Bifidobacterium longum with significant improvement of cognitive memory ability for regulating circadian rhythm and application thereof

Abstract

The application discloses a long bifidobacterium with the function of regulating circadian rhythm and improving cognitive memory ability and application thereof, and belongs to the technical field of microorganisms. The long bifidobacterium GDMCC No: 65850 can effectively reduce the expression disorder of rhythm genes caused by sleep deprivation, thereby relieving the cognitive defects of mice with circadian rhythm disorder. In addition, the intervention of the long bifidobacterium GDMCC No: 65850 can regulate the changes of intestinal flora health and intestinal metabolite levels, promote the generation of neuroprotective metabolites, and reduce the decrease of cognitive ability caused by circadian rhythm disorder. The long bifidobacterium of the application expands the application range of the long bifidobacterium as probiotics, and has great application prospect in products for relieving the decrease of cognitive memory ability induced by circadian rhythm disorder.

Description

Technical Field

[0001] This invention relates to a strain of Bifidobacterium longum that can significantly improve cognitive memory by regulating diurnal rhythms and its applications, belonging to the field of microbial technology. Technical Background Modern lifestyles, such as social jet lag, shift work, and delayed bedtime, frequently disrupt people's normal sleep-wake cycles, causing misalignment between the natural environment and the endogenous biological clock, as well as between different circadian rhythm oscillators within the body (i.e., circadian rhythm disorder). Increasing clinical evidence suggests that long-term circadian rhythm disorder can lead to various complications, such as obesity, diabetes, and sleep disorders. Circadian rhythm disorder can also affect brain function, accelerating the development of various chronic diseases, including cognitive impairment, and impacting people's daily learning, work, and life. Mild cognitive impairment is a transitional condition between normal cognitive function and dementia, mainly manifested as memory loss, poor concentration, decreased thinking ability, and mood and emotional changes. Melatonin is considered an effective drug for treating circadian rhythm disorder, as it can alleviate rhythm-induced cognitive impairment by regulating the expression of rhythm genes. However, long-term supplementation with exogenous melatonin inevitably produces side effects such as dizziness, drowsiness, and hormonal imbalances, therefore, safer and more effective treatment strategies are needed.

[0002] In recent years, an increasing number of studies have revealed the link between host circadian rhythms, cognitive abilities, and gut microbiota. Research indicates that sleep deprivation-induced circadian rhythm disruption significantly alters the composition of the mouse gut microbiota, particularly significantly reducing the levels of beneficial lactobacilli (Lactobacillus family). Lactobacillaceae Akkermania ( ) Akkermansia The abundance of ) (Microbiological Research, 2023, 268: 127292; Gut Microbes, 2023, 15(2):2252764). When fecal microbiota from sleep-deprived mice was transplanted into the intestines of healthy antibiotic-treated mice, the cognitive abilities of the recipient mice were significantly worse than those of the control mice (Brain, Behavior, and Immunity, 2023, 108: 98-117), but the cognitive impairment of the mice was observed to be repaired after transplantation of fecal microbiota from healthy mice (Brain, Behavior, and Immunity, 2023, 108: 98-117; Nutritional Neuroscience, 2023, 26(3): 254-264). Therefore, gut microbiota may be an important target for regulating host circadian rhythms and cognitive levels.

[0003] However, it is currently unknown whether Bifidobacterium longum can alleviate cognitive impairment caused by circadian rhythm disorders. Although some existing technologies have disclosed that Bifidobacterium longum can alleviate cognitive decline caused by Alzheimer's disease (CN118256399A) and aging (Scientific Reports, 2019, 9(1): 11814) to some extent, cognitive decline caused by circadian rhythm disorders differs from cognitive impairment caused by the above two factors in terms of scope of influence, pathogenesis, and symptoms. First, different types of cognitive impairment differ in their apparent symptoms: Alzheimer's disease (AD) is a very common neurodegenerative disease among the elderly, with most AD patients being over 65 years of age. In contrast, circadian rhythm disorders have a wider range of effects, affecting people of all ages and occupations, such as adolescents with irregular schedules, adults working shifts, and the elderly with declining physical function. Circadian rhythm disorders can cause systemic damage to the body. Furthermore, cognitive impairment caused by Alzheimer's disease is a manifestation of neurodegenerative disease, characterized by irreversibility and progressive worsening; cognitive impairment caused by aging is progressive and irreversible, requiring long-term intervention and management; while insomnia-induced cognitive impairment is temporary and can be alleviated by improving sleep. Secondly, different types of cognitive impairment differ in their mechanisms. Circadian rhythm genes are key regulators of biological rhythms, and the main manifestation of circadian rhythm disorders is the dysregulation of rhythm gene expression. Studies have shown that rhythm genes play an important role in regulating neurogenesis and cognitive function. Bmal1 The absence of these cells increases the activation of microglia and astrocytes and promotes neuroinflammation, while reducing the host's cognitive level. Bmal1 The absence of LPS can also enhance LPS-induced NF-κB pathway activation by increasing ROS levels (The FASEB Journal, 2020, 34(5): 6570-6581). In contrast, in a published invention (Scientific Reports, 2019, 9(1):11814), *Bifidobacterium longum* NK46 mainly alleviates AD-induced cognitive decline by regulating gut microbiota to reduce LPS levels in feces and blood, thereby inhibiting LPS-mediated NF-κB pathway activation. In another invention (CN118256399A), *Bifidobacterium longum* CGMCC No.24068 mainly alleviates AD-induced cognitive decline by reducing D levels in feces and blood. Galactose-induced amyloid protein Aβ1 in mouse hippocampus 42. Accumulation and enhancement of neurotransmitter (acetylcholine) levels can alleviate age-related cognitive decline. Therefore, cognitive impairment caused by circadian rhythm disorders differs from cognitive impairment caused by AD and age-related cognitive decline in both mechanism and appearance.

[0004] Given that there is currently no direct evidence that Bifidobacterium longum improves cognitive impairment induced by circadian rhythm disorders through the regulation of circadian rhythm mechanisms, research into the role of Bifidobacterium longum in regulating circadian rhythms and cognitive abilities and its application is of great significance. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a strain of *Bifidobacterium longum* that can alleviate circadian rhythm disorders and their induced cognitive and memory decline, along with its applications. Currently, it is unknown whether *Bifidobacterium longum* can alleviate cognitive impairment caused by circadian rhythm disorders, and its role in improving rhythm-related gut microbiota and intestinal metabolites is also unclear. Therefore, there is a need to develop a new probiotic agent that can alleviate circadian rhythm disorders and cognitive and memory decline to fill the current technological gap.

[0006] This invention provides a strain of Bifidobacterium longum that can alleviate cognitive impairment induced by circadian rhythm disorders by regulating circadian rhythms. Bifidobacterium longum GDMCC No. 65850 and its application in the preparation of products for regulating biological circadian rhythms, improving cognitive and memory impairment caused by sleep rhythm disorders, regulating intestinal flora imbalance and abnormal synthesis of metabolites.

[0007] In one embodiment, the Bifidobacterium longum was deposited on January 23, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65850, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] In one embodiment, the Bifidobacterium longum ( Bifidobacterium longum Growth characteristics of GDMCC No: 65850: This strain is a facultative anaerobic bacterium. When inoculated into the culture medium, it should be cultured in an anaerobic chamber at 37°C for at least 24 hours.

[0009] In one embodiment, the Bifidobacterium longum ( Bifidobacterium longum GDMCC No. 65850 is a Gram-positive bacterium. When grown on MRS solid medium, it forms smooth, raised, round colonies that are white with neat edges. In MRS liquid medium, it grows in a uniformly turbid manner, and the cells form a white precipitate after prolonged standing. The optimal growth temperature is 37°C.

[0010] The present invention also provides a microbial preparation containing the aforementioned Bifidobacterium longum GDMCC No: 65850.

[0011] In one embodiment, the amount of Bifidobacterium longum GDMCC No: 65850 added to the microbial preparation is not less than 5 × 10⁻⁶. 9 CFU / g or 5×10 9CFU / mL.

[0012] The present invention also provides products containing the aforementioned Bifidobacterium longum GDMCC No: 65850 or the aforementioned microbial preparation.

[0013] In one embodiment, the product includes food, medicine, or health products.

[0014] The present invention also provides the use of the aforementioned Bifidobacterium longum GDMCC No: 65850 or the aforementioned microbial preparation in the preparation of a drug having the effect of regulating circadian rhythm.

[0015] In one embodiment, the drug can treat circadian rhythm disorders caused by sleep deprivation or insomnia.

[0016] In one embodiment, the amount of *Bifidobacterium longum* GDMCC No: 65850 added to the drug is not less than 5 × 10⁻⁶. 9 CFU / g or 5×10 9 CFU / mL.

[0017] In one embodiment, the pharmaceutical product comprises Bifidobacterium longum GDMCC No: 65850 and pharmaceutically permissible carriers and / or pharmaceutical excipients.

[0018] In one embodiment, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0019] In one embodiment, the dosage form of the drug is granules, capsules, tablets, pills, or oral liquid.

[0020] In one implementation, the application includes at least one of the following functions: (a) Regulating the circadian rhythm of organisms; (b) Improve anxiety-like mood caused by circadian rhythm disorder; (c) Improves the decline in exploration and memory abilities caused by circadian rhythm disorders.

[0021] The present invention also provides a drug for regulating circadian rhythms, the drug containing the Bifidobacterium longum GDMCC No: 65850.

[0022] In one embodiment, the amount of *Bifidobacterium longum* GDMCC No: 65850 added to the drug is not less than 5 × 10⁻⁶. 5 CFU / g or 5×10 9 CFU / mL.

[0023] This invention also provides the application of the above-mentioned Bifidobacterium longum GDMCC No: 65850 in the preparation of a drug having at least one of the following functions: (a) Alleviating abnormal expression of circadian rhythm-related genes in the ileum and hypothalamus caused by circadian rhythm disorder; (b) Alleviate anxiety-like behavior and impaired exploratory and memory functions caused by circadian rhythm disruption; (c) Alleviating gut microbiota dysbiosis caused by circadian rhythm disruption; (d) Alleviate changes in intestinal metabolites caused by circadian rhythm disruption.

[0024] The present invention also provides the use of the aforementioned Bifidobacterium longum GDMCC No: 65850 in the preparation of health products that help improve memory decline caused by insomnia or in the preparation of health products that help improve sleep.

[0025] Beneficial effects: This invention provides a strain of *Bifidobacterium longum* that alleviates cognitive impairment caused by circadian rhythm disruption and improves changes in gut microbiota composition and gut metabolite levels caused by circadian rhythm disruption. Bifidobacterium longum GDMCC No: 65850, using the Bifidobacterium longum provided by this invention ( Bifidobacterium longum GDMCC No. 65850, when applied to sleep-deprived mice, significantly alleviated the decline in cognitive memory levels and improved changes in gut microbiota composition and gut metabolite levels caused by circadian rhythm disruption. Specifically, compared to the model group: (1) The disordered expression of circadian rhythm genes in the ileum and hypothalamus of mice with circadian rhythm disorder was improved.

[0026] (2) In the water maze test, the shortest time for mice with circadian rhythm disorder to find the center of the platform was shortened from 35.40±5.50s to 12.20±8.40s.

[0027] (3) In the water maze test, the proportion of time spent in the third quadrant of mice with circadian rhythm disorder increased from 15.67±4.93% to 34.57±8.45%.

[0028] (4) In the open field test, the proportion of time spent in the central region of mice with circadian rhythm disorder increased from 30.35±4.57% to 44.28±5.87%, and the trajectory of activity in the central region increased significantly.

[0029] (5) In the Y maze test, the frequency of alternation of the three arms of mice with circadian rhythm disorder increased from 46.96±4.61% to 57.08±2.05%.

[0030] (6) In the elevated cross maze test, the percentage of open arm time in mice with circadian rhythm disorder increased from 17.07±0.28% to 29.97±5.19%.

[0031] (7) It regulates the gut microbiota disorder caused by circadian rhythm disorder, and the relative abundance of Proteobacteria decreased from 4.53±0.85% to 2.05±0.61%, restoring the gut microbiota to a near-healthy level.

[0032] (8) It improves the changes in the levels of intestinal metabolites in mice caused by circadian rhythm disorder, upregulates the content of metabolites such as 2'-deoxyguanosine, guanosine, inosine, and N,N-dimethylglycine, and downregulates the content of metabolites such as sphingosine.

[0033] Therefore, Bifidobacterium longum ( Bifidobacterium longum GDMCC No. 65850 has great application potential in the preparation of products that alleviate circadian rhythm disorders and related cognitive and memory decline.

[0034] Preservation of biological materials Bifidobacterium longum GDMCC No: 65850, taxonomically named Bifidobacterium longum It was deposited on January 23, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65850. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology. Attached Figure Description

[0035] Figure 1 The regulatory effect of Bifidobacterium longum GDMCC No: 65850 on the expression of circadian rhythm-related genes in sleep-deprived mice: (A) ileum Bmal1 mRNA expression levels; (B) in the ileum Per3 mRNA expression levels; (C) in the ileum Cry1 mRNA expression levels; (D) in the hypothalamus Bmal1 mRNA expression levels; (E) in the hypothalamus Per3 mRNA expression levels; (F) in the hypothalamus Cry1 mRNA expression levels.

[0036] Figure 2 The following are the results of different groups of experimental mice in the water maze behavioral test: (A) Changes in escape time during the five-day positioning and navigation test; (B) Representative swimming trajectory map and trajectory heat map of the space exploration test on the sixth day; (C) Escape time during the space exploration test on the sixth day; (D) Percentage of time spent in the third quadrant during the space exploration test on the sixth day.

[0037] Figure 3The performance of mice in different groups in the open field behavioral test: (A) Representative movement trajectories of the open field test; (B) Percentage of time spent by mice in the central area of ​​the open field.

[0038] Figure 4 The performance of mice in different groups in other behavioral tests: (A) Y-maze transition frequency; (B) Percentage of time spent in open arms of the elevated cross maze.

[0039] Figure 5 The regulatory effects of GDMCC No. 65850 on the gut microbiota of sleep-deprived mice: (A) α-diversity; (B) changes in the abundance of Proteobacteria; (C) β-diversity.

[0040] Figure 6 To investigate the regulatory effect of GDMCC No. 65850 on intestinal metabolite levels in sleep-deprived mice: (A) Volcano plot analysis of the blank group vs. the model group; (B) Volcano plot analysis of the GDMCC No. 65850 intervention group vs. the model group.

[0041] In the above images: *, **, and *** represent comparisons with the blank group, respectively. p Values ​​less than 0.05, 0.01, and 0.001. Detailed Implementation

[0042] The following describes embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0043] The C57BL / 6J male mice used in the following examples were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. RNA extraction, reverse transcription kits, and the ChamQ Universal SYBR qPCR Master Mix fluorescent dye were purchased from Nanjing Novizan Biotechnology Co., Ltd. The fecal DNA extraction kit was purchased from MP Biomedicals, Inc. (USA). The gel extraction kit was purchased from Biomiga, Inc. (USA). The melatonin used in the following examples was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0044] The culture media involved in the following examples are as follows: MRS liquid culture medium (1 L): peptone 10 g, beef extract 10 g, yeast powder 5 g, anhydrous glucose 20 g, anhydrous sodium acetate 2 g, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.5 g, manganese sulfate monohydrate (MnSO4·H2O) 0.25 g, diammonium hydrogen citrate 2 g, dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O) 2.6 g, Tween 80 1 mL.

[0045] MRS solid medium (1 L): 2% agar powder added to MRS liquid medium.

[0046] The following examples illustrate the preparation method of the Bifidobacterium longum GDMCC No: 65850 bacterial suspension: (1) Strain screening, activation and purification A fecal sample was collected from an 84-year-old woman in Wuxi City, Jiangsu Province, using a disposable sterile stool sampler. 1 g of the sample was homogenized with PBS (containing 0.05% cysteine) and serially diluted. 10 samples were selected. -7 ~10 -9 Diluted solutions were spread onto MRS solid medium containing 1% nystatin and mupirocin and incubated anaerobically at 37°C for 48 h. Colony morphology was observed and recorded. Single colonies were picked and streaked onto MRS solid medium for purification and incubated upside down in an anaerobic incubator at 37°C for 48 h. Several single colonies were then inoculated into 5 mL of MRS liquid medium and incubated anaerobicly at 37°C for 24 h. Gram staining was performed, and Gram-positive bacteria were selected. After catalase analysis, catalase-positive strains were discarded, and catalase-negative strains were retained.

[0047] (2) Preservation and identification of strains After two generations of activation, single colony cultures were preserved. 500 μL of the culture was transferred to a preservation tube, and an equal volume of 60% sterile glycerol was added. The mixture was thoroughly mixed, labeled, and stored at -80°C. One tube of glycerol culture was used for 16S rDNA identification: centrifuged at 6000 rpm for 3 min, the supernatant was discarded, and the bacterial sludge was resuspended in 500 μL of sterile water. This centrifugation and resuspension process was repeated twice. The resulting bacterial suspension could be used as a DNA template for PCR amplification. The bacterial PCR amplification reaction system (50 μL) consisted of: 2.5 μL of 27F forward primer, 2.5 μL of 1492R reverse primer, 25 μL of Taq enzyme, 10 μL of bacterial suspension template, and 10 μL of sterile water. The PCR reaction conditions were: ① 94℃, 5 min; ② 94℃, 30 s; ③ 55℃, 30 s; ④ 72℃, 1 min; ⑤ 72℃, 10 min; ⑥ 12℃, 2 min (②-④ were repeated for 30 cycles). The PCR amplification products were sent to a professional sequencing company. The sequenced results were compared with the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed a new strain with a nucleic acid sequence similarity of 99% to *Bifidobacterium longum*, named *Bifidobacterium longum* GDMCC No: 65850, and deposited at the Guangdong Provincial Microbial Culture Collection Center on January 23, 2025, with accession number GDMCC No: 65850.

[0048] (3) Expand the system cultivation The identified and activated Bifidobacterium longum GDMCC No: 65850 bacterial suspension, which had been activated for more than 2 generations, was used at 2% ( v / v The inoculum was added to 1 L of MRS liquid medium, shaken well, and incubated in a 37°C anaerobic incubator for 36 h. The culture was then incubated at 8000× g Centrifuge at 4°C for 15 min, discard the supernatant, wash twice with sterile physiological saline, and finally resuspend in a certain volume of 30% glycerol and aliquot into several 5 mL EP tubes. Use one tube for strain identification to ensure the correctness of the strain; use another tube for dilution and plate spreading to determine the concentration of the bacterial suspension; store the remaining tubes at -80°C.

[0049] (4) Preparation of bacterial suspension for animal gavage A tube of glycerol bacterial solution was taken from a -80℃ freezer and stored at 4℃ and 8000× g Centrifuge for 10 min under the specified conditions, discard the supernatant, wash twice with sterile physiological saline, resuspend, and then dilute to 5 × 10⁻⁶. 9 The concentration of CFU / mL was used to obtain the required Bifidobacterium longum GDMCC No: 65850 bacterial suspension for gavage.

[0050] Example 1: Bifidobacterium longum GDMCC No: 65850 can alleviate sleep deprivation-induced abnormal expression of circadian rhythm-related genes in the ileum and hypothalamus of mice. Twenty male SPF-grade C57BL / 6J mice, aged 6-8 weeks and weighing 20±2 g, were selected and housed in an experimental environment with a temperature of 22±2℃ and a relative humidity of 50±5%, receiving a 12-hour light cycle daily and free access to food and water. After one week of acclimatization feeding, all mice were randomly divided into four groups (n=5 per group) for 28 days of treatment. The grouping and treatment methods are shown in Table 1.

[0051] Table 1. Animal grouping and treatment methods

[0052] Note: During the treatment period, except for the control group, all mice were deprived of sleep for 19 hours a day for 28 consecutive days.

[0053] The animal model was created using a modified multi-platform tank sleep deprivation method. The platform method (MMPM) allows for simultaneous sleep deprivation of multiple animals without affecting their free movement and feeding. Each sleep deprivation device consists of one large water tank (40×30×20 cm) and eight small platforms (3 cm in diameter, 4 cm high, spaced 3 cm apart). The small platforms are fixed inside the large water tank, with the water level 1-2 cm below the platform plane. The platforms are approximately 4 cm apart, allowing mice to move easily between them and maintain free movement, eating, and drinking. When mice enter REM sleep, muscle relaxation makes them prone to falling into the water; therefore, to avoid falling off the platforms, the mice maintain an upright posture and cannot sleep normally. During sleep deprivation, the water at the bottom of the tank should be kept clear, and the temperature should be maintained at 20-25°C. Control group mice are placed in a specially designed tank with four large platforms (12 cm in diameter, 4 cm high), large enough for them to sleep normally. The sleep deprivation cycle is from 2:00 PM to 9:00 AM the following day.

[0054] Mice were euthanized with isoflurane after the final sleep deprivation (9:00 AM). Fresh tissue samples (ileum and hypothalamus), 50-100 mg each, were collected from the mice. 1 mL of Trizol and two zirconium beads were added, and the mixture was homogenized at 65-70 Hz for 30 seconds at 4°C, repeated 3-5 times. The homogenate was then incubated overnight at 4°C. 0.2 mL of chloroform was added, and the mixture was vortexed for 15 seconds, followed by incubation at room temperature for 15 minutes until separation. (4°C, 12000×) g Centrifuge for 15 min; at this point, the RNA will be contained in the colorless aqueous supernatant. Transfer the supernatant (containing RNA) to a new tube, add one volume of pre-chilled isopropanol (4°C), mix well, and incubate at -20°C for at least 30 min to precipitate the RNA. Centrifuge at 4°C, 12000× g Centrifuge for 10 min. At this point, total RNA will appear as a white gel-like precipitate at the bottom of the EP tube. Aspirate the supernatant using a pipette, retaining the precipitate. Add 1 mL of pre-chilled 75% ethanol (prepared with DEPC water) to wash the precipitate and incubate at room temperature for 5 min. Gently vortex to wash the precipitate, then centrifuge at 12000 rpm / min for 5 min at 4°C, discarding the supernatant. Vacuum or air dry the RNA tube for 5-10 min until the RNA becomes translucent. Add 20-50 μL of DEPC water to resuspend the RNA, vortexing gently. Finally, incubate the resuspended solution at 55-60°C for 10-15 min, then immediately place it on ice. The resulting total RNA solution should be stored at -80°C.

[0055] RNA quality was assessed and concentration determined using a NanoDrop 2000 microphotometer. RNA purity (A260 / A280) should be between 1.8 and 2.0. Subsequently, it was reverse transcribed into cDNA using the Novizan RNA Reverse Transcription Kit, followed by real-time quantitative PCR. Samples were mixed with the fluorescent dye ChamQ Universal SYBR qPCR Master Mix. The qPCR system consisted of 5 μL Mix, 1 μL cDNA, 0.5 μL forward primer, and 0.5 μL reverse primer, with ddH2O added to a total volume of 10 μL. The thermal cycling protocol included initial denaturation at 95°C for 30 s, 40 denaturation cycles at 95°C for 5 s, and annealing / extension at 60°C for 30 s. After PCR amplification, melting curve analysis (range 65°C to 95°C, in 0.5°C increments) was performed to verify the specificity of the amplified fragments. The assay was performed using a CFX384 Real-Time System (Bio-Rad, USA) with real-time quantitative gene amplification. Each sample was analyzed in triplicate, and housekeeping genes were used. GAPDH As an internal reference, through 2 - Ct Calculate the relative change level of the target gene, where ΔCt=(Ct 目的基因 -Ct 管家基因 ) 实验组 -(Ct 目的基因 -Ct 管家基因 ) 对照组 Table 2 lists the sequences of the relevant primers.

[0056] Table 2 Primer sequences for mouse rhythm genes

[0057] Bmal1 , Per3 , Cry1 These genes are key components of the core clock transcription / translation feedback loop, and their normal expression plays a vital role in maintaining circadian rhythm balance and preserving host learning and memory abilities. Related research indicates that in the forebrain... Bmal1 The absence of these cells can impair hippocampal function, leading to deficits in both short-term and long-term memory. Per3 , Cry1 Mutations and abnormal expression of these genes can affect the transcription and translation of other core clock genes, leading to severe circadian rhythm disruptions, resulting in decreased sleep and cognitive impairment. qPCR results are as follows... Figure 1 As shown, compared with the blank control group, the ileum of the sleep deprivation model group had higher concentrations of [unclear text - possibly related to blood or fluid]. Bmal1 The mRNA expression level was significantly reduced by 37.00% ( p <0.001), Per3 The mRNA expression level increased significantly by 70.49% ( p <0.001), Cry1 The mRNA expression level increased significantly by 27.55% ( p <0.01); Hypothalamus Bmal1 The mRNA expression level increased significantly by 25.96% ( p <0.05), Per3 The mRNA expression level increased significantly by 36.34% ( p <0.05), Cry1 The mRNA expression level was significantly reduced by 34.13% ( p <0.01). This shows that after sleep deprivation, the ileum and hypothalamus... Bmal1 , Per3 , Cry1 Abnormal gene expression indicates circadian rhythm disruption in mice. Following intervention with GDMCC No. 65850, compared to the model group, the ileum... Bmal1 The mRNA expression level increased by 51.32% ( p <0.01), Per3 The mRNA expression level decreased by 76.61% ( p <0.001), Cry1 The mRNA expression level decreased by 67.74% ( p <0.001); Hypothalamus Bmal1 The mRNA expression level decreased by 20.34% ( p <0.05), Per3 The mRNA expression level decreased by 9.72%, but this was not statistically significant. Cry1 The mRNA expression level increased by 48.76% ( p <0.05). Therefore, GDMCC No: 65850 can inhibit central and peripheral rhythm disorders caused by sleep deprivation and improve cognition in mice.

[0058] After melatonin intervention ( Figure 1 Compared with the model group, the ileum Bmal1 The mRNA expression level increased by 132.22% ( p <0.001), Per3 The mRNA expression level decreased by 78.36% ( p <0.001), Cry1 The mRNA expression level decreased by 55.20% ( p <0.001); Hypothalamus Bmal1 The mRNA expression level decreased by 38.25% ( p <0.001), Per3 The mRNA expression level decreased by 46.75% ( p <0.001), Cry1 The mRNA expression level increased by 46.74% ( p <0.05). Especially for the ileum and hypothalamus. Cry1 In terms of gene expression, GDMCC No. 65850 and melatonin have similar regulatory effects. The results indicate that GDMCC No. 65850 and melatonin exhibit similar rhythm regulation effects, both capable of modulating the expression of rhythm genes in the ileum and hypothalamus to some extent.

[0059] Example 2: Bifidobacterium longum GDMCC No: 65850 can improve cognitive and memory behaviors in sleep-deprived mice. Animal models were established according to the method in Example 1, and behavioral tests were conducted on mice on day 15 after sleep deprivation. The open field test and elevated cruciate maze were used to evaluate the mice's anxieties, while the water maze and Y-maze were used to evaluate their cognitive functions. All mice were transferred to the behavioral testing room at least 30 minutes before the start of the tests to acclimatize to the conditions. All tests were conducted between 8:00 and 17:00.

[0060] (1) Water Maze The spatial learning and memory abilities of mice were assessed using a water maze test. The test was conducted in a circular tank with a diameter of 120 cm and a height of 35 cm. The maze was divided into four quadrants. A platform was placed in the center of the first quadrant and hidden 1 cm below the water surface. Milk powder was added to the water to make it opaque. The water maze test included a five-day orientation and navigation test and a one-day spatial exploration test. In the orientation and navigation test, each mouse was trained twice daily, with a 2-3 hour interval between tests. Each test involved placing the mouse in the water from a different quadrant. The escape time (60 seconds) was recorded for each mouse to find the hidden platform. If a mouse could not find the platform within 60 seconds, the escape time was recorded as 60 seconds. The mouse was then guided to the platform and observed standing on it for 10 seconds. On day 6, the hidden platform was removed to conduct a spatial exploration experiment and a memory retention test. Each mouse was placed in water from the third quadrant, and the movement path of the mouse within 60 seconds, the time required to reach the center of the original platform for the first time (escape time), and the time spent in the original platform location and quadrant were recorded.

[0061] like Figure 2 As shown in Figure A, after 5 consecutive days of orientation and navigation experiments, the time it took for mice in each group to find the platform decreased progressively. Specifically, on day 5, compared to the model group, the escape time of the GDMCC No. 65850 intervention group was significantly reduced by 71.96% (…). Figure 2 A, p <0.001 indicates that GDMCC No. 65850 intervention significantly improved the decline in spatial learning ability in mice caused by rhythm disorder. In the spatial exploration test on day 6, compared to the control group, the model group mice required a longer path ( Figure 2 B) and time ( Figure 2 C, p <0.001, blank group 4.27±2.14 s vs model 35.40±5.50 s) found the center of the original platform, and the exploration time in the third quadrant where the original platform is located was also significantly shorter than that of the blank group ( Figure 2 D, p <0.05, blank 32.67±2.03% vs model 15.67±4.93%. After intervention with GDMCC No: 65850, the speed and accuracy of mice finding the original platform location were significantly improved, as evidenced by a significant shortening of the path taken to find the center of the original platform ( Figure 2 B) Escape time significantly reduced ( Figure 2 C, p<0.01, GDMCC No: 65850 12.20±8.40 s vs model 35.40±5.50 s), in the third quadrant ( Figure 2 D, p The exploration time of GDMCC No. 65850 (34.57±8.45% vs. model 15.67±4.93%) was significantly increased (<0.05), indicating that GDMCC No. 65850 intervention can improve the decline in spatial learning and cognitive abilities caused by rhythm disorder.

[0062] (2) Open field The open field test is used to assess exploratory activity and anxiety-like behavior in mice. Mice are placed in the center of an open field (50×50cm, 30cm high) and allowed to explore freely for 8 minutes. The number of times the mice explore the central area (33×33cm), the time spent exploring it, and the exploration path throughout the field are recorded.

[0063] The results of the open field test are as follows Figure 3 As shown, from Figure 3 As shown in Figure A, the model group mice mainly roamed the perimeter of the open field, while the control group and the GDMCC No. 65850 intervention group mice preferred to roam in the central area of ​​the open field. The percentages of time spent in the central area of ​​the open field by the three groups of mice (control group, model group, and GDMCC No. 65850 intervention group) were 44.84±5.15%, 30.35±4.57%, and 44.28±5.87%, respectively. Figure 3 B), compared with the model group, the GDMCC No: 65850 intervention group ( p <0.05) The proportion of time spent in the middle area of ​​the open field increased significantly, indicating that GDMCC No: 65850 can alleviate anxiety caused by rhythm disorder.

[0064] (3) Y Maze Mice were placed individually in the center of a Y-maze (3 arms, each 30 cm long, spaced 120° apart) and allowed to explore freely for 5 minutes. The number of times each mouse entered all arms and the frequency of alternation between the three arms were recorded. A mouse was only counted as entering an arm when it completely entered one of the arms.

[0065] The results of the Y-maze experiment are as follows: Figure 4 As shown in Figure A, the percentages of time spent in the central area of ​​the open field by the three groups of mice (blank group, model group, and GDMCC No. 65850 intervention group) were 59.75±2.89%, 46.96±4.61%, and 57.08±2.05%, respectively. Compared with the model group, the GDMCC No. 65850 intervention group (blank group, model group, and GDMCC No. 65850 intervention group) p<0.01) The proportion of time spent in the middle area of ​​the open field increased significantly, indicating that GDMCC No: 65850 can alleviate the decline in spatial memory and cognitive ability caused by rhythm disorder.

[0066] (4) Elevated cross maze The elevated maze has a "+" structure, consisting of two open arms (25×5×0.5 cm), two closed arms (25×5×16 cm), and a central platform (5×5×0.5 cm). Each mouse is placed in the central platform area, facing one of the open arms, and is allowed to move freely for 5 minutes. The number of times a mouse enters an open arm and the percentage of time spent on an open arm are recorded (when the tip of a mouse's nose enters an arm, it is counted as one entry into that arm).

[0067] The results of the elevated cross maze experiment are as follows Figure 4 As shown in B, the percentages of time spent in the open arms of the elevated cross maze in the three groups of mice (blank group, model group, and GDMCC No.: 65850 intervention group) were 25.65±2.07%, 17.07±0.28%, and 29.97±5.19%, respectively. Compared with the model group, the GDMCC No.: 65850 intervention group ( p <0.01) The proportion of open arm activity time in the elevated cross maze was significantly increased, indicating that GDMCC No: 65850 can alleviate anxiety-like behavior in mice caused by rhythm disorder.

[0068] Melatonin can reduce the exploration paths of mice with circadian rhythm disorders in a water maze. Figure 2 B) and positioning time ( Figure 2 C, 7.4 ± 2.42 s, p <0.001, increases the mice's activity in the central region of the open field ( Figure 3 43.71±1.74%, p <0.05) and the elevated cross-maze open arm ( Figure 4 B, 33.28±2.86%, p The exploration time was <0.001), and the alternation frequency of mice in the Y maze was increased. Figure 4 A, 59.25±1.85%, p <0.01). In the water maze, Y-maze, elevated cross maze, and open field tests, GDMCC No. 65850 showed cognitive enhancement and anxiety relief effects similar to melatonin. Therefore, GDMCC No. 65850 has the potential to replace medication (melatonin) in alleviating circadian rhythm disorders and their induced cognitive decline.

[0069] Example 3: Bifidobacterium longum GDMCC No: 65850 can improve the gut microbiota of sleep-deprived mice. An animal model was established according to the method in Example 1. Fresh feces were collected from mice at the end of the 5th week, and the feces were tested using MP. The kit was used to extract total DNA from mouse fecal samples. The specific operating steps were as follows, mainly following the kit instructions. Using the total DNA from each sample as a template, PCR amplification of the V3-V4 region was performed using primers (341F: 5'-CCTAYGGGRBGCASCAG-3', 806R: 5'-GGACTACNNGGGTATCTAAT-3'). The PCR system (50 μL) consisted of: 2 μL DNA template, 1.5 μL 341F, 1.5 μL 806R, 20 μL ddH2O, and 25 μL Taq mix. The PCR program was: 95℃, 5 min; 95℃, 30 s; 50℃, 30 s; 72℃, 30 s, 30 cycles; 72℃, 10 min. Prepare a 2.0% agarose gel and perform electrophoresis at 120V for 30-40 min. After electrophoresis, recover the target bands from the gel according to the QIA quick Gel Extraction Kit instructions. Measure and record the concentration and purity of the recovered DNA using a NanoDrop 2000 microphotometer. Construct a library using the TurSeqDNA LT Sample Preparation Kit and its instructions. Finally, sequence the data using an Illumina MiSeq sequencer according to the MiSeq Regent Kit and its instructions. Process the sequence data using the QIIME2 analysis channel and perform principal coordinate analysis (PCoA) using the Lianchuan Cloud website (https: / / www.omicstudio.cn / tool).

[0070] Alpha diversity and beta diversity are used to assess changes in the diversity of fecal microbiota. Figure 5 ). The α-diversity was characterized by the Shannon index, and the results showed ( Figure 5 A), the Shannon index in the model group was significantly increased ( p <0.001, which may be related to the proliferation of conditionally pathogenic bacteria in the gut. After intervention with GDMCC No. 65850, the Shannon index significantly decreased to near-normal levels. p <0.001). β diversity was assessed using PCoA, and the results showed that ( Figure 5 C), significant differences were found among the blank group, the model group, and the GDMCC No: 65850 intervention group. p <0.001). In terms of relative abundance changes at the phylum level, the relative abundance of Proteobacteria in the model group was significantly higher than that in the control group (…). p<0.001), Proteobacteria includes a variety of pathogens such as Escherichia coli, Salmonella, and Vibrio cholerae. After intervention with GDMCC No. 65850, the relative abundance of Proteobacteria decreased significantly ( p <0.001), indicating that GDMCC No: 65850 can reduce the relative abundance of harmful bacteria in the gut, improve gut microbiota dysbiosis caused by rhythm disorders, and restore gut microbiota health.

[0071] Example 4: Bifidobacterium longum GDMCC No: 65850 can improve the synthesis of intestinal metabolites in sleep-deprived mice. An animal model was established according to the method in Example 1. Fresh feces were collected from mice at the end of the 5th week. A pre-cooled methanol-acetonitrile-water mixture (2:2:1, v / v / v) and two zirconium beads were added, and the mixture was vortexed for 30 s. The tissue was then lysed at 4°C and 65-70 Hz for 30 s, repeated 3-5 times. The tissue was extracted by sonication in an ice-water bath for 20 min, followed by standing at -20°C for 30 min. The tissue was then centrifuged at 13000 rpm / min at 4°C for 15 min, and 200 μL of the supernatant was used for LC-MS analysis. Column conditions: ACQUITY UPLC BEH Amide column (Water, 1.7 μm, 2.1 mm × 100 mm), mobile phases A and B were aqueous and acetonitrile, respectively. The eluent for positive mode was 0.01% acetic acid + H₂O (mobile phase A), and the eluent for negative mode was 50% ACN + 50% IPA (mobile phase B). The temperature was 35℃, the flow rate was 0.3 mL / min, and the injection volume was 2 μL. Raw data processing was performed using Compound Discover software, followed by data analysis using the metabolic analysis website (https: / / www.metaboanalyst.ca).

[0072] Volcano plot analysis revealed 60 metabolites with significant differences between the control group and the model group (FC>1.5 or <0.67, and...). p <0.05), sleep deprivation-induced circadian rhythm disturbances lead to the downregulation of 43 metabolites, including 2'-deoxyguanosine and phenylacetaldehyde, and the upregulation of 17 metabolites, including sphingosine. Figure 6 A). However, GDMCC No: 65850 intervention can reduce sphingosine levels and increase 2'-deoxyguanosine and phenylacetaldehyde levels ( Figure 6B). Studies have shown that excessive accumulation of sphingosine has neurotoxicity and can impair host cognitive function. 2'-Deoxyguanosine is one of the precursors of DNA synthesis, participating in DNA replication and repair processes, and plays a crucial role in maintaining normal cellular physiological functions. These findings indicate that circadian rhythm disruption leads to changes in the levels of intestinal metabolites in mice, and that GDMCC No. 65850 intervention can regulate these metabolic changes, thereby promoting host health. Furthermore, compared with the model group, GDMCC No. 65850 intervention can also increase the levels of adenosine, guanosine, inosine, and N,N-dimethylglycine (DMG). Figure 6 (B) Adenosine can integrate cues between sleep / wake behavior and circadian rhythms, and the adenosine signaling pathway is involved in the repair of damaged intestinal barrier. Guanosine, inosine, and N,N-dimethylglycine have anti-inflammatory and immunomodulatory effects, which can reduce inflammatory responses and protect nervous system function. In summary, GDMCC No. 65850 intervention can promote the production of neuroprotective metabolites and reduce cognitive decline caused by circadian rhythm disruption.

[0073] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Bifidobacterium longum ( Bifidobacterium longum GDMCC No. 65850, characterized in that, The Bifidobacterium longum was deposited on January 23, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65850, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A microbial preparation containing the Bifidobacterium longum GDMCC No: 65850 as described in claim 1.

3. The microbial preparation as described in claim 2, characterized in that, In the microbial preparation, the number of *Bifidobacterium longum* GDMCC No: 65850 cells is not less than 5 × 10⁻⁶. 9 CFU / g or 5×10 9 CFU / mL.

4. A product containing *Bifidobacterium longum* GDMCC No: 65850 as described in claim 1 or the microbial preparation as described in claim 2 or 3, characterized in that, The products include food, medicine, or health products.

5. The use of the *Bifidobacterium longum* GDMCC No: 65850 of claim 1 or the microbial preparation of claim 2 or 3 in the preparation of a medicament for regulating circadian rhythm disorders caused by insomnia.

6. The application as described in claim 5, characterized in that, The drug also contains a drug carrier and / or pharmaceutical excipients.

7. The application as described in claim 5 or 6, characterized in that, The drug has at least one of the following functions: (a) Regulating the circadian rhythm of organisms; (b) Improve anxiety-like mood caused by circadian rhythm disorder; (c) Improves the decline in exploration and memory abilities caused by circadian rhythm disorders.

8. A drug for regulating circadian rhythm disorders caused by insomnia, characterized in that, Contains the Bifidobacterium longum GDMCC No: 65850 as described in claim 1.

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