Application of bifidobacterium breve NX-5 in preparation of drugs for relieving anxiety and improving sleep

Abstract

The invention discloses application of bifidobacterium breve NX-5 in preparation of drugs for relieving anxiety and improving sleep, and belongs to the technical field of microorganisms. The invention discloses application of bifidobacterium breve NX-5 in preparation of drugs for relieving anxiety and improving sleep, a bacterial suspension of the bifidobacterium breve NX-5 can significantly reduce the movement distance, mania time and active time of zebra fish in an in-vivo insomnia model, and significantly increase the standing time, and has the potential of being applied in vivo to relieve anxiety and improve sleep. The bifidobacterium breve NX-5 disclosed by the invention has a huge potential application prospect in the aspects of relieving anxiety and improving sleep.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to the application of Bifidobacterium breve NX-5 in the preparation of drugs for relieving anxiety and improving sleep. Background Technology

[0002] Sleep is closely related to our lives; however, with rapid societal development, people face increasing pressure, and insomnia has gradually become a common phenomenon. Insomnia may be accompanied by symptoms such as anxiety and depression. Currently, the most common clinical medications used to relieve anxiety and treat sleep disorders are benzodiazepines. However, these medications may have varying degrees of side effects, such as dizziness, fatigue, drowsiness, poor concentration, fear, irritability, hallucinations, and even temporary amnesia and hangovers, excessively prolonging sleep time. They do not truly achieve the expected effects of relieving anxiety and improving sleep quality. Therefore, choosing natural, side-effect-free ingredients is an important approach to relieving anxiety and treating sleep disorders.

[0003] Gamma-aminobutyric acid (GABA) is an important neurotransmitter. In recent years, the research and development of GABA-rich foods has become a hot topic both domestically and internationally. Studies have shown that GABA can effectively relieve mental stress and regulate mood, and has a certain alleviating effect on anxiety and insomnia. Other studies have shown that sleep disorders can lead to metabolic disturbances, accompanied by mild changes in the gut microbiota. Recently, some studies have reported preliminary evidence that the gut microbiota affects sleep. Mice that have been given antibiotics for a long time exhibited sleep structure disorders accompanied by changes in gut neurotransmitters. Furthermore, studies have found that dietary probiotics can effectively improve stress-affected sleep. These results suggest that remodeling the microbiota may improve host sleep quality through GABA.

[0004] However, current research and application of probiotics in relieving anxiety and improving sleep are limited. Domestic manufacturers have long relied on imported probiotic strains, and these foreign strains may not be suitable for the gastrointestinal physiology of Chinese residents. Furthermore, the lack of strong scientific evidence regarding the functions of probiotics severely hinders the promotion of probiotics and their products. Therefore, in-depth exploration of the functions of probiotic strains, and the screening of novel probiotic strains with independent intellectual property rights, specific functional properties, and suitability for the physiological characteristics of the Chinese population, are crucial for enhancing the core competitiveness of Chinese probiotic manufacturers and promoting the development of probiotic products in my country.

[0005] Therefore, providing the application of Bifidobacterium breve NX-5 in the preparation of drugs to relieve anxiety and improve sleep is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the application of Bifidobacterium breve NX-5 in the preparation of drugs for relieving anxiety and improving sleep.

[0007] Zebrafish share a similar nervous system with mammals. Most of the molecules and cellular groups that play a role in sleep regulation in mammals are also present in zebrafish. The responses of various hypnotic drugs, such as melatonin agonists and gamma-aminobutyric acid (GABA) modulators, in zebrafish are also similar to those in mammals. GABA is a naturally occurring non-protein amino acid and an important inhibitory neurotransmitter in the mammalian central nervous system. Pentyltetrazole (PTZ) is a GABA inhibitor; low doses of PTZ can induce bipolar disorder, characterized by both mania and depression, thereby causing insomnia.

[0008] The zebrafish insomnia model exhibits abnormal behavior, including a significant increase in total movement distance, manic duration, and active duration, which can be observed using a behavior analyzer.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The application of Bifidobacterium breve NX-5 in the preparation of drugs for relieving anxiety and improving sleep, wherein the accession number of Bifidobacterium breve NX-5 is CGMCC No. 20113 (see patent number 202110372822.2).

[0011] The described Bifidobacterium NX-5 significantly reduced the movement distance, agitation time, and active time of zebrafish in a PTZ-induced zebrafish insomnia model, while significantly increasing the resting time, demonstrating good probiotic effects in relieving anxiety and improving sleep.

[0012] Furthermore, the application of the aforementioned Bifidobacterium NX-5 in the preparation of fermented dairy products and other foods.

[0013] Furthermore, the Bifidobacterium NX-5 is a bacterial suspension.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of Bifidobacterium breve NX-5 in the preparation of drugs for relieving anxiety and improving sleep. Bifidobacterium breve NX-5 is isolated and screened from the feces of healthy infants and can significantly reduce the movement distance, agitation time, and active time of zebrafish in a zebrafish insomnia model, and significantly increase the resting time. It has the potential to relieve anxiety and improve sleep in vivo. This provides a theoretical reference and guidance for the development of probiotic preparations for relieving anxiety and improving sleep using Bifidobacterium breve NX-5. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 The attached figure shows the effect of the present invention's Bifidobacterium NX-5 on the movement trajectory of a PTZ-induced zebrafish insomnia model;

[0017] Wherein, A: normal group; B: model group; C: positive control group; D: 1×10 4 CFU / mL Bifidobacterium breve 15701; E: 1×10 5 CFU / mL Bifidobacterium breve 15701; F: 1×10 6 CFU / mL Bifidobacterium breve 15701; G: 1×10 4 CFU / mL Bifidobacterium breve NX-5; H: 1×10 5 CFU / mL Bifidobacterium breve NX-5; I: 1×10 6 CFU / mL Bifidobacterium breve NX-5;

[0018] Figure 2 The attached figure shows the effect of Bifidobacterium breve NX-5 of the present invention on the movement distance of a PTZ-induced zebrafish insomnia model;

[0019] Figure 3 The attached figure shows the effect of Bifidobacterium breve NX-5 of the present invention on the manic duration of a PTZ-induced zebrafish insomnia model;

[0020] Figure 4 The attached figure shows the effect of Bifidobacterium breve NX-5 of the present invention on the active time of a PTZ-induced zebrafish insomnia model;

[0021] Figure 5 The attached figure shows the effect of Bifidobacterium breve NX-5 of the present invention on the resting time of the PTZ-induced zebrafish insomnia model. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] The zebrafish is an AB lineage zebrafish.

[0024] Oxazepam and pentylenetetrazol (PTZ) were purchased from Sigma-Aldrich, Inc., USA; Bifidobacterium breve 15701 (ATCC15701) was purchased from Beijing BioBio Biotechnology Co., Ltd.

[0025] Example 1: Preparation of Bifidobacterium breve NX-5 bacterial suspension (cells)

[0026] After activation and culture, *Bifidobacterium breve* NX-5 was inoculated into BS liquid medium and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C and 6000 rpm for 10 min to obtain a bacterial pellet. The bacterial pellet was washed twice with PBS, and the cells were resuspended in PBS to adjust the cell concentration to 1 × 10⁻⁶ cells / mL. 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.

[0027] Example 2: Preparation of Bifidobacterium breve 15701 bacterial suspension (cells)

[0028] After activation and culture, *Bifidobacterium breve* 15701 was inoculated into BS liquid medium and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C and 6000 rpm for 10 min to obtain a bacterial pellet. The bacterial pellet was washed twice with PBS, and the cells were resuspended in PBS to adjust the cell concentration to 1 × 10⁻⁶. 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.

[0029] Example 3: The effect of Bifidobacterium breve NX-5 on improving sleep and anxiety in a zebrafish insomnia model.

[0030] Healthy wild-type AB strain zebrafish that had reached 6 days post-fertilization (dpf) were selected and placed in 96-well cell culture plates. The experiment included a normal control group, a model group, a positive control group (oxazepam), and a Bifidobacterium breve 15701 intervention group (1×10⁻⁶). 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL), Bifidobacterium breve NX-5 intervention group (1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL), with 25 replicates per group and one fish per well. Both the normal and model groups received PBS; the positive control group received oxazepam solution (30 μg / mL); and the Bifidobacterium breve 15701 intervention group received 1×10⁻⁶ CFU / mL.4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL Bifidobacterium breve 15701; Bifidobacterium breve NX-5 intervention group was added with 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL Bifidobacterium breve NX-5, 150 μL per well; after incubation at 28℃ for 24 h, 50 μL of PBS was added to the normal group, and 1×10⁻⁶ CFU / mL Bifidobacterium breve 15701 intervention group was added to the model group, positive control group, and Bifidobacterium breve 15701 intervention group. 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL), Bifidobacterium breve NX-5 intervention group (1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 PTZ (60 mM) was added to each well at a concentration of CFU / mL, 20 μL per well. After incubation at 28°C for 2 h, the 96-well cell culture plate was placed in a zebrafish behavior analysis system (EthoVision XT10.4, Nordas Information Technology Co., Ltd.) for trajectory tracking. The light source was turned off to allow the zebrafish to acclimatize to the environment for 5 min. Then, the behavior trajectory analysis software was turned on to collect the zebrafish's behavior trajectory within 5 min in the dark environment. The movement distance of the zebrafish was counted. At the same time, the zebrafish were divided into three movement states according to their swimming speed: stationary (<4 mm / s), active (4-20 mm / s), and agitated (>20 mm / s). The duration of agitation, activity, and stationary states of the zebrafish were also recorded.

[0031] Data were statistically processed using SPSS 19.0 software. Experimental data are expressed as mean ± SEM values. T-tests were used for analysis, comparing the results with the normal group: ### p<0.005, compared with the model group: &&& p<0.001; using one-way ANOVA, compared with the model group: *P<0.05, **P<0.01, ***P<0.005.

[0032] The experimental results of the effect of Bifidobacterium breve NX-5 on improving sleep and anxiety in a zebrafish insomnia model are as follows: Figure 1-5 As shown.

[0033] Depend on Figure 1 and Figure 2As can be seen, the red area represents the area covered by the zebrafish's movement path. Compared with the normal group, the movement trajectory of the zebrafish in the model group increased, indicating that the activity level of the zebrafish in the model group increased. At the same time, compared with the normal group (42.97±2.77cm), the movement distance of the zebrafish in the model group (72.91±2.44cm) increased significantly (p<0.005), indicating that the zebrafish insomnia model was successfully established.

[0034] Compared with the model group, the zebrafish in the positive control group (oxazepam) exhibited reduced movement trajectories, indicating that oxazepam can reduce the activity level of zebrafish in the PTZ-induced zebrafish insomnia model. Simultaneously, the movement distance of the zebrafish in the positive control group was 46.30±3.05 cm, significantly different from that in the model group (72.91±2.44 cm) (P<0.005). Therefore, oxazepam has an anxiolytic effect, consistent with clinical findings. Compared with the model group, the concentration of Bifidobacterium breve 15701 was 1×10⁻⁶. 6 At CFU / mL, the zebrafish movement distance was 62.78±2.62 cm, which was significantly different from the model group (72.91±2.44 cm) (P<0.05). Additionally, the *Bifidobacterium breve* NX-5 group (1×10⁻⁶) showed significantly different movement distances. 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 The reduced movement trajectory of zebrafish (CFU / mL) indicates that *Bifidobacterium breve* NX-5 can decrease the activity level of zebrafish in a PTZ-induced zebrafish insomnia model; simultaneously, the concentration of *Bifidobacterium breve* NX-5 was 1×10⁻⁶. 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 At CFU / mL, the movement distances of zebrafish were 58.14±2.53 cm, 56.42±2.85 cm, and 48.30±3.29 cm, respectively, all significantly different from the model group (72.91±2.44 cm) (P<0.01). Therefore, these results indicate that at the same concentration, *Bifidobacterium breve* NX-5 has a stronger inhibitory effect on PTZ-induced anxiety in zebrafish than *Bifidobacterium breve* 15701, and possesses the potential to alleviate anxiety.

[0035] Depend on Figure 3 , Figure 4 and Figure 5It can be seen that, compared with the normal group (frenzy time: 7.97±0.98s, active time: 31.18±2.33s, still time: 260.85±2.21s), the frenzy time (24.41±0.66s) and active time (62.06±2.98s) of the zebrafish in the model group were significantly increased (p<0.005), while the still time (213.53±2.98s) was significantly decreased (p<0.005), indicating that the zebrafish insomnia model was successfully established.

[0036] Compared with the model group (frenzy time: 24.41±0.66s, active time: 62.06±2.98s, resting time: 213.53±2.98s), the positive control group zebrafish showed significantly reduced frenzy time (11.56±0.59s) and active time (33.94±2.84s) (p<0.005), and significantly increased resting time (254.50±3.15s) (p<0.005), indicating that oxazepam has an anti-anxiety and hypnotic effect, consistent with clinical results. Compared with the model group (frenzy time: 24.41±0.66s, active time: 62.06±2.98s, resting time: 213.53±2.98s), the concentration of Bifidobacterium breve 15701 was 1×10⁻⁶. 6 At a concentration of CFU / mL, the duration of agitation (20.15±0.91s) and active period (49.62±2.70s) in zebrafish were significantly reduced (p<0.05), while the resting period (230.23±2.95s) was significantly increased (p<0.01). Furthermore, compared to the model group (agitation time: 24.41±0.66s, active period: 62.06±2.98s, resting period: 213.53±2.98s), a concentration of 1×10⁻⁶ CFU / mL significantly reduced the agitation time (20.15±0.91s) and resting period (49.62±2.70s) in zebrafish (p<0.05). 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 At CFU / mL, the duration of agitation in zebrafish (1×10⁻⁶) 4 CFU / mL group: 17.49±1.03s, 1×10 5 CFU / mL group: 15.50±0.90s, 1×10 6 CFU / mL group: 14.35±0.75s) and active time (1×10 4 CFU / mL group: 42.47±3.54s, 1×10 5 CFU / mL group: 41.06±2.81s, 1×10 6 The CFU / mL group (34.99±4.09s) was significantly reduced (p<0.01), and the resting time (1×10) was also significantly reduced. 4 CFU / mL group: 240.04±3.22s, 1×105 CFU / mL group: 243.44±2.98s, 1×10 6 The CFU / mL group (250.66±4.23s) was significantly increased (p<0.005). Therefore, the above results indicate that at the same concentration, *Bifidobacterium breve* NX-5 has a stronger inhibitory effect on PTZ-induced sleep in zebrafish than *Bifidobacterium breve* 15701, and has the potential to improve sleep.

[0037] Therefore, the above results indicate that Bifidobacterium breve NX-5 can significantly reduce the manic and active time and significantly increase the resting time in zebrafish in an in vivo insomnia model, demonstrating the effects of relieving anxiety and improving sleep.

[0038] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of Bifidobacterium breve NX-5 in the preparation of drugs for relieving anxiety and improving sleep, characterized in that, The preservation number of the Bifidobacterium breve NX-5 is CGMCC No. 20113.

2. The application of Bifidobacterium breve NX-5 according to claim 1 in the preparation of drugs for relieving anxiety and improving sleep, characterized in that, The Bifidobacterium NX-5 was a bacterial suspension.

3. The application of the Bifidobacterium NX-5 as described in claim 1 in the preparation of fermented dairy products and other foods.

4. The application of Bifidobacterium breve NX-5 according to claim 3 in the preparation of fermented dairy products and other foods, characterized in that, The Bifidobacterium NX-5 was a bacterial suspension.

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