Lactobacillus rhamnosus strain night-09 and its use in improving sleep and anxiety

Abstract

This invention relates to the field of biotechnology, specifically disclosing a strain of *Lactobacillus rhamnosus* Night-09 and its application in improving sleep and anxiety. This invention discovers a novel lactic acid bacterium that, through multi-target and multi-system synergistic effects, corrects sleep-wake system disorders and mood regulation system imbalances, thereby simultaneously achieving the dual effects of anti-anxiety and sleep promotion. Specifically, this invention provides a strain of *Lactobacillus rhamnosus* (… Lacticaseibacillus rhamnosus Night-09, with accession number CGMCC NO. 36239, is a strain of *Lactobacillus rhamnosus* Night-09 that can simultaneously and effectively improve insomnia symptoms and anxiety levels in animals with insomnia and anxiety. This provides a new, safe, and long-lasting natural conditioning approach for people with insomnia and anxiety, enriching the germplasm resources of functional probiotics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a strain of Lactobacillus rhamnosus Night-09 and its application in improving sleep and anxiety. Background Technology

[0002] With the accelerated pace of modern life and continuously increasing mental stress, insomnia and generalized anxiety disorder have become prevalent psychosomatic health problems, often occurring comorbidly. Epidemiological data shows that many adults suffer from chronic insomnia, with over 50-70% of insomnia sufferers also experiencing anxiety, and the incidence rate is increasing year by year. Approximately 60-80% of anxiety disorder patients also have sleep disorders. Insomnia and anxiety form a vicious cycle through the hypothalamus-pituitary-adrenal (HPA) axis, central neurotransmitter imbalances, and chronic neuroinflammation, severely reducing quality of life, inducing cognitive decline, metabolic disorders, and cardiovascular risks, among other health risks, which has attracted widespread attention.

[0003] Current clinical and mass-market interventions for insomnia and anxiety mainly fall into two categories: prescription chemical drugs and melatonin-based dietary supplements. Both approaches have significant safety shortcomings and application limitations. For example, sedative-hypnotic drugs such as benzodiazepines and selective serotonin reuptake inhibitors (SSRIs), while fast-acting, have high dependence rates and side effects such as withdrawal rebound, neurocognitive impairment, and abuse risks, making long-term use unsuitable. Exogenous melatonin, as a commonly used sleep aid, has a high incidence of short-term adverse reactions (headache 38.6%, gastrointestinal discomfort 26.3%, etc.), and long-term continuous use can increase the risk of heart failure by 90%, with rebound effects easily occurring upon discontinuation. Therefore, developing safe and effective natural interventions has significant clinical and social value.

[0004] Studies have shown that the gut microbiota-brain axis may be a core regulatory target for insomnia and anxiety. The gut-brain axis is a bidirectional communication mechanism between gut microbes and the central nervous system, involving multiple pathways including neural, endocrine, immune, and metabolic processes. Gut microbiota can influence host brain function through multiple target regulatory mechanisms, thereby regulating mood, cognition, and sleep behavior. It is non-addictive and has no side effects, making it an ideal alternative intervention pathway. Compared to live bacteria, probiotic metabolites have greater advantages in application. They have better stability, can tolerate more demanding environments, are highly safe, and do not need to colonize and reproduce in the gut, but rather exert their effects directly through bacterial components and / or metabolites, making them suitable for a wider range of people. However, sleep and anxiety involve different neural circuits (sleep-wake center vs. mood regulation center). Currently, dedicated strains or metabolites that can improve both insomnia and alleviate anxiety are scarce, and further research is needed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a new method that can both improve insomnia and relieve anxiety.

[0006] This invention provides a strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus Night-09, with accession number CGMCC NO. 36239.

[0007] To develop a safer and more effective method that can simultaneously improve insomnia and alleviate anxiety, this invention conducted research and found that γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the central nervous system and has a clear physiological role in relieving insomnia and anxiety. However, the efficacy of oral GABA supplements is controversial: 1) GABA molecules are large and difficult to directly cross the blood-brain barrier to enter the central nervous system; 2) Some studies show that oral GABA may exert its effects indirectly through the gut-brain axis (such as the vagus nerve and local intestinal effects); 3) Other studies suggest that its effects may come from the placebo effect or its influence on the peripheral nervous system.

[0008] Based on this, the invention initially focused on the characteristic that various lactic acid bacteria can convert glutamate into γ-aminobutyric acid (GABA), aiming to develop probiotic strains that improve sleep and alleviate anxiety by screening high-GABA-producing strains. However, during the functional screening and validation of high-GABA-producing lactic acid bacteria strains in animal models, it was found that the effects of post-biotic inactivation of lactic acid bacteria strains with the same GABA content on promoting sleep were inconsistent. Most high-GABA-producing strains did not show significant sleep-promoting effects from post-biotic inactivation, while a strain of *Lactobacillus rhamnosus* Night-09 showed significant sleep-promoting effects from post-biotic inactivation. Further research revealed that its live bacteria were also effective, and this effect was strain-specific and not entirely dependent on the GABA it produced. Night-09 intervention can simultaneously and effectively improve sleep in animal models of insomnia and anxiety and reduce anxiety levels, providing a new, safe, and long-lasting natural conditioning approach for people with insomnia and anxiety.

[0009] The *Lactobacillus rhamnosus* Night-09 strain provided by this invention, which promotes sleep and improves insomnia and anxiety, was isolated from traditionally fermented cow's milk from Tianshan Ranch, Wusu City, Ili Prefecture, Xinjiang Uygur Autonomous Region. It was deposited on October 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC NO. 36239, and classified as *Lactobacillus rhamnosus*. Lacticaseibacillus rhamnosus .

[0010] The present invention also provides a microbial agent comprising the above-mentioned *Lactobacillus rhamnosus* ( Lacticaseibacillus rhamnosus Night-09 and / or its successors.

[0011] In this invention, metabiotics refer to non-living microorganisms and / or their cell components, which may or may not contain their metabolites.

[0012] The bacterial agent of the present invention can be a bacterial liquid (e.g., live bacterial suspension, bacterial fermentation broth or post-biotic after bacterial fermentation broth inactivation) or a solid freeze-dried powder.

[0013] The microbial agent of the present invention may also contain other microbial strains with similar or different functions. It may be a solid microbial agent or a liquid microbial agent. Those skilled in the art can prepare the microbial agent according to methods known in the art.

[0014] This invention also provides the above-mentioned Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus The use of Night-09 or its antibacterial agents in the preparation of products that improve sleep and / or anxiety.

[0015] This invention has discovered that Lactobacillus rhamnosus Night-09 has a specific neurotransmitter regulation pattern, which can regulate the level of neurotransmitters in the brain, promote the recovery of 5-HT, melatonin and GABA levels, and downregulate glutamate and histamine, reversing the sleep-wake system disorder and mood regulation system imbalance in the pathological state of insomnia and anxiety.

[0016] Specifically, Lactobacillus rhamnosus Night-09 improves sleep and / or anxiety by increasing serotonin levels in the hypothalamus, increasing melatonin levels in brain tissue and / or brain serum, increasing γ-aminobutyric acid levels in the hypothalamus, decreasing glutamate levels in the hypothalamus, and / or decreasing histamine levels in the hypothalamus.

[0017] The present invention also provides a medicament for improving sleep and / or anxiety, comprising the above-mentioned *Lactobacillus rhamnosus* ( Lacticaseibacillus rhamnosus Night-09 or fungicide.

[0018] The medicine of the present invention also includes pharmaceutically acceptable excipients.

[0019] The present invention also provides a food for improving sleep and / or anxiety, comprising the above-mentioned *Lactobacillus rhamnosus* ( Lacticaseibacillus rhamnosus Night-09 or fungicide.

[0020] The Lactobacillus rhamnosus Night-09 of this invention has the effect of improving sleep and can be prepared as a health food.

[0021] The food products of this invention also include food-grade additives acceptable in the food industry.

[0022] Preferably, the food is a dairy product.

[0023] Those skilled in the art can select appropriate excipients to use in combination with Lactobacillus rhamnosus Night-09 or its inoculum, based on the product type, required dosage form, application method, and other common knowledge in the field.

[0024] Preferably, Lactobacillus rhamnosus Night-09 or its agent can be added to dairy products (such as liquid milk, yogurt, milk powder, cheese, etc.) to further enhance their comprehensive effects.

[0025] The beneficial effects of this invention are at least as follows: This invention provides a novel lactic acid bacteria strain, Lactobacillus rhamnosus Night-09, with good safety profile. It can effectively improve insomnia symptoms and anxiety levels in animals with insomnia and anxiety. Both its live bacteria and metabiotics can significantly promote sleep, providing a new, safe, and long-lasting natural conditioning approach for people with insomnia and anxiety, and enriching the germplasm resources of functional probiotics. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 The effects of Night-09 postbiotic or live bacteria intervention on sleep duration in zebrafish.

[0028] Figure 2 The effect of Night-09 live bacteria intervention on sleep in rats with an insomnia and anxiety model.

[0029] Figure 3 To examine the effects of Night-09 live bacteria intervention on motor ability and anxiety-like behavior in rats with an insomnia-anxiety model in an open field experiment.

[0030] Figure 4 To examine the effect of Night-09 live bacteria intervention on anxiety-like behavior in an insomnia-anxiety model rat using the elevated cross maze test.

[0031] Figure 5 The effects of Night-09 intervention on neurotransmitters.

[0032] In all figures (if any), ns represents P > 0.05. This means P < 0.05. This means P < 0.01. This means P < 0.001. This means P < 0.0001. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0035] Example 1. Screening of lactic acid bacteria strains producing high levels of γ-aminobutyric acid (GABA) 1.1 Lactic acid bacteria fermentation 175 strains of lactic acid bacteria derived from traditional fermented foods were streaked onto MRS agar plates for activation. Single colonies were then inoculated into MRS broth and anaerobically cultured at 37°C for 24 h. Three biological replicates were set up for each strain. The bacterial suspension was inoculated at a 10% inoculum into MRS medium containing 0.1% monosodium glutamate (MSG), and aerobic fermentation was carried out for 24 h. After centrifugation at 7000 rpm for 5 min, the supernatant was collected for GABA content detection.

[0036] 1.2 Determination of GABA content in fermentation supernatant by high performance liquid chromatography Transfer 1.0 mL of the fermentation supernatant sample to a 50 mL centrifuge tube, add 10 mL of extraction buffer, sonicate for 30 min, shake for 2 min, centrifuge at 5000 rpm for 8 min, transfer the supernatant to a 25 mL volumetric flask, dilute to volume with water, and shake well for derivatization. Prepare γ-aminobutyric acid (GABA) standard working solutions at concentrations of 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 20.0 mg / L, 50.0 mg / L, and 100.0 mg / L, freshly prepared, and derivatized together with the sample.

[0037] Accurately transfer 1.0 mL of the sample solution and the standard working solution into a 2 mL centrifuge tube. Add 0.2 mL of sodium bicarbonate solution (0.04 g / mL) and 0.4 mL of dansyl chloride derivatizing reagent (2 mg / mL dansyl chloride dissolved in acetonitrile, freshly prepared before use). Mix well and incubate at 70 °C for 20 min. Cool with water and filter through a microporous membrane. Use for high-performance liquid chromatography (HPLC) detection (column: C18, 250 mm × 4.6 mm, 5 μm; mobile phase: 6.8 g / L sodium acetate trihydrate solution and 80% (v / v) acetonitrile solution mixed at a ratio of 65:35; column temperature: 30 °C; flow rate: 1 mL / min; separation time: 20 min; detector wavelength: 216 nm; detector temperature: 30 °C; injection volume: 10 μL). Plot a standard curve based on the results of the standard working solution detection. Determine the concentration of γ-aminobutyric acid in the test solution based on the standard curve according to the sample detection results. The GABA yields of the 14 high-GABA-producing and well-growing strains are shown in Table 1.

[0038] Table 1. Yield of high-GABA-producing strains

[0039] Example 2. Screening sleep-promoting bacterial strains based on a zebrafish model and verifying their sleep-promoting efficacy. 2.1 Screening for sleep-inducing effects using inactivated bacterial solutions (post-biotics) Fourteen experimental bacterial strains screened in Example 1 were streaked onto MRS agar plates for activation. Single colonies were inoculated into MRS broth and anaerobically cultured at 37°C for 24 hours. The bacterial suspension was then inoculated at a 10% inoculation rate into MRS medium containing 0.1% monosodium glutamate and aerobically cultured for 24 hours. The OD of the bacterial suspension was measured. 600 Record the bacterial concentration. The culture medium was then pasteurized at 70°C for 40 min to inactivate the bacteria. The inactivated bacterial solution was mixed and dispensed, and stored at -20°C until it was used in the zebrafish intervention experiment.

[0040] This embodiment uses 4-month-old female zebrafish (type AB). Prior to the experiment, the zebrafish underwent a one-week acclimatization period in a recirculating aquatic system at 27°C and pH 7.0 (±0.5), and were fed twice daily. They were fasted for 24 hours before the formal experiment to ensure the accuracy of the results.

[0041] During the experiment, each zebrafish was kept in a separate black beaker containing 200 mL of water. 1 mL of OD was added to each bacterial strain experimental group. 600The zebrafish were treated with a sterilized bacterial solution of 1:1, followed by a positive control group (melatonin group) with added melatonin to achieve a final concentration of 10 μmol / L. The blank control group (control group) consisted of water. Each group had four replicates. At the start of the experiment, each zebrafish was isolated in a beaker for 30 minutes to allow them to acclimatize to the new environment. After the acclimatization period, the beakers were placed in the Noldus behavioral observation system for two consecutive hours of sleep behavior observation. The observation process employed two cycles of 30 minutes of light followed by 30 minutes of darkness to simulate the diurnal rhythm. After the experiment, the sleep behavior data of the zebrafish were statistically analyzed and calculated using the JavaScript event function of Ethovision v17.5 software. "Total sleep duration" was defined as the sum of the durations of all sleep events (speed less than 1 cm / s and duration exceeding 6 seconds) within the test period, reflecting the overall amount of sleep accumulated. "Average single sleep duration" was defined as the average duration of all sleep events.

[0042] Through postbiotic intervention and sleep behavior monitoring of the 14 experimental bacterial strains mentioned above in zebrafish, it was found that not all high-GABA-producing strains had a sleep-inducing effect, nor did all *Lactobacillus rhamnosus* strains have a sleep-inducing effect. Specifically, it was found that zebrafish treated with one *Lactobacillus rhamnosus* strain, Night-09, had significantly longer sleep duration than the control group, while other strains did not have this effect. This indicates that the postbiotic solution of Night-09 inactivated bacterial solution has the effect of promoting sleep in zebrafish, and this effect is strain-specific and not entirely dependent on the GABA produced.

[0043] 2.2 Evaluation of the efficacy of live Lactobacillus rhamnosus Night-09 bacteria and post-biotics in promoting sleep in zebrafish The Night-09 strain was activated by streaking onto MRS agar plates, and single colonies were inoculated into MRS broth and anaerobically cultured at 37°C for 24 h. The bacterial suspension was then inoculated into MRS medium at a 10% inoculum and aerobically cultured for 24 h. The OD of the bacterial suspension was measured. 600 Record the bacterial concentration. Mix the culture medium thoroughly, centrifuge half of the culture medium at 5000 rpm for 8 min, resuspend the viable bacterial pellet in PBS, and measure the OD of the resuspended solution. 600 And adjust the concentration to OD 600 =1, immediately used for zebrafish intervention experiments (Night-09 live bacteria). The other half of the culture medium was pasteurized at 70℃ for 40 min to inactivate the bacteria, and the concentration was adjusted to OD. 600 =1, used for zebrafish intervention experiment (Night-09 post-genetic agent).

[0044] The zebrafish experimental methods, conditions, and control group setup were the same as described in Section 2.1, with six replicates per group. Since the Night-09 live bacteria intervention group did not show a sleep-promoting effect within a 2-hour timeframe, the intervention period was extended to 24 hours (2-hour sleep behavior observation was conducted from hour 23 to 24). Experimental results are shown below. Figure 1 Statistical analysis of the data results was performed using the Kruskal-Wallis nonparametric test and Dunn's multiple comparison test. Two hours after intervention with Night-09 inactivated bacterial solution, the total sleep duration of zebrafish significantly increased (p<0.01 compared to the control group) and the average duration of a single sleep episode (p<0.05 compared to the control group), achieving the same effect as the melatonin group (p>0.05). Two hours of intervention with Night-09 live bacteria had no significant sleep-promoting effect, while 24 hours of intervention significantly increased the total sleep duration of zebrafish (p<0.0001 compared to the control group) and the average duration of a single sleep episode (p<0.01 compared to the control group).

[0045] Example 3. Study on the efficacy of Lactobacillus rhamnosus Night-09 in improving insomnia and anxiety. 3.1 Construction and intervention of animal models of insomnia and anxiety Thirty-two 7-week-old male SD rats were randomly divided into four groups (n=8 per group): a normal control group, a model control group, a Night-09 intervention group, and a positive drug control group (melatonin group). The rats were ear-tagged. The animals were housed at 24±2℃ and 55±10% relative humidity, maintaining a 12-hour light-dark cycle. During the experiment, bedding and water were changed twice a week, and feed was changed daily. The rats were weighed regularly, and their weight was recorded to observe any differences in weight changes among the groups. The rats' daily activity, diet, and water intake were also observed.

[0046] After a week of acclimatization, all animals underwent EEG and EMG electrode implantation surgery: all rats underwent aseptic surgery to implant EEG and EMG electrodes, and immediately after the operation, they were injected intraperitoneally with sodium penicillin for 3 consecutive days. After a week of recovery, modeling and intervention experiments were conducted.

[0047] Model establishment: For three consecutive days, rats in the model control group, Night-09 intervention group and positive drug control group were injected intraperitoneally with chlorophenylalanine (PCPA) (400 mg / kg body weight) at a fixed time (8:00 am) to establish the model. The normal control group was injected with an equal volume of solvent.

[0048] After modeling, for 7 consecutive days, all groups were administered gavage intervention at a fixed time (8:00 AM) each day: the normal group and the model group were administered physiological saline (1 ml / 100g body weight); the Night-09 group was administered Lactobacillus rhamnosus Night-09 bacterial suspension (1×10⁶ viable bacteria count) resuspended in physiological saline. 10 CFU / kg body weight); positive drug group received melatonin by gavage (10 mg / kg body weight).

[0049] 3.2 Sleep monitoring based on EEG and EMG After the modeling and intervention experiments, sleep monitoring was conducted on each group of animals. Before monitoring, the animals were placed in plexiglass boxes to acclimatize to the environment for more than 24 hours. Subsequently, the EEG / EMG base and signal transmission line were connected, and the signal was amplified. Monitoring data from ZT0 to ZT24 were collected in the software Sirenia Acquisition (Pinnacle Technology, Michigan, USA), including 0.5-49Hz EEG signals and 10-100Hz EMG signals. After monitoring, data analysis and statistics were performed using Sirenia SleepPro (Pinnacle Technology, Michigan, USA) software (version 3.2.2), with 10 seconds as an analysis unit, to determine the wakefulness, non-rapid eye movement (NREMS) sleep, and rapid eye movement (REMS) sleep states. The characteristics of awakening are high-amplitude EMG and low-amplitude EEG signals; the characteristics of NREMS are low-amplitude EMG and high-amplitude EEG signals, with delta waves (0.5-4Hz) dominating; the characteristics of REMS are low-amplitude EMG and low-amplitude EEG signals, with theta waves (4-8Hz) dominating.

[0050] Statistical analysis of the data results was performed by first using the Shapiro-Wilk test to determine the normality of the data, followed by the One-way ANOVA test and Turkey's multiple comparison test to determine the statistical significance of differences between groups. GraphPadPrism 10.6.0 software was used for analysis and plotting. All data are expressed as mean ± standard deviation.

[0051] The results are as follows Figure 2As shown, PCPA modeling prolonged sleep latency by approximately 3 times in the model group (p<0.01 compared to the blank control group), while melatonin intervention significantly reduced sleep latency (p<0.01 compared to the model group), reaching the level of the normal group (p>0.05), indicating the successful establishment of the insomnia model. Night-09 intervention also significantly reduced sleep latency in rats by approximately 2.6 times (p<0.01 compared to the model group), reaching similar levels to the normal group (p>0.05) and the melatonin group (p>0.05). This indicates that Night-09 intervention can effectively improve sleep onset difficulties in insomnia-prone animals.

[0052] 3.3 Behavioral experiments to assess anxiety levels 3.3.1 Open Field Experiment The open field test is a classic behavioral experimental method for assessing an animal's capacity for autonomous activity and anxiety-like behavior. It reflects the animal's anxiety level by observing and recording the distance and duration of movement in the central area of ​​the open field; lower values ​​indicate higher anxiety. Three hours before the experiment, the test rats were moved to the behavioral laboratory to acclimatize after modeling and intervention. A 50×50cm open field box (Hunan Baizhong Biotechnology Co., Ltd.) was used, divided into nine 3×3 grids, with the central grid defined as the central area. During the experiment, the test rats were gently placed in the center of the open field box with their backs to the experimenter, allowing them to explore freely for 5 minutes. The exploration trajectory was recorded by a camera and analyzed using a behavioral tracking system. After each round of exploration, the area was thoroughly cleaned with 75% ethanol to eliminate any residual odor, and the next animal was tested only after the alcohol had completely evaporated.

[0053] Statistical analysis of the data results was performed by first using the Shapiro-Wilk test to determine the normality of the data, followed by the One-way ANOVA test and Turkey's multiple comparison test to determine the statistical significance of differences between groups. GraphPadPrism 10.6.0 software was used for analysis and plotting. All data are expressed as mean ± standard deviation.

[0054] Experimental results are as follows Figure 3As shown, there was no significant difference in the total distance traveled in the open field among the experimental groups, suggesting that the serotonin (5-HT) depletion induced by PCPA modeling did not significantly affect the rats' motor ability. The model group showed a significant reduction in distance and time traveled in the central area of ​​the open field compared to the normal group, approximately 6.3-fold and 11.9-fold, respectively, indicating that PCPA modeling significantly increased the rats' anxiety levels. Night-09 intervention significantly increased the distance and time traveled in the central area of ​​the open field (compared to the model group, p<0.05), with increases of approximately 2.3-fold (distance) and 7.2-fold (time), indicating that Night-09 intervention can effectively alleviate anxiety symptoms.

[0055] 3.3.2 Elevated Cross Maze Experiment The elevated cross maze test is a classic behavioral test for assessing anxiety-like behavior in rodents. The elevated cross maze is 50 cm above the ground and consists of two open arms (50 cm long and 10 cm wide) without side rails and two closed arms (50 cm long, 10 cm wide, and 30 cm high) with high side rails. The number of times an animal enters the open arms and the duration of its stay reflect its anxiety level; lower values ​​indicate higher anxiety. Three hours before the experiment, rats are transferred to the behavioral laboratory to acclimatize after modeling and intervention. During the experiment, the rats are gently placed on the central platform of the elevated cross maze with their heads facing the open arms and released to explore freely for 5 minutes. The exploration trajectory is recorded by a camera and analyzed using a behavioral tracking system. After each round of exploration, the area is thoroughly cleaned with 75% ethanol to eliminate any residual odor, and the next animal is tested only after the alcohol has completely evaporated.

[0056] Statistical analysis of the data results was performed by first using the Shapiro-Wilk test to determine the normality of the data, followed by the One-way ANOVA test and Turkey's multiple comparison test to determine the statistical significance of differences between groups. GraphPadPrism 10.6.0 software was used for analysis and plotting. All data are expressed as mean ± standard deviation. The number of times the experimental open arm was entered and the dwell time in the open arm are presented as percentages, calculated using the following formulas: Open arm entry count (percentage) = (Number of times the test animal entered the open arm / Total number of times the test animal entered the open and closed arms) × 100%; Open arm dwell time (percentage) = (Dwell time of test animal in open arm / Total dwell time) × 100%.

[0057] Experimental results are as follows Figure 4As shown, the number of open arm entry attempts and the open arm dwell time in the model group were significantly reduced by approximately 3.5 times and 5.6 times, respectively, compared to the normal group. However, Night-09 intervention significantly increased the number of open arm entry attempts and the open arm dwell time in rats (p<0.001 compared to the model group), with an increase of approximately 2.8 times (number of open arm entry attempts) and 7.7 times (open arm dwell time), indicating that Night-09 intervention can improve anxiety levels.

[0058] 3.4 Neurotransmitter testing reveals mechanisms for improving insomnia and anxiety. To explore the neurobiological basis of Night-09 intervention in improving insomnia and anxiety, this study measured the hypothalamic tissue and serum neurotransmitter levels of rats in each group after modeling and intervention.

[0059] 3.4.1 Sample preparation Accurately weigh the rat hypothalamus and cortex. Add 50 μL of ultrapure water to the hypothalamus tissue and 100 μL of ultrapure water to the cortex tissue. Homogenize the tissues separately using an ultrasonic homogenizer in an ice-water bath. Take 30 μL of the homogenate and add 50 μL of mass spectrometry-grade acetonitrile containing 0.2% formic acid (containing acetaminophen as an internal standard). Vortex thoroughly and incubate on ice for 30 min to allow complete protein precipitation. Centrifuge at 21000g, 4℃ for 20 min, and transfer the supernatant to a sample vial for analysis. The density of rat brain tissue is 1 g / mL. Therefore, the total volume of the hypothalamus or cortex homogenate is the sum of the mass of added ultrapure water and the weight of the brain tissue divided by 1 g / mL.

[0060] 3.4.2 Enzyme-linked immunosorbent assay (ELISA) The levels of melatonin in rat hypothalamic serum and histamine (HA) in the cortex were detected using enzyme-linked immunosorbent assay (ELISA). Hypothalamic melatonin, γ-aminobutyric acid (GABA), glutamate (Glu), and serotonin (5-HT) were also detected using ELISA. The experimental procedures were performed according to the kit instructions in Table 2 below.

[0061] Table 2

[0062] Statistical analysis of the data results was performed by first using the Shapiro-Wilk test to determine the normality of the data, followed by the One-way ANOVA test and Turkey's multiple comparison test to determine the statistical significance of differences between groups. GraphPadPrism 10.6.0 software was used for analysis and plotting. All data are expressed as mean ± standard deviation.

[0063] Test results as follows Figure 5As shown, compared with the normal control group, the hypothalamic 5-HT level in the model group rats was significantly lower (p<0.001), indicating that PCPA, as a tryptophan hydroxylase inhibitor, depleted brain 5-HT by inhibiting 5-HT synthesis. After Night-09 intervention, the 5-HT level significantly rebounded compared with the model group (p<0.05), recovering to 80.8% of the normal level. The positive control melatonin also significantly increased the 5-HT level (p<0.01), with similar effects to the Night-09 group (p<0.05). 5-HT is a precursor to melatonin, the core hormone for sleep regulation. Melatonin shows a consistent trend in brain tissue and serum: the melatonin content in the brain tissue of the model group was significantly lower than that of the control group (p<0.0001), with a decrease of more than 60%, indicating that PCPA severely inhibits the melatonin synthesis pathway. After Night-09 intervention, the melatonin level in the brain significantly rebounded (p<0.01), recovering to 71.0% of the normal level, which was comparable to the effect of direct melatonin intervention (recovering to 77.7% of the normal level) (p>0.05 compared with the melatonin group). Serum melatonin levels in the model group were also significantly lower than those in the normal group (p<0.0001), while both the Night-09 group and the melatonin group significantly increased serum melatonin levels (p<0.0001 and p<0.001), recovering to 94.1% and 92.3% of the normal level, respectively.

[0064] Hypothalamic GABA levels showed a similar trend. The model group exhibited a significantly lower level of GABA compared to the normal group (p<0.0001), while both the Night-09 and melatonin groups significantly reversed GABA depletion (p<0.05 and p<0.01), restoring levels to 84.7% and 86.9% of normal, respectively. This suggests that Night-09 has the potential to enhance GABAergic neurotransmission in the hypothalamus of insomniac rats. Hypothalamic glutamate (Glu) analysis showed that the model group had a significantly higher Glu level than the normal group (p<0.001), while both the Night-09 and melatonin groups significantly reduced Glu levels (both p<0.05), restoring levels to 63.7% and 63.5% of normal, respectively. This suggests that Night-09 may improve the excitability imbalance in insomnia by regulating glutamatergic activity. Histamine (HA), a key neurotransmitter in the arousal system, was significantly elevated in the model group compared to the normal group (p<0.0001), reflecting overactivation of the arousal system after PCPA depletion of 5-HT. Both the Night-09 group and the melatonin group significantly inhibited the increase of HA (both p<0.05), restoring it to 77.2% and 77.9% of the normal level, respectively. This indicates that Night-09 can inhibit the overactive histaminergic system and help restore sleep-wake balance.

[0065] The above results indicate that Night-09 intervention, through multi-target and multi-system synergistic effects, reverses the depletion of 5-HT, melatonin, and GABA in insomnia-anxious animals, inhibits the overactive glutamatergic and histaminergic systems, and corrects sleep-wake system disorders and mood regulation system imbalances, thereby achieving the dual effects of anti-anxiety and sleep promotion.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus Night-09, characterized in that, The accession number is CGMCC NO. 36239.

2. A microbial agent, characterized in that, Including the Lactobacillus rhamnosus described in claim 1 ( Lacticaseibacillus rhamnosus Night-09 and / or its successors.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is a bacterial liquid or a solid freeze-dried powder.

4. The *Lactobacillus rhamnosus* as described in claim 1 ( Lacticaseibacillus rhamnosus The use of Night-09 or the microbial agent according to claim 2 or 3 in the preparation of products that improve sleep and / or anxiety.

5. The application according to claim 4, characterized in that, Improve sleep and / or anxiety by increasing serotonin levels in the hypothalamus, increasing melatonin levels in brain tissue and / or brain serum, increasing GABA levels in the hypothalamus, decreasing glutamate levels in the hypothalamus, and / or decreasing histamine levels in the hypothalamus.

6. A medicine for improving sleep and / or anxiety, characterized in that, Including the Lactobacillus rhamnosus described in claim 1 ( Lacticaseibacillus rhamnosus Night-09 or the microbial agent as described in claim 2 or 3.

7. The drug according to claim 6, characterized in that, It also includes pharmaceutically acceptable excipients.

8. A food product for improving sleep and / or anxiety, characterized in that, Including the Lactobacillus rhamnosus described in claim 1 ( Lacticaseibacillus rhamnosus Night-09 or the microbial agent as described in claim 2 or 3.

9. The food product according to claim 8, characterized in that, It also includes food-grade additives.

10. The food product according to claim 8 or 9, characterized in that, The food product in question is a dairy product.

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