group.

[0063] 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 Lactobacillus plantarum GOLDGUT-HNU082, characterized in that: The strain is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO.33075 and the deposit date December 16, 2024. The taxonomic name is Lactiplantibacillus plantarum.

2. Use of Lactobacillus plantarum GOLDGUT-HNU082 as claimed in claim 1 in the preparation of a medicament for alleviating depression.

3. The use according to claim 2, characterized in that: The depression is caused by unpredictable mild stress.

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