Lactobacillus plantarum with sleep improving effect and application thereof

Abstract

The invention provides lactobacillus plantarum with a sleep improving effect and application of the lactobacillus plantarum, and belongs to the field of microorganisms and biological medicines. The preservation number of the lactobacillus plantarum is CGMCC (China General Microbiological Culture Collection Center) The lactobacillus plantarum GM11 with a remarkable sleep improving effect is separated from samples such as plateau dairy products, plateau salt lakes and Sichuan special fermented food (pickles, bean halves and the like) for the first time, the awakening activity amount and the awakening total time of a zebra fish insomnia model can be remarkably reduced, and the rapid movement distance in the movement track of the zebra fish insomnia model is reduced; the slow movement distance is increased, and the excited state of the insomnia zebra fish is relieved. The strain has the potential of being developed into mental probiotics for improving sleep quality. The lactobacillus plantarum GM11 can independently form a preparation, can also be used as an additive to be added into other medicines and health-care foods for improving the sleep quality, and has a very wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and biomedicine, and particularly relates to a plant lactobacillus with sleep improving effect and a use thereof. Background Art

[0002] Sleep is a periodic and reversible resting phenomenon regulated by the sleep visual center. It is a common physiological phenomenon in higher vertebrates. Generally speaking, adults need 7-9 hours of sleep every day. Sleep plays many important roles for the human body, mainly including eliminating fatigue, protecting the brain, enhancing immunity, promoting growth and development, and delaying aging. Sleep disorders are manifestations of abnormal sleep quantity and abnormal behavior during sleep. Insomnia is the most common sleep disorder. Insomnia refers to a subjective experience that despite having a suitable sleep opportunity and sleep environment, one is still dissatisfied with the sleep time and (or) sleep quality, and affects daytime function or causes physical discomfort. The main clinical manifestations are difficulty falling asleep, sleep maintenance disorders, early awakening, decreased sleep quality and reduced total sleep time. With the rapid development of social economy, the pace of life has accelerated, and the pressure of work and study has increased. The number of people with insomnia has increased year by year and has become younger. According to epidemiological surveys and studies, about 27% of people in the world have sleep problems to varying degrees. The China Sleep Research Society also has relevant survey data showing that the incidence of insomnia in people over 18 years old in my country is as high as 38.2%. Insomnia not only affects people's daily work and study, but also leads to a decline in the quality of life. Long-term insomnia may also induce psychological illnesses such as irritability, anger, low mood, anxiety and depression, as well as physical diseases such as cardiovascular and cerebrovascular diseases, obesity, diabetes, and hypertension.

[0003] At present, the commonly used non-drug relief measures to improve sleep quality include psychotherapy, health education, relaxation training, family intervention, physical therapy, etc., but they all have certain defects in application. Drugs that improve sleep quality mainly include benzodiazepines, such as diazepam, clonazepam, etc., non-benzodiazepines, such as zopiclone, eszopiclone, etc., and melatonin receptor agonists, such as ramelteon, melatonin sustained-release tablets, etc. Although drug treatment has achieved gratifying results, most individuals who receive treatment have not fully achieved the purpose of symptom relief. A considerable number of patients cannot tolerate the adverse reactions of existing drug treatments, such as feeling sleepy and lack of energy during the day after use, which affects their daily work and life. At the same time, long-term use of drugs such as benzodiazepines may also cause dependence, and withdrawal symptoms may occur when the drug is discontinued. Therefore, it is particularly important to develop and find better solutions or alternative measures to improve sleep quality, which has become a research topic that needs to be solved urgently.

[0004] In recent years, the role of intestinal microorganisms in health and disease has become a research hotspot. It is a key factor in the formation of the gut-brain axis. Gut microorganisms, the intestines and the brain may communicate bidirectionally through a variety of direct or indirect pathways such as neural, immune, endocrine and metabolic pathways. Gut microorganisms are an important factor in many central nervous system disorders. They can regulate sleep by regulating the host immune system, leading to changes in the intestinal and systemic immune status, regulating endocrine levels, and affecting vagus nerve afferents. Probiotics refer to a class of living microorganisms that can have a beneficial effect on the host when ingested in sufficient quantities. The role of probiotics in the prevention and treatment of central nervous system diseases has also attracted great attention from scholars at home and abroad. Probiotics that can regulate central nervous system function and behavior are also defined as "psychoprobiotics", which may provide us with new ideas and new directions for improving insomnia. Screening for psychoprobiotics and their compositions with sleep-improving effects has great scientific and practical significance for the improvement and auxiliary treatment of sleep disorders and other central nervous system-related diseases.

[0005] The patent application with publication number CN114480213A discloses a strain of fermented Lactobacillus E15 that produces γ-aminobutyric acid and its application in the preparation of a drug for improving sleep. The strain can produce γ-aminobutyric acid under fermentation conditions, and can reduce the movement distance, manic time, and active time of zebrafish in an in vivo insomnia model, and increase the static time. However, the effect of the invention on improving sleep still needs to be improved. Summary of the invention

[0006] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a Lactobacillus plantarum with significantly improved sleep improvement effect and its use.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The invention provides a plant lactobacillus strain, and its preservation number is CGMCC No.25063.

[0009] The above-mentioned Lactobacillus plantarum (named: GM11) is deposited in the General Microbiology Center of the Microbial Culture Collection Management Committee, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is June 10, 2022, and the deposit number is CGMCC No.25063.

[0010] The present invention also provides use of the above-mentioned Lactobacillus plantarum in preparing a medicine for improving sleep quality.

[0011] The present invention also provides use of the above-mentioned Lactobacillus plantarum in preparing health food for improving sleep quality.

[0012] The invention also provides a bacterial agent, which is prepared from the above-mentioned Lactobacillus plantarum.

[0013] Bacterial agents refer to live bacterial preparations made from beneficial microorganisms.

[0014] Furthermore, in the bacterial agent, the number of viable Lactobacillus plantarum is ≥ 1.6×10 5 CFU / mL.

[0015] Furthermore, in the bacterial agent, the number of viable Lactobacillus plantarum is ≥ 2×10 5 CFU / mL.

[0016] Furthermore, in the bacterial agent, the number of viable Lactobacillus plantarum is ≥ 1.6×10 7 CFU / mL.

[0017] Furthermore, in the bacterial agent, the number of viable Lactobacillus plantarum is ≥ 1.6×10 7 CFU / mL and <1.6×10 8 CFU / mL.

[0018] The present invention also provides use of the above-mentioned bacterial agent in preparing a medicine for improving sleep quality.

[0019] The present invention also provides use of the bacterial agent in preparing health food for improving sleep quality.

[0020] The present invention also provides a medicine for improving sleep quality, which is prepared by taking the above-mentioned Lactobacillus plantarum and / or the above-mentioned bacterial agent as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0021] The present invention also provides a health food for improving sleep quality, which is prepared by using the above-mentioned Lactobacillus plantarum and / or the above-mentioned bacterial agent as active ingredients and adding auxiliary materials or auxiliary ingredients acceptable in the food field.

[0022] The present invention has achieved the following beneficial effects:

[0023] The present invention is the first to isolate Lactobacillus plantarum GM11 with a significant sleep-improving effect from samples such as plateau dairy products, plateau salt lakes, and Sichuan specialty fermented foods (kimchi, bean curd, etc.).

[0024] The present invention firstly finds through model animal research that plant lactobacillus GM11 has the effect of improving sleep, can significantly reduce the amount of awakening activity and the total awakening time of the zebrafish insomnia model, reduce the fast movement distance in the movement trajectory of the zebrafish insomnia model, increase the slow movement distance, and relieve the excited state of the insomnia zebrafish. The strain has the potential to be developed into a psychoactive probiotic for improving sleep quality. Plant lactobacillus GM11 can form a preparation alone, and can also be added as an additive to other medicines and health foods for improving sleep quality, and has a very wide application prospect.

[0025] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0026] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The invention discloses the effect of Lactobacillus plantarum GM11 on the amount of wakefulness activity in a PTZ-induced zebrafish insomnia model.

[0028] Figure 2 The present invention shows the effect of Lactobacillus plantarum GM11 on the total wakefulness time of the PTZ-induced zebrafish insomnia model.

[0029] Figure 3 The present invention shows the influence of Lactobacillus plantarum GM11 on the movement trajectory of the PTZ-induced zebrafish insomnia model. DETAILED DESCRIPTION

[0030] The reagents and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0031] Example 1 Strain screening and bacterial solution (microbial agent) preparation

[0032] 1. Strain Isolation and Bacterial Liquid Preparation

[0033] Various types of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, etc. were screened from sources such as plateau dairy products, plateau salt lakes, and Sichuan specialty fermented foods (kimchi, bean paste, etc.). After physical and chemical analysis, category identification, and testing of some safety indicators, the GM11 strain was selected from the strains that met the basic requirements in the preliminary tests.

[0034] 2. Identification and preservation

[0035] The specific identification characteristics of the GM11 strain are as follows:

[0036] (1) Morphological characteristics

[0037] Gram-positive, no spores. Curved rods or short rods, mostly short rods with blunt ends, arranged singly or in pairs.

[0038] (2) Physiological and biochemical characteristics

[0039] The colonies are medium-sized, convex, milky white, opaque, moist and smooth on the surface, with neat edges and rounded shapes. ① It can utilize maltose, cellobiose, mannitol, lactose, esculin, sorbitol and sucrose. ② The H2O2 catalase, nitrate reduction, gelatin liquefaction and indole tests are all negative.

[0040] (3) Sequence analysis of 16S rDNA

[0041] The 16S rDNA sequence of GM11 is as follows (SEQ ID NO.1):

[0042]

[0043] The BLAST tool was used to perform homology comparison on the sequence in the gene library on the NCBI website. The results showed that the 16S rDNA of GM11 had a sequence similarity of more than 99.93% with that of Lactobacillus plantarum. Combined with the morphological and physiological and biochemical characteristics, the strain GM11 was identified as a strain of Lactobacillus plantarum.

[0044] After identification, the GM11 strain was found to be Lactobacillus plantarum and was deposited in the General Microbiology Center of the China Culture Collection Administration on June 10, 2022; the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit number is: CGMCC No.25063.

[0045] The beneficial effects of the present invention are further illustrated by experiments below.

[0046] Experimental Example 1 Study on the effect of Lactobacillus plantarum GM11 on improving sleep

[0047] 1. Experimental methods

[0048] (1) Experimental animals

[0049] Wild-type AB strain zebrafish were selected 5 days after fertilization, and zebrafish with normal development were selected under a dissecting microscope for grouping and processing. Zebrafish were raised in 28°C fish water (water quality: 200 mg of instant sea salt was added to 1L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3), and the experimental animal use license number was: SYXK (Zhejiang) 2022-0004. The feeding and management met the requirements of the international AAALAC certification (certification number: 001458).

[0050] (2) Maximum Tolerated Concentration (MTC) determination

[0051] Zebrafish were randomly selected in a 6-well plate, 30 zebrafish were fed in each well, and the feeding system in each well was 3 mL. Sample aqueous solutions (concentrations shown in Table 1) were given respectively, and a normal control group was set up at the same time. The sample addition amount in the normal control group was 0. After 1 day of treatment at 28°C, the activity status of zebrafish was observed to determine the MTC of Lactobacillus plantarum GM11 on zebrafish.

[0052] (3) Experimental animal grouping

[0053] Zebrafish were randomly selected in a 6-well cell culture plate, and 30 zebrafish were fed in each well, with a feeding system of 3 mL in each well. Zebrafish were randomly divided into a normal control group, an insomnia model group, a melatonin group (positive control), a low-dose group, a medium-dose group, and a high-dose group of Lactobacillus plantarum GM11. The melatonin group was given a melatonin solution of 125 μg / mL in the culture system. The low-dose group, the medium-dose group, and the high-dose group of Lactobacillus plantarum GM11 were given the corresponding concentration of bacterial solution in the culture system according to the MTC results. After adding the sample treatment for 24 hours, the zebrafish were transferred to a 96-well plate for subsequent experiments.

[0054] (4) Construction of zebrafish insomnia model

[0055] From the above groups of zebrafish, 10 zebrafish were randomly selected from each group and transferred to a 96-well plate. Except for the normal control group, the other experimental groups were stimulated with 20 μL of 5 mM pentetrazol (PTZ) aqueous solution for 1 h to establish the zebrafish insomnia model.

[0056] (5) Evaluation of sleep improvement efficacy

[0057] The behavior analyzer was used to record the amount of awakening activity, total awakening time, and movement trajectory of zebrafish within 1 hour. The behavior analyzer parameter settings defined movement speed <4 mm / s as slow movement, movement speed between 4-20 mm / s as medium-speed movement, and movement speed >20 mm / s as fast movement. The amount of awakening activity refers to the sum of the average amount of movement within 1 hour, and the total awakening time refers to the sum of the time the zebrafish is in motion within 1 hour.

[0058] (6) Statistical analysis

[0059] All data results were expressed as mean ± SE. One-way analysis of variance was performed using SPSS (Windows version 21.0, IBM Corp., Armonk, NY, USA) for inter-group comparisons. P < 0.05 was considered statistically significant.

[0060] 2. Experimental results

[0061] Zebrafish is a model organism that can be used to study sleep, and its genome sequence similarity with humans reaches 87%. Compared with other mammals used in experiments, zebrafish has advantages such as transparent body, easy observation, less medication, easy operation, and short cycle. Zebrafish, like humans, have a normal sleep cycle. Stimulant drugs can cause zebrafish to quickly lose sleep. For example, pentylenetetrazol (PTZ) is a γ-aminobutyric acid (GABA) inhibitor. GABA is a naturally occurring non-protein amino acid and an important inhibitory neurotransmitter in the central nervous system of mammals. Small doses of PTZ can induce bipolar disorder with both mania and depression, thus creating a zebrafish insomnia model.

[0062] (1) MTC determination

[0063] The concentrations of Lactobacillus plantarum GM11 were set as follows: 1.6×10 5 , 1.6×10 6 , 1.6×10 7 , 1.6×10 8 and 1.6×10 9 CFU / mL, of which 1.6×10 8 and 1.6×10 9 CFU / mL concentration, all zebrafish died (Table 1). Under the experimental conditions, the MTC of Lactobacillus plantarum GM11 on zebrafish was determined to be 1.6×10 7 CFU / mL. Set 1.6×10 7 CFU / mL is for the high-dose group; 1.6×10 6 CFU / mL for the medium-dose group; 1.6×10 5 CFU / mL for the low-dose group.

[0064] Table 1. Results of the experimental study on the concentration of samples for improving sleep (n=30)

[0065]

[0066] (2) Effect of Lactobacillus plantarum GM11 on sleep improvement

[0067] The amount of wakefulness activity (monitored by the distance the zebrafish moved, reflecting its activity level in the wakeful state; in the insomniac state, the amount of wakefulness activity of the zebrafish usually increased significantly) and the total wakefulness time (recording the length of time the zebrafish was awake during the experiment, which is an important parameter for measuring sleep efficiency and sleep structure disorders) were used as evaluation indicators for the sample's effect on improving sleep.

[0068] Using a behavioral analyzer to observe within 1 hour, the awakening activity of the zebrafish insomnia model group was 613±90.4mm, which was much higher than the normal control group 32.0±7.78mm, and the difference was statistically significant (P<0.01). The awakening activity of the melatonin positive control group was 176±25.8mm, which significantly reduced the awakening activity of insomnia zebrafish compared with the insomnia model group (P<0.01). Lactobacillus plantarum GM111.6×10 5 CFU / mL low dose group, 1.6×10 6 CFU / mL medium dose group, 1.6×10 7 The awakening activity of zebrafish in the high-dose group was 421±53.5, 478±89.7, and 285±56.9 mm, respectively. Compared with the insomnia model group, the Lactobacillus plantarum GM111 was 1.6×10 7 CFU / mL high-dose group can significantly reduce the amount of zebrafish awakening activity (P<0.05) ( Figure 1 ).

[0069] The total time of zebrafish awakening activity was observed using a behavioral analyzer. The total time of awakening activity in the insomnia model group was 16.0±2.34s, which was much higher than that in the normal control group (1.11±0.271s). Compared with the normal control group, the difference was statistically significant (P<0.01). The total time of awakening activity in the positive control melatonin group was 4.58±0.588s, which significantly reduced the total time of awakening activity in insomnia zebrafish compared with the insomnia model group (P<0.01). 5 CFU / mL low dose group, 1.6×10 6 CFU / mL medium dose group, 1.6×10 7 The total time of zebrafish awakening activity in the high-dose group was 11.4±1.49s, 13.0±2.26s, and 7.80±1.55s, respectively. Compared with the insomnia model group, the total time of zebrafish awakening activity in the high-dose group was 11.4±1.49s, 13.0±2.26s, and 7.80±1.55s, respectively. 7 The high-dose group of CFU / mL significantly reduced the total time of zebrafish awakening activity (P<0.05) ( Figure 2 ).

[0070] Depend on Figure 3 The zebrafish movement trajectory diagram (black line is slow movement distance, green line is medium speed movement distance, and red line is fast movement distance) shows that the fast movement distance of zebrafish in the insomnia model group is significantly longer than that of the normal control group, and the slow movement distance is significantly reduced compared with the normal control group. 7 The high-dose CFU / mL group can significantly reduce the fast movement distance of zebrafish, increase the slow movement distance, make insomnia zebrafish calmer, and significantly alleviate their hyperactive state.

[0071] In summary, the present invention successfully isolated a Lactobacillus plantarum GM11. Zebrafish experiments showed that Lactobacillus plantarum GM111.6×10 7 The CFU / mL high-dose group can significantly reduce the amount of awakening activity and the total time of awakening activity of insomnia zebrafish, reduce the fast movement distance of insomnia zebrafish, increase the slow movement distance, make the insomnia zebrafish state calmer, and alleviate its excited state. Plant lactobacillus GM11 has a significant improvement effect on insomnia. Therefore, the plant lactobacillus GM11 in the present invention has the potential to be developed into a psychoactive probiotic for improving sleep quality, and has a very broad application prospect.

Claims

1. A strain of Lactobacillus plantarum, characterized in that Its deposit number is CGMCC No.25063.

2. Use of the plant lactobacillus according to claim 1 in preparing a medicine for improving sleep quality.

3. Use of the plant lactobacillus according to claim 1 in preparing a health food for improving sleep quality.

4. A bacterial agent, characterized in that The invention is prepared from the plant lactobacillus described in claim 1.

5. Use of the bacterial agent according to claim 4 in the preparation of a medicine for improving sleep quality.

6. Use of the bacterial agent according to claim 4 in preparing a health food for improving sleep quality.

7. A drug for improving sleep quality, characterized in that: The invention is prepared by taking the plant lactobacillus described in claim 1 and / or the bacterial agent described in claim 4 as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

8. A health food for improving sleep quality, characterized in that: The invention is prepared by taking the plant lactobacillus described in claim 1 and / or the bacterial agent described in claim 4 as active ingredients and adding auxiliary materials or supplementary ingredients acceptable in the food field.

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