Probiotics for regulating intestinal flora and improving insomnia and anxiety and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to probiotics for regulating intestinal flora and improving insomnia and anxiety and application of the probiotics. The probiotics are plant lactobacillus, and the preservation number of the probiotics is GDMCC No: 67148. According to the scheme provided by the invention, insomnia and anxiety related scale scores can be effectively reduced, and insomnia and anxiety symptoms can be relieved; moreover, the diversity of intestinal flora can be adjusted, the composition of key phylum and bacteria genus is optimized (for example, the abundance of beneficial bacteria is increased, and the abundance of potential harmful bacteria is reduced), and a complete intervention logic of strain-intestinal flora-symptom improvement is formed. The traditional Chinese medicine composition is high in safety, convenient to take, good in compliance, low in cost and easy to popularize, and a new effective way is provided for insomnia anxiety intervention.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to probiotics that regulate gut microbiota to improve insomnia and anxiety, and their applications. Background Technology

[0002] In contemporary society, insomnia and anxiety have become common health problems due to multiple factors such as life stress and environmental changes. These symptoms not only manifest as sleep abnormalities such as difficulty falling asleep, sleep maintenance disorders, and early awakening, but are also often accompanied by psychological and cognitive impairments such as unexplained tension, irritability, poor concentration, and memory decline. Some people may also experience physical reactions such as decreased appetite and weight fluctuations. Long-term insomnia and anxiety can disrupt the neuroendocrine balance, increase the risk of mental illness and physical diseases of the cardiovascular and digestive systems, seriously affecting people's physical and mental health and quality of life, and has become one of the public health problems that urgently need to be addressed.

[0003] Currently, interventions for insomnia and anxiety are mainly divided into two categories: pharmacological treatment and non-pharmacological treatment. Pharmacological treatment primarily uses sedative-hypnotic drugs and anti-anxiety medications, which can quickly relieve symptoms, but have potential side effects such as drug dependence, drowsiness, and memory loss. The safety and tolerability of long-term use require careful evaluation, limiting its clinical application. Non-pharmacological treatments include cognitive behavioral therapy, psychological counseling, and exercise intervention. While they do not carry the risk of drug side effects, they suffer from long treatment cycles (usually requiring 4-8 weeks to take effect), high requirements for therapist expertise, high intervention costs, and poor patient compliance (requiring regular in-person treatment), making large-scale application difficult.

[0004] In recent years, the "gut-brain axis" regulatory mechanism between gut microbiota and the central nervous system has become a research hotspot. Studies have confirmed that gut microbiota can influence brain function and emotional state through multiple pathways, including neural, immune, and metabolic pathways, providing new directions for the intervention of insomnia and anxiety. Probiotics, as a safe means of regulating gut microbiota balance, have shown potential in the intervention of insomnia and anxiety. However, existing research mostly focuses on general probiotic strains, lacking specific probiotic strains adapted to the physiological characteristics (such as gut microbiota structure and metabolic patterns) and lifestyle (such as portability and ease of administration) of the target population. Furthermore, existing research has not deeply elucidated the specific mechanisms by which probiotics improve insomnia and anxiety, especially regarding the regulatory patterns of probiotics on the diversity of gut microbiota and the composition of key genera in the target population. This makes it difficult to establish clear evidence of a "strain-gut microbiota-improvement of insomnia and anxiety," hindering the clinical translation and application of probiotics in the intervention of insomnia and anxiety.

[0005] Therefore, there is an urgent need to develop a safe, efficient, and convenient intervention program for insomnia and anxiety, especially to screen for specific probiotic strains with clear mechanisms of action, and to achieve precise intervention for insomnia and anxiety by regulating the balance of gut microbiota. This would address the pain points of existing treatments, such as large side effects, poor compliance, and insufficient targeting, and provide a new and effective way to safeguard the mental health of the population. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides an insomnia and anxiety intervention program centered on *Lactobacillus plantarum* GDMCC No: 67148. This program explicitly uses *Lactobacillus plantarum* GDMCC No: 67148 as the dedicated probiotic strain. *Lactobacillus plantarum* GDMCC No: 67148 improves insomnia and anxiety by regulating gut microbiota diversity and optimizing the composition of key phyla and genera, forming a complete intervention logic of "strain-gut microbiota-symptom improvement".

[0007] The technical solution of this invention is as follows: On the one hand, the present invention provides a strain of *Lactobacillus plantarum*, the preservation number of which is GDMCCNo: 67148.

[0008] In another aspect, the present invention provides the application of the aforementioned *Lactobacillus plantarum* in the preparation of sleep aid products.

[0009] Specifically, the products include, but are not limited to: pharmaceuticals, probiotic preparations, or health products.

[0010] Specifically, the product includes fermentation broth of *Lactobacillus plantarum*, fermentation broth supernatant, fermentation broth precipitate, lyophilized powder, and / or bacterial suspension.

[0011] More specifically, the fermentation broth can be the original mixture obtained after *Lactobacillus plantarum* has completed fermentation culture in a liquid medium. It contains bacterial cells, all beneficial substances produced by bacterial metabolism (such as GABA, bacteriocins, organic acids, etc.), and unconsumed culture medium components. After centrifugation or filtration to separate the solid and liquid components of the fermentation broth, two parts are obtained: the liquid part is called the fermentation broth supernatant, which mainly contains soluble metabolites and residual culture medium components, but contains little or no bacterial cells; the solid part is the fermentation broth precipitate, whose main components are the bacterial cells themselves and any insoluble substances that may be attached to them. If a high-concentration bacterial suspension is rapidly frozen at extremely low temperatures and then vacuum dehydrated, lyophilized powder can be obtained. This form greatly extends the shelf life of the strains, facilitates transportation and storage, and is one of the most common end-product forms of probiotic products. A bacterial suspension usually refers to a homogeneous liquid mixture formed by resuspending bacterial cells in a sterile buffer or protectant. It can be a direct concentrate of the fermentation broth or prepared by rehydrating and activating lyophilized powder, facilitating precise dosage experiments or the preparation of liquid formulations.

[0012] In another aspect, the present invention provides a health product comprising the aforementioned *Lactobacillus plantarum*.

[0013] Specifically, the health products also include excipients.

[0014] More specifically, the excipients include, but are not limited to, one or more of the following: prebiotics, preservatives, flavoring agents, and diluents.

[0015] Preferably, the prebiotics include, but are not limited to: fructooligosaccharides, galactooligosaccharides, inulin, or resistant dextrin; and / or, the preservatives include, but are not limited to: trehalose, skim milk powder, or glycerin; and / or, the flavoring agent is fruit powder, sweetener, or flavoring; and / or, the diluents include, but are not limited to: maltodextrin or starch.

[0016] Specifically, the health products include, but are not limited to, tablets, capsules, granules, powders, or oral liquids.

[0017] In another aspect, the present invention provides a drug comprising the aforementioned *Lactobacillus plantarum*.

[0018] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0019] Specifically, the dosage forms of the drug include, but are not limited to: injections, tablets, capsules, granules, oral liquids, or powders.

[0020] The beneficial effects of this invention are as follows: (1) After short-term intervention, the scores of anxiety and insomnia-related scales of the subjects in the probiotic group were significantly lower than those in the placebo group, which could quickly relieve insomnia and anxiety symptoms and break the vicious cycle of "anxiety-insomnia".

[0021] (2) No adverse reactions were observed during the intervention, and there was no drug dependence or neurotoxicity. It can be taken for a long time and is suitable for the physiological health needs of the population.

[0022] (3) The technical solution of the present invention optimizes the diversity and composition of the gut microbiota, increases the abundance of beneficial bacteria and reduces the abundance of potentially harmful bacteria, and clarifies the association between gut microbiota and insomnia and anxiety symptoms, providing scientific support for the intervention effect.

[0023] Preservation instructions: Biomaterials: Lactiplantibacillus plantarum ; Category Naming: Lactiplantibacillus plantarum ; Accession number: GDMCC No: 67148; Preservation period: October 22, 2025; Preservation institution: Guangdong Provincial Center for Microbial Culture Collection; Abbreviation of depositary institution: GDMCC; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0024] Figure 1 A flowchart for clinical subject recruitment and intervention.

[0025] Figure 2 Anxiety and depression levels were measured in the MP and MB groups. (A) HAMA score. (B) AIS score. HAMA: Hamilton Anxiety Rating Scale; AIS: Athens Insomnia Scale. MP: Placebo group (n=56); MB: Probiotic group (n=57). Data are presented as median (interquartile range). The Shapiro-Wilk test was used to analyze the normality of the data between the two groups, and the Mann-Whitney U test was used for comparisons of continuous variables. Statistical significance was set at p<0.05.

[0026] Figure 3 To analyze the diversity of fecal microbiota in the MP and MB groups of the subjects; MP: placebo group (n=35); MB: probiotic group (n=35); statistical significance was set as p<0.05.

[0027] Figure 4 Fecal microbiota diversity analysis was performed between the MP and MB groups of the subjects (principal coordinate analysis); MP: placebo group (n=35); MB: probiotic group (n=35); statistical significance was set at p<0.05.

[0028] Figure 5 Fecal microbiota diversity analysis (α diversity index) was performed between the MP and MB groups of subjects; MP: placebo group (n=35); MB: probiotic group (n=35); data are expressed as median (interquartile range); the Mann-Whitney U test was used to compare the α diversity index; statistical significance was set at p<0.05.

[0029] Figure 6 Fecal microbiota diversity analysis of subjects in the MP and MB groups (Venn plot); MP: placebo group (n=35); MB: probiotic group (n=35); statistical significance was set at p<0.05.

[0030] Figure 7 The fecal microbiota were analyzed at the phylum and genus levels for the MP and MB groups (phylum-level classification composition); MP: placebo group (n=35); MB: probiotic group (n=35); statistical significance was set at p<0.05.

[0031] Figure 8 The phylum and genus levels of fecal microbiota were analyzed for the MP and MB groups of the subjects (relative abundance of Firmicutes (A) and Bacteroidetes (B)); MP: placebo group (n=35); MB: probiotic group (n=35); statistical significance was set at p<0.05.

[0032] Figure 9 The fecal microbiota were analyzed at the phylum and genus levels (genus-level taxonomic composition) for the MP and MB groups of the subjects; MP: placebo group (n=35); MB: probiotic group (n=35); statistical significance was set at p<0.05.

[0033] Figure 10 The phylum and genus levels of fecal flora were analyzed in the MP and MB groups of the subjects (relative abundance of Bifidobacterium (A), Bacteroides (B), Coprococcus (C), Prevotella (D), Akkermansia (E) and Lactobacillus (F); MP: placebo group (n=35); MB: probiotic group (n=35); statistical significance was set at p<0.05.

[0034] Figure 11 Differential species analysis of fecal microbiota between the MP and MB groups (LEfSe analysis); MP: placebo group (n=35); MB: probiotic group (n=35).

[0035] Figure 12 Differential species analysis of fecal microbiota between the MP and MB groups was performed (using a horizontal random forest analysis); MP: placebo group (n=35); MB: probiotic group (n=35).

[0036] Figure 13 Correlation analysis was performed between gut microbiota and anxiety and insomnia. Spearman correlation analysis revealed the association between gut microbiota and anxiety and insomnia scores. Red indicates a positive correlation, and blue indicates a negative correlation. * p <0.05,** p <0.01, *** p <0.001. Detailed Implementation

[0037] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0038] Example 1 1.1 Baseline characteristics of subjects (1) Experimental subjects The study included healthy students at Nanchang University and participants experiencing insomnia and anxiety due to exam preparation. A questionnaire survey was conducted on campus to select 120 participants with symptoms of insomnia and anxiety.

[0039] (2) Inclusion criteria Insomnia and Anxiety Group: 1. Gender not limited, age ≥18 years old, ≤30 years old. 2. Hamilton Anxiety Rating Scale (score ≥7 points). 3. AIS-8 Insomnia Scale (score >6 points). 4. Clinical manifestations are mainly depressed mood, accompanied by at least 4 of the following: ① Frequent inexplicable tension and anxiety; ② Likes to complain, easily agitated, gets angry and holds grudges over trivial matters; ③ Poor concentration, decreased memory, fatigue, and reduced energy; ④ Accompanied by a series of physical symptoms, such as sleep disorders, early awakening, insomnia, excessive dreaming, and excessive sleep, poor appetite, and weight loss trend; ⑤ Always looks sad and unwilling to talk to others; ⑥ Reduced activity and slow reaction; ⑦ Feeling guilty, self-blaming, and low self-esteem. 5. Has not taken probiotics or other medications within the past month. 6. Signed informed consent.

[0040] (3) Exclusion criteria 1. Smoking or excessive alcohol consumption; 2. Receiving antidiarrheal medications, bulk-forming agents, probiotics, antispasmodics, and antibiotics within 30 days prior to enrollment; 3. Having severe heart, lung, kidney, or liver dysfunction or metabolic diseases, or having undergone radiotherapy or chemotherapy.

[0041] (4) Experimental grouping and intervention 1. Placebo group (MP group, n=60): Subjects with insomnia and anxiety were given a placebo (2 grams per packet, containing only maltodextrin), 2 packets per day for 2 weeks.

[0042] 2. Probiotic Group (MB Group, n=60): Subjects suffering from insomnia and anxiety were treated with probiotics (Lactobacillus plantarum GDMCC No: 67148), 2 packets per day (2 grams per packet, live bacteria count of 1×10⁻⁶). 10 Colony forming units (CFU, using maltodextrin as a carrier matrix) were collected for two consecutive weeks.

[0043] This study recruited 221 participants. After tiered screening based on exclusion / inclusion criteria, 52 participants met the exclusion criteria, 12 refused to participate, and 37 did not meet the inclusion criteria, ultimately enrolling 120 participants with insomnia and anxiety symptoms. Participants were randomly assigned to either the placebo group (MP group, n=60) or the probiotic group (MB group, n=60). At the end of the trial, 113 participants completed the clinical study (56 in the MP group and 57 in the MB group). Figure 1 The results showed that the mean age of the subjects in both the MP and MB groups was 21 years (p=0.4548) (Table 1). Regarding gender distribution, the proportion of males in the placebo group and the probiotic group was 50.00% and 49.12%, respectively. There was no significant difference in mean body mass index (BMI) between the two groups (p=0.4078), which were 22.51±2.73 and 22.11±2.31, respectively. Furthermore, the HAMA scores of the two groups were 16.00 (12.00-20.50) and 16.00 (11.50-21.00), respectively (p=0.7776), and the AIS scores were 12.00 (9.00-13.75) and 11.00 (9.00-15.00), respectively (p=0.8591), indicating no significant difference in the severity of anxiety and insomnia between the two groups. In summary, the two groups in this study had similar baseline characteristics and were highly homogeneous.

[0044] Table 1. Basic characteristics of all subjects

[0045] Note: BMI: Body Mass Index; HAMA: Hamilton Anxiety Rating Scale; AIS: Athens Insomnia Scale. MP: Placebo group (n=60); MB: Probiotic group (n=60). Data are expressed as mean ± standard deviation and median (interquartile range). " / " indicates no data available. The Shapiro-Wilk test was used to analyze the normality of the two groups. Unpaired t-tests or Mann-Whitney U tests were used for comparisons of continuous variables. Statistical significance was set at p<0.05.

[0046] 1.2 *Lactobacillus plantarum* GDMCC No: 67148 significantly improved insomnia and anxiety symptoms in subjects. After a 2-week intervention, the effects of *Lactobacillus plantarum* GDMCC No: 67148 on insomnia and anxiety in the subjects were evaluated. Results showed that, compared to the placebo group, the HAMA score of the MB group treated with probiotics was significantly lower (MP vs MB = 16.50 vs 12.00, p = 0.0011). Figure 2 In the A) and AIS scores (MP vs MB = 11.00 vs 8.00, p<0.0001) Figure 2 The levels of B) were significantly reduced, indicating that Lactobacillus plantarum GDMCC No: 67148 can effectively alleviate the insomnia and anxiety symptoms of the subjects.

[0047] 1.3 *Lactobacillus plantarum* GDMCC No: 67148 altered gut microbiota diversity in subjects with insomnia and anxiety. Prognosis: Fecal samples were collected from both groups, and high-throughput sequencing of the 16S rDNA gene V4 region was performed on fecal samples from the MP and MB groups after intervention.

[0048] Sequencing primers: 515F (SEQ ID NO.1): 5'-GTGYCAGCMGCCGCGGTAA-3'; 806R (SEQ ID NO. 2): 5'-GGACTACNVGGGTWTCTAAT-3'.

[0049] The specific steps are as follows: After the clinical trials are completed, the samples will be frozen in a laboratory freezer at -80°C. They will then be sent to Shanghai Paisenno Company for high-temperature testing. Throughput sequencing. a. Add 3 mL of sterile PBS to the fecal sample and mix by pipetting and vortexing; b. Let stand at room temperature for 5 min, discard the supernatant, add 2 mL of sterile PBS to the precipitate, let stand and collect the supernatant; centrifuge the supernatant at 6000 rpm for 3-5 min, and when the solution is no longer turbid, aspirate 1 mL of the supernatant; c. Centrifuge the supernatant at 8000 rpm for 2 min, discard the supernatant, add 200 μL of GA, vortex to resuspend the bacterial cells, add 20 μL of proteinase K, and continue to mix; d. Add 0.2 g of glass beads and 200 μL of GB to the above suspension, vortex to mix, and incubate in a water bath for 10 min; e. Add 200 μL of anhydrous ethanol, vortex to mix, carefully aspirate the supernatant into the adsorption column, being careful not to aspirate the glass beads, centrifuge at 10000 rpm for 1 min at 25 ℃, and discard the waste liquid; f. Add 600 μL of PW, centrifuge at 10000 rpm for 1 min at 25 ℃. min. Discard the waste liquid; g. Repeat the previous step; h. Centrifuge at 10000 rpm for 2 min at 25℃, remove the adsorption column and air dry in a new 1.5 mL EP container for 15-20 min; i. Add 40 μL of elution buffer to the center of the adsorption membrane in the adsorption column, incubate at 25℃ for 2 min, then centrifuge at 10000 rpm for 2 min at 25℃; j. Repeat the above steps to obtain high-concentration genomic DNA. After extracting genomic DNA, the DNA concentration was measured using the NanoDrop system and the DNA molecule size was estimated using agarose gel electrophoresis. The V3-V4 region of the 16S rRNA gene was amplified using the sample genomic DNA as a template, and sequenced to obtain OTUs. Species diversity was analyzed based on the number of OTUs and other data, and community structure data were statistically analyzed at multiple levels, including kingdom, phylum, class, order, family, genus, and species. Based on this, cluster analysis can be used to explore a series of information such as OTUs and species composition, and further study the differences between samples in the population.

[0050] The sparse curves indicate that the abundance of microorganisms in both groups has reached a plateau, and the sequencing depth is sufficient to cover the vast majority of microbial diversity. Figure 3 However, the overall diversity of the MP group was higher than that of the MB group. Principal coordinate analysis (PCoA) showed some overlap between the two groups, but there was a significant difference in β diversity. Figure 4 Alpha diversity analysis showed that the Shannon index in the MB group was significantly lower than that in the MP group (p = 0.044). Figure 5Furthermore, although the Chao1 index (p = 0.1), Goods_coverage (p = 0.28), and Observed_species (p = 0.091) of the MB group were slightly lower than those of the MP group, the differences were not statistically significant, suggesting that α-diversity was reduced in the MB group. Notably, the Venn plot showed that a total of 3291 OTUs were obtained from the sequencing samples, with 740 OTUs in total across both groups. The MP group had 1496 unique OTUs, while the MB group had 1055 unique OTUs. Figure 6 These results suggest that *Lactobacillus plantarum* GDMCC No: 67148 may improve insomnia and anxiety symptoms in subjects by modulating gut microbiota diversity.

[0051] 1.4 *Lactobacillus plantarum* GDMCC No: 67148 improves gut microbiota composition in subjects with insomnia and anxiety Further analysis of the species composition after intervention was conducted. At the phylum level, the fecal microbiota of the subjects was mainly dominated by Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. Figure 7 Compared with the MP group, the relative abundance of Bacteroidetes was significantly decreased in the MB group (p<0.01), while the relative abundance of Actinobacteria and Proteobacteria increased, and the relative abundance of Firmicutes showed no significant difference. Figure 8 Subsequently, analysis at the genus level revealed that the fecal microbiota was mainly composed of Bifidobacterium (Bifidobacterium genus). Bifidobacterium ), Broutella spp. Blautia ), Collins genus ( Collinsella ) and Bacteroides ( Bacteroides )composition( Figure 9 Compared with the MP group () Figure 10 Bifidobacterium spp. (p<0.01) and Lactobacillus spp. (MB group) Lactobacillus The relative abundance of Bacteroides (p<0.05) was significantly increased, while that of Prevotella (p<0.01) and Prevotella (p<0.05) was significantly increased. Prevotella (p<0.05) significantly reduced. Coprococcus The relative abundance showed an increasing trend but was not statistically significant, Akkermania ( Akkermansia There was no significant difference between the two groups, and the levels remained low. In summary, these results indicate that *Lactobacillus plantarum* GDMCC No: 67148 improves insomnia and anxiety in subjects by increasing beneficial bacteria (*Bifidobacterium* and *Lactobacillus*) and decreasing pathogenic bacteria (*Bacteroides*).

[0052] In addition, differential species analysis was performed. LEfSe analysis showed that the MB group was enriched with beneficial bacteria, such as Bifidobacterium spp. ( Bifidobacterium Lactobacillus ( ) Lactobacillus ) and Clostridium ( Clostridium The MP group is enriched with harmful bacteria, such as Bacteroides ( ); while the MP group is enriched with harmful bacteria, such as Bacteroides spp. ( ); Bacteroides ) and Fusobacterium genus ( Fusobacterium () Figure 11 Random forest analysis further validated this result, showing an increase in beneficial bacteria (). Figure 12 The results suggest that intervention with Lactobacillus plantarum GDMCC No: 67148 may increase the level of lactobacillus in feces, which may alleviate insomnia and anxiety.

[0053] 1.5 Correlation analysis between gut microbiota and insomnia and anxiety symptoms in subjects To explore the association between gut microbiota and insomnia and anxiety in the subjects, Spearman correlation analysis was performed. The results showed that the HAMA score was negatively correlated with Bifidobacterium, Akkermansia, and Brouthus, and positively correlated with Bacteroides; the AIS score was negatively correlated with Bifidobacterium and Brouthus, and positively correlated with Bacteroides, but none of these correlations reached statistical significance. Figure 13 These data suggest a link between insomnia and anxiety in the subjects and gut microbiota imbalance.

[0054] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A strain of *Lactobacillus plantarum*, characterized in that, The preservation number of the *Lactobacillus plantarum* is GDMCC No:67148.

2. The application of the *Lactobacillus plantarum* as described in claim 1 in the preparation of sleep aid products.

3. The application according to claim 2, characterized in that, The product is a drug, probiotic preparation, or health product.

4. The application according to claim 2, characterized in that, The product includes fermentation broth of Lactobacillus plantarum, fermentation broth supernatant, fermentation broth precipitate, freeze-dried powder and / or bacterial suspension.

5. A health product, characterized in that, The health product includes *Lactobacillus plantarum* as described in claim 1.

6. The health product according to claim 5, characterized in that, The health products also include excipients.

7. The health product according to claim 5, characterized in that, The health products mentioned are in tablet, capsule, granule, powder or oral liquid form.

8. A drug, characterized in that, The drug includes *Lactobacillus plantarum* as described in claim 1.

9. The medicament according to claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients.

10. The medicament according to claim 8 or 9, characterized in that, The dosage form of the drug is injection, tablet, capsule, granule, oral liquid or powder.

Images