Lactobacillus plantarum PB13 and its application in improving sleep function

Abstract

This invention belongs to the field of microbial technology, specifically relating to *Lactobacillus plantarum* PB13 and its application in improving sleep function. This invention provides a *Lactobacillus plantarum* PB13 strain, whose taxonomic name is... Lactobacillus plantarum The preservation number is GDMCC No: 67088. This invention also provides the application of *Lactobacillus plantarum* PB13 in the preparation of a drug for improving sleep, wherein improving sleep includes shortening sleep latency and prolonging sleep duration.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to Lactobacillus plantarum PB13 and its application in improving sleep function. Background Technology

[0002] In modern society, sleep problems have become a widespread concern. A significant number of people suffer from insomnia, poor sleep quality, and staying up late, which can lead to the following far-reaching harms: In the short term, it can cause fatigue, memory loss, and irritability the next day, thus affecting work and study efficiency; in the long term, it can lead to a weakened immune system and significantly increase the risk of depression, anxiety, cardiovascular disease, and Alzheimer's disease. Furthermore, sleep problems are a major cause of accidents such as traffic accidents. Existing sleeping pills have significant limitations; common side effects include drowsiness, dizziness, and memory loss the next day. More seriously, long-term use of sleeping pills can easily lead to drug tolerance, resulting in decreased efficacy and the need for constantly increasing dosages; it can also lead to physiological and psychological dependence, and withdrawal symptoms and rebound insomnia may occur upon discontinuation, treating only the symptoms, not the root cause.

[0003] Improving sleep has always been a goal for many, and probiotics offer a novel and promising approach. These special probiotics contain high levels of GABA (gamma-aminobutyric acid) and 5-HT, two neurotransmitters that play a crucial role in regulating mood and sleep.

[0004] GABA is an inhibitory neurotransmitter that reduces excitatory signals in the brain, helping to relax tense emotions and lower anxiety levels. With busy and stressful days, many people find it difficult to relax at night, which can lead to difficulty falling asleep. High-GABA-producing probiotics can help calm the brain, creating a more ideal environment for sleep. On the other hand, serotonin (5-HT) is a precursor to dopamine, crucial for mood stability and emotional balance. Insufficient serotonin levels can lead to mood swings and feelings of depression, which can also negatively impact sleep quality. By consuming high-serotonin probiotics, people can increase their body's serotonin levels, improve mood, and consequently improve sleep.

[0005] Research suggests that psychoactive probiotics can provide sleep-improving benefits by interacting with the body's gut microbiota. This interaction makes it easier for the body to absorb key neurotransmitter precursors, thereby improving mental health and sleep. Psychoactive probiotics may be a promising approach to improving sleep and emotional health by providing high levels of GABA and 5-HT to help people better manage stress, anxiety, and mood swings, thus enjoying deeper, more restorative sleep.

[0006] Invention CN121825807A, entitled "A strain of *Lactobacillus plantarum* and its application in the preparation of γ-aminobutyric acid and improvement of sleep," discloses that this strain not only has good acid and bile salt tolerance but also produces GABA. The fermentation supernatant of *Lactobacillus plantarum* XBMU-SN-23 (containing GABA) can significantly improve sleep quality in insomnia-prone mice by upregulating GABA, 5-HT, IL-1, and MT, downregulating Glu and DA, and regulating the expression of 5-HT1A receptor, GABAARα1, and GABAARγ2 receptor proteins, thereby modulating the neurotransmitter system and improving sleep quality. However, the effect of *Lactobacillus plantarum* XBMU-SN-23 on improving insomnia in mice is not ideal, possibly due to the strain's lack of intestinal adhesion and colonization capabilities.

[0007] The invention CN 119859598 A, entitled "A strain of Lactobacillus plantarum B534 and its application in the preparation of functional products that help improve sleep and regulate mood," states that using a zebrafish insomnia model induced by the GABA inhibitor PTZ, Lactobacillus plantarum B534 can help improve sleep, but the amount and duration of arousal activity are still significantly higher than those in the normal control group.

[0008] Invention CN 120082467 A, entitled "A strain of *Lactobacillus plantarum* and its application in the preparation of products relieving insomnia," discloses that *Lactobacillus plantarum* GD 06, under the model conditions of 6 mg PCPA / 20 g mice, has a bacterial suspension of 0.8 mL / 30 g mice, and the bacterial suspension concentration is 2.5 × 10⁻⁶. 8 CFU / mL can alleviate insomnia symptoms in mice, but the sleep time is significantly lower than that of the control group (NC group). Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus plantarum PB13 and its application in improving sleep function.

[0010] To solve the above-mentioned technical problems, the present invention provides a *Lactobacillus plantarum* PB13 strain, the taxonomic name of which is... lactiplantibacillus plantarum The accession number is GDMCC No: 67088.

[0011] The present invention also provides the application of *Lactobacillus plantarum* PB13 in the preparation of drugs for improving sleep: improving sleep includes shortening sleep latency and prolonging sleep duration.

[0012] As an improvement to the application of the present invention: improving sleep also includes increasing central 5-HT and GABA levels.

[0013] The beneficial effects of this invention are mainly reflected in the following aspects: *Lactobacillus plantarum* PB13 can significantly improve various sleep problems, including shortening sleep latency (shortening sleep latency), prolonging total sleep duration, and alleviating insomnia symptoms. As a probiotic, *Lactobacillus plantarum* PB13 can also exert other health benefits while improving sleep; for example, it enhances its survival and colonization ability in the gastrointestinal tract to better exert its effects. In addition, *Lactobacillus plantarum* PB13 can also help regulate the balance of intestinal flora and may also have benefits such as enhancing immunity. Attached Figure Description

[0014] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0015] Figure 1 This is a colony diagram of *Lactobacillus plantarum* PB13. Figure 2 The adhesion results of Lactobacillus plantarum PB13; Figure 3 The number of sleeping mice in different experimental groups; Figure 4 The sleep latency of different experimental groups; Figure 5 Total sleep duration for different experimental groups; Figure 6 Central 5-HT and GABA levels in different experimental groups. Detailed Implementation

[0016] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: Example 1: Isolation, culture and identification of Lactobacillus plantarum PB13 Pickled vegetables from Guizhou were used as samples. After serial dilution with physiological saline, the samples were plated on MRS medium and incubated at 37°C. Single colonies were picked and expanded. Sequencing and homology comparison were performed to obtain *Lactobacillus plantarum* PB13. The 16S nucleotide sequence of *Lactobacillus plantarum* PB13 is shown in SEQ ID NO.1.

[0017] Morphological identification of *Lactobacillus plantarum* PB13: Gram-positive, non-spore-forming, short rod-shaped or elliptical cylindrical, with blunt, rounded ends. Colonies are milky white to pale yellow, opaque, with a smooth, moist surface, regular edges, and slightly papillary margins. Figure 1 As shown.

[0018] The preservation information of *Lactobacillus plantarum* PB13 of the present invention is as follows: Collection Name: lactiplantibacillus plantarumDepository: Guangdong Provincial Center for Microbial Culture Collection, Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Date of deposit: October 13, 2025, Accession number: GDMCC No: 67088.

[0019] Example 2: Determination of the acid and bile salt tolerance of Lactobacillus plantarum PB13 I. Acid resistance test: The activation (activation expansion) of Lactobacillus plantarum PB13 was carried out by inoculating the bacterial culture of Lactobacillus plantarum PB13 into MRS liquid medium at a volume ratio of 1:10 and culturing at 37°C for 24 hours.

[0020] The activated and expanded *Lactobacillus plantarum* PB13 bacterial suspension was inoculated into MRS liquid medium at a rate of 1% (v / v). After incubation at 37°C until the logarithmic growth phase, the bacterial cells were collected by centrifugation for 5 min. The cells were washed twice with phosphate-buffered saline (pH 6.8), and then resuspended in MRS liquid medium at pH 3.0 and pH 1.0, respectively. The initial viable count was corrected to approximately 10⁻⁶. 8 CFU / mL, incubated at 37℃ for 3 h. Viable bacteria in the 0 h and 3 h samples were counted using the plating method, and their survival rate was determined (after incubation at 37℃ for 48 h). The survival rate was calculated using the following formula:

[0021] N0 represents the viable cell count (CFU / mL) of the test strain at 0 h; N t The number of viable bacteria (CFU / mL) of the test strain after 3 hours.

[0022] Table 1 Results of acid tolerance of the strains

[0023] Note: After culturing strain B534 at pH=1 for 3 hours, the survival rate was less than 30%.

[0024] II. Determination of tolerance to bile salts (sodium taurocholate) The activated and expanded *Lactobacillus plantarum* PB13 bacterial suspension was inoculated into MRS liquid medium at a rate of 1% (v / v), and cultured at 37°C until the logarithmic growth phase. The medium was then vortexed to adjust the initial viable count to approximately 10⁻⁶. 8 CFU / mL. 10% (v / v) of the samples were inoculated into MRS liquid medium containing 0.3% and 0.5% (m / v) ox bile salts, respectively, with MRS liquid medium without ox bile salts as a control. The samples were incubated at 37°C for 3 hours. The viable bacterial count was then determined using the plating method to assess the survival rate (after 3 hours of incubation at 37°C). The survival rate was calculated using the following formula:

[0025] N0 represents the viable cell count (CFU / mL) of the test strain at 0 h; N t The number of viable bacteria (CFU / mL) of the test strain after 3 hours.

[0026] Table 2 Results of strain tolerance to bile salts

[0027] Example 3: Determination of the adhesion ability of Lactobacillus plantarum PB13 1) Caco-2 cells (human colorectal adenocarcinoma cells) were passaged to the third generation. After digestion with 0.25% trypsin (containing EDTA), the cells were centrifuged at 1000 rpm for 5 min, and then resuspended in 5 mL of DMEM medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and 100 mg / mL streptomycin) until the cells were in a single-cell suspension. An appropriate amount of cells was counted using a hemocytometer, and the cells were diluted to a concentration of 1×10⁻⁶ cells in DMEM medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and 100 mg / mL streptomycin). 6 1 mL of the sample was seeded into a 24-well cell culture plate containing cell spreaders and cultured at 37°C with 5% CO2 for 2 days.

[0028] 2) The *Lactobacillus plantarum* PB13 strain was activated for three generations, and 3 mL of the bacterial culture was taken out to determine the OD value of the bacterial culture. 600 Collect bacterial cells by centrifugation at 4000 g for 10 min at room temperature, resuspend in 5 mL of DMEM complete medium (without antibiotics) containing 10% fetal bovine serum, and dilute to a bacterial concentration of 2 × 10⁻⁶. 8 CFU / mL.

[0029] Take 1 mL of the bacterial strain dilution and inoculate it into a 24-well cell culture plate containing cell spreaders obtained in step 1). Incubate at 37°C with 5% CO2 for 2 h, with at least two replicates for each sample. After incubation, slowly aspirate the culture medium, wash three times with PBS, and fix with 100% methanol for 8 min. Remove the cell spreaders and let them stand for 20 min, then Gram-stain and mount with neutral resin. Observe under a light microscope, selecting at least 20 fields of view for each bacterial species, counting and taking the average value. Results are as follows: Figure 2 As shown: Lactobacillus plantarum PB13 can adhere to Caco-2 cells.

[0030] It should be noted that Lactobacillus plantarum B534 does not demonstrate the ability to adhere to Caco-2 cells.

[0031] The results of Examples 2 and 3 above show that PB13 can survive in the extreme environment of the gastrointestinal tract with a high survival rate and has a strong ability to adhere and colonize in the intestine.

[0032] Example 4: Establishment of a mouse insomnia model and intervention with PB13 (1) Preparation of WT mice: Forty male BALB / c mice (20.05±0.20 g, 8 weeks old) were purchased. The mice were housed in a clean environment with a room temperature of 26±0.5°C, humidity of 50-60%, and a 12 / 12h day / night cycle. After one week of acclimatization, the mice were randomly divided into three groups: a control group, a model group, and a probiotic group (Lactobacillus plantarum PB13).

[0033] (2) Intestinal colonization of Lactobacillus plantarum PB13: Lactobacillus plantarum PB13 was inoculated into liquid MRS medium and incubated statically at 37°C for 20–24 h. The cells were then collected by centrifugation at 8000 rpm for 5 min, washed twice with physiological saline, and resuspended in physiological saline to a final concentration of approximately 1 × 10⁻⁶. 9 CFU / mL.

[0034] PB13 intervention group (probiotic group): WT mice were treated by gavage with 200 μL of bacterial solution per mouse, repeated daily for 2 weeks. After 2 weeks of gavage treatment, an insomnia model was established in the mice. Insomnia was induced by intraperitoneal injection of p-chlorophenylalanine PCPA (350 mg / kg, 0.2 mL) for 2 consecutive days. During the insomnia model induction period (2 days), the PB13 intervention group (probiotic group) continued to be gavaged with the same amount of bacterial solution daily.

[0035] Model group: The 200 μL bacterial solution administered by gavage was replaced with the same volume of physiological saline (200 μL), and the rest was the same as the PB13 intervention group. That is, during the insomnia model induction period, the model group was still administered the same amount of physiological saline by gavage every day.

[0036] Control group: Induction of insomnia was cancelled, that is, the intraperitoneal injection of p-chlorophenylalanine PCPA in mice was cancelled, and the rest was the same as the model group.

[0037] On day 17 of the experiment, all mice in the PB13 intervention group, model group, and control group were intraperitoneally injected with sodium pentobarbital (42 mg / kg, 0.2 mL) to conduct a sleep quality test. The sleep latency and total sleep duration of the mice were recorded.

[0038] After the total sleep duration was recorded (i.e., after the mice woke up), brain tissue was collected from the mice to detect central 5-HT and GABA levels. Specifically, the mice were euthanized by cervical retraction and immediately decapitated. The skull was rapidly dissected and the whole brain was removed under ice bath conditions. The entire process was performed on ice to slow down neurotransmitter degradation. After adding 10 mL of PBS, the brain tissue was homogenized using a tissue homogenizer. After homogenization, the tissue was centrifuged, and the supernatant was collected as the test sample. Then, the central 5-HT and GABA levels in the brain tissue of each group of mice were detected using an ELISA kit.

[0039] like Figure 3 As shown, there was little difference in the number of mice falling asleep between the model group and the PB13 intervention group compared to the control group.

[0040] according to Figure 4 Compared with the control group, the sleep latency of mice in the model group was significantly increased (P < 0.0001); compared with the model group, the sleep latency of mice in the PB13 intervention group was significantly decreased (P < 0.05); indicating that Lactobacillus plantarum PB13 has a good sleep-aiding effect.

[0041] The sleep latency period refers to the time it takes for a mouse to enter sleep, that is, from the time the mouse receives an intraperitoneal injection of sodium pentobarbital until the righting reaction disappears.

[0042] according to Figure 5 Compared with the control group, the total sleep duration of mice in the model group was significantly reduced (P<0.05); compared with the model group, the total sleep duration of mice in the PB13 intervention group was significantly restored (P<0.05); this indicates that Lactobacillus plantarum PB13 can effectively alleviate sleep deprivation caused by insomnia in mice.

[0043] Total sleep duration refers to the period from when a mouse enters sleep until it wakes up, that is, from when the righting reflex disappears until the righting reflex returns.

[0044] according to Figure 6 It was found that, compared with the control group, the central 5-HT and GABA levels in the model group mice were significantly decreased, while the central GABA level in the PB13 intervention group mice was significantly restored (P<0.05).

[0045] (3) Conclusion Based on the above results, it can be concluded that the sleep condition of mice was improved after treatment with Lactobacillus plantarum PB13.

[0046] Comparative experiment: Lactobacillus plantarum XBMU-SN-23, Lactobacillus plantarum B534, and Lactobacillus plantarum GD 06 were subjected to corresponding experiments according to the methods described in this invention: The central GABA reversion ability of *Lactobacillus plantarum* XBMU-SN-23, *Lactobacillus plantarum* B534, and *Lactobacillus plantarum* GD 06 was significantly lower than that of *Lactobacillus plantarum* PB13 of the present invention. Testing showed that the GABA content of these three strains was significantly lower than that of the control group (slightly higher than that of the model group); while the GABA content of the PB13 intervention group of the present invention was higher than that of the control group.

[0047] Furthermore, although the total sleep duration of Lactobacillus plantarum XBMU-SN-23, Lactobacillus plantarum B534, and Lactobacillus plantarum GD 06 was higher than that of the model group, it was significantly lower than that of the control group; while the total sleep duration of the PB13 intervention group of the present invention was significantly higher than that of the control group.

[0048] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Lactobacillus plantarum PB13, characterized in that: The taxonomic name of strain PB13 is lactiplantibacillus plantarum The accession number is GDMCC No: 67088.

2. The use of *Lactobacillus plantarum* PB13 as described in claim 1 in the preparation of a drug for improving sleep, characterized in that: Improving sleep includes shortening sleep latency and extending sleep duration.

3. The application according to claim 2, characterized in that: Improving sleep also includes increasing central 5-HT and GABA levels.

Images