A composition of lactic acid bacteria strains for improving sleep quality and its application.

Abstract

This invention relates to the field of probiotics technology, and in particular to a composition of lactic acid bacteria strains for improving sleep quality and its application. The composition includes *Bifidobacterium breve* BBr-28 strain, *Bifidobacterium animalis* subsp. *lactobacter* PS23 strain, *Bifidobacterium longum* subsp. *infant* strain BR631 strain, and *Chemobacter paracasei* strain BR176 strain. The lactic acid bacteria strain composition of this invention possesses physiological activity that improves sleep quality, and therefore can be used to prepare compositions that enhance sleep quality to address sleep problems.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, and in particular to a composition of lactic acid bacteria strains that improve sleep quality and its application. Background Technology

[0002] Sleep quality not only affects physical health but can also lead to premature aging, making it a crucial factor in maintaining normal daily functioning. However, with the prevalence of smartphones and other electronic devices, many people continue to use their phones before bed, such as browsing social media, watching TV shows, or playing games. These behaviors significantly reduce sleep quality and contribute to a year-on-year increase in insomnia. Therefore, insomnia is no longer limited to the elderly; it is increasingly affecting younger people, peaking in those aged 20 to 40.

[0003] Research on the health benefits of probiotics is extensive. Generally, strains with specific health benefits for humans are called functional probiotics (Guidelines for the evaluation of probiotics information; Report of joint FAO / WHO working group on drafting guidelines for the evaluation of probiotics in food; London Ontario, Canada April 30 and May 1, 2002: 1-7). Various functional probiotics are publicly available; however, the specific efficacy of functional probiotics lies in the strain specificity rather than the species. That is, even the same species, if different strains, may have different, or even opposite, effects.

[0004] In light of this, there is an urgent need to develop a safe and long-term edible nutritional supplement that can improve sleep quality and address sleep problems. Since lactic acid bacteria are generally considered safe, identifying compositions containing lactic acid bacteria strains with sleep-enhancing activity is a pressing goal. Summary of the Invention

[0005] The purpose of this application is to address the shortcomings of the prior art by providing a composition of lactic acid bacteria strains that improve sleep quality and its application.

[0006] The above-mentioned inventive objective of this application is achieved through the following technical solutions:

[0007] One objective of this invention is to provide a composition of lactic acid bacteria strains that improve sleep quality. This composition includes *Bifidobacterium breve* strain BBr-28 (CGMCC NO. 32485), *Bifidobacterium animalis subsp. lactis* strain PS23 (CGMCC NO. 32691), *Bifidobacterium longum subsp. infantis* strain BR631 (CGMCC NO. 26243), and *Lacticaseibacillus paracasei* strain BR176 (CGMCC NO. 21563).

[0008] In the above technical solution, the ratio of the strains of Bifidobacterium breve BBr-28, Bifidobacterium animalis subsp. lactis PS23, Bifidobacterium longum subsp. infantis BR631, and Bifidobacterium paracasei BR176 is 1:(0.5-1.5):(0.5-1.5):(0.5-1.5).

[0009] A second objective of this invention is to provide the application of the above-mentioned lactic acid bacteria strain composition in the preparation of compositions for improving sleep quality.

[0010] In the above technical solution, the effective viable count of the lactic acid bacteria strain composition is equal to or greater than 5 × 10⁻⁶. 9 CFU / g.

[0011] A third objective of this invention is to provide a composition for improving sleep quality, the composition comprising the above-mentioned lactic acid bacteria strain composition and a carrier.

[0012] In the above technical solution, the ratio of the strains of Bifidobacterium breve BBr-28, Bifidobacterium animalis subsp. lactis PS23, Bifidobacterium longum subsp. infantis BR631, and Bifidobacterium paracasei BR176 is 1:1:1:1.

[0013] In the above technical solution, the effective viable count of all lactic acid bacteria strains in the composition is equal to or greater than 5 × 10⁻⁶. 9 CFU / g.

[0014] In the above technical solution, the carrier is a physiologically acceptable excipient or diluent.

[0015] In the above technical solution, the carrier is a pharmaceutically acceptable excipient or diluent.

[0016] In the above technical solution, the dosage form of the composition is tablets, capsules or powder.

[0017] The beneficial effects of this application are:

[0018] This invention provides a composition of lactic acid bacteria strains for improving sleep quality, comprising: Bifidobacterium breve BBr-28 strain with accession number CGMCC NO.32485; Bifidobacterium animalis subsp. lactis PS23 strain with accession number CGMCC NO.32691; Bifidobacterium longum subsp. infantis BR631 strain with accession number CGMCC NO.26243; and Lacticaseibacillus paracasei BR176 strain with accession number CGMCC NO.21563. This invention conducted experiments on the above-mentioned lactic acid bacteria strain composition and the composition prepared with a carrier. The Pittsburgh Sleep Quality Index (PSQI) was used to assess the sleep of subjects. Blood samples were collected from subjects before and two weeks after taking the compositions in the experimental and control groups to detect changes in serotonin levels. The improvement in sleep quality was also observed using a wearable medical watch sleep analyzer and a PSQI questionnaire. The results showed that: 1) the experimental group containing four lactic acid bacteria strains showed a significantly greater increase in serotonin than the control group containing only a single lactic acid bacteria strain; 2) the experimental group containing four lactic acid bacteria strains showed a greater improvement in sleep quality than the control group containing only a single lactic acid bacteria strain; 3) taking the composition in the experimental group significantly improved the sleep quality of the subjects. This demonstrates that the lactic acid bacteria strain composition of this invention has physiological activity that improves sleep quality and can be used to prepare compositions that improve sleep quality. Attached Figure Description

[0019] Figure 1 The results of the experiment in Example 2 show changes in serotonin levels in subjects before and after taking the composition of the experimental and control groups, respectively.

[0020] Figure 2 This is a comparison of serotonin levels in subjects of Example 2 before and after taking the composition of the experimental and control groups, respectively.

[0021] Figure 3 The results of the experiment on the percentage of deep sleep and light sleep in the subjects of Example 2 before and after taking the composition of the experimental group.

[0022] Figure 4The results of the experiment on the percentage of deep sleep and light sleep in the subjects of Example 2 before and after taking the composition of the control group.

[0023] Figure 5 The results show the changes in the proportion of deep sleep and light sleep in the subjects of Example 2 after they took the composition of the experimental group.

[0024] Figure 6 The results show the changes in the proportion of deep sleep and light sleep in the subjects of Example 2 after they took the composition of the control group.

[0025] Figure 7 The results of PSQI changes in the subjects of Example 2 before and after taking the experimental group composition. Detailed Implementation

[0026] The microbial strains involved in the following examples are as follows:

[0027] 1. Bifidobacterium breve BBr-28 strain

[0028] Deposit date: 2024 / 11 / 04

[0029] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0030] Address of depositary institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Accession number: CGMCC No. 32485

[0031] Classification and nomenclature: Bifidobacterium breve

[0032] 2. Bifidobacterium animalis subsp. lactis PS23 strain

[0033] Preservation date: 2024 / 11 / 18

[0034] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0035] Address of depositary institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Accession number: CGMCC No. 32691

[0036] Classification and nomenclature: *Bifidobacterium animalis* subsp. *lactis*

[0037] 3. Bifidobacterium longum subsp. infantis BR631 strain

[0038] Deposit date: 2022 / 12 / 25

[0039] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0040] Address of depositary institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Accession number: CGMCC No. 26243

[0041] Classification and nomenclature: Bifidobacterium longum subsp. infantis

[0042] 4. *Chemotrophus paracasei* strain BR176

[0043] Deposit date: 2020 / 12 / 28

[0044] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0045] Address of depositary institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Accession number: CGMCC No. 21563

[0046] Classification and nomenclature: *Lacticaseibacillus paracasei*

[0047] The freeze-dried cultures of the above-mentioned strains have been deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. Detailed information on the deposit is shown in Table 1.

[0048] Table 1. Preservation information of the lactic acid bacteria strains of the present invention

[0049]

[0050] The present application will be further described in detail below with reference to the accompanying drawings.

[0051] Example 1:

[0052] Research on the health benefits of probiotics is extensive. Generally, strains with specific health benefits for humans are called functional probiotics (Guidelines for the evaluation of probiotics information; Report of joint FAO / WHO working group on drafting guidelines for the evaluation of probiotics in food; London Ontario, Canada April 30 and May 1, 2002: 1-7). It is important to note that the specific efficacy of these functional probiotics lies in the strain, not the species. That is, even the same species, different strains, may have different, or even opposite, effects.

[0053] Based on this, the first embodiment of the present invention proposes a composition of lactic acid bacteria strains for improving sleep quality, including Bifidobacterium breve BBr-28 strain, Bifidobacterium animalis subsp. lactis PS23 strain, Bifidobacterium longum subsp. infantis BR631 strain, and Bifidobacterium paracasei BR176 strain.

[0054] The ratio of Bifidobacterium BBr-28, Bifidobacterium animalis subsp. lactis PS23, Bifidobacterium longum subsp. infantis BR631, and Bifidobacterium paracasei BR176 strains is 1:(0.5–1.5):(0.5–1.5):(0.5–1.5). Preferably, the ratio of the above four strains is 1:1:1:1.

[0055] The properties of the lactic acid bacteria strains of the present invention are as follows:

[0056] The taxonomic characteristics of the strains were confirmed based on 16S rRNA sequence analysis and API bacterial identification system analysis results. The morphological and general characteristics of the lactic acid bacteria strains of this invention are detailed in Table 2:

[0057] Table 2. Morphological and general properties of the lactic acid bacteria strains of the present invention

[0058]

[0059]

[0060] The method for preparing the lactic acid bacteria strain composition of the present invention:

[0061] *Bifidobacterium breve* strain BBr-28, *Bifidobacterium animalis* subsp. *lactamase* strain PS23, and *Bifidobacterium longum* subsp. *infant* strain BR631 were isolated from breast milk; *Chemotrophus paracasei* strain BR176 was isolated from infant feces. All four isolated lactic acid bacteria strains were stored at -80°C with 20% glycerol. For use, each strain was inoculated into Difco at a 1% (v / v) inoculation rate. TM The cultures were incubated in MRS (De Man, Rogosa, and Sharpe) medium at 37°C for 20 hours. Afterward, the four cultures were centrifuged at 4°C for 20 minutes each. The supernatant was discarded, and the resulting precipitates were freeze-dried to obtain the bacterial powder of each lactic acid bacteria strain. The centrifuged precipitate could be mixed with a protective carrier before freeze-drying to maintain optimal bacterial activity.

[0062] The above-mentioned lactic acid bacteria strain composition has physiological activity that improves sleep quality. Therefore, the above-mentioned lactic acid bacteria strain composition has the purpose of improving sleep quality and can be used to prepare compositions that improve sleep quality.

[0063] Example 2:

[0064] A second embodiment of the present invention provides a composition for improving sleep quality, comprising a lactic acid bacteria strain composition having an active effect on improving sleep quality and a carrier. The lactic acid bacteria strain composition is the same as in Example 1.

[0065] The carrier may be a physiologically acceptable excipient or diluent. Alternatively, the carrier may be a pharmaceutically acceptable excipient or diluent. Specifically, the carrier may comprise oligosaccharides, sugar alcohols, sodium carboxymethyl cellulose, ethyl cellulose, galactooligosaccharides, stearic acid, magnesium stearate, potato starch, or a combination thereof. Further, the oligosaccharides may comprise fructooligosaccharides, isomaltose oligosaccharides, xylooligosaccharides, stachyose, mannose, arabinose, or a combination thereof; the sugar alcohols may comprise erythritol, sorbitol, isomaltitol, xylitol, mannitol, or a combination thereof.

[0066] In the composition of the present invention for improving sleep quality, the effective viable count of the lactic acid bacteria strain composition is equal to or greater than 1 × 10⁻⁶. 6 In a preferred embodiment, the effective viable count of the lactic acid bacteria strain composition is equal to or greater than 5 × 10⁻⁶ CFU / g. 9 CFU / g.

[0067] The dosage form of the composition of the present invention for improving sleep quality can be tablets, capsules or powder for convenient oral administration by the user.

[0068] The method for preparing the composition for improving sleep quality according to the present invention:

[0069] Step 1: Prepare the lactic acid bacteria strain composition (same as in Example 1).

[0070] *Bifidobacterium breve* strain BBr-28, *Bifidobacterium animalis* subsp. *lactamase* strain PS23, and *Bifidobacterium longum* subsp. *infant* strain BR631 were isolated from breast milk; *Chemotrophus paracasei* strain BR176 was isolated from infant feces. All four isolated lactic acid bacteria strains were stored at -80°C with 20% glycerol. For use, each strain was inoculated into Difco at a 1% (v / v) inoculation rate. TM The cultures were incubated in MRS (De Man, Rogosa, and Sharpe) medium at 37°C for 20 hours. Afterward, the four cultures were centrifuged at 4°C for 20 minutes each. The supernatant was discarded, and the resulting precipitates were freeze-dried to obtain the bacterial powder of each lactic acid bacteria strain. The centrifuged precipitate could be mixed with a protective carrier before freeze-drying to maintain optimal bacterial activity.

[0071] Step 2: Prepare a composition for improving sleep quality.

[0072] The composition of the present invention is obtained by uniformly mixing the lactic acid bacteria powder and the carrier in step 1, wherein the effective viable count of the lactic acid bacteria strain is equal to or greater than 5 × 10⁻⁶. 9 CFU / g.

[0073] Physiological activity experiments and analyses of the compositions of the present invention:

[0074] The composition of the present invention was formulated into a capsule dosage form for convenient oral administration. The 30 participants in the trial were divided into two groups: an experimental group and a control group, wherein:

[0075] Experimental group: Take the capsules of the composition of the present invention twice a day, once between 2-3 pm and once 2 hours before bedtime, for 2 weeks.

[0076] Control group: The capsules taken contained only the powder of Bifidobacterium animalis subsp. lactis PS23, and the rest of the administration method was the same as that of the experimental group.

[0077] The Pittsburgh Sleep Quality Index (PSQI) questionnaire was used to assess the sleep quality of the participants. The PSQI, proposed by American scholars Buysse et al., has seven dimensions and a higher PSQI score indicates poorer sleep quality. A PSQI score greater than 5 is considered indicative of sleep disorder.

[0078] Before and 2 weeks after taking the combination of the experimental and control groups, blood samples were collected from the subjects to detect changes in serotonin levels, and the improvement in sleep quality was observed using a wearable medical watch sleep analyzer and a PSQI questionnaire.

[0079] (a) Effects of taking the composition of the present invention on serotonin:

[0080] Serotonin, also known as 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter that regulates mood, appetite, and sleep. Studies indicate that serotonin deficiency can lead to lethargy, mood swings, anxiety, insufficient melatonin, or insomnia. Therefore, increasing serotonin levels can help improve sleep quality.

[0081] Before and two weeks after administration of the experimental and control groups, approximately 5 ml of blood was collected from each subject for serotonin testing. The collected blood was centrifuged to separate the serum. Serum was processed using serotonin ELISA kits (Abnova) according to the manufacturer's instructions. After HRP (horseradish peroxidase) catalysis to produce TMB (3,3',5,5'-Tetramethyl-benzidine) colorimetric reaction, the absorbance was measured using an ELISA Reader at OD 450 nm, and the serotonin concentration was calculated using a standard curve. The test results are shown below. Figure 1 and Figure 2 .

[0082] Figure 1 a represents the test results of serotonin changes in subjects before and after taking the experimental group's composition; Figure 1 b represents the test results of serotonin changes in subjects before and after taking the control group's composition. Figure 1 a and Figure 1 As can be seen from b, after 2 weeks of taking the experimental and control group compositions, the serotonin levels in the blood of the subjects increased significantly compared to before taking the compositions.

[0083] Figure 2 The results of changes in serotonin levels after subjects took the experimental and control groups of the combination were compared, and the values ​​are presented as Mean ± SD. Statistical analysis was performed using a paired t-test, and p < 0.05 was considered statistically significant. Figure 2As shown, the changes in serotonin levels (i.e., the difference between serotonin levels before and after administration) in the experimental group were compared with those in the control group. The experimental group showed a greater increase in serotonin levels than the control group, and the difference was statistically significant (p = 0.0075).

[0084] Therefore, the experimental group containing four strains of lactic acid bacteria showed a significantly better increase in serotonin than the control group containing only a single strain of lactic acid bacteria.

[0085] 1. Effects of taking the composition of the present invention on deep sleep:

[0086] The sleep cycle includes light sleep, deep sleep, and rapid eye movement (REM) sleep. Deep sleep helps the brain rest, repair itself, and restore energy, and it also helps strengthen declarative memory. Furthermore, during deep sleep, the pituitary gland secretes growth hormone, which helps body tissues grow and cells regenerate. Sufficient deep sleep is crucial to allow the brain and body enough time to repair; therefore, more deep sleep indicates better sleep quality.

[0087] Sleep quality was observed in subjects using a wearable medical watch sleep analyzer before and two weeks after administration of the experimental and control groups. Results are shown below. Figure 3 and Figure 4 .

[0088] Figure 3 a and Figure 3 b represents the results of the experiment showing the proportion of deep sleep and light sleep before and after the subjects took the experimental group's composition. Figure 3 a and Figure 3 As can be seen from b, after taking the composition in the experimental group, the proportion of light sleep decreased while the proportion of deep sleep increased. Therefore, the combination in the experimental group did improve the sleep quality of the subjects, and the difference was significant.

[0089] Depend on Figure 4 a and Figure 4 b indicates that the combination used by the subjects in the control group also improved sleep quality.

[0090] In addition, a comparison was made between subjects who experienced improved sleep quality (i.e., those with an increased percentage of deep sleep) after taking the experimental group's composition and subjects who experienced improved sleep quality after taking the control group's composition. The results are as follows: Figure 5 and Figure 6 As shown. By Figure 5As can be seen, the experimental group showed a greater increase in the proportion of deep sleep and a greater reduction in the proportion of light sleep than the control group after taking the combination, with significant differences (p = 0.0201). Therefore, the experimental group containing four strains of lactic acid bacteria is more effective in improving sleep quality than the control group containing only a single strain of lactic acid bacteria.

[0091] 2. Effects of taking the composition of the present invention on PSQI:

[0092] The Pittsburgh Sleep Quality Index (PSQI) has seven dimensions: sleep quality, sleep latency, sleep duration, sleep efficiency, sleep disturbance, use of sleeping pills, and daytime activity impairment. A higher PSQI score indicates poorer sleep quality.

[0093] Two weeks after taking the composition of the experimental group of this invention, the sleep quality of the subjects was assessed again using the Pittsburgh Sleep Quality Index Questionnaire, and the results were as follows: Figure 7 As shown. By Figure 7 The results showed that, compared with the group before taking the composition, the PSQI was significantly reduced after 2 weeks of taking the composition (p = 0.006), indicating that taking the composition could significantly improve the sleep quality of the subjects.

[0094] In summary, the composition of the present invention has physiological activity that improves sleep quality. It should be noted that, compared to the control group (composition containing only a single lactic acid bacteria strain), the composition containing four lactic acid bacteria strains (experimental group) significantly improved sleep quality, indicating that the lactic acid bacteria strain combination of the present invention has unexpected efficacy.

[0095] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of this application.

Claims

1. A composition of lactic acid bacteria strains for improving sleep quality, characterized in that: The lactic acid bacteria strain composition includes *Bifidobacterium breve* strain BBr-28 with accession number CGMCC NO. 32485, *Bifidobacterium animalis* subsp. *lactum* strain PS23 with accession number CGMCC NO. 32691, *Bifidobacterium longum* subsp. *infant* strain BR631 with accession number CGMCC NO. 26243, and *C. paracasei* strain BR176 with accession number CGMCC NO. 21563. The ratio of the strains of Bifidobacterium breve BBr-28, Bifidobacterium animalis subsp. lactis PS23, Bifidobacterium longum subsp. infantis BR631, and Bifidobacterium paracasei BR176 is 1:(0.5~1.5):(0.5~1.5):(0.5~1.5).

2. The use of the lactic acid bacteria strain composition as described in claim 1 in the preparation of a composition for improving sleep quality.

3. The application according to claim 2, characterized in that: The effective viable count of the lactic acid bacteria strain composition is equal to or greater than 5 × 10⁻⁶. 9 CFU / g.

4. A composition for improving sleep quality, characterized in that, The composition comprises the lactic acid bacteria strain composition of claim 1 and a carrier.

5. The composition according to claim 4, characterized in that: The ratio of the strains of Bifidobacterium breve BBr-28, Bifidobacterium animalis subsp. lactis PS23, Bifidobacterium longum subsp. infantis BR631, and Bifidobacterium paracasei BR176 was 1:1:1:

1.

6. The composition according to claim 4, characterized in that: In the composition, the effective viable count of all lactic acid bacteria strains is equal to or greater than 5 × 10⁻⁶. 9 CFU / g.

7. The composition according to claim 4, characterized in that: The carrier is a physiologically acceptable excipient or diluent.

8. The composition according to claim 4, characterized in that: The carrier is a pharmaceutically acceptable excipient or diluent.

9. The composition according to claim 4, characterized in that: The composition is available in tablet, capsule or powder form.

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