A lactobacillus plantarum and a fermented liquid of spina date seed, and application thereof

Abstract

The present application belongs to the field of microbial biotechnology, and specifically relates to a lactobacillus plantarum and a zizyphus jujuba mill. fermentation liquor and application thereof. The present application obtains a lactobacillus plantarum KLDS 1.0386 through extensive screening, and the preservation number is CGMCC NO.30809, which can regulate mood and improve sleep. The lactobacillus plantarum KLDS 1.0386 of the present application can be used to improve depression and prevent and treat mental diseases. The lactobacillus plantarum can be used to ferment zizyphus jujuba mill, greatly improve the content of flavonoids, GABA and indole-3-lactic acid in the zizyphus jujuba mill fermentation liquor, and improve the effects of improving sleep, resisting depression and improving mental diseases of the zizyphus jujuba mill fermentation liquor. The zizyphus jujuba mill fermentation liquor of the present application can also regulate intestinal flora and restore it to normal state.

Description

Technical Field

[0001] This invention belongs to the field of microbial biotechnology, specifically to a Lactobacillus plantarum and its jujube seed fermentation broth, as well as their applications. Background Technology

[0002] Depression is a complex neuropsychiatric disorder characterized by low mood, anhedonia, and impaired cognitive function. Its onset is not caused by a single factor but is the result of multiple factors, including genetics, environment, neuroendocrine disorders, and gut microbiota imbalance. Depression is characterized by high incidence, heavy disease burden, and complex pathological mechanisms. Major depressive disorder has a high prevalence among people with common physical illnesses such as cancer, neurodegenerative diseases, and cardiovascular diseases. Current treatments for depression mainly include medication, physical therapy, and psychotherapy. Medication is commonly used to treat depression, such as SSRIs (Selective Serotonin Reuptake Inhibitors), SNRIs (Serotonin-Norepinephrine Reuptake Inhibitors), and NaSSAs (Noradrenergic and Specific Serotonergic Antidepressants). Common medications include fluoxetine, venlafaxine, and bupropion. Different classes of drugs have different characteristics, but all share issues such as slow onset of action and unstable efficacy. These antidepressants have many adverse reactions, especially in the early stages of medication, with symptoms being more pronounced, mainly manifested as gastrointestinal reactions such as nausea, vomiting, and indigestion.

[0003] Probiotics, as a safe and effective new intervention method, can alleviate depressive symptoms by regulating the gut-brain axis. (Lactobacillus plantarum (...) Lactiplantibacillus plantarum, L. plantarum Lactobacillus plantarum has shown great application potential in improving mood disorders and alleviating depression. CN202311514364.7 discloses that Lactobacillus plantarum JYLP-326 can improve low mood and depressive tendencies in mice with chronic unpredictable mild stimulation (CUMS), regulate the levels of mood-related neurons, proteins, and inflammatory factors in the hippocampus of CUMS mice, and has potential neuroprotective effects on the mouse hippocampus. CN202210810812.7 discloses that Lactobacillus plantarum GM11 has the potential to improve negative emotions such as anxiety and depression as a probiotic. CN202610044953.0 discloses that Lactobacillus plantarum KLDS 1.0386 can effectively improve depressive-like behaviors.

[0004] Furthermore, as natural substances that combine medicinal and nutritional properties, some food-medicine homologous substances have been shown to have the potential to improve mood. For example, jujube seed has significant potential in regulating central nervous system function and improving mood disorders. However, the low utilization rate of active ingredients in food-medicine homologous substances limits their intervention effects. Microbial fermentation technology can promote the release of their active substances. Summary of the Invention

[0005] The purpose of this invention is to provide a more efficient antidepressant-like behavior of *Lactobacillus plantarum* that can enhance the antidepressant effect of jujube seed.

[0006] The objective of this invention is achieved through the following technical solution: In one aspect, the present invention discloses a *Lactobacillus plantarum* strain, wherein the *Lactobacillus plantarum* strain is *Lactobacillus plantarum* KLDS 1.0386 (… lactobacillus plantarum KLDS 1.0386), accession number CGMCC NO. 30809, accession date: May 29, 2024, accession location: China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Beijing.

[0007] In one aspect, the present invention discloses a composition comprising the aforementioned *Lactobacillus plantarum* KLDS 1.0386.

[0008] In some embodiments, the composition is a food, functional food, health food, dietary supplement, or pharmaceutical.

[0009] In one aspect, the present invention discloses the application of Lactobacillus plantarum KLDS 1.0386 in the preparation of mood-regulating products.

[0010] In one aspect, the present invention discloses the application of Lactobacillus plantarum KLDS 1.0386 in the preparation of drugs for improving depression and preventing and treating mental illnesses.

[0011] In this invention, the improvement of depression and prevention of mental illness includes improving hopeless behavior, alleviating anhedonia, and enhancing exploratory abilities. The *Lactobacillus plantarum* KLDS 1.0386 can downregulate CORT and CRH levels in the body, increase 5-HT and BDNF levels, alleviate nerve cell damage, and protect neurons.

[0012] In this invention, the *Lactobacillus plantarum* KLDS 1.0386 is capable of... In some embodiments, the drug also contains pharmaceutically acceptable excipients.

[0013] In this invention, the content of *Lactobacillus plantarum* KLDS 1.0386 in the aforementioned product / drug is greater than 10. 7 CFU / dose, preferably greater than 10 8 CFU / dose, more preferably, greater than 10 9 CFU / dosage. The dosage is in mL or g.

[0014] In one aspect of the present invention, the application of *Lactobacillus plantarum* KLDS 1.0386 in fermented jujube seed is disclosed, wherein *Lactobacillus plantarum* KLDS 1.0386 can significantly increase the content of flavonoids, GABA and indole-3-lactic acid in the fermentation broth of jujube seed.

[0015] In one aspect, the present invention discloses a jujube seed fermentation broth, which is obtained by fermentation with Lactobacillus plantarum KLDS 1.0386.

[0016] In one aspect, the present invention discloses the application of the aforementioned jujube seed fermentation liquid in the preparation of sleep-regulating products.

[0017] In one aspect, the present invention discloses the application of the aforementioned jujube seed fermentation broth in the preparation of mood-regulating products.

[0018] In one aspect, the present invention discloses the application of the aforementioned jujube seed fermentation liquid in the preparation of drugs for improving depression and preventing and treating mental illnesses.

[0019] In one aspect, the present invention discloses the application of Lactobacillus plantarum KLDS 1.0386 of the present invention in regulating mood, improving depression, preventing and treating mental illnesses and / or improving sleep.

[0020] In one aspect, this invention discloses the application of jujube seed fermented with Lactobacillus plantarum KLDS 1.0386 in regulating mood, improving depression, preventing and treating mental illnesses, and / or improving sleep.

[0021] Beneficial effects This invention obtained a strain of *Lactobacillus plantarum* KLDS 1.0386 through extensive screening, which can regulate mood and improve sleep. *Lactobacillus plantarum* KLDS 1.0386 of this invention can be used to improve depression and prevent mental illnesses. Specifically, *Lactobacillus plantarum* of this invention can be used to ferment jujube seeds, significantly increasing the content of flavonoids, GABA, and indole-3-lactic acid in the jujube seed fermentation broth, thereby enhancing the antidepressant and mental illness-improving effects of the jujube seed fermentation broth. The jujube seed fermentation broth of this invention can also improve sleep and regulate intestinal flora, restoring it to a normal state. Attached Figure Description

[0022] Figure 1 The movement of mice in each treatment group is shown in Figure A, where A represents the trajectory of each group of mice and B represents the total distance traveled by each group of mice.

[0023] Figure 2 The effects of each treatment on despair behavior in CUMS mice, where A represents the immobility time of mice in the forced swimming test and B represents the percentage of immobility time of mice in the tail suspension test.

[0024] Figure 3 Effects of different doses of Lactobacillus plantarum KLDS 1.0386 on anhedonia in mice.

[0025] Figure 4 Serum hormone levels under different treatments, where A represents the serum CORT level and B represents the serum CRH level.

[0026] Figure 5 The levels of serum neurotransmitters under different treatments, where A represents the serum dopamine level and B represents the serum norepinephrine level.

[0027] Figure 6 The neurotransmitter levels in brain tissue under different treatments, where A represents the BDNF content in brain tissue and B represents the serotonin (5-HT) content in brain tissue.

[0028] Figure 7 Effects of different treatments on the morphology and arrangement of nerve cells in the hippocampus of CUMS mice.

[0029] Figure 8 Effects of different treatments on Nissl bodies in hippocampal neurons of CUMS mice. A represents Nissl staining results (40 ×), B represents the number of neurons in the CA1 region, and C represents the number of neurons in the CA3 region.

[0030] Figure 9 The movement of mice under different treatments: A is the trajectory route map of each group of mice, B is the trajectory route heatmap of each group of mice, and C is the total trajectory distance of each group of mice.

[0031] Figure 10 The effects of different treatments on the tail suspension test and forced swimming test in mice, where A represents the immobility time of mice in the tail suspension test and B represents the percentage of immobility time of mice in the forced swimming test.

[0032] Figure 11 The effects of different treatments on the anhedonia experiment in mice.

[0033] Figure 12 The levels of CORT, CRH, and ACTH in the serum of mice under different treatments.

[0034] Figure 13 The levels of DA, NE, and 5-HT in the serum of mice under different treatments.

[0035] Figure 14 The levels of neurotransmitters (NDNF, 5-hydroxytryptamine, GABA) in the brain tissue of mice under different treatments.

[0036] Figure 15 Effects of different treatments on the gut microbiota of mice.

[0037] Figure 16 Changes in total saponin and soluble flavonoid content before and after fermentation with Lactobacillus plantarum.

[0038] Figure 17 Changes in GABA content before and after fermentation of Lactobacillus plantarum.

[0039] Figure 18 Changes in tryptophan and indole-3-lactic acid content before and after fermentation with Lactobacillus plantarum.

[0040] Figure 19 The content of indole-3-lactic acid in the brains of mice under different treatments. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0042] In this invention, the strain *Lactobacillus plantarum* KLDS 1.0386 ( lactobacillus plantarum KLDS1.0386 L. plantarum KLDS 1.0386 It was isolated from traditional fermented dairy products in Inner Mongolia, with accession number CGMCC NO.30809, accession date: May 29, 2024, and accession location: China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Beijing.

[0043] Example 1: 1.1 Strain activation The *Lactobacillus plantarum* KLDS 1.0386 strain, stored at -80℃, was inoculated into MRS solid medium for activation. Single colonies were picked and cultured in liquid culture, and then passaged 2-3 times at 2% to obtain stable and highly active bacterial cells.

[0044] 1.2 Animal Experiments Mice were placed in an environment of 20–23°C and 54–56% humidity, using a 12-hour light-dark cycle, and provided with free access to water and food. All animal experimental procedures were strictly conducted in accordance with the guidelines of the Animal Experiment Ethics Committee of Northeast Agricultural University. Before the formal experiment began, the experimental mice underwent a 7-day environmental acclimatization feeding period. After the acclimatization feeding, all mice were randomly assigned to different groups for a 5-week intervention experiment. In the 6th week of the experiment, behavioral indicators were measured in each group of mice, and samples were collected and processed after the measurements were taken. The mice were grouped as shown in Table 1, into a blank control group (NC), a model group (MC), and a test group (low-dose group LP, medium-dose group MP, and high-dose group HP). The blank control group mice were housed in an environment free from external interference, and during the experimental period, they were only provided with routine feeding such as cage cleaning, bedding replacement, and basic food and water supply, without any experimental intervention factors. The other groups of mice were used to establish a depression model according to the CUMS model construction protocol (Table 2).

[0045] Table 1: Experimental Groups and Treatments ; Table 2: Modeling methods for CUMS models ; 1.3 Measurement of depressive-like behavior After modeling, mice underwent experiments including autonomous exploration, despair, and anhedonia. The results were analyzed using the behavioral analysis software Smart 3.0 after the tests.

[0046] Open field test: Mice in each group were gently placed in the central area of ​​a black open field test chamber measuring 50 cm × 50 cm × 35 cm to eliminate interference from the starting position at the edge of the chamber. The environment was kept quiet, with soft and constant lighting throughout the test to avoid stress on the mice's behavior from external factors. The mice's spontaneous movement trajectory was recorded over 6 minutes. The total distance traveled and the number of crossings were measured using a behavioral analysis system. The total distance traveled reflects the mice's physical activity level. The behavioral parameters in the central area were negatively correlated with anxiety-like emotions, which were used to comprehensively assess the mice's depressive state.

[0047] Tail suspension test: Before the experiment, tape was attached to the hanging hooks to facilitate the attachment and removal of mice from each group, reducing the suspension time. Each mouse was secured with medical tape 2-3 cm from the tail end. The mice were then suspended from the tape-attached hooks, with the suspension height adjusted to ensure a suitable distance between the mouse's head and the bottom of the enclosure, preventing its hind limbs from touching the bottom and allowing its body to hang naturally. To avoid behavioral interference between mice, opaque partitions were used to completely isolate adjacent suspended mice. The experiment was conducted in a quiet environment without additional stimulation for 6 minutes. The time it took for the mice to stop struggling and remain still was recorded. This indicator was positively correlated with hopeless-like behavior and can be used as an evaluation criterion for depressive-like phenotypes.

[0048] Forced swimming test: Before the experiment, ensure the water temperature is maintained at 23 ± 1℃. At the start of the experiment, slowly place each group of mice into a cylindrical container with a diameter of 11 cm and a height of 30 cm. Fill the container with clean water to a height of 25 cm, ensuring that the mice's hind limbs are completely off the bottom. Record the last 4 minutes of the 6-minute period as the valid recording time, and calculate the immobility time. The immobility state is defined as ceasing active struggling and swimming, and maintaining a floating, still state. Only maintain the necessary head above water for breathing. In this experiment, prolonged immobility time is generally considered a manifestation of enhanced despair-like behavior in animals and is positively correlated with depressive-like phenotypes.

[0049] ; Sugar water preference test: The experiment used 1% sucrose water. After fasting and water restriction for 12 h, a 24 h test was conducted. At 12 h, the horizontal positions were exchanged, and the preference index was used to assess anhedonia and depressive-like behavior in mice.

[0050] ; 1.4 Determination of hormone and neurotransmitter levels in mouse serum The levels of CORT (corticosterone), CRH (corticotropic hormone-releasing hormone), DA (dopamine), and NE (norepinephrine) were determined using enzyme-linked immunosorbent assay (ELISA).

[0051] 1.5 Determination of neurotrophic factors and 5-HT levels in mouse brain tissue After centrifuging the supernatant of whole brain homogenate from mice, the levels of 5-HT (5-hydroxytryptamine) and BDNF (brain-derived neurotrophic factor) were measured using an ELISA kit.

[0052] 1.6 Pathological examination of mouse hippocampal tissue Brain tissue was immediately removed and fixed in 4% paraformaldehyde for 24 hours. Paraffin embedding, sectioning, dewaxing, rehydration, tissue staining, dehydration and clearing, and mounting were then performed. Hematoxylin and eosin were used for H&E staining, and 1% toluidine blue solution was used for Nissl staining. After all staining was completed, the tissue was observed using a Slide Viewer 2.5.

[0053] Example 1 Test Results: See Figure 1 The movement paths of representative mice from different groups can be observed. Mice in the NC group moved in both the central and peripheral regions, while mice in the MC group moved only in the peripheral region and did not enter the central region. The total distance traveled by the mice during the experiment can also be observed. Compared to the NC group, the total distance traveled by the MC group was significantly decreased (p<0.05), from 9168.76 ± 1544.71 mm to 2960.94 ± 1215.02 mm. Compared to the MC group, the total distance traveled by the LP, MP, and HP groups all showed an increasing trend, with the HP group showing the most significant increase (p<0.05), rising by 128.56%. L. plantarum Following KLDS 1.0386 intervention, spontaneous activity in mice increased, and the number of times they entered the central region increased (Table 3), with the high-dose group showing the most significant effect (p<0.05). This indicates... L. plantarum KLDS 1.0386 showed a dose-dependent effect on improving spontaneous activities and exploratory behaviors, with the high-dose group being the most effective.

[0054] Table 3. Effects of different L. plantarum species on the number of times mice enter the central region in their autonomous exploration ability. ; Note: Different letters indicate significant differences. See Figure 2 The figures represent the experimental results of mice exhibiting despair behavior. Compared to the NC group, the MC group showed significantly increased immobility time during tail suspension and forced swimming (p<0.05). Different dosages... L. plantarum After intervention with KLDS 1.0386, immobility time was reduced in all mice, and the reduction in immobility time was greatest in the HP group compared to the LP and MP groups. These results indicate that KLDS 1.0386 has the ability to alleviate despair behavior in CUMS mice, with the high-dose group showing the most significant improvement.

[0055] See Figure 3 The value is the saccharide preference index of mice in the saccharide preference experiment. The results showed that the saccharide preference index of the NC group was 79.39 ± 4.31%, while the saccharide preference index of the MC group was significantly lower, at 52.19 ± 5.90% (p<0.05). L. plantarumFollowing KLDS 1.0386 intervention, the sucrose preference index in mice significantly increased (p<0.05), and the sucrose preference index in the HP group recovered to 77.40 ± 4.23%, showing no significant difference from the NC group (p>0.05). These results indicate... L. plantarum KLDS 1.0386 has the ability to alleviate anhedonia in CUMS mice, a result consistent with the aforementioned open field, tail suspension, and forced swimming experiments, further demonstrating... L. plantarum KLDS 1.0386 has an ameliorative effect on depressive-like behavior in CUMS mice.

[0056] See Figure 4 CORT and CRH are key regulators of the HPA axis. Compared to the NC group, the serum CORT and CRH levels in the MC group mice were significantly increased after 35 days of CUMS induction, increasing by 42.49% and 62.60%, respectively (p < 0.05). Compared with the MC group, all dose groups after probiotic intervention reduced the serum CORT and CRH levels in mice, with the medium and high dose groups showing significant reductions (p < 0.05), and the high dose group showing the largest decrease, to 163.81 ± 15.03 ng / mL and 735.17 ± 55.28 pg / mL, respectively. These results suggest that KLDS 1.0386 intervention may alleviate the CUMS-induced HPA axis overactivation by downregulating CORT and CRH levels.

[0057] See Figure 5 As important neurotransmitters in the central nervous system, DA and NE play an irreplaceable role in regulating the body. Figure 5 As experimental results showed, compared with the NC group, the DA level in the MC group mice decreased to 59.14 ± 11.06 pg / mL after CUMS stimulation (p<0.05). Mice were administered the drug via gavage. L. plantarum After KLDS 1.0386, DA levels recovered to some extent in all groups, with the most significant recovery observed in the HP group, reaching 81.83 ± 7.52 pg / mL (p<0.05). (Note: This likely refers to a specific treatment or treatment regimen.) L. plantarum KLDS 1.0386 demonstrated the ability to restore reduced dopamine levels in depressed mice. Norepinephrine (NE) is a neurotransmitter that plays a crucial role in the body. Compared to the NC group, the MC group showed a significant decrease in NE levels, reducing them to 62.97 ± 14.52 ng / mL (p < 0.05). L. plantarum Mice administered KLDS 1.0386 via gavage showed elevated NE levels, and the HP group significantly restored NE levels to 88.53 ± 10.79 ng / mL (p<0.05), indicating that... L. plantarumKLDS 1.0386 increased NE levels in CUMS-depressed mice. Overall, CUMS stimulation significantly decreased DA and NE levels in mice, while gavage... L. plantarum After KLDS 1.0386, especially after HP intervention, serum DA and NE levels in CUMS mice were significantly restored, indicating that this strain can effectively improve the insufficient production of amine neurotransmitters in depressed mice.

[0058] See Figure 6 5-HT and its receptors are involved to varying degrees in the pathogenesis and treatment of depression. Compared with the NC group, the 5-HT level in depressed mice in the MC group was reduced by 32.39% (p<0.05). L. plantarum Mice treated with KLDS 1.0386 showed increased 5-HT levels, especially at medium and high doses. L. plantarum KLDS 1.0386 significantly restored 5-HT levels in the mouse hippocampus, restoring 22.29% and 35.13% respectively (p<0.05). BDNF is a growth factor that plays an important role in regulating synaptic plasticity, neurogenesis, and neuronal health. After CUMS stimulation in mice, BDNF levels in brain tissue significantly decreased by 11.42% (p<0.05). L. plantarum KLDS 1.0386 intervention resulted in a recovery of BDNF levels in mouse brain tissue, with the MP and HP groups showing significant recovery of BDNF levels in CUMS-treated mouse brain tissue (p<0.05). The results indicated that CUMS stimulation significantly decreased 5-HT and BDNF levels in mouse brain tissue, while medium and high doses... L. plantarum After intervention with KLDS 1.0386, all of the above indicators showed significant recovery, indicating that... L. plantarum KLDS 1.0386 may play a role in alleviating depressive-like behaviors by regulating neurotransmitter levels in the brain.

[0059] See Figure 7 The H&E staining results for each group of mice show that the NC group had a greater number of hippocampal neurons with intact cell morphology, uniform staining, clear nucleoli, and dense and regular cell arrangement. The MC group showed a significant decrease in the number of hippocampal neurons, with deeper staining, marked nuclear pyknosis, incomplete cell structure, and loose and disordered cell arrangement. Compared with the MC group, all dose groups showed varying degrees of improvement in hippocampal neuronal damage in the CA1 and CA3 regions, characterized by increased cell number, more intact structure, and a decrease in the number of cells with nuclear pyknosis. This indicates that KLDS 1.0386 intervention can alleviate hippocampal neuronal damage in CUMS mice.

[0060] See Figure 8In the NC group mice, Nissl bodies stained evenly and densely in neurons, with uniform distribution. Neuronal cell bodies were plump, structurally intact, and nucleoli were clearly visible, indicating intact neuronal structure. In contrast, the MC group mice showed a reduction in neurons and a significant increase in the number of necrotic neurons. Compared to the MC group, different doses... L. plantarum KLDS 1.0386 mice showed an increase in the number of neurons in the CA1 and CA3 regions of the hippocampus, with an increase in structurally intact neurons. The HP group showed a significant increase in the number of neurons in the CA3 region (p<0.05). These results further illustrate... L. plantarum KLDS 1.0386 has a protective effect on hippocampal neurons, and the protective effect is more obvious in the high-dose group.

[0061] Example 2: Lactobacillus plantarum KLDS 1.0386 fermented jujube seed broth relieves depression. 2.1 Preparation of Unfermented Jujube Seed Liquid Take 10 g of sucrose, 0.1 g of ascorbic acid, and 50 g of jujube seed powder and add them to an Erlenmeyer flask. Then add distilled water (jujube seed powder: distilled water (m:v) = 1:18) and stir well. Perform sonication for 40 min. After sonication, place the sample in liquid nitrogen for quick freezing, store it in a -80 ℃ freezer for 12 h, and then freeze-dry it for storage.

[0062] 2.2 Preparation of fermented jujube seed liquid Activated L. plantarum KLDS 1.0386 was centrifuged at 6000 rpm for 10 min, the supernatant was discarded, the bacterial sludge was washed with sterile physiological saline, and resuspended to 1×10⁹ CFU / mL. The bacterial suspension was inoculated into the cooled unfermented jujube seed liquid at a 5% (v / v) inoculation rate and incubated at 37 ℃ for 48 h. After incubation, the fermentation broth was flash-frozen in liquid nitrogen and then freeze-dried at -80 ℃ for 12 h.

[0063] 2.3 Animal Testing Arrangement and Procedure The experimental groups and information are shown in Table 4, and the CUMS modeling method is the same as in Table 2. Among them, the unfermented jujube seed liquid was dissolved with freeze-dried powder to a concentration of 3 times the original concentration, and the fermented jujube seed liquid was treated in the same direction.

[0064] Table 4. Grouping and drug administration of mice with depression relieved by Lactobacillus plantarum fermentation broth in jujube seed. ; 2.4 Measurement of depressive-like behavior in mice The specific testing procedure for measuring depressive-like behavior in mice is the same as in Example 1.

[0065] See Figure 9 ,from Figure 9The map in Figure A shows the route of mice in an open field. It can be seen that the NC group mice were active in the open field, exhibiting significant exploratory behavior, moving between the central and peripheral areas. In contrast, the MC group mice showed significantly reduced activity, almost never appearing in the central area, exhibiting pronounced peripheral behavior, and showing a decreased willingness to explore, demonstrating a clear decline in autonomous exploration ability. Compared to the MC group, the UZSS, FZSS, and POS groups showed significantly increased autonomous activity and exploratory behavior, with more pronounced activity in the central area. Compared to the UZSS group, the FZSS group mice showed even more pronounced activity in the central area, a wider range of movement, and less activity close to the edge of the open field. Figure 9 Figure B shows the heatmap of the mice's route in the open field. Mice in the NC group showed a continuous movement trajectory with heatmap signal in the central region, while mice in the MC group showed a lack of central region signal, exhibiting significant activity inhibition and avoidance behavior. Compared to the MC group, mice in the UZSS, FZSS, and POS groups showed increased motor activity and significantly enhanced central region heatmap signal. The total distance traveled and the number of times mice entered the central region in the open field are shown in the figure. Figure 9 Tables C and 5 show that, compared with the NC group, the total distance traveled by mice in the MC group decreased from 12770.84 ± 2045.50 mm to 4046.71 ± 822.02 mm (p<0.05), and the number of times they entered the central region decreased from 8.55 ± 1.59 to 1.55 ± 1.01 (p<0.05), further demonstrating that CUMS can lead to a decrease in the spontaneous activity ability of mice. Compared with the model group, after intervention with unfermented jujube seed liquid and fermented jujube seed liquid, the total distance traveled by mice significantly increased to 8650.86 ± 1398.93 mm and 10920.88 ± 2114.13 mm (p<0.05), and the number of times they entered the central region significantly increased to 4.55 ± 1.01 and 6.44 ± 1.33 (p<0.05). The total distance traveled and the number of times they entered the central region were both higher in the FZSS group than in the unfermented group, indicating that all intervention groups can effectively improve the mice's willingness to explore. The fermented jujube seed liquid group of L. plantarum was more effective than the unfermented jujube seed liquid group.

[0066] Table 5: Statistics on the number of times mice from different groups entered the central region ; See Figure 10 For further observation L. plantarum The effect of fermented jujube seed on improving despair behavior in CUMS mice was evaluated using the tail suspension test and forced swimming test. Figure 10As shown in Figure A, the immobility time of mice in the NC group was 35.83 ± 9.17 s, while the immobility time of mice in the MC group with tail suspension was prolonged by 77.59 ± 6.30 s (p<0.05), exhibiting a clear state of behavioral despair. Compared with the MC group, the immobility time of mice in each intervention group was significantly shortened (p<0.05). Compared with the UZSS group, the immobility time of mice in the FZSS group was shortened by 7.68 ± 8.81 s. The results of the tail suspension experiment are as follows. Figure 3-16 As shown in b), the immobility time of mice in the NC group was 20.87 ± 2.73%, while the immobility time in the MC group was significantly prolonged to 44.12 ± 4.03% (p<0.05), indicating a clear state of behavioral despair. Compared with the MC group, the immobility time of mice in the intervention groups decreased by 25.41%, 43.60%, and 58.65%, respectively (p<0.05). Compared with the unfermented jujube seed group, the immobility time of mice in the intervention groups was significantly reduced. L. plantarum After intervention with fermented jujube seed extract, the immobility time of mice was significantly shortened (p<0.05). This indicates that, compared with the UZSS group, the FZSS group mice had shorter forced swimming and tail suspension immobility times, and the fermentation treatment enhanced the effect of jujube seed extract on improving hopelessness behavior in depressed mice.

[0067] See Figure 11 The saccharide preference test is a classic method for evaluating anhedonia in CUMS mice, directly reflecting changes in the mice's reward response. The saccharide preference index of the NC group mice was 77.66 ± 2.49%, indicating normal reward system function and no anhedonia. Compared to the NC group, the saccharide preference index of the MC group mice was significantly lower at 46.44 ± 3.63% (p<0.05), indicating anhedonia-like behavior and a decreased ability to perceive and experience pleasure from sweet substances. Compared to the MC group, the saccharide preference index of mice in all intervention groups significantly increased (p<0.05), with the FZSS group showing an increase to 70.54 ± 6.00%, closer to the NC group. This indicates that all intervention groups effectively improved anhedonia caused by CUMS, with the FZSS group showing better results than the UZSS group.

[0068] 2.5 Determination of serum and tissue hormone and neurotransmitter levels in mice Referring to sections 1.4 and 1.5 of Example 1, the levels of CORT (corticosterone), CRH (corticotropic hormone-releasing hormone), DA (dopamine), NE (norepinephrine), and 5-HT (serotonin) in mouse serum, and the levels of 5-HT (serotonin), BDNF (brain-derived neurotrophic factor), and GABA in brain tissue were measured using an ELISA kit.

[0069] Test results as follows Figure 12As shown, compared with the NC group, the serum levels of CORT, CRH, and ACTH in the MC group were significantly increased, by 54.84%, 25.80%, and 62.92%, respectively (p<0.05), indicating that CUMS modeling led to HPA axis dysfunction and abnormal stress hormone secretion in mice, which is consistent with the typical physiological characteristics of depressive model animals. After intervention, the levels of CORT, CRH, and ACTH in all groups were significantly decreased (p<0.05). Among them, compared with the UZSS group, the serum hormone levels in the FZSS group were even lower, at 147.60 ± 16.32 ng / mL, 652.16 ± 34.14 pg / mL, and 44.34 ± 3.84 pg / mL, respectively, with CORT and CRH levels significantly decreased by 34.07% and 16.19%, respectively (p<0.05). The above results indicate that both unfermented and fermented jujube seeds can alleviate CUMS-induced overactivation of the HPA axis, with the fermented group showing better overall regulatory effects on CRH, ACTH, and CORT.

[0070] Monoamine neurotransmitters are key substances in the central nervous system involved in the regulation of mood, motivation, sleep, appetite, and stress response. Measurement results are as follows... Figure 13 As shown in the figure. Compared with the NC group, the serum levels of DA, NE, and 5-HT in the MC group mice were significantly decreased (p<0.05), decreasing from 126.20 ± 8.94 ng / mL, 112.47 ± 16.26 pg / mL, and 201.75 ± 13.91 ng / mL to 68.25 ± 8.03 ng / mL, 51.01 ± 5.94 pg / mL, and 170.60 ± 14.31 ng / mL, respectively. Compared with the MC group, the serum levels of DA and NE in all intervention groups were significantly increased (p<0.05), with the FZSS group showing an increase of 54.97% and 81.57%, respectively. The 5-HT levels in the FZSS and POS groups were significantly higher than those in the MC and UZSS groups (p<0.05), with the FZSS group showing an increase of 13.48%. The results showed that both unfermented jujube seed and fermented jujube seed broth could improve the decrease in monoamine neurotransmitters caused by CUMS, and the effect of fermented jujube seed broth was more significant.

[0071] To further evaluate the effects of jujube seed extract before and after fermentation on the brain tissue of CUMS mice, the results are as follows: Figure 14As shown, compared with the NC group, the BDNF content in the brain tissue of mice in the MC group decreased by 30.11% (p<0.05), suggesting that CUMS can lead to impaired neurotransmitter-related functions in the mouse brain. After intervention, the BDNF content in all groups increased, with the fermented jujube seed liquid group and POS group showing a significant increase of 22.94% and 43.76% respectively compared with the MC group (p<0.05). The 5-HT content in the brain tissue of mice in the NC group was 333.53 ± 15.73 pg / mL, while that in the MC group was 196.57 ± 36.77 pg / mL, showing a significant decrease (p<0.05); after intervention, the 5-HT content in all groups of mice increased significantly (p<0.05). L. plantarum The 5-HT levels in the jujube seed fermentation broth group and the POS group recovered to a state where there was no significant difference compared with the NC group (p>0.05). Figure 14 In the figure, C represents the GABA content in the brain tissue of mice in each group. The GABA concentration in the brain tissue of mice in the NC group was 6.24 ± 0.62 ng / mL, while that in the MC group decreased to 3.02 ± 0.27 ng / mL (p<0.05). After intervention, the GABA content in the UZSS group increased to 3.92 ± 0.34 ng / mL, the FZSS group increased to 5.67 ± 0.38 ng / mL, and the POS group increased to 6.17 ± 0.42 ng / mL. Compared with the MC group, the FSZZ content increased significantly by 87.92% (p<0.05). These results indicate that all interventions can alleviate the decrease in BDNF, 5-HT, and GABA levels in mouse brain tissue caused by CUMS, and the improvement effect of the fermented jujube seed broth is more prominent.

[0072] 2.6 Mouse 16S rRNA Sequencing Analysis After modeling, feces from each group of mice were collected, flash-frozen in liquid nitrogen, and then stored at -80 °C for later testing, avoiding repeated freeze-thaw cycles. After genomic DNA extraction and quality testing, the target variable regions were amplified using high-fidelity polymerase, and sequencing was performed after library construction and quality control. Based on the 16S rDNA hypervariable region sequencing results, microorganisms were classified according to sequence similarity to obtain information on microbial diversity. Simultaneously, OTUs (Operational Taxonomic Units) were used as taxonomic units to assess sample differences.

[0073] Mouse feces were analyzed using 16S rRNA detection technology. Figure 15 The image shows the alpha diversity of the gut microbiota in mouse contents and PCoA analysis based on OTU samples. Alpha diversity is primarily used to reflect the diversity and richness of the microbial community in mouse contents. Figure 15Figures A and B show that, compared to the NC group, the Chao1 and Shannon indices in the gut of MC group mice decreased (p>0.05). Compared to the MC group, after intervention with unfermented jujube seed, both the Chao1 and Shannon indices showed an increasing trend, with the Shannon index significantly increasing in the fermented jujube seed group. This indicates that both unfermented and fermented jujube seed can increase the richness and diversity of gut microbiota in CUMS mice, with fermented jujube seed intervention showing a more significant effect on increasing gut diversity. Figure 15 As shown in Figure C, the cumulative contribution rate of PC1 (40.43%) and PC2 (32.66%) was 73.09%, indicating that these two principal axes can reflect the differences in gut microbiota structure among samples. On PC1, the coordinates of the NC group and the FZSS group were similar. On PC2, the coordinates of the NC group and the FZSS group were closest, followed by the UZSS group and the MC group. Among the four groups, the coordinates of the NC group and the FZSS group on both the PC1 and PC2 axes were very close, indicating that their gut microbiota species composition and abundance were most similar. This shows that compared with unfermented jujube seed liquid, jujube seed fermentation broth intervention can change the gut microbiota of mice, restoring it to a state close to that of normal mice.

[0074] Example 3: Effects of Lactobacillus plantarum fermentation on changes in the composition of jujube seed. 3.1 Changes in total saponins and total flavonoids in the fermentation broth after fermentation of jujube seeds with Lactobacillus plantarum KLDS 1.0386 Sour jujube seeds contain abundant saponins, and total saponins have been shown to have an effect in relieving depression. Figure 16 Figure A represents the total saponin content. The fermentation process significantly reduced the total saponin content of jujube seed (p<0.05), from 0.43 ± 0.02 mg / mL to 0.28 ± 0.02 mg / mL. Furthermore, flavonoids in jujube seed also exhibit significant neuroprotective effects; therefore, the content of soluble total flavonoids in the fermentation broth was measured. The results are as follows: Figure 16 As shown in Figure B, after fermentation, the flavonoid content increased significantly (p<0.05), from 332.76 ± 20.87 μg / mL to 525.03 ± 29.05 μg / mL, an increase of 57.78%.

[0075] 3.2 Changes in GABA content in fermentation broth after fermentation of jujube seed with Lactobacillus plantarum KLDS 1.0386 Glutamic acid is one of the main amino acids in jujube kernels, and it can be converted into GABA by glutamate decarboxylase from lactic acid bacteria. Decreased GABA activity can lead to increased nerve excitability, increasing the risk of anxiety, depression, and stress responses. Figure 17The values ​​represent the GABA content in jujube seeds before and after fermentation. The GABA content before fermentation was 8.55 ± 1.02 μg / mL, and the content after fermentation increased to 15.40 ± 1.31 μg / mL, which was a highly significant increase of 80.00% (p<0.05).

[0076] 3.3 Changes in indole-3-lactic acid content in fermentation broth after fermentation of jujube seed with Lactobacillus plantarum KLDS 1.0386 The contents of tryptophan and ILA (indole-3-lactic acid) in jujube seed extract before and after fermentation were quantitatively determined by LC-MS / MS. The results are as follows: Figure 18 The results showed that the tryptophan content in the unfermented jujube seed broth was 23.10 ± 2.48 μg / mL, while the tryptophan content in the fermented jujube seed broth decreased significantly to 3.12 ± 1.12 μg / mL (p<0.05). The ILA content in the unfermented jujube seed broth was 44.23 ± 2.18 ng / mL, while the ILA content in the fermented jujube seed broth was 3613.47 ± 79.48 ng / mL, showing a highly significant increase (p<0.05). This indicates that the fermentation process may promote the increase of ILA in the jujube seed fermentation broth by regulating the conversion of tryptophan to ILA.

[0077] Example 4: High indole-3-lactic acid in the fermentation broth of *Lactobacillus plantarum* KLDS 1.0386 jujube seed can promote the increase of indole-3-lactic acid in the brain. To further investigate the regulatory effect of significantly increased ILA content in fermentation broth on CUMS mice, the ILA content in mouse brain tissue was detected using LC-MS / MS. The results are as follows: Figure 19 As shown, compared with the NC group, the ILA content in the brain tissue of mice in the MC group was significantly decreased (p<0.05). After intervention, the ILA content in the brain tissue of mice recovered to varying degrees. Among them, the ILA content in the brain tissue of mice in the fermentation group increased to 22.00 ± 1.33 ng / mL (p<0.05), which was significantly higher than that in the non-fermentation group (p<0.05). This indicates that the intervention of jujube seed fermentation liquid can effectively promote the increase of ILA content in the brain and improve the excitability and mental state of mice.

[0078] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A type of Lactobacillus plantarum, characterized in that, The Lactobacillus plantarum is Lactobacillus plantarum KLDS 1.0386, with accession number CGMCC NO. 30809, accession date: May 29, 2024, accession location: China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Beijing.

2. A composition, characterized in that, The composition includes Lactobacillus plantarum KLDS1.0386 as described in claim 1.

3. The combination according to claim 2, characterized in that, The composition is a food, functional food, health food, dietary supplement, or medicine.

4. The application of Lactobacillus plantarum according to claim 1 in the preparation of mood-regulating products.

5. The application of *Lactobacillus plantarum* according to claim 1 in the preparation of drugs for improving depression and preventing and treating mental illnesses, characterized in that, The measures to improve depression and prevent mental illness include improving hopeless behavior, alleviating anhedonia, and enhancing exploratory abilities.

6. The application of *Lactobacillus plantarum* according to claim 1 in fermented jujube seed, characterized in that, The *Lactobacillus plantarum* KLDS 1.0386 significantly increased the content of flavonoids, GABA, and indole-3-lactic acid in the fermentation broth of jujube seed.

7. A fermented liquid of jujube kernels, characterized in that, The jujube seed fermentation liquid is obtained by fermentation using the *Lactobacillus plantarum* as described in claim 1.

8. The application of the jujube seed fermentation liquid according to claim 7 in the preparation of sleep-regulating products.

9. The application of the jujube seed fermentation liquid according to claim 7 in the preparation of mood-regulating products.

10. The application of the jujube seed fermentation broth according to claim 7 in the preparation of drugs for improving depression and preventing mental illnesses.

Images