A probiotic composition and uses thereof

Abstract

The present application relates to the field of microorganisms, in particular to a probiotic composition and application thereof. The probiotic composition comprises Lactobacillus plantarum 3 strain with the preservation number of CGMCC No. 19523 and Lactobacillus plantarum YIT2004 strain with the preservation number of CGMCC No. 39600. The probiotic composition provided by the present application realizes synergistic effect in helping to improve sleep quality, can not only effectively shorten sleep latency, improve sleep effect and prolong sleep time, but also can up-regulate GABA level in the brain, so as to play a role in helping to improve sleep state and relieve nerves. Compared with existing clinical anxiolytic chemical drugs, the probiotic composition of the present application is not only high in safety, but also not easy to produce dependence, is suitable for daily conditioning and intervention of people with poor sleep and insomnia, and makes up for the defects of traditional anxiolytic drugs which are limited in long-term application.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically, to a probiotic composition and its application. Background Technology

[0002] With rapid socio-economic development and an increasingly fast pace of life, sleep disorders have become a prevalent public health problem worldwide. Insomnia is no longer limited to specific age groups; its prevalence among young and middle-aged adults and adolescents is showing an upward trend year by year. It is often closely linked to and exacerbates emotional problems such as anxiety and depression, severely reducing people's quality of life and affecting their daily work and study efficiency.

[0003] Sleep occupies nearly one-third of a person's life, playing an irreplaceable role in fatigue repair, memory consolidation, metabolic homeostasis maintenance, and the regulation of multiple physiological functions. Current research confirms that quality sleep helps reduce the risk of various chronic diseases; while chronic sleep deprivation impairs cognitive abilities, emotional regulation, immune balance, and reproductive health, causing multiple negative impacts on physical and mental functions. Insomnia, the most common type of sleep disorder in clinical practice, is typically characterized by prolonged sleep latency, difficulty maintaining sleep, early awakening, and a decline in overall sleep quality. Epidemiological data shows that approximately 30% of adults worldwide experience varying degrees of sleep abnormalities, with 6-10% meeting the clinical diagnostic criteria for insomnia. Sleep disorders not only easily induce or aggravate various psychosomatic illnesses such as anxiety, depression, and diabetes, but can also cause functional damage to brain tissue. The brain is a high-energy-consuming organ, and its physiological functions are highly dependent on normal mitochondrial energy metabolism. Existing research indicates that insomnia can cause damage and dysfunction of mitochondrial structure in brain tissue, further inducing oxidative stress, promoting excessive accumulation of reactive oxygen species (ROS), and ultimately exacerbating damage and functional decline of brain nerve cells.

[0004] Currently, clinical interventions for insomnia primarily rely on chemical sedative-hypnotic drugs. However, long-term use can easily lead to neurological damage, drug dependence, drug tolerance, and other toxic side effects, limiting their application and long-term safety. Therefore, developing safe, gentle, and stable-acting sleep improvement and insomnia intervention programs with no significant side effects has significant clinical application value and meets real market demand. Summary of the Invention

[0005] This invention first provides a probiotic composition comprising *Lactobacillus plantarum* with accession number CGMCC No. 19523. Lactobacillus plantarum LP3 strain and Lactobacillus plantarum with accession number CGMCC No. 39600 were also tested. Lactiplantibacillus plantarum )YIT2004 strain (abbreviated as LPYIT2004).

[0006] In this invention, *Lactobacillus plantarum* (Lactobacillus plantarum Strain 3 was deposited on March 30, 2020, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 19523. This strain has also been published in Chinese patent application CN202310020827.8.

[0007] In this invention, *Lactobacillus plantarum* ( Lactiplantibacillus plantarum YIT2004, taxonomically named Lactiplantibacillus plantarum It was deposited on July 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 39600.

[0008] The present invention also provides the use of the probiotic composition described herein in helping to improve or assist in improving sleep.

[0009] The present invention also provides the use of the probiotic composition described herein in the preparation of products for improving or assisting in improving sleep.

[0010] The probiotic composition provided by this invention achieves a synergistic effect in improving sleep quality. It not only effectively shortens sleep latency, improves sleep onset, and prolongs sleep duration, but also upregulates the level of gamma-aminobutyric acid (GABA) in the brain, thereby contributing to improved sleep and nerve relaxation. Compared to existing clinical chemical hypnotic drugs, the probiotic composition of this invention is not only highly safe but also less likely to cause dependence, making it suitable for daily conditioning and intervention for people with poor sleep or insomnia, thus overcoming the limitations of traditional hypnotic drugs in long-term use.

[0011] Information on the preservation of biological materials: Lactobacillus plantarum ( Lactobacillus plantarum 3. Taxonomic naming Lactobacillus plantarum, It was deposited on March 30, 2020, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, accession number: CGMCC No. 19523. This strain has also been published in Chinese patent application CN202310020827.8.

[0012] Lactobacillus plantarum ( Lactiplantibacillus plantarum YIT2004, taxonomically named Lactiplantibacillus plantarumIt was deposited on July 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, accession number: CGMCC No. 39600.

[0013] Classification and Nomenclature Notes: In 2022, according to the latest international revision of the taxonomic status of Lactobacillus family, *Lactobacillus plantarum* (… Lactobacillus plantarum It was reclassified into the genus *Lactobacillus plantarum* and officially renamed *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum The correction of this strain's name has been clearly stated in the "Announcement on the Update of the List of Microbial Strains that Can Be Used in Food and the List of Microbial Strains that Can Be Used in Infant Food (No. 4 of 2022)" issued by the Department of Food Safety Standards and Monitoring and Evaluation of the National Health Commission of the People's Republic of China. Therefore, the "Lactobacillus plantarum (...") referred to in this specification and claims is incorrect. Lactobacillus plantarum ")" all refer to *Lactobacillus plantarum* ( Lactiplantibacillus plantarum ). Attached Figure Description

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0015] Figure 1 This represents the results of prolonged sleep time induced by sodium pentobarbital in each group of mice in the embodiments of the present invention.

[0016] Figure 2 The results of the sodium barbital sleep latency experiment in each group of mice in the embodiments of the present invention are shown.

[0017] Figure 3 The results show the weight changes of mice in each group in this embodiment of the invention.

[0018] Figure 4 The results show the detection of GABA content in the brains of mice in each group in this embodiment of the invention. Detailed Implementation

[0019] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0020] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0021] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B; "A, B, and / or C" includes A, B, and C existing individually, as well as any combination of A+B, A+C, B+C, and A+B+C, for a total of seven parallel solutions.

[0022] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, and do not exclude additional, uncited members, elements, or method steps.

[0023] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0024] The numerical values ​​involved in this invention include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 1% can fluctuate within ±0.05%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 75% can fluctuate within ranges of ±1%, ±2%, ±5%, etc.

[0025] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0026] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0027] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0028] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0029] In this invention, insomnia refers to a sleep-wake disorder in which an individual, despite having a suitable sleep environment and conditions, still experiences persistent difficulty falling asleep, difficulty maintaining sleep, or early awakening, resulting in a decline in sleep quality and / or quantity, and accompanied by daytime functional impairment. This concept covers the following two categories: (1) Clinically defined insomnia: meeting the authoritative diagnostic criteria of the International Classification of Sleep Disorders (ICSD) or the Diagnostic and Statistical Manual of Mental Disorders (DSM), characterized by at least 3 episodes of sleep difficulty per week for more than 3 months, causing significant distress or impairment in social, occupational, educational, academic, behavioral, and other important functional areas; (2) General insomnia: not meeting the above clinical diagnostic criteria, but exhibiting abnormal objective sleep parameters such as prolonged sleep latency, increased nighttime awakenings, reduced total sleep time, or decreased sleep efficiency, or subjectively feeling poor sleep quality, and having a perceptible impact on daytime mental state, cognitive function, or quality of life. The above definition is based on a combination of objective sleep parameters (such as sleep latency, number of awakenings, total sleep time, sleep efficiency, etc.) and subjective sleep quality evaluation, without distinguishing between primary or secondary causes, nor limited to a specific course or severity. Any condition meeting any of the above criteria falls within the scope of "insomnia" as described in this invention.

[0030] In this invention, "functional food" refers to food that, based on ordinary food, is scientifically formulated with added or enriched specific nutrients and active substances (such as probiotics, dietary fiber, plant extracts, etc.) to supplement dietary nutrition, regulate bodily physiological functions, and maintain or improve health. In some embodiments of this invention, the product is a functional food that helps improve sleep.

[0031] In this invention, "health food" refers to food that claims to have specific health functions or is intended to supplement vitamins and minerals. Its core characteristics are: suitable for specific populations, capable of regulating bodily functions, not intended to treat diseases, and not causing any acute, subacute, or chronic harm to the human body. In some embodiments of this invention, the product is a health food with the function of improving sleep (or assisting in improving sleep).

[0032] This invention first provides a probiotic composition comprising *Lactobacillus plantarum* with accession number CGMCC No. 19523. Lactobacillus plantarum )3 strains and Lactobacillus plantarum with accession number CGMCC No. 39600 Lactiplantibacillus plantarum ) YIT2004 strain.

[0033] This invention is the first to discover that combining the two strains mentioned above exhibits a significant synergistic effect in improving sleep quality. Under the premise of the same total viable bacterial count, it is also significantly superior to using either single strain alone or other combinations of the same strain. This combination can more effectively achieve the following effects: (1) prolonging sleep duration, optimizing sleep structure, and exerting a calming and soothing effect; (2) relieving mental fatigue and physical weakness, and improving the body's behavioral activity and reaction agility. Furthermore, the strains used in this invention are all recognized safe-grade probiotics, exhibiting good safety when applied to sleep improvement products, and there is no risk of developing drug resistance.

[0034] In some embodiments, the viable count ratio of *Lactobacillus plantarum* strain YIT2004 to *Lactobacillus plantarum* strain 3 (LP3) is (1-5):1. At this ratio, the synergistic effect of the two strains in regulating the sleep-wake cycle and improving behavioral performance is further enhanced.

[0035] In some specific implementations, the ratio of viable bacteria of Lactobacillus plantarum strain YIT2004 to Lactobacillus plantarum strain 3 (LP3) can be 1:1, 2:1, 3:1, 4:1, 5:1, etc.

[0036] In some specific embodiments, the proportion of viable bacteria can be determined by the colony forming units (CFU) of the strain.

[0037] In some embodiments, the probiotic composition is an oral preparation.

[0038] In some embodiments, the oral formulation includes any one of the following dosage forms: powder, capsule, tablet, granule, ointment, pill, or liquid.

[0039] In some embodiments, the powder includes any one of ordinary powder and lyophilized powder.

[0040] Those skilled in the art can use common knowledge to prepare the probiotic composition of the present invention into various specific oral dosage forms as described above. As an example, the lyophilized powder can be prepared by the following method: Lactobacillus plantarum strain YIT2004 and Lactobacillus plantarum strain 3 (LP3) are cultured separately, the bacterial cells are collected by centrifugation, resuspended with a lyophilization protectant and then freeze-dried under vacuum, and finally the lyophilized powders of the two strains are compounded in proportion.

[0041] Those skilled in the art can use common sense to identify other adjuvants applicable to the probiotic compositions of the present invention.

[0042] In some embodiments, the probiotic composition further comprises a functional adjuvant, including stachyose and / or mannose oligosaccharides. In some preferred embodiments, the functional adjuvant comprises a mixture of stachyose and mannose oligosaccharides. In some further preferred embodiments, the functional adjuvant comprises a mixture of stachyose and mannose oligosaccharides in a mass ratio of (2-4):1. This functional adjuvant can further promote the colonization and efficacy of probiotics.

[0043] In some embodiments, the probiotic composition further comprises a lyophilization protectant, which includes one or more combinations of skim milk, trehalose, sucrose, sorbitol, and polyvinylpyrrolidone.

[0044] In some embodiments, the probiotic composition also includes other functional components that are beneficial for improving insomnia.

[0045] As an example, the functional components may be selected from one or more of the following categories: (1) Amino acids and their derivatives, preferably one or more of GABA, L-theanine, glycine and tryptophan. GABA is the main inhibitory neurotransmitter in the central nervous system and can reduce the excitability of neurons; theanine can promote the secretion of GABA in the brain and reduce the secretion of stress-related cortisol. The amino acids and their derivatives can further enhance the inhibitory nerve signal input of the central nervous system and shorten the sleep latency in synergistically, based on the regulation of gut-brain axis nerve signals by probiotics. (2) Melatonin and 5-hydroxytryptophan, which can be combined with the compound bacteria of the present invention to form a multi-link synergistic system covering pre-sleep mood regulation, sleep rhythm synchronization and sleep preparation. (3) Plant extracts, preferably one or more of jujube seed extract, longan flower water extract, saffron extract, hops extract, withaniasomnifera root and leaf extract and egg yolk hydrolysate. The plant extract functional components can be used as auxiliary components of the probiotic composition to form a multi-target synergistic network with probiotics in the neurotransmitter regulation pathway of the gut-brain axis. (4) Microecological preparations, preferably one or more of postbiotics and other probiotic strains of psychoactive bacteria. To form a multi-level systemic regulatory effect in the upstream microbiota remodeling and downstream neurotransmitter regulation links of the gut-brain axis. (5) Minerals, preferably one or more of magnesium (such as magnesium glycinate or magnesium citrate) and zinc. Magnesium, as a natural agonist of GABA receptors, can help improve insomnia by enhancing GABA neurotransmission. The mineral functional components can be used as auxiliary components of the probiotic composition to supplement the trace elements required for sleep regulation together with probiotics.

[0046] In some embodiments, the above-mentioned functional components may be applied together with the probiotics in the same formulation (such as compound powder, capsules or oral liquid), or they may be applied separately with the probiotics in different formulations at the same or different application times.

[0047] The probiotic composition described in this invention can be prepared into pharmaceutical, health food, or functional food formulations according to actual application needs, adapting to different compliance scenarios and market application forms. Appropriate excipients and preparation processes can be rationally selected based on the regulatory requirements, dosage form requirements, and usage scenarios of the corresponding product category.

[0048] In some specific embodiments, when the probiotic composition is prepared as a pharmaceutical product, pharmaceutically acceptable carriers and adjuvants may be selected, including but not limited to one or more of fillers, disintegrants, binders, lubricants, flow aids, flavoring agents, preservatives, sustained-release matrix materials, and coating materials.

[0049] In some specific embodiments, when the probiotic composition is prepared as a health food, excipients and processing aids permitted for use in health foods may be selected, including but not limited to one or more of the following: food-grade fillers, diluents, sweeteners, acidulants, flavorings, anti-caking agents, humectants, encapsulation carriers, and freeze-drying protectants.

[0050] In some specific embodiments, when the probiotic composition is prepared as a functional food, conventional food raw materials and food-grade processing aids may be selected, including but not limited to one or more of cereal flour, milk powder, sugar alcohol, dietary fiber, thickener, emulsifier, antioxidant, excipient, and flavor modifier.

[0051] Those skilled in the art can combine the above embodiments with common sense to obtain more embodiments of the probiotic composition of the present invention.

[0052] This invention also provides the use of the probiotic composition in improving or assisting in improving sleep. In other words, this invention also provides a method for improving or assisting in improving sleep, the method comprising: administering the probiotic composition to a subject. The following description of related applications is also applicable to further explanation and limitation of the method described in this invention.

[0053] The present invention also provides the use of the probiotic composition described herein in the preparation of products for improving or assisting in improving sleep.

[0054] In some embodiments, the product includes any one of pharmaceuticals, health foods, and functional foods.

[0055] In some embodiments, the probiotic composition is used for at least one of the following: (I) preventing, treating or improving insomnia; (II) sedating and calming the nerves; (III) preparing a medicine for (I) and / or (II).

[0056] In some implementations, the improvement or aiding improvement of sleep includes an improvement in at least one of the following indicators: (i) an increase in sleep duration; (ii) an increase in sleep efficiency; and (iii) a reduction in sleep latency.

[0057] In some embodiments, the probiotic composition is used to increase GABA levels in the brain.

[0058] In some embodiments, the GABA content in the brain refers to the GABA content in brain tissue.

[0059] In some embodiments, the probiotic composition is administered to the subject orally.

[0060] The subjects to which the application described in this invention is applicable include, but are not limited to, humans, as well as non-human mammals used for scientific research, such as laboratory animals such as mice, rats, rabbits, pigs, and dogs.

[0061] Those skilled in the art can combine the above embodiments with common sense to obtain more embodiments related to the application of the present invention.

[0062] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0063] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0064] Example 1: Strain Identification The 16S rRNA gene sequence of strain YIT2004 was determined, and the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO. 1.

[0065] SEQ ID NO. 1: Based on 16S rRNA, it was identified as *Lactobacillus plantarum*. Lactiplantibacillus plantarum ), named Lactobacillus plantarum ( Lactiplantibacillus plantarum YIT2004. This bacterium was deposited on July 7, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC No. 39600.

[0066] Example 2 Mouse Experiment I. Experimental Methods 1. Experimental animals: 50 male C57 mice, 6 weeks old, randomly divided into 5 groups.

[0067] 2. Experimental Grouping (1) Blank group: The same volume of physiological saline was administered by gavage daily.

[0068] (2) Probiotic treatment group: LP3 group: Daily gavage administration of a suspension of Lactobacillus plantarum 3 (LP3) strain (dose of 1 billion CFU).

[0069] LPYIT2004 group: Daily gavage administration of a suspension of Lactobacillus plantarum strain YIT2004 (dose of 1 billion CFU).

[0070] LP3+LPAY01 group: Daily gavage administration of a suspension of Lactobacillus plantarum 3 (LP3) strain and Lactobacillus plantarum AY01 strain (LPAY01 for short) (total dose of 1 billion CFU, of which the ratio of LP3 to LPAY01 dose (CFU) is 1:1).

[0071] Lactobacillus plantarum ( Lactobacillus plantarum AY01 was deposited on September 27, 2022, at the China Center for Type Culture Collection (CCTCC, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province), with accession number CCTCC M20221517. This strain has also been published in Chinese Patent Application No. CN202211438725.X.

[0072] LP3+LPYIT2004 group: Daily gavage administration of a suspension of Lactobacillus plantarum 3 (LP3) and Lactobacillus plantarum YIT2004 (total dose of 1 billion CFU, with a LP3 to LPYIT2004 dose (CFU) ratio of 1:1).

[0073] The preparation method of probiotic powder suspension is as follows: After the probiotic powder is fully dissolved in sterile PBS, the bacteria are counted using the plate count method to make the suspension concentration reach the corresponding dose (CFU).

[0074] 3. Helps improve sleep quality. The mice were subjected to direct sleep experiments, prolonged sodium pentobarbital sleep time experiments, subthreshold sodium pentobarbital hypnotic experiments, and sodium barbital sleep latency experiments, in accordance with the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)".

[0075] (1) Direct sleep experiment After daily oral administration of the drug, mice in each group were observed to see if they experienced direct sleep. Sleep status was determined based on the disappearance and recovery of the righting reflex: when a mouse was placed in a supine position, if it could not spontaneously return to a normal position within 1 minute (i.e., the righting reflex disappeared), it was considered to have entered a sleep state; conversely, when the mouse regained the ability to right itself, sleep was considered to have terminated. The number of mice that fell asleep in each group and the sleep duration (the time interval from the disappearance to the recovery of the righting reflex) were recorded.

[0076] (2) Experiment on prolonging sodium pentobarbital sleep time Mice were intraperitoneally injected with sodium pentobarbital (50 mg / kg) 30 minutes after the last gavage. The time interval between the disappearance of the righting reflex (sleep initiation) and its recovery (wakefulness) was observed, and the duration of sleep was calculated.

[0077] (3) Subthreshold dose hypnotic experiment with sodium pentobarbital Mice were injected intraperitoneally with sodium pentobarbital (30 mg / kg) 30 minutes after the last gavage. Mice were considered to have fallen asleep if the righting reflex disappeared for more than 1 minute. The number of animals that fell asleep within 30 minutes was recorded.

[0078] (4) Sodium barbital sleep latency test Mice were injected intraperitoneally with sodium barbital (300 mg / kg) 30 minutes after the last gavage. The time from the injection of sodium barbital to the disappearance of the righting reflex was recorded, and the sleep latency was determined.

[0079] 4. Changes in mouse body weight Mice were weighed before gavage, and then measured weekly.

[0080] 5. Determination of GABA content in mouse brain tissue Twenty-four hours after the last administration, mice were euthanized by dislocation, and their brains were removed, rinsed with ice-cold physiological saline, blotted dry with filter paper, and weighed. 0.1 g of brain tissue was weighed, and 0.5 mL of tissue lysis buffer was added. The tissue was then homogenized (4°C) and centrifuged at 4000 r / min for 10 min. The supernatant was collected, diluted 50 times, and the level of GABA in the mouse brain tissue was determined by ELISA according to the kit instructions.

[0081] II. Experimental Results 1. Direct Sleep Experiment Within 30 minutes after daily gavage, no mice in the probiotic treatment group experienced sleep, indicating that Lactobacillus plantarum strain 3 (LP3) and Lactobacillus plantarum strain YIT2004 had no direct hypnotic effect on mice.

[0082] 2. Experiment on prolonging sodium pentobarbital sleep time The results of prolonging the sleep time induced by sodium pentobarbital in each group of mice are as follows: Figure 1 As shown, compared with the blank group, the probiotic treatment groups significantly prolonged the sleep time induced by sodium pentobarbital in mice. The sleep time of the LP3 group, LPYIT2004 group, LP3+LPAY01 group, and LP3+LPYIT2004 group was prolonged by 10.67 min, 15.08 min, 20.97 min, and 22.65 min, respectively. This indicates that both Lactobacillus plantarum strain 3 and Lactobacillus plantarum strain YIT2004 can effectively increase the sleep time of mice when used alone or in combination. Among them, the combination of LP3+LPYIT2004 group showed the best effect in improving sleep, which was not only significantly better than the single strains but also better than combinations involving other commercially available strains.

[0083] 3. Subthreshold dose hypnotic experiment with sodium pentobarbital The results of the subthreshold dose experiment of sodium pentobarbital in each group of mice are shown in Table 1. The sleep rates of mice in the LP3 group, LPYIT2004 group, LP3+LPAY01 group and LP3+LPYIT2004 group were 80%, 80%, 80% and 90%, respectively, indicating that probiotics can significantly improve the sleep rate of mice, and the combination of Lactobacillus plantarum strain 3 and Lactobacillus plantarum strain YIT2004 has the best effect.

[0084] Table 1

[0085] 4. Sodium barbital sleep latency test The results of the sodium barbital sleep latency experiment in each group of mice are as follows: Figure 2 As shown, compared with the control group, the probiotic treatment groups significantly shortened the sleep latency of mice. Specifically, the sleep latency of the LP3 group, LPYIT2004 group, LP3+LPAY01 group, and LP3+LPYIT2004 group was shortened by 1.69 min, 2.46 min, 2.65 min, and 2.87 min, respectively. This indicates that both *Lactobacillus plantarum* strain 3 and *Lactobacillus plantarum* strain YIT2004, whether used alone or in combination, can effectively shorten the sleep latency of mice. The combination of two strains showed the best effect in improving sleep and was superior to combinations involving other commercially available strains.

[0086] 5. Mouse body weight change graph Mice were weighed before gavage, and their weight was measured weekly thereafter. Weight changes over 30 days were recorded as follows: Figure 3 As shown, the weight change trends of mice in each group were basically the same, and there was no significant difference between the groups, indicating that probiotics do not affect the normal growth of mice.

[0087] 6. Comparison of GABA content in mouse brains GABA is a major inhibitory neurotransmitter; increasing its levels helps reduce the excitability of the nervous system, thereby promoting sleep. Figure 4 It can be seen that, compared with the blank group, the probiotic treatment groups can significantly increase the content of the inhibitory neurotransmitter GABA in the brain tissue of mice. Among them, the combination group involving Lactobacillus plantarum 3 (LP3) strain and Lactobacillus plantarum YIT2004 strain (LP3+LPYIT2004 group) has better effect.

[0088] III. Experimental Conclusions Both *Lactobacillus plantarum* strain 3 (LP3) and *Lactobacillus plantarum* strain YIT2004 significantly prolonged sleep duration, increased the number of mice falling asleep, and shortened sleep latency in mice. The combined intervention group of the two strains showed the best overall improvement. Furthermore, this study confirmed that probiotics do not have a direct hypnotic effect, but they possess a clear sleep-improving efficacy; and the combined use of strains exhibits a synergistic effect, which is not only superior to either strain administered alone, but also significantly superior to other combinations of similar strains.

[0089] Lactobacillus plantarum strain 3 (LP3) and Lactobacillus plantarum strain YIT2004 can enhance the central nervous system inhibitory effect by increasing the GABA content in the mouse brain, thereby exerting a sedative and calming effect, which is one of the core mechanisms by which they improve sleep in mice.

[0090] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A probiotic composition comprising *Lactobacillus plantarum* with accession number CGMCC No. 19523 (… Lactobacillus plantarum )3 strains and Lactobacillus plantarum with accession number CGMCC No. 39600 Lactiplantibacillus plantarum ) YIT2004 strain.

2. The probiotic composition according to claim 1, wherein, The ratio of viable counts of Lactobacillus plantarum strain YIT2004 to Lactobacillus plantarum strain 3 was (1-5):

1.

3. The probiotic composition according to claim 1 or 2, wherein it is an oral preparation.

4. The probiotic composition according to claim 3, wherein, The oral preparations include any one of the following dosage forms: powder, capsule, tablet, granule, ointment, pill, and liquid; wherein the powder includes any one of the following: ordinary powder and lyophilized powder.

5. The probiotic composition according to claim 1 or 2, further comprising a functional adjuvant, said functional adjuvant including stachyose and / or mannose oligosaccharides.

6. The probiotic composition according to claim 5, wherein, The functional additives include a mixture of stachyose and oligomannose in a mass ratio of (2-4):

1.

7. The probiotic composition according to claim 1 or 2, further comprising a lyophilization protectant, said lyophilization protectant comprising one or more combinations of skim milk, trehalose, sucrose, sorbitol, and polyvinylpyrrolidone.

8. Use of the probiotic composition according to any one of claims 1-7 in the preparation of a product for improving or helping to improve sleep.

9. The application according to claim 8, wherein, The products include any one of pharmaceuticals, health foods, and functional foods.

10. The application according to claim 8, wherein, The improvement or improvement in sleep mentioned includes an improvement in at least one of the following indicators: (i) Increased sleep duration; (ii) Improved sleep efficiency; (iii) Shortening of the sleep latency period.

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