Use of lactobacillus plantarum JYLP-326 in preparation of product for improving mood

Abstract

The present invention relates to the technical field of microorganisms, in particular to the use of Lactobacillus plantarum JYLP-326 in the preparation of a product for improving mood. The Lactobacillus plantarum JYLP-326 is deposited in China General Microbiological Culture Collection Center on 27 June 2019, with the deposit address of Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number of CGMCC No.18038. The product for improving the mood contains the Lactobacillus plantarum JYLP-326. Experimental results show that the Lactobacillus plantarum JYLP-326 can significantly improve the low mood and depression tendency of chronic unpredictable mild stress (CUMS) model mice, adjust the levels of emotion-related nerve cells, proteins and inflammatory factor in hippocampus regions of the CUMS mice, and have a potential neuroprotective effect on the hippocampus regions of the mice.

Description

Application of Lactobacillus plantarum JYLP-326 in preparing products for improving mood Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to the use of Lactobacillus plantarum JYLP-326 in the preparation of a product for improving mood. Background Art

[0002] Depression is a common psychological problem, significantly impacting daily life, work, and study. If depression persists for a long time, it can have a range of consequences. Physiologically, prolonged depression can weaken the immune system and increase the risk of infection. It can also increase adrenaline and cortisol levels in the blood, increasing the risk of heart disease and stroke. It can also lead to digestive problems such as excessive stomach acid, stomach cramps, and diarrhea. In terms of mental health, prolonged depression can be a precursor to depression and can even contribute to anxiety disorders.

[0003] As an emotional symptom, low mood should be assessed for severity first. If the low mood is already quite serious and is accompanied by symptoms of slow thinking and decreased willpower, it is necessary to see a doctor to confirm whether it has reached the level of depression. For patients with mild depression, psychological counseling and treatment can gradually improve the patient's irrational cognitive patterns and rebuild the cognitive system, which can relieve anxiety and improve mood to a certain extent, but psychotherapy is slow to take effect and the efficacy varies from person to person. For patients with severe depression who have obvious anxiety and mania, antidepressants are needed. Antidepressants include selective serotonin reuptake inhibitors, selective norepinephrine reuptake inhibitors, tricyclic antidepressants, monoamine oxidase inhibitors, and drugs targeting multiple neurotransmitters, but these drugs are prone to side effects, such as drowsiness, dry mouth, blurred vision, constipation, rapid heartbeat, difficulty urinating, and orthostatic hypotension.

[0004] At the same time, a growing number of scientists are exploring the use of probiotics to improve mood and treat depression. Chinese patent CN110791451A discloses a strain of Lactobacillus plantarum JYLP-326 and its applications and products for improving sleep. The patent discloses that Lactobacillus plantarum JYLP-326 can increase GABA levels in the hypothalamus, potentially helping to alleviate insomnia. Further research is needed to determine whether probiotics can effectively improve mood and treat depression, and their mechanisms of action.

[0005] Summary of the Invention

[0006] To address the technical issue of unclear effects and mechanisms of action of probiotics in improving mood and treating depression, this application provides a use of Lactobacillus plantarum JYLP-326 in the preparation of a product that improves mood. The specific technical solution is as follows:

[0007] Lactobacillus plantarum JYLP-326 was deposited in the General Microbiology Center of China Culture Collection Administration on June 27, 2019. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 18038.

[0008] Furthermore, the types of products include medicines.

[0009] Furthermore, the mood-improving product is a product that regulates mood-related nerve cells, proteins and inflammatory factors in the hippocampus of CUMS mice.

[0010] Furthermore, the mood-improving product is a product that regulates the TLR4-MyD88-NF-κB signaling pathway and inflammatory factors in the colon of CUMS mice.

[0011] Furthermore, the mood-enhancing product is a product that regulates colonic inflammation in CUMS mice.

[0012] Furthermore, the mood-enhancing product is a product that inhibits the RAGE-TLR4 signaling pathway.

[0013] Furthermore, the mood-enhancing product is a product that regulates the intestinal microbial flora of CUMS mice.

[0014] The present application also provides a product for improving mood, wherein the ingredients of the product include Lactobacillus plantarum JYLP-326, and the preservation number of Lactobacillus plantarum JYLP-326 is CGMCC No.18038.

[0015] Furthermore, the content of Lactobacillus plantarum JYLP-326 in the product is 1.0×10 10 ~2.5×10 10 cfu / g.

[0016] The present application also provides a method for preparing the above-mentioned product, comprising the following steps:

[0017] (1) Lactobacillus plantarum JYLP-326 was activated on MRS solid medium, and after anaerobic culture for 24 hours, a single colony was picked and inoculated into MRS liquid medium, and then cultured at 37°C for 24 hours to obtain a bacterial solution; the concentration of the bacterial solution was adjusted, and bacterial powder was obtained by low-temperature centrifugation, washing, coating, and vacuum freeze-drying;

[0018] (2) Compounding the Lactobacillus plantarum JYLP-326 prepared in step (1) with isomaltooligosaccharide to obtain a product for improving mood.

[0019] Furthermore, the components of the MRS liquid culture medium include: 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of dipotassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, and 1000 mL of distilled water; 15 g / L of agar is added to the MRS liquid culture medium to obtain an MRS solid culture medium.

[0020] The beneficial effects of this application are:

[0021] 1. The application provides an application for the use of Lactobacillus plantarum JYLP-326 in the preparation of products for improving mood. Experimental results show that Lactobacillus plantarum JYLP-326 has high activity and strong antioxidant capacity, can effectively inhibit the growth of various pathogens, significantly improve the low mood and depressive tendencies of chronic unpredictable mild stimulation (CUMS) model mice, regulate the levels of emotion-related neurons, proteins and inflammatory factors in the hippocampus of CUMS mice, and have a potential neuroprotective effect on the hippocampus of mice.

[0022] 2. The application of Lactobacillus plantarum JYLP-326 in the preparation of mood-enhancing products. Experimental results show that Lactobacillus plantarum JYLP-326 effectively enhances the integrity of the intestinal barrier and reduces the expression of TLR4, MyD88, and p-p65, indicating its potential in regulating intestinal inflammation. Experimental verification shows that treatment with Lactobacillus plantarum JYLP-326 and the RAGE inhibitor FPS-ZM1 significantly reduced the expression of HMGB1, RAGE, and TLR4 proteins in the hippocampus of mice, suggesting that Lactobacillus plantarum JYLP-326 may improve low mood and instability by inhibiting the RAGE-TLR4 signaling pathway.

[0023] 3. Intestinal flora is closely related to mood changes. Treatment with Lactobacillus plantarum JYLP-326 can effectively increase the abundance of Lactobacillus and reduce the presence of Sutterella and Eubacterium, reversing to a certain extent the changes in intestinal flora associated with low mood and instability.

[0024] Preservation Instructions

[0025] The present application's Lactobacillus plantarum JYLP-326 was deposited in the General Microbiology Center of the China Culture Collection Administration on June 27, 2019. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 18038. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0027] FIG1 is a graph showing the growth curve of Lactobacillus plantarum JYLP-326 in Example 1-2, the study of probiotic properties, and the effect on the mood of CUMS mice.

[0028] FIG2 is a graph showing the effects of Lactobacillus plantarum JYLP-326 on emotion-related neurons, proteins, and inflammatory factors in the hippocampus of CUMS mice in Example 3.

[0029] FIG3 is a graph showing the effects of Lactobacillus plantarum JYLP-326 on the TLR4-MyD88-NF-κB signaling pathway and inflammation in the colon of CUMS mice in Example 4.

[0030] FIG4 is a graph showing the effects of Lactobacillus plantarum JYLP-326 and RAGE-specific inhibitor FPS-ZM1 on the emotions of CUMS mice and the emotion-related neurons in the hippocampus in Example 5.

[0031] FIG5 is a graph showing the effects of Lactobacillus plantarum JYLP-326 and RAGE-specific inhibitor FPS-ZM1 on the intestinal microbial flora of CUMS mice in Example 6. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of this application.

[0033] The Lactobacillus plantarum JYLP-326 used in the following examples was collected by the applicant and deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection on June 27, 2019, with the deposit number CGMCC No. 18038. Other relevant information is disclosed in patent CN110791451A.

[0034] Example 1 Study on the Growth Curve and Probiotic Characteristics of Lactobacillus plantarum JYLP-326

[0035] 1. Growth curve of Lactobacillus plantarum JYLP-326

[0036] Lactobacillus plantarum JYLP-326 was inoculated into MRS liquid medium and cultured in a shaker at 37°C. OD values ​​were measured every two hours. The results are shown in Figure 1A. The growth curve shows that Lactobacillus plantarum JYLP-326 entered the logarithmic phase at 2 hours and reached the stationary phase at 12 hours, indicating that the bacterium has good activity.

[0037] 2. Study on the probiotic properties of Lactobacillus plantarum JYLP-326

[0038] Lactobacillus plantarum JYLP-326 was inoculated into MRS liquid culture medium and cultured in a 37° C. constant temperature incubator. The culture was terminated when the OD value was equal to 0.6, and the strain culture solution was retained for later use.

[0039] (1) Acid and bile salt resistance test

[0040] ① Acid resistance test: Take 100 μL of strain culture solution and dilute it with PBS buffer solution for 10 1 , 10 3 , 10 5 times, centrifuge at 6000 g for 3 min, and discard the supernatant; add PBS buffer with pH = 3, 4, 5, and 7 respectively, let it stand for 4 hours, mix well, take 10 μL to spread on the plate, and culture in a constant temperature incubator at 37°C for 12 to 48 hours, count the viable bacteria, and make experimental records. The results are shown in Figure 1B.

[0041] ② Bile salt tolerance test: Take 100 μL of strain culture solution and dilute it with PBS buffer solution. 1 , 10 3 , 10 5 times, centrifuge at 6000 g for 3 min, and discard the supernatant; add culture medium containing 0, 0.1%, 0.2%, and 0.3% ox bile salts, respectively, and culture in a constant temperature incubator at 37°C for 12 to 48 hours. Mix well, take 10 μL and spread on a plate, and culture in a constant temperature incubator at 37°C for 12 to 48 hours. Count the viable bacteria and make experimental records. The results are shown in Figure 1C.

[0042] The results of acid and bile salt resistance experiments showed that Lactobacillus plantarum JYLP-326 had strong tolerance to pH 3-7 environment and 0-0.3% bile salt concentration environment.

[0043] (2) Determination of free radical scavenging ability

[0044] ①DPPH free radical scavenging ability determination

[0045] Take 2 mL of the supernatant from the strain culture and add 2 mL of DPPH methanol solution (concentration 0.2 mmol / L). Incubate in the dark at room temperature for 30 minutes. Measure the absorbance of the supernatant at 517 nm. Use 2 mL of DPPH methanol solution added to 2 mL of deionized water as a control. Calculate the DPPH radical scavenging rate according to the following formula:

[0046] ② Determination of hydroxyl radical scavenging ability

[0047] Take a mixed solution of 1 mL of 2 mmol / L ferrous sulfate solution, 1 mL of 6 mmol / L hydrogen peroxide, and 1 mL of 6 mmol / L salicylic acid, add 1 mL of the strain culture supernatant, let it stand for 30 minutes, and measure the absorbance at 510 nm. Add 1 mL of distilled water to the mixed solution as a control, and calculate the scavenging rate of hydroxyl free radicals according to the following formula:

[0048] ③Determination of superoxide free radical scavenging ability

[0049] Add 2 mL of 150 mmol / L Tris-HCl solution (pH = 8.0) to 0.5 mL of the strain culture solution, and then add 1 mL of 1.2 mmol / L pyrogallol solution. React at room temperature for 30 minutes, measure the absorbance at 325 nm, and calculate the superoxide radical scavenging rate according to the following formula:

[0050] Among them, A00 represents the absorbance without strain culture fluid and pyrogallol; A01 represents the absorbance without strain culture fluid but with pyrogallol; A10 represents the absorbance with strain culture fluid but without pyrogallol; A11 represents the absorbance with strain culture fluid and pyrogallol.

[0051] The results of free radical scavenging ability are shown in FIG1D . Lactobacillus plantarum JYLP-326 exhibits a strong antioxidant capacity, with a superoxide radical scavenging capacity of 54.3%.

[0052] (3) Antibacterial experiment

[0053] The Oxford cup method was used to test the inhibitory effect against common pathogens. Using a long-handled cotton swab, absorb an appropriate amount of pathogen culture, press to remove excess water, and gently spread the pathogen onto the test plate using a 360° rotation. Place the Oxford cup in the appropriate position, then pipette 200 μL of the strain culture into the cup. Place the plate in a 4°C refrigerator to diffuse for 24 hours. After incubation at 30°C for 48 hours, measure and record the diameter of the inhibition zone.

[0054] The experimental results are shown in Figure 1E. Lactobacillus plantarum JYLP-326 significantly inhibited the growth of pathogenic bacteria Salmonella typhimurium ATCC 13311, Salmonella enteritidis ATCC 13076, Shigella flexneri ATCC 12022, Listeria monocytogenes ATCC 19111, and Enterococcus faecalis ATCC 29212, with inhibition zone diameters ranging from 9 to 14.67 mm.

[0055] Example 2 Effect of Lactobacillus plantarum JYLP-326 on the emotional state of CUMS mice

[0056] The chronic unpredictable mild stimulation (CUMS) model is a widely used animal model for studying depression and other psychiatric disorders, simulating the condition of patients with depression. By establishing a CUMS model to mimic the condition of patients with depression, CUMS mice were then fed with Lactobacillus plantarum JYLP-326 to explore the pathophysiological mechanisms of Lactobacillus plantarum JYLP-326 in treating depression. The overall experimental scheme is shown in Figure 1F.

[0057] 1. Establishment of the CUMS Mouse Model

[0058] Forty-eight eight-week-old male C57BL / 6 mice were used for the experiment, and 36 of them were randomly selected to establish the CUMS mouse model. The CUMS model was established as follows: eight stimuli were randomly assigned over 28 days: empty cage for 17 hours, tilted cage at 45° for 17 hours, wet cage for 17 hours, food deprivation for 14 hours, night light exposure for 12 hours, hot water swimming for 5 minutes, water deprivation for 16 hours, and tail clamping for 5 minutes. The stimuli were presented discontinuously to prevent the mice from anticipating them. The remaining 12 mice served as the control group (C) and were housed normally without any stimuli.

[0059] 2. Experimental Treatment

[0060] Starting from week 5, 36 CUMS model mice were randomly divided into three groups: model group (M), low-dose treatment group (ML), and high-dose treatment group (MH), with 12 mice in each group. Each of the four groups of mice was housed in two cages. The control group and model group mice were fed a normal diet without any other treatment. The low-dose treatment group mice were gavage-treated with 1×10 7 The mice in the high-dose treatment group were gavaged with 1×10 CFU of Lactobacillus plantarum JYLP-326 every day. 9CFU of Lactobacillus plantarum JYLP-326 for one month. During the experiment, feces of all mice were collected.

[0061] 3. Open Field Test (OFT)

[0062] The experiment was conducted in a quiet environment. The animal was placed in the center of the bottom of the box while video and timing were performed. After observing for 3–5 minutes, video recording was stopped. The inside and bottom of the box were cleaned to prevent residual information from the previous animal (such as urine, feces, and odor) from affecting the next test results. The animal was then replaced and the experiment continued.

[0063] The test results are shown in Figure 1G. Compared with the normal mice in the control group, the total movement distance of the CUMS mice in the model group was significantly shorter (P < 0.05). However, the total movement distance of the mice in the low-dose treatment group and the high-dose treatment group showed moderate improvement.

[0064] 4. Sugar Preference Test (SPT)

[0065] First, acclimation training was conducted. For the first 24 hours, two bottles of 1% sucrose solution were placed in each cage. After 24 hours, one of the bottles was replaced with pure water. Following acclimation, testing was performed: a bottle of 1% sucrose solution and a bottle of pure water were placed in each cage, with free access to water. The mice were fasted. After 12 hours, the remaining fluid volume was measured, and the sucrose solution, pure water, and total fluid consumption were recorded. Sugar preference was calculated using the following formula:

[0066] The test results are shown in Figure 1H. As assessed by the sugar preference test, CUMS mice in the model group exhibited aversion and decreased sucrose preference. While the sucrose preference of the control group mice was 77.27±1.94%, the sucrose preference level of the model group mice decreased to 31.80±3.09%. Administration of Lactobacillus plantarum JYLP-326 significantly reversed this sucrose preference (P<0.05).

[0067] 5. Tail Suspension Test (TST)

[0068] The mouse's tail was fixed to the top of the experimental box using medical tape, and the mouse's behavior was recorded with a video camera for 6 minutes. The behavior was analyzed using Tail Suspension Scan™ (Clever Sys Inc, VA, USA) analysis software, and the time during which the mouse's limbs and trunk were completely immobile within the last 4 minutes was counted.

[0069] The test results are shown in Figure 1I. Compared with the control group, the mice in the model group showed a longer immobility time in the tail suspension test (P<0.05), while the immobility time of the mice in the high-dose treatment group was significantly shorter than that of the mice in the model group.

[0070] Example 3 Effects of Lactobacillus plantarum JYLP-326 on emotion-related neurons, proteins, and inflammatory factors in the hippocampus of CUMS mice

[0071] Mice were sacrificed and samples were collected. Immunohistochemistry was used to detect cells associated with neuroinflammation. Western blot analysis was performed to measure the expression of 5-HT1A receptors and the rate-limiting enzymes for 5-HT synthesis (tryptophan hydroxylase 2 and phosphorylated tryptophan hydroxylase 2, TPH2 / p-TPH2) in the brain. RT-PCR was used to detect the expression of inflammatory cytokines IL-6, IL-1β, and TNF-α in the hippocampus. The results are shown in Figure 2.

[0072] First, immunohistochemistry was used to observe the expression of GFAP (astrocyte marker) and Iba-1 (microglia marker) in the mouse hippocampus (Figure 2A). Compared with the control group, the percentage of GFAP-positive neurons and Iba1-positive neurons in the CUMS model group were significantly increased (P < 0.05, Figure 2B, C). Compared with the model group, the results of the ML and MH groups showed that treatment with Lactobacillus plantarum JYLP-326 significantly reduced the expression of GFAP and Iba-1 in the mouse hippocampus (P < 0.05).

[0073] Then, 5-HT levels in the hippocampus and intestine of mice were measured to analyze the connection between emotional instability and its internal molecular mechanisms (Figure 2D, E). The 5-HT level in the hippocampus of CUMS mice in the model group (269 μg / g) was much lower than that in the normal mice in the control group (396.30 μg / g, P < 0.01, Figure 2D). Compared with the M group, the 5-HT levels in the ML and MH groups treated with Lactobacillus plantarum JYLP-326 were significantly increased (337.37 μg / g, 364.33 μg / g, P < 0.01, Figure 2D). At the same time, the 5-HT level in the colon of mice in the model group was much lower than that in the control group (P < 0.01, Figure 2E), while Lactobacillus plantarum JYLP-326 treatment induced a significant increase in 5-HT levels in the colon of the ML and MH groups (P < 0.01, Figure 2E), which was consistent with the trend in the hippocampus.

[0074] In addition, the effect of Lactobacillus plantarum JYLP-326 on the expression levels of 5-HT-related proteins was detected by immunoblotting (Figure 2F). The results showed that the expression of p-TPH2 / TPH2 in the hippocampus of CUMS mice in the model group was significantly lower than that in the control group (P<0.05, Figure 2G), and the expression of 5-HT1 receptors was also significantly reduced (P<0.01, Figure 2H). However, the high-dose treatment group treated with Lactobacillus plantarum JYLP-326 significantly restored these changes to normal levels (P<0.01). Overexpression of inflammatory cytokines in brain tissue is also considered to be one of the hallmarks of emotional instability. Therefore, the mRNA expression levels of IL-6, IL-1β, and TNF-α were detected by reverse transcription polymerase chain reaction (RT-PCR) (Figures 2I-K). Compared with the control group, CUMS mice in the model group showed overexpression of inflammatory factors (P<0.01). However, after treatment with Lactobacillus plantarum JYLP-326, both the low-dose treatment group (P<0.05) and the high-dose treatment group (P<0.01) showed significant improvement. These results suggest that Lactobacillus plantarum JYLP-326 has the potential to improve depression and instability and has a neuroprotective effect on the hippocampus of mice.

[0075] Example 4 Effects of Lactobacillus plantarum JYLP-326 on the Colonic TLR4-MyD88-NF-κB Signaling Pathway and Inflammation in CUMS Mice

[0076] In patients with mood instability, intestinal 5-HT deficiency leads to impaired intestinal motility, manifested as shortened ileal villi. Increased expression of proinflammatory cytokines promotes the development of mood through the brain-gut axis. Mice were sacrificed and samples were collected. Ileal villi were examined using HE staining, and RT-PCR was used to detect the expression of inflammatory factors such as IL-6, IL-1β, and TNF-α. TLR4, MyD88, p65, and p-p65 proinflammatory pathways were also detected. The experimental results are shown in Figure 3.

[0077] As shown in Figure 3A, in the colon of CUMS mice in the model group, mucosal epithelial cells fell off and the intestinal gland structure was partially destroyed, while in the intestinal tissues of mice in the control group, low-dose treatment group and high-dose treatment group, there was no mucosal epithelial cell shedding and the intestinal gland structure was intact. In addition, the levels of inflammatory factors TNF-α, IL-6 and IL-1β were studied by RT-PCR. The results showed that compared with the control group, the colon inflammatory factors in mice in the model group were significantly increased (P<0.01). Figures 3B-D show that in the colon of CUMS mice in the model group, the expression of TNF-α, IL-6 and IL-1β was significantly higher than that in the low-dose treatment group and the high-dose treatment group (P<0.01).

[0078] Western blot analysis was used to assess the expression of key proteins in the TLR4-MyD88-NF-κB inflammatory pathway (Figure 3E). Compared with the control group, the expression of TLR4, MyD88, and p-p65 in the model group was significantly increased (P < 0.05, Figures 3F-H). In addition, the expression of proteins related to intestinal permeability in the colon tissue of mice was assessed. Compared with the control group, the expression of ZO-1 and Occludin in the model group was reduced. However, treatment with Lactobacillus plantarum JYLP-326 significantly increased the expression of ZO-1 and Occludin in both the low-dose and high-dose treatment groups (Figures 3I-K). These Western blot results indicate that Lactobacillus plantarum JYLP-326 effectively enhances the integrity of the intestinal barrier and reduces the expression of TLR4, MyD88, and p-p65, indicating its potential in regulating intestinal inflammation.

[0079] Example 5 Effects of RAGE-specific inhibitor FPS-ZM1 on emotional behavior of CUMS mice

[0080] In order to verify the involvement of the RAGE-TLR4 pathway in improving depression and instability with Lactobacillus plantarum JYLP-326, additional experiments were performed. 60 8-week-old male C57BL / 6 mice were selected as experimental mice, 48 of which were selected to establish a CUMS mouse model according to the method of Example 2, and the remaining 12 were normal groups. Starting from the 5th week, the 48 CUMS model mice were randomly divided into 3 groups again, namely, a model group (M group), a low-dose treatment group (ML group), a high-dose treatment group (MH group) and an inhibitor group (MI group), and the experiments in Examples 2-4 were repeated to study the effects of RAGE-TLR4 inhibition on macrophage / microglial polarization and the improvement of emotional instability symptoms in CUMS mice. The experimental results are shown in Figure 4.

[0081] To evaluate the effects of the FPS-ZM1 inhibitor, we performed OFT, SPT, and TST tests on mice (Figures 4A, B, and C). According to the OFT test results, compared with the model group, the parameter values ​​of the low-dose treatment group, high-dose treatment group, and FPS-ZM1 inhibitor group treated with Lactobacillus plantarum JYLP-326 were improved (P < 0.01). In the SPT test, the sucrose preference of the model group decreased to 34.25 ± 4.26% compared with the control group (P < 0.05). The results of the TST test showed that compared with Lactobacillus plantarum JYLP-326, the FPS-ZM1 inhibitor showed a better effect, and the immobility time of mice in the high-dose treatment group and the inhibitor group was significantly less than that of the model group CUMS mice. Therefore, both Lactobacillus plantarum JYLP-326 and the FPS-ZM1 inhibitor significantly improved low mood and instability, and helped to reverse depressive tendencies.

[0082] Immunohistochemistry was used to examine the expression of GFAP (an astrocyte marker) and Iba1 (a microglial marker) in the hippocampus of each group of mice (Figure 4D). Compared with the control group, the percentage of GFAP- and Iba1-positive neurons in the model group was significantly decreased (P < 0.05). Results from the high-dose treatment group and the inhibitor group showed that inhibition of inflammation significantly decreased the expression of GFAP and Iba1 (P < 0.01, Figure 4E,F). Furthermore, the expression levels of HMGB1, RAGE, and TLR4 proteins in the hippocampus of CUMS mice were also assessed. Compared with the control group, the expression levels of HMGB1, RAGE, and TLR4 proteins in the hippocampus of the model group CUMS mice were significantly increased (P < 0.01), whereas treatment with Lactobacillus plantarum JYLP-326 and FPS-ZM1 significantly decreased the expression of HMGB1, RAGE, and TLR4 proteins in the hippocampus of the mice (P < 0.01, Figure 4G-J).

[0083] Example 6 Effect of Lactobacillus plantarum JYLP-326 on the intestinal flora of CUMS mice

[0084] Gut microbiota is closely associated with mood changes. During the experiment, feces from all mice were collected and analyzed by 16S rRNA high-throughput sequencing. The results are shown in Figure 5.

[0085] The Chao1 index and observed_species index showed differences in the α-diversity index of the intestinal flora between the different groups, indicating that low mood and instability disrupted the balance of the intestinal flora to a certain extent (Figures 5A, B). The Chao1 index and observed_species index of the low-dose and high-dose treatment groups were slightly higher than those of the model group (P < 0.05), indicating that Lactobacillus plantarum JYLP-326 can restore the diversity of the intestinal flora disrupted by low mood and instability. In addition, PCoA analysis of Bray-Curtis dissimilarity showed that the β-diversity of the intestinal flora of mice in the ML, MH, and MI groups was similar to that of the C group, but different from that of the M group, further demonstrating that emotional instability affects the structure of the human intestinal flora and that the probiotic Lactobacillus plantarum JYLP-326 can reshape the intestinal microbial spectrum (Figure 5C). The Venn diagram showed that a total of 3114 operational taxonomic units (OTUs) were identified in all samples, of which 377 OTUs were shared by all groups. 705, 607, 512, 497, and 416 unique OTUs were detected in groups C, M, ML, MH, and MI, respectively (Figure 5D). These results indicate that administration of Lactobacillus plantarum JYLP-326 improved the intestinal microbiota of CUMS mice, resulting in a pattern similar to that of normal mice. At the phylum level, the dominant microbiota in different groups were mainly Firmicutes, Proteobacteria, Bacteroidetes, and Verrucomicrobia, which accounted for 97.79%, 91.41%, 93.29%, 94.25%, and 92.96% of the sequencing results of the three groups, respectively (Figure 5E). Notably, the relative abundance of Proteobacteria (25.62%) in the M group was significantly higher than that in the ML, MH, and MI groups (P < 0.001, Figure 5G), but this increase could be restored by the administration of Lactobacillus plantarum JYLP-326 or the FPS-ZM1 inhibitor. In contrast, the relative abundance of Firmicutes (58.97%) in the M group was lower than that in the MH group (78.64%), while the relative abundance of Bacteroidetes (6.02%) in the M group was lower than that in the MI group (13.92%). At the genus level, the dominant bacterial groups in different groups were mainly Lactobacillus, Akkermansia, and Sutterella (Figure 5F). The fecal microbial profiles of mice in group M showed relatively high levels of Sutterella (13.73%) and Blautia (4.74%), whereas the fecal microbial profiles of mice in group ML, MH, and MI showed low levels of Sutterella and Blautia (Fig. 5I, K).However, the presence of Lactobacillus in the M group (20.46%) was lower than that in the C group (26.06%), the MH group (29.51%), and the MI group (30.97%) (Figure 5J). These findings suggest that treatment with Lactobacillus plantarum JYLP-326 effectively increased the abundance of Lactobacillus and reduced the presence of Sutterella and Eubacterium spp., reversing, to some extent, the changes in the intestinal microbiota associated with low mood and instability.

[0086] Example 7 Preparation of Mood-Improving Products

[0087] (1) Preparation of MRS liquid culture medium and MRS solid culture medium:

[0088] The components of MRS liquid medium include: 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, and 1000 mL of distilled water; 15 g / L of agar is added to the MRS liquid medium to obtain an MRS solid medium.

[0089] (2) Lactobacillus plantarum JYLP-326 was activated on MRS solid culture medium, and after anaerobic culture for 24 hours, a single colony was picked and inoculated into MRS liquid culture medium, and then cultured at 37°C for 24 hours to obtain a bacterial liquid; Lactobacillus plantarum JYLP-326 powder was obtained by low-temperature centrifugation, washing, coating and vacuum freeze-drying.

[0090] (3) The powder of Lactobacillus plantarum JYLP-326 was compounded with isomaltooligosaccharide (purchased from Baolingbao Biotechnology Co., Ltd.) to obtain a product that improves mood.

[0091] According to different dosage requirements, by adjusting the compound ratio of Lactobacillus plantarum JYLP-326 powder and isomaltooligosaccharide, products with Lactobacillus plantarum JYLP-326 contents of 10 billion / g, 15 billion / g, 20 billion / g, and 25 billion / g can be obtained.

[0092] Although the present application has been described in detail with reference to the accompanying drawings and in conjunction with preferred embodiments, the present application is not limited thereto. Without departing from the spirit and substance of the present application, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present application, and such modifications or substitutions shall be within the scope of the present application. Any changes or substitutions that can be easily conceived by persons skilled in the art within the technical scope disclosed in the present application shall be within the scope of protection of the present application.

Claims

1. An application of Lactobacillus plantarum JYLP-326 in preparing a product for improving mood, characterized in that: Lactobacillusp lantarum JYLP-326 was deposited in the General Microbiology Center of China National Committee for the Preservation of Microbiological Cultures on June 27, 2019. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 18038.

2. The use according to claim 1, characterized in that The types of products described include pharmaceuticals.

3. The use according to claim 1, characterized in that The mood-improving product is a product that regulates mood-related nerve cells, proteins and inflammatory factors in the hippocampus of CUMS mice.

4. The use according to claim 1, characterized in that The mood-improving product is a product that regulates the TLR4-MyD88-NF-κB signaling pathway and inflammatory factors in the colon of CUMS mice.

5. The use according to claim 1, characterized in that The mood-improving product is a product that inhibits the RAGE-TLR4 signaling pathway.

6. The use according to claim 1, characterized in that The mood-improving product is a product that regulates the intestinal microbial flora of CUMS mice.

7. A product for improving mood, characterized in that The product comprises Lactobacillus plantarum JYLP-326, and the preservation number of Lactobacillus plantarum JYLP-326 is CGMCC No.18038.

8. The product according to claim 7, characterized in that The content of Lactobacillus plantarum JYLP-326 in the product is 1.0×10 10 ~2.5×10 10 cfu / g.

9. The method for preparing the product according to claim 7 or 8, characterized in that: The following steps are involved: (1) Activating Lactobacillus plantarum JYLP-326 on MRS solid culture medium, picking a single colony after anaerobic culture for 24 hours and inoculating it into MRS liquid culture medium, and then culturing it at 37° C. for 24 hours to obtain a bacterial solution; adjusting the concentration of the bacterial solution, and obtaining bacterial powder by low-temperature centrifugation, washing, coating and vacuum freeze-drying; (2) Compounding the Lactobacillus plantarum JYLP-326 prepared in step (1) with isomaltooligosaccharide to obtain a mood-improving product.

10. The preparation method according to claim 9, characterized in that: The components of MRS liquid culture medium include: 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, and 1000 mL of distilled water; 15 g / L agar is added to the MRS liquid culture medium to obtain the MRS solid culture medium.

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