Inactivated lactobacillus plantarum LP4 metagen preparation and application thereof

Abstract

The invention relates to the technical field of metagen preparations, in particular to an inactivated lactobacillus plantarum LP4 metagen preparation and application thereof. The preparation method comprises the following steps: centrifuging fermentation liquor of lactobacillus plantarum LP4 to obtain a thallus precipitate, resuspending the thallus precipitate to obtain a bacterial suspension with the cell concentration of 1 * 10 < 10 > CFU / ml, and carrying out thermal inactivation to obtain the metagen preparation. The invention provides the treatment effect of relieving depression for the first time, and proves that the inactivated lactobacillus plantarum LP4 has the same curative effect as fluoxetine, can replace fluoxetine, can effectively relieve symptoms of depression patients, improves the life quality of the depression patients, and has a good application prospect. And a safer and more effective treatment scheme is provided for depression patients.

Description

Technical Field

[0001] The invention relates to the technical field of postbiotic preparations, and in particular to an inactivated Lactobacillus plantarum LP4 postbiotic preparation and application thereof. Background Art

[0002] Depression is a common mental illness with a complex pathogenesis that may involve multiple system disorders such as neuroendocrine, immune, and metabolic systems. Recent studies have found that patients with depression often have intestinal dysfunction. The main treatment drug is fluoxetine, which has many side effects, including intestinal reactions (nausea, vomiting, diarrhea, indigestion, etc.), nervous system reactions (headache, dizziness, insomnia, fatigue, etc.), mental disorders, etc. Therefore, it is urgent to find and develop safe natural antidepressant drugs.

[0003] Probiotics are live microorganisms that are beneficial to human health when taken in sufficient quantities and comply with national regulations on food-use bacteria. Lactic acid bacteria, as a type of probiotic, has been certified as a "Generally Recognized as Safe" (GRAS) by the U.S. Food and Drug Administration and has the Qualified Presumption of Safty (QPS) by the European Food Safety Authority (EFSA). As a lactic acid bacterium that is widely distributed in nature, researchers have studied the depression-relieving function of Lactobacillus plantarum. For example, Chinese invention patent application CN 111110705 A found that Lactobacillus plantarum LP45 can relieve the abnormal expression of BDNF and GABA, improve brain memory, and relieve anxiety and depression-like behaviors. Chinese invention patent application CN117327631 A found that Lactobacillus plantarum strain 10A-2 can sustainably provide the endogenous necessary amount of cobalamin, phosphatidylserine, and acetylcholine during intestinal reproduction and growth. It has the effects of preventing, alleviating and treating anxiety and depression, and can effectively maintain a healthy nervous system. These prior arts confirm the prospect of plant lactobacillus in the preparation of antidepressant drugs, but the only drawback is that the antidepressant effects achieved in the above studies are inseparable from live plant lactobacillus bacteria.

[0004] Studies have shown that for people with impaired immune systems or sensitive people, taking live probiotics directly may cause abdominal discomfort, flatulence, diarrhea and other symptoms, and in severe cases may increase the risk of infection. Moreover, if live probiotics and antibiotics are taken at the same time, the live probiotics will be interfered with and inhibited by the antibiotics, affecting their efficacy.

[0005] Postbiotics are a general term for the metabolites of probiotics after probiotics have been processed, including inactivated bacteria and metabolites. Compared with probiotics, postbiotics are safer, more adaptable to sensitive populations, and not interfered with or inhibited by antibiotics. However, there is currently a lack of research on the use of Lactobacillus plantarum postbiotics to alleviate depression. Summary of the invention

[0006] In view of the current technical problem of lack of Lactobacillus plantarum postbiotics with the effect of alleviating depression, the present invention provides an inactivated Lactobacillus plantarum LP4 postbiotic preparation and application thereof.

[0007] In a first aspect, the present invention provides a postbiotic preparation of inactivated Lactobacillus plantarum LP4, which is prepared according to the following method: Lactobacillus plantarum ( Lactobacillus plantarum ) The fermentation broth of LP4 was centrifuged to obtain bacterial pellets, and the bacterial pellets were resuspended to obtain a cell concentration of 1×10 10 CFU / ml bacterial suspension, and heat-inactivation to obtain inactivated Lactobacillus plantarum LP4 postbiotic preparation; Among them, Lactobacillus plantarum LP4 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 18, 2019. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 17955, and the classification name is Lactobacillus plantarum Lactobacillus plantarum .

[0008] Furthermore, the thermal inactivation method is heating in a water bath at 80°C.

[0009] In a second aspect, the present invention provides a use of an inactivated Lactobacillus plantarum LP4 postbiotic preparation in the preparation of an antidepressant drug.

[0010] Furthermore, the antidepressant drug contains the inactivated Lactobacillus plantarum LP4 postbiotic preparation as the sole active ingredient, or contains a pharmaceutical composition containing the inactivated Lactobacillus plantarum LP4 postbiotic preparation and other commonly used antidepressants (such as fluoxetine) as active ingredients.

[0011] Furthermore, the antidepressant drug also includes pharmaceutically acceptable excipients.

[0012] Furthermore, the antidepressant is in oral dosage form, which may be a liquid dosage form or a solid dosage form, including powders, lyophilized powders, granules, tablets, capsules, pills or suspensions and the like.

[0013] The beneficial effects of the present invention are: The present invention proposes for the first time that the inactivated Lactobacillus plantarum postbiotic preparation has a therapeutic effect on alleviating depression, and confirms that the inactivated Lactobacillus plantarum LP4 postbiotic has the same degree of curative effect as fluoxetine, can replace fluoxetine, can effectively alleviate the symptoms of patients with depression, and improve their quality of life, providing a safer and more effective treatment plan for patients with depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 It is a SEM photo of live Lactobacillus plantarum LP4 and the inactivated Lactobacillus plantarum LP4 postbiotics of Example 1.

[0016] Figure 2 This is a statistical chart of the number of times each group of mice crossed the squares in the open field test.

[0017] Figure 3 This is a statistical chart of the number of times each group of mice stood upright in the open field test.

[0018] Figure 4 This is a statistical chart of the sucrose preference index results of each group of mice in the sucrose preference experiment.

[0019] Figure 5 This is a statistical chart of the immobility time of each group of mice in the forced swimming test.

[0020] Figure 6 This is a statistical chart of the immobility time of each group of mice in the tail suspension test.

[0021] Figure 7 It is the statistical chart of the results of serum 5-HT in each group of mice.

[0022] Figure 8 It is the statistical graph of the serum ROS results of each group of mice.

[0023] Fig. 9 It is the statistical chart of serum ACTH results of each group of mice.

[0024] Fig.10 It is the statistical chart of the results of serum CRH in each group of mice.

[0025] Fig.11 It is the statistical chart of the results of serum adrenaline in each group of mice.

[0026] Fig.12 It is the statistical chart of the results of serum norepinephrine in each group of mice.

[0027] Fig.13 This is the result graph of serum dopamine in each group of mice.

[0028] Fig.14 It is the result graph of serum T3 of each group of mice.

[0029] Fig.15 This is the PCA analysis of the intestinal flora of each group of mice.

[0030] Fig.16 This is the result graph of the top 10 bacterial genera in terms of intestinal flora abundance in each group of mice.

[0031] Fig.17 These are the HE staining images and GFAP immunofluorescence staining images of the hippocampus of mice in each group.

[0032] Fig.18 It is the statistical graph of the average optical density of GFAP immunofluorescence in the hippocampus of each group of mice.

[0033] Fig.19 It is the PCA analysis of cecal contents, serum and brain samples.

[0034] Fig. 20 It is the KEGG tertiary pathway statistics of cecal contents, serum and brain metabolome.

[0035] Fig.21 This is a comparison chart of the changes in tryptophan metabolites in the cecal contents of each group of mice.

[0036] Fig. 22 This is a comparison chart of the changes in tryptophan metabolites in the serum of each group of mice.

[0037] Fig.23 This is a comparison chart of the changes in tryptophan metabolites in the brains of each group of mice.

[0038] Fig.24 It is the tryptophan metabolic pathway of cecal contents, serum and brain metabolome.

[0039] Figure 2~Figure 19 and Figure 21~Figure 24 In the figure, LP4 represents the live Lactobacillus plantarum LP4 group, and inactivated LP4 represents the inactivated Lactobacillus plantarum LP4 group. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings 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 should fall within the scope of protection of the present invention.

[0041] The Lactobacillus plantarum LP4 used in the present invention has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on June 18, 2019, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No. 17955, and the classification name being Lactobacillus plantarum Lactobacillus plantarum ; And, the Lactobacillus plantarum LP4 has been disclosed in Chinese invention patent CN 117137042 B.

[0042] Example 1 The activated Lactobacillus plantarum LP4 was inoculated into MRS liquid culture medium, cultured at 37°C for 24 hours, and then centrifuged at 8000 r / min at 4°C for 3 minutes to obtain bacterial precipitate; The bacterial precipitate was washed twice with distilled water and resuspended in physiological saline to adjust the cell concentration to 1×10 10 CFU / ml, and bacterial suspension was obtained; The bacterial suspension was heat-inactivated in an 80°C water bath for 5 minutes, and the MRS plate live bacteria counting method was used to verify that no colonies grew, confirming that Lactobacillus plantarum LP4 had been inactivated, thereby obtaining the inactivated Lactobacillus plantarum LP4 postbiotic preparation.

[0043] Example 2 2.5% glutaraldehyde was used to fix the live Lactobacillus plantarum LP4 and the inactivated Lactobacillus plantarum LP4 postbiotics treated in Example 1, and then 30%, 50%, 70%, and 100% ethanol gradient dehydration was performed in sequence, each gradient was 10 minutes, and after dehydration, the sample was critical point dried in a supercritical dryer for about 1 hour. After drying, the sample was fixed on the sample stage with conductive tape, gold was sprayed, and electron microscopy was performed.

[0044] The SEM photos of the live Lactobacillus plantarum LP4 and the inactivated Lactobacillus plantarum LP4 postbiotics of Example 1 are shown in FIG. Figure 1 As shown in A and B. It can be seen that the inactivated Lactobacillus plantarum LP4 postbiotics of Example 1 have a complete cell wall structure like the live Lactobacillus plantarum LP4 bacteria, except that the inactivated Lactobacillus plantarum LP4 postbiotics of Example 1 have depressions on the bacterial surface.

[0045] Example 3 1. Experimental animals and groups Sixty 7-week-old male C57BL / 6J mice were randomly divided into five groups after 1 week of adaptation: normal group, model group, inactivated Lactobacillus plantarum LP4 group, live Lactobacillus plantarum LP4 group and fluoxetine treatment group, with 12 mice in each group and 3 mice in each cage. The feeding conditions were the same: 12h dark / light cycle, room temperature 23℃±1℃, humidity 50%±5%, free drinking water and food. The normal group was placed in a separate room.

[0046] 2. Feeding method Drug treatment was carried out from the first day of the stress test until the mice were killed. The normal group and the model group were gavaged with 200 μL of normal saline / day / mouse, the inactivated Lactobacillus plantarum LP4 group was gavaged with 200 μL of the inactivated Lactobacillus plantarum LP4 postbiotic preparation prepared in Example 1 / day / mouse, the live Lactobacillus plantarum LP4 group was gavaged with 200 μL of the bacterial suspension prepared in Example 1 / day / mouse, and the fluoxetine group was gavaged with 20 mg fluoxetine / day / kg mouse. The gavage time was fixed every day.

[0047] 3. Chronic Unpredictable Mild Stress (CUMS) Every day, 4 hours after intragastric administration, CUMS treatment was performed to prevent mice from vomiting. The normal group was not treated with CUMS, while the other groups were treated with CUMS.

[0048] CUMS-treated mice were randomly exposed to one of the stressors every day, and the same stressor was not applied for two consecutive days. The stressor program lasted for 5 weeks. The stressors included: ① no food and water for 24 hours; ② cage tilted at 30° for 24 hours; ③ light all night; ④ tail clamped for 1 minute (1 cm from the tip of the tail); ⑤ crowded group living for 24 hours; ⑥ wet cage for 24 hours: 200 ml of water added to 100 g of sawdust bedding; ⑦ physical restraint for 2 hours.

[0049] 4. Behavioral testing The open field test was performed on the 36th day, the sucrose preference test was performed on the 38th day, the forced swimming test was performed on the 40th day, and the tail suspension test was performed on the 42nd day.

[0050] (1) Open field test (OFT) To evaluate the possible effects of drug treatment on spontaneous locomotor activity, the open field test was performed on each group of mice. The test was performed under bright and quiet ambient room light. The open field apparatus consisted of a four-sided black wooden box (96×96×50 cm), with the floor divided into 16 equal squares by white lines, and the central 4 squares were defined as the central area. Mice were placed individually in the central area and allowed to freely explore the unfamiliar arena for 5 min. During this period, the total number of squares crossed by the mice and the number of rearing times (hind legs upright) were manually recorded. The open field apparatus was cleaned and dried after each mouse entered the room.

[0051] The results are as follows Figure 2 and Figure 3 As shown, compared with the normal group, the model group had significantly lower times of crossing squares and standing upright in the open field, indicating that the mice in the model group had stronger symptoms of depression. After intervention with inactivated Lactobacillus plantarum LP4 postbiotics, the times of crossing squares and standing upright were significantly increased, and there were no significant differences between the inactivated Lactobacillus plantarum LP4 group, the live Lactobacillus plantarum LP4 group, and the fluoxetine group.

[0052] (2) Sucrose preference experiment 72 hours before the test, mice were trained to adapt to 1% sucrose solution: 2 bottles of 1% sucrose solution were placed in each cage, and 1 bottle of 1% sucrose was replaced with water after 24 hours for 24 hours. After the adaptation, the mice were fasted but not watered for 24 hours, and then the sucrose preference experiment was conducted. Each cage of mice was free to eat two bottles of 100 ml of 1% sucrose solution and 100 ml of tap water. After 12 hours, the sugar water consumption and water consumption were recorded, and the sugar water preference index (%) was calculated = sugar water consumption / (sugar water consumption + water consumption).

[0053] The results are as follows Figure 4 As shown, compared with the normal group, the sucrose preference index of the model group was significantly reduced, indicating that the mice in the model group had stronger depressive symptoms. After the intervention of inactivated Lactobacillus plantarum LP4 postbiotics, the sucrose preference index was significantly increased, and there was no significant difference between the inactivated Lactobacillus plantarum LP4 group, the live Lactobacillus plantarum LP4 group and the fluoxetine group.

[0054] (3) Forced swim test (FST) The mice were placed individually in a transparent glass cylinder 25 cm high and 10 cm in diameter, which contained 10 cm of fresh water, and the forced swimming test was performed for 6 minutes at 23°C ± 2°C. After the first two minutes of intense struggle, the immobility time in the next 4 minutes was recorded, and the mice were considered immobile when they floated in the water without struggling and only made necessary movements.

[0055] The results are as follows Figure 5 As shown, compared with the normal group, the immobility time of the model group in the forced swimming test was significantly increased, indicating that the mice in the model group had stronger depressive symptoms. After the intervention of inactivated Lactobacillus plantarum LP4 postbiotics, the immobility time was significantly reduced, and there was no significant difference between the inactivated Lactobacillus plantarum LP4 group and the fluoxetine group.

[0056] (4) Tail suspension test The acoustically and visually isolated mice were suspended 50 cm above the ground by placing tape approximately 1 cm from the tip of the mouse's tail. The time the mouse remained motionless was recorded during the 6-min test period.

[0057] The results are as follows Figure 6As shown, compared with the normal group, the immobility time of the model group in the tail suspension test was significantly increased, indicating that the mice in the model group had stronger depressive symptoms. After intervention with inactivated Lactobacillus plantarum LP4 postbiotics, live Lactobacillus plantarum LP4 and fluoxetine, the immobility time of the mice was reduced.

[0058] 5. Serum index determination After the behavioral test, blood was collected from the retro-orbital venous plexus of mice to measure serum biochemical indices.

[0059] (1) 5-HT The mouse serum 5-HT was measured using a kit. The results are shown in Figure 7 As shown in Table 1 below, compared with the normal group, the 5-HT concentration in the serum of the model group increased significantly. After intervention with inactivated Lactobacillus plantarum LP4 postbiotics, the 5-HT concentration in the serum of mice decreased significantly, which was better than that of the live Lactobacillus plantarum LP4 group and the fluoxetine group.

[0060] Table 1 Statistical table of serum 5-HT in each group of mice

[0061] (2) ROS The kit was used to measure the ROS in mouse serum. Figure 8 As shown in Table 2 below, compared with the normal group, the ROS concentration in the serum of the model group increased significantly, indicating that the mice in the model group had stronger depressive symptoms. After intervention with live Lactobacillus plantarum LP4, inactivated Lactobacillus plantarum LP4 postbiotics and fluoxetine, the ROS concentration in the serum of the mice decreased, indicating that live Lactobacillus plantarum LP4 and inactivated Lactobacillus plantarum LP4 postbiotics had an inhibitory effect, and live Lactobacillus plantarum LP4 had the best inhibitory effect, followed by inactivated Lactobacillus plantarum LP4 postbiotics.

[0062] Table 2 Statistical table of serum ROS in each group of mice

[0063] (3) ACTH The mouse serum ACTH was measured using a kit. Fig. 9 As shown in Table 3 below, compared with the normal group, the ACTH concentration in the serum of the model group was significantly increased. After intervention with inactivated Lactobacillus plantarum LP4 postbiotics, the serum ACTH content of mice decreased and was more inclined to the normal group.

[0064] Table 3 Statistical table of serum ACTH in each group of mice

[0065] (4) CRH The mouse serum CRH was measured using a kit. Fig.10 As shown in Table 4 below, compared with the normal group, the CRH level in the serum of the model group was significantly reduced. After intervention with inactivated Lactobacillus plantarum LP4 postbiotics, the CRH level in the serum of mice was significantly increased, which was better than that of the live Lactobacillus plantarum LP4 group and the fluoxetine group.

[0066] Table 4 Statistical table of serum CRH in each group of mice

[0067] (5) Adrenaline The test kit was used to measure the serum adrenaline of mice. The results were as follows: Fig.11 As shown in Table 5 below, compared with the normal group, the adrenaline in the serum of the model group was significantly reduced. After intervention with inactivated Lactobacillus plantarum LP4 postbiotics and live Lactobacillus plantarum LP4, the adrenaline content in the serum of mice was significantly increased, which was better than that of the fluoxetine group.

[0068] Table 5 Statistical table of serum adrenaline in each group of mice

[0069] (6) Norepinephrine The kit was used to measure the level of norepinephrine in mouse serum. Fig.12 As shown in Table 6 below, compared with the normal group, the norepinephrine in the serum of the model group was significantly reduced. After intervention with inactivated Lactobacillus plantarum LP4 postbiotics and live Lactobacillus plantarum LP4, the norepinephrine content in the mouse serum was significantly increased, both better than the fluoxetine group, and the effect of inactivated Lactobacillus plantarum LP4 postbiotics was better than that of live Lactobacillus plantarum LP4.

[0070] Table 6 Statistical table of serum norepinephrine in each group of mice

[0071] (7) Dopamine The kit was used to measure the dopamine in mouse serum. Fig.13 As shown in Table 7 below, compared with the normal group, the dopamine in the serum of the model group was significantly reduced. After intervention with inactivated Lactobacillus plantarum LP4 postbiotics, the dopamine content in the serum of mice increased, which was better than that of the live Lactobacillus plantarum LP4 group and the fluoxetine group.

[0072] Table 7 Statistical table of serum dopamine in each group of mice

[0073] (8) T3 The mouse serum T3 was measured using the kit. The results are shown in Fig.14As shown in Table 8 below, compared with the normal group, the serum T3 of the model group was significantly increased. After intervention with inactivated Lactobacillus plantarum LP4 postbiotics, the serum T3 content of mice was significantly reduced, which was better than that of the live Lactobacillus plantarum LP4 group and the fluoxetine group.

[0074] Table 8 Statistical table of serum T3 in each group of mice

[0075] 6. Determination of cecal content indicators After the behavioral test was completed and blood was collected, the mice were anesthetized and killed, and the cecal contents of the mice were quickly separated, quickly frozen with liquid nitrogen, and stored at -80°C until detection.

[0076] (1) Different bacterial genera After DNA extraction using the kit, the extracted genomic DNA was detected by 1% agarose gel electrophoresis. According to the specified sequencing region, specific primers 338F and 806R with barcodes were synthesized. After amplification, Shanghai Meiji Biotechnology Co., Ltd. determined and analyzed bioinformatics. Fig.15 As shown, the PCA diagram shows that there are significant differences in the intestinal flora between the inactivated Lactobacillus plantarum LP4 group and the model group, and it is more inclined to the normal group, indicating that the inactivated Lactobacillus plantarum LP4 postbiotics have a significant regulatory effect on the intestinal flora, and can alleviate depression by changing the composition of the intestinal flora of chronic unpredictable mild stress mice.

[0077] The top 10 bacterial genera at the genus level are as follows Fig.16 As shown in Figure 2, it can be seen that the inactivated Lactobacillus plantarum LP4 postbiotic can increase the number of bacteria producing short-chain fatty acids. Muribaculaceae and Allobaculum , reducing harmful bacteria Desulfovibrio and norank_f_ Oscillospiraceae .

[0078] 7. Determination of hippocampal indexes (1) HE staining The hippocampus tissue of the mice was taken to prepare paraffin-embedded sections, and then HE staining was performed. The hippocampus tissue after HE staining was examined under a microscope.

[0079] like Fig.17As shown in the figure, the brain tissue structure of mice in the normal group was normal, the neurons in the hippocampus were neatly arranged and tightly arranged in the visual field, the Nissl body structure was clear and abundant in number; the neurons were less degenerated, the cell bodies were pyknotic and darkly stained; and the glial cells were few. The brain tissue structure of mice in the model group was severely abnormal, a large number of neurons were degenerated, the cell bodies were pyknotic and darkly stained, the Nissl body structure disappeared, and the glial cells increased compared with the normal group (red arrows). After intervention with live Lactobacillus plantarum LP4, inactivated Lactobacillus plantarum LP4 postbiotics and fluoxetine, the brain tissue structure returned to normal, the neurons in the hippocampus were neatly arranged and tightly arranged in the visual field, the Nissl body structure was clear and abundant in number; the neurons were less degenerated, the cell bodies were pyknotic and darkly stained; and the glial cells were few.

[0080] (2) Immunofluorescence single label staining The hippocampus tissue of mice was taken to prepare paraffin-embedded sections, and GFAP was immunofluorescently stained (TSA method). Three 200-fold fields of view were randomly selected from each section in each group for photography, and each photo was analyzed using Image-Pro Plus 6.0 software to obtain the cumulative optical density value (IOD) and the pixel area (AREA) of each photo, and the mean optical density value IOD / AREA (Mean Density) was calculated.

[0081] like Fig.17 As shown in the figure, after GFAP immunofluorescence staining, the red fluorescence density of the hippocampus in the model group increased compared with the normal group, indicating that the number of astrocytes in the model group increased and an inflammatory response occurred in the brain. Compared with the model group, after intervention with live Lactobacillus plantarum LP4, inactivated Lactobacillus plantarum LP4 postbiotics and fluoxetine, the red fluorescence density of the hippocampus decreased, indicating that the number of astrocytes decreased and the inflammatory response in the brain was alleviated. The statistical results of the average optical density values ​​are shown in the figure. Fig.18 As shown, the average optical density value of immunofluorescence in each group represents the density of astrocytes. Compared with the normal group, the optical density of the model group increased, indicating an increase in astrocytes and an inflammatory response in the brain; compared with the model group, the optical density of the inactivated Lactobacillus plantarum LP4 group, the live Lactobacillus plantarum LP4 group and the fluoxetine group decreased, indicating fewer astrocytes and a reduced inflammatory response in the brain.

[0082] 8. Comprehensive analysis of cecal contents, serum, and brain (1) Sample comparison analysis Non-targeted metabolomics analysis was performed on the cecal contents, serum and brain tissue of mice in the normal group, model group, inactivated Lactobacillus plantarum LP4 group and live Lactobacillus plantarum LP4 group (sample analysis / data acquisition method was LC-MS). Principal component analysis (PCA) was performed on the three metabolomes. A set of variables that may be correlated was converted into a set of linearly uncorrelated variables through orthogonal transformation. The converted set of variables is called principal components. By performing principal component analysis on the samples, we can preliminarily understand the overall metabolic differences between the samples in each group and the degree of variability between the samples within the group.

[0083] like Fig.19 As shown, in the cecal contents and serum, the metabolome of the model group was separated compared with the normal group, and after the intervention of live Lactobacillus plantarum LP4 and inactivated Lactobacillus plantarum LP4 postbiotics, the metabolome was further separated, indicating that live Lactobacillus plantarum LP4 and inactivated Lactobacillus plantarum LP4 postbiotics can affect the metabolism of intestinal flora and mice.

[0084] (2) KEGG tertiary pathway statistics KEGG pathway enrichment was performed on differential metabolism to obtain Fig. 20 KEGG pathway projection statistics are shown. Among them, the tryptophan metabolism pathway is enriched in the cecal contents, serum and brain metabolome. Tryptophan metabolism is an important way for the intestinal flora to regulate depression through the brain-gut axis. This shows that live Lactobacillus plantarum LP4 and inactivated Lactobacillus plantarum LP4 postbiotics may relieve depression in mice through the tryptophan metabolism pathway.

[0085] (3) Analysis of tryptophan metabolic pathway The substances related to the tryptophan metabolic pathway in each metabolome were extracted one by one, and the normal group, the inactivated Lactobacillus plantarum LP4 group, the live Lactobacillus plantarum LP4 group and the model group were subjected to univariate analysis to find out the substances with significant differences (such as Figures 21 to 23 ), and draw metabolic pathway diagrams according to KEGG pathways (such as Fig.24 As shown in the figure, compared with the change trend of "model group vs normal group", "LP4 vs model group" and "inactivated LP4 vs model group" of multiple metabolites have opposite trends, indicating that live Lactobacillus plantarum LP4 and inactivated Lactobacillus plantarum LP4 postbiotics can alleviate the change trend of the model group and relieve depression in mice through the tryptophan metabolic pathway.

[0086] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A postbiotic preparation of inactivated Lactobacillus plantarum LP4, characterized in that: Prepared as follows: Lactobacillus plantarum ( Lactobacillus plantarum ) The fermentation broth of LP4 was centrifuged to obtain bacterial pellets, and the bacterial pellets were resuspended to obtain a cell concentration of 1×10 10 CFU / ml bacterial suspension, and heat-inactivation to obtain inactivated Lactobacillus plantarum LP4 postbiotic preparation; Among them, Lactobacillus plantarum LP4 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 18, 2019. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.17955, and the classification name is Lactobacillus plantarum Lactobacillus plantarum .

2. The inactivated Lactobacillus plantarum LP4 postbiotic preparation according to claim 1, characterized in that The heat inactivation method is heating in a water bath at 80°C.

3. Use of the inactivated Lactobacillus plantarum LP4 postbiotic preparation as claimed in claim 1 or 2 in the preparation of antidepressant drugs.

4. The use according to claim 3, characterized in that The antidepressant drug contains a postbiotic preparation of killed Lactobacillus plantarum LP4 as the only active ingredient.

5. The use according to claim 3, characterized in that The antidepressant drug has a pharmaceutical composition comprising an inactivated Lactobacillus plantarum LP4 postbiotic preparation and fluoxetine as active ingredients.

6. The use according to claim 3, characterized in that The antidepressant drug also includes pharmaceutically acceptable excipients.

7. The use according to claim 3, characterized in that Antidepressants are taken orally.

Images