Probiotic composition with functions of resisting anxiety and enhancing memory and concentration and application thereof

Abstract

The invention provides a probiotic composition with functions of resisting anxiety and enhancing memory and concentration and application of the probiotic composition, and belongs to the technical field of functional microorganisms. The probiotic composition provided by the invention is prepared from a lactobacillus mucus strain A21196, a pediococcus pentosaceus strain A20001 and a lactobacillus plantarum strain A21337, wherein the ratio of the viable count of the lactobacillus mucus strain A21196 to the viable count of the pediococcus pentosaceus strain A20001 to the viable count of the lactobacillus plantarum strain A21337 to the viable count of the lactobacillus plantarum strain A21196 to the viable count of the pediococcus pentosaceus strain A20001 to the viable count of the lactobacillus plantarum strain A21337 is (0.5-1.5) to (0.5-1.5) to (0.5-1.5). Experiments prove that the probiotic composition has good functions of resisting anxiety and depression and enhancing memory and concentration, and a new means is provided for treatment of neurological diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microorganisms, and specifically relates to a probiotic composition having the functions of relieving anxiety and enhancing memory and concentration, and an application thereof. Background Art

[0002] Anxiety neurosis, referred to as anxiety disorder, is a neurosis characterized by anxiety. It manifests as a fearful and uneasy mood without factual basis, clear objective object and specific concept content, as well as autonomic nervous system symptoms, muscle tension, and motor restlessness. This disease is divided into two forms: panic disorder and generalized anxiety. At present, the animal models for studying anxiety mainly include models based on animal non-conditioning, which can be divided into exploratory behavior models and social behavior models according to their behavioral characteristics. The exploratory behavior models mainly include the more classic elevated cross maze experiment and open field experiment.

[0003] The human gut is home to trillions of microorganisms that play an important role in digestion, immunity, and overall health, and malfunction of any part can lead to problems such as disease. Increasing research evidence shows that there is a relationship between the gut microbiome and the host, including the gut-brain axis, which is a two-way communication between the gastrointestinal system and the central nervous system to maintain the body's homeostatic environment. Gut microorganisms play a vital role in human health. They influence the blood-brain barrier, myelin neurogenesis, and other neurodevelopmental processes, and regulate cognitive processes such as synaptic plasticity, learning, and memory. The gut-brain axis promotes two-way communication between the gut microbiota and metabolites and has become a promising target for the treatment of depression. A comprehensive understanding of the complex molecular relationship between gut microbiota and anxiety disorders is essential for the development of new treatments. Although modulation of the gut microbiota has attracted considerable attention as a potential therapeutic target in depression research, the functional role of specific bacteria and pharmacologically active metabolites in depression treatment remains largely unexplored.

[0004] At present, there are not many reports on the use of intestinal probiotic preparations in improving anxiety and enhancing cognitive functions such as memory. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a probiotic composition, which is compounded by specific strains of fermentative Lactobacillus mucilaginosus, Pediococcus pentosaceus and Lactobacillus plantarum, and has the functions of anti-anxiety and enhancing memory and concentration.

[0006] The invention provides a probiotic composition, comprising a Limosilactobacillusfermentum strain A21196, a Pediococcus pentosaceus strain A20001 and a Lactiplantibacillus plantarum strain A21337, the viable bacteria count ratio of which is (0.5-1.5):(0.5-1.5):(0.5-1.5);

[0007] The deposit number of the fermented Lactobacillus mucilaginosus strain A21196 is GDMCC No: 62959;

[0008] The deposit number of the Pediococcus pentosaceus strain A20001 is GDMCC No: 64674;

[0009] The deposit number of the plant lactobacillus strain A21337 is GDMCC No: 64677.

[0010] Preferably, the ratio of viable bacteria count of the fermented Lactobacillus mucilaginosus strain A21196, the Pediococcus pentosaceus strain A20001 and the Lactobacillus plantarum strain A21337 is (0.8-1.2):(0.8-1.2).

[0011] Preferably, the ratio of viable bacteria count of the fermented Lactobacillus mucilaginosus strain A21196, the Pediococcus pentosaceus strain A20001 and the Lactobacillus plantarum strain A21337 is 1:1:1.

[0012] The invention provides a probiotic agent, wherein the active ingredients include the probiotic composition.

[0013] Preferably, the probiotics are in the form of aqueous solutions and / or powders.

[0014] Preferably, the total live bacteria concentration in the probiotic is 1×10 10 CFU / mL~100×10 10 CFU / mL or 1×10 10 CFU / g~100×10 10 CFU / g.

[0015] The present invention provides a medicine comprising the probiotic composition or the probiotic agent and a pharmaceutically acceptable auxiliary material.

[0016] Preferably, the total viable bacteria concentration in the drug is 1×10 10 CFU / mL~100×10 10 CFU / mL or 1×10 10 CFU / g~100×1010 CFU / g.

[0017] The present invention provides use of the probiotic composition or the probiotic agent in preparing a medicament for preventing and / or treating at least one of the following neurological diseases or indications: anxiety, depression and cognitive dysfunction.

[0018] Preferably, the dosage form of the drug includes at least one of the following: powder, tablet, oral solution, capsule and drops.

[0019] The present invention provides a probiotic composition, comprising fermented mucus lactobacillus strain A21196, Pediococcus pentosaceus strain A20001 and plant lactobacillus strain A21337, with a viable cell count ratio of (0.5-1.5): (0.5-1.5): (0.5-1.5); the fermented mucus lactobacillus strain A21196 has a preservation number of GDMCC No: 62959; the Pediococcus pentosaceus strain A20001 has a preservation number of GDMCC No: 64674; the plant lactobacillus strain A21337 has a preservation number of GDMCC No: 64677. The present invention compounds strain A21196, strain A20001 and strain A21337 according to the above-mentioned specific viable cell count ratio, and the prepared probiotic composition is verified for anti-depressive behavior, anti-anxiety and learning and memory improvement functions. The present invention uses a mouse model of chronic unpredictable mild stress as an experimental object, and uses sucrose preference test, tail suspension test, and forced swimming test to evaluate the antidepressant behavior of the probiotic composition. The results of the sucrose preference test show that compared with the model group, after gavage of the probiotic composition preparation, the mouse sugar preference value is significantly increased, indicating that the strain probiotic composition preparation of the present invention has the function of improving the anhedonia of depressed mice. The results of the tail suspension experiment show that compared with the model group, the immobility time of the mice in the probiotic composition preparation group is significantly shortened. At the same time, the results of the forced swimming experiment show that compared with the model group, the immobility time of the mice in the probiotic composition preparation group is significantly shortened. This shows that the probiotic composition preparation strain can effectively alleviate the depressive-like behavior of mice caused by CUMS. The results of the open field experiment show that compared with the model group, the time for the mice in the probiotic composition preparation group to enter the center of the open field is significantly increased. At the same time, the results of the elevated cross maze experiment show that compared with the model group, the number of times the mice in the probiotic composition preparation group entered the open arm and the percentage of time in the open arm increased significantly, and the time in the closed arm was significantly shortened. These results show that the probiotic composition preparation strain can effectively alleviate the anxiety behavior of mice. The present invention also carried out a new object recognition experiment to evaluate the efficacy of the probiotic composition on the memory and cognitive ability of the mouse model. The results showed that compared with the model group, the percentage of time for the probiotic composition preparation group mice to explore new objects increased significantly, indicating that the probiotic composition preparation strain can effectively improve the cognitive behavior of mice and improve the memory of mice. In addition, the present invention also measured the levels of 5-hydroxytryptamine (5-HT) and brain-derived neurotrophic factor (BDNF) in the serum of mice in each experimental group. The results showed that compared with the model group, the probiotic composition preparation group mice serum 5-HT and BDNF content increased significantly. It can be seen that the probiotic composition can be used to develop and prepare drugs for the treatment of neurological diseases including anxiety, depression and cognitive impairment, providing a new means for the treatment of neurological diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The results are as follows: the effect of the probiotic composition preparation on the body weight of CUMS mice;

[0021] Figure 2 The results show that the probiotic composition preparation significantly improves the sucrose preference of CUMS mice;

[0022] Figure 3 The probiotic composition preparation significantly shortened the tail suspension immobility time of CUMS mice;

[0023] Figure 4 The probiotic composition preparation significantly shortened the forced swimming immobility time of CUMS mice;

[0024] Figure 5 The results show that the probiotic composition preparation significantly increases the exploration ability of CUMS mice in an open field;

[0025] Figure 6 The results of the probiotic composition preparation on CUMS mice in the elevated plus maze;

[0026] Figure 7 The results showed that the probiotic composition preparation significantly increased the time CUMS mice spent exploring new objects;

[0027] Figure 8 The probiotic composition preparation significantly increased the serum 5-HT level in mice;

[0028] Fig. 9 The probiotic composition preparation significantly increased the serum BDNF level in mice;

[0029] Fig.10 The results showed that the probiotic composition preparation significantly increased the BDNF level in the mouse hippocampus.

[0030] Biomaterial Deposit Information

[0031] Limosilactobacillusfermentum strain A21196 was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 11, 2022, with the abbreviation of GDMCC. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 62959.

[0032] Pediococcus pentosaceus strain A20001 was deposited in Guangdong Microbiological Culture Collection Center on May 23, 2024, with the abbreviation of GDMCC. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64674.

[0033] Lactobacillus plantarum strain A21337 was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 23, 2024, with the abbreviation of GDMCC. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64677. DETAILED DESCRIPTION

[0034] The invention provides a probiotic composition, comprising a fermented Lactobacillus mucoides strain A21196, a Pediococcus pentosaceus strain A20001 and a Lactobacillus plantarum strain A21337, the viable bacteria count ratio of which is (0.5-1.5):(0.5-1.5):(0.5-1.5); the fermented Lactobacillus mucoides strain A21196 has a preservation number of GDMCC No: 62959; the Pediococcus pentosaceus strain A20001 has a preservation number of GDMCC No: 64674; and the Lactobacillus plantarum strain A21337 has a preservation number of GDMCC No: 64677.

[0035] In the present invention, the ratio of the viable cell counts of the fermented Lactobacillus mucilaginosus strain A21196, the Pediococcus pentosaceus strain A20001 and the Lactobacillus plantarum strain A21337 can be (0.8-1.2):(0.8-1.2):(0.8-1.2), or can be 1:1:1.

[0036] In the present invention, the compounding of three specific strains of the fermented mucus lactobacillus strain A21196, the pediococcus pentosaceus strain A20001 and the plant lactobacillus strain A21337 is more advantageous in anti-depression, anti-anxiety and improving learning and memory abilities compared with the common strain compounding scheme, indicating that the above three specific strains have a synergistic function. And the fermented mucus lactobacillus strain A21196, the pediococcus pentosaceus strain A20001 and the plant lactobacillus strain A21337 of the present invention are compounded according to a specific ratio, and compared with the probiotic composition formed by the control ratio, the effect is stronger in anti-depression, anti-anxiety and improving learning and memory abilities.

[0037] The invention provides a probiotic agent, wherein the active ingredients include the probiotic composition.

[0038] In the present invention, the dosage form of the probiotics preferably includes aqueous solution and / or powder. The total live bacteria concentration in the probiotics is preferably 1×10 10 CFU / mL~100×10 10 CFU / mL or 1×10 10 CFU / g~100×10 10 CFU / g, can be 5×10 10 CFU / mL~80×1010 CFU / mL or 5×10 10 CFU / g~80×10 10 CFU / g, or 10×10 10 CFU / mL~50×10 10 CFU / mL or 10×10 10 CFU / g~50×10 10 CFU / g.

[0039] In the present invention, the preparation method of the aqueous solution of the probiotic agent comprises the following steps: inoculating the activated fermented Lactobacillus mucosa strain A21196, Pediococcus pentosaceus strain A20001 and Plant Lactobacillus plantarum strain A21337 in MRS liquid culture medium for cultivation to obtain seed solution, inoculating the seed solution in MRS liquid culture medium for expansion culture to obtain three bacterial solutions for mixing and compounding according to the above-mentioned live bacteria ratio, adding glycerol, and obtaining the aqueous solution of the probiotic agent. The culture temperature can be 36-38°C, or 37°C. The culture time can be 2-4 hours or 3 hours. During the culture, the inoculation amount can be 3%-6%, or 4%-5%. The temperature of the expansion culture can be 36-38°C, or 37°C. The inoculation amount during the expansion culture can be 5%-10%, or 8%. The amount of glycerol added preferably accounts for 30%-70% of the total volume of the bacterial solution, or 40%-60%, or 50%. The preparation method of the probiotic powder is preferably to expand the culture according to the above method, obtain three bacterial solutions according to the above live bacteria ratio, mix and compound, separate the solid and liquid, obtain bacterial precipitation, mix the bacterial precipitation with skim milk with a volume concentration of 40%, and vacuum freeze-dry to obtain the powder. The present invention does not specifically limit the type of the lyophilization protective agent, and lyophilization protective agents known in the art can be used, such as skim milk, sucrose, glucose, etc.

[0040] The present invention provides use of the probiotic composition or the probiotic agent in preparing a medicament for preventing and / or treating at least one of the following neurological diseases or indications: anxiety, depression and cognitive dysfunction.

[0041] In the present invention, the anxiety disorder refers to a neurotic disorder characterized by widespread, persistent anxiety or recurrent panic, often accompanied by autonomic nervous system symptoms and motor tension. The anxiety disorder is manifested as a person's fear and worry about many upcoming things, such as work, study, health, etc., tension, irritability, difficulty concentrating, and decreased thinking ability. Insomnia, fatigue, loss of appetite, restlessness, walking back and forth, smoking, rubbing hands and other nervous behaviors. The present invention evaluates the efficacy of the probiotic composition or its preparation in anti-anxiety behavior through an open field test and an elevated cross maze test. The results show that compared with the model group, the time for the probiotic composition preparation group mice to enter the center of the open field was significantly increased (p < 0.05), and the elevated cross maze test results showed that compared with the model group, the number of times the probiotic composition preparation group mice entered the open arm and the percentage of time in the open arm was significantly increased (p < 0.0001), and the time in the closed arm was significantly shortened (p < 0.0001), indicating that the probiotic composition preparation strain can effectively alleviate the anxiety behavior of the model mice. Therefore, the probiotic composition or the preparation thereof has the medicinal effect of treating anxiety.

[0042] In the present invention, the cognitive dysfunction refers to one or more impairments in cognitive functions in memory, language, visual space, execution, calculation, and understanding and judgment, including memory impairment (especially mainly in the decline of recent memory), disorientation (not knowing the time, place, and people), language impairment (including difficulty in speaking and not being able to find accurate words to express one's meaning), decreased visual-spatial ability, decreased calculation ability, and decreased ability to find and solve problems. In an embodiment of the present invention, a new object recognition experiment was carried out to evaluate the efficacy of the probiotic composition or its preparation in improving cognition and memory. The results showed that compared with the model group, the percentage of time for mice in the probiotic composition preparation group to explore new objects was significantly increased (p < 0.0001), indicating that the probiotic composition preparation strain can effectively improve the cognitive behavior of model mice and improve the memory of mice. It can be seen that the probiotic composition and its preparation provided by the present invention have good therapeutic efficacy in treating diseases related to cognitive dysfunction.

[0043] In the present invention, depression, also known as depressive disorder, is characterized by significant and persistent low mood as the main clinical feature and is the main type of mood disorder. The degree of low mood in patients with depression is disproportionate to their situation. The negative emotional state can range from melancholy to grief, inferiority and depression, and even pessimism and world-weariness. There may be attempts or behaviors to commit suicide; even stupor (a state of high psychomotor inhibition, and often maintaining a fixed posture. The patient is silent, does not eat or drink, does not move, and verbal and motor behaviors are in a state of complete inhibition). Some patients with depression have obvious anxiety and motor agitation (obvious restlessness and excessive limb activity, accompanied by anxiety. In the embodiment of the present invention, a chronic unpredictable mild stress mouse model (CUMS) was constructed, and the sucrose preference test, tail suspension test, and forced swimming test were used to evaluate the anti-depressant effect of the probiotic composition or its preparation. The results of the sucrose preference experiment showed that compared with the model group, the sugar preference value of mice increased significantly after gavage of the probiotic composition preparation, indicating that the probiotic composition preparation of the strain of the present invention has the function of improving the anhedonia of depressed mice. The results of the tail suspension experiment showed that compared with the model group, the immobility time of mice in the probiotic composition preparation group was significantly shortened. The results of the forced swimming experiment showed that compared with the model group, the immobility time of mice in the probiotic composition preparation group was significantly shortened. This shows that the probiotic composition preparation can effectively alleviate the depressive-like behavior of mice caused by CUMS. At the same time, the probiotic composition preparation can increase the body weight of depressed mice caused by CUMS, saying that the probiotic composition and its preparation have good safety for taking and have no effect on the growth of mice.

[0044] In the present invention, in order to further explore the mechanism of the probiotic composition in anti-anxiety, anti-depression and improving cognitive dysfunction, the levels of 5-HT and BDNF in the serum of the mouse model were detected and it was found that compared with the model group, the probiotic composition preparation can significantly improve the levels of 5-HT and BDNF. 5-hydroxytryptamine (5-HT) is an important neurotransmitter in the body, widely distributed in the central nervous system, involved in regulating emotions, sleep, appetite and behavior, etc., and is crucial to maintaining the normal physiological functions of the body. Brain-derived neurotrophic factor (BDNF) is a protein that promotes the growth and survival of neurons, participates in cognitive processes such as synaptic plasticity, learning and memory, and plays an important role in the development and functional maintenance of the nervous system. 5-HT and BDNF synergistically regulate cognitive function. 5-HT regulates the activity of neurons by binding to its receptors, thereby affecting the expression and secretion of BDNF, increasing cognitive processes such as learning and memory, and enhancing neural network function.

[0045] The present invention provides a medicine comprising the probiotic composition or the probiotic agent and a pharmaceutically acceptable auxiliary material.

[0046] In the present invention, the total viable bacteria concentration in the drug is preferably 1×10 10 CFU / mL~100×10 10 CFU / mL or 1×10 10 CFU / g~100×10 10 CFU / g, can be 5×10 10 CFU / mL~80×10 10 CFU / mL or 5×10 10 CFU / g~80×10 10 CFU / g, or 10×10 10 CFU / mL~50×10 10 CFU / mL or 10×10 10 CFU / g~50×10 10 CFU / g. The dosage form of the drug preferably includes at least one of the following: powder, tablet, oral solution, capsule and drops. The present invention has no special restrictions on the preparation method of the drug, and the preparation method of the drug known in the art can be used.

[0047] The following is a detailed description of a probiotic composition with anti-anxiety and memory and concentration enhancing functions and its application provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] A method for preparing a probiotic composition preparation

[0050] The activated fermentation lactobacillus strain A21196 was inoculated into MRS liquid culture medium at an inoculation rate of 3% and cultured for 3 hours to obtain seed liquid, and the seed liquid was inoculated into MRS liquid culture medium at an inoculation rate of 8% and cultured at 37°C to the logarithmic phase to obtain the bacterial liquid of the fermentation lactobacillus strain A21196.

[0051] The activated Pediococcus pentosaceus strain A20001 was inoculated into MRS liquid culture medium at an inoculation rate of 3% and shaken for 3 hours to prepare a seed solution, and the seed solution was inoculated into MRS liquid culture medium at an inoculation rate of 8% to expand the culture to the logarithmic phase to obtain a large amount of Pediococcus pentosaceus strain A20001 bacterial solution.

[0052] The activated Lactobacillus plantarum strain A21337 was inoculated into MRS liquid culture medium and cultured at 37° C. for 3 hours to obtain seed solution. The seed solution was inoculated into MRS liquid culture medium and cultured at 37° C. to the logarithmic phase to obtain a bacterial solution of Lactobacillus plantarum strain A21337.

[0053] The formula of MRS liquid culture medium is glucose 20g / L, beef extract 10g / L, peptone 10g / L, yeast powder 5g / L, dipotassium hydrogen phosphate 2g / L, diammonium citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.58g / L, manganese sulfate 0.25g / L, Tween-80 0.1%, and the pH value is 6.6-6.8.

[0054] The prepared bacterial solution of Lactobacillus fermentum strain A21196, the bacterial solution of Pediococcus pentosaceus strain A20001 and the bacterial solution of Lactobacillus plantarum strain A21337 were mixed in a volume ratio of 1:1:1, and after centrifugation, the bacterial mud precipitate was mixed with a skim milk aqueous solution with a volume concentration of 40%, and freeze-dried to obtain a probiotic composition powder. When used, it was diluted to a target concentration using a counting method to obtain a probiotic composition resuspension.

[0055] Example 2

[0056] Construction of the Chronic Unpredictable Mild Stress (CUMS) Model in Mice and Validation of the Probiotic Combination Preparation

[0057] 1. Construction of CUMS Animal Model

[0058] Thirty-six healthy 6-week-old SPF-grade male C57BL / 6J mice, weighing 18-22 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., and the experimental animal production license is SCXK (Beijing) 2019-0010. The mice were housed in a constant temperature and humidity barrier environment (temperature 22±1℃, humidity 55±10%), with a light-dark alternation time of 12h:12h, and free food and water. After a week of adaptation, the mice were randomly grouped according to their weight, and the grouping is shown in Table 1. The following 1-3 different stimulations were used every day: restraint for 4h, crowding for 4h, tail clamping for 1min, ice water swimming for 5min, wet bedding for 24h, lighting all night for 24h, continuous darkness for 24h, no bedding for 24h, fasting for 24h, water deprivation for 24h, cage tilting for 24h, noise stimulation for 10min, and foreign body stimulation for 4h; the stimulation was irregular every day, and the model was continuously established for 4 weeks, and the mice were weighed once a week. After the modeling, the mice were subjected to behavioral tests to evaluate whether the modeling was successful. The sucrose preference test (SPT), tail suspension test (TST), and forced swim test (FST) were used to evaluate the depressive-like behavior of mice. The open field test (OFT) and elevated plus maze test (EPM) were used to evaluate the anxiety behavior of mice. The novel object recognition test (NOR) was used to evaluate the learning and cognitive abilities of mice.

[0059] Table 1 Treatment methods of mice in each group

[0060]

[0061] 2. Mouse Weight Statistics

[0062] The mice were modeled according to the above modeling method, and the weight of the mice was counted once a week.

[0063] The results are as follows Figure 1 The experimental results showed that compared with the normal group, the weight of mice in the CUMS model group decreased significantly (p < 0.0001). Compared with the model group, the weight loss trend of mice after intragastric administration of the probiotic composition preparation did not slow down significantly, indicating that intragastric administration of the probiotic composition preparation had no effect on the weight of depressed mice caused by CUMS.

[0064] 3. Sucrose preference experiment

[0065] Before the formal test, all mice were first trained with sugar water for 3 days, that is, they were given a bottle of drinking water containing 1% sucrose and a bottle of regular drinking water, and the positions of the two bottles were swapped from time to time. After the training period, all mice were fasted and deprived of water, and a formal sugar water test was conducted. Two bottles of drinking water containing 1% sucrose and regular drinking water of the same volume were prepared. After 24 hours, the sugar water consumption (g) of each mouse was counted, and the sugar water preference value was calculated according to Formula I.

[0066] Sugar water preference value = (mass of sugar water consumed / total mass of liquid consumed) × 100% Formula I.

[0067] The results are as follows Figure 2 As shown, compared with the normal group, the sugar preference value of mice in the CUMS model group was significantly reduced (p < 0.0001), indicating that the model was successfully constructed. In addition, after intragastric administration of the probiotic composition preparation, the sugar preference value of mice was significantly increased compared with the model group (p < 0.001), indicating that the probiotic composition preparation of the present invention has the function of improving the anhedonia of depressed mice.

[0068] 4. Tail suspension test

[0069] The mouse tail was fixed with tape at the 1 / 3 of the back, and hung on a stand. The timing and video recording were started immediately, and the video recording was stopped after 6 minutes of observation. The immobility time of the mouse in the last 4 minutes was counted using Smart 3.0 analysis software. Immobility refers to the state in which the animal gives up active struggle and is completely immobile.

[0070] The results are as follows Figure 3 As shown in the figure, compared with the normal group, the immobility time of mice in the CUMS model group was significantly increased (p < 0.001), indicating that the model was successfully constructed. Compared with the model group, the immobility time of mice in the probiotic composition preparation group was significantly shortened (p < 0.01). This shows that the probiotic composition preparation strain can effectively alleviate the depressive-like behavior of mice caused by CUMS.

[0071] 5. Forced swimming test

[0072] The mice were placed in a transparent experimental bucket (filled with sterile water about 30 cm high) for a 6-minute swimming test. The Smart 3.0 analysis software was used for real-time video recording and analysis, and the immobility time of the mice in the last 4 minutes (immobility, the judgment standard was immobility of the limbs or only slight movement of the hind limbs) was calculated.

[0073] The results are as follows Figure 4As shown in the figure, compared with the normal group, the immobility time of mice in the CUMS model group was significantly increased (p < 0.0001), indicating that the model was successfully constructed. Compared with the model group, the immobility time of mice in the probiotic composition preparation group was significantly shortened (p < 0.001). This shows that the probiotic composition preparation can effectively alleviate the depressive-like behavior of mice caused by CUMS.

[0074] 6. Open field experiment

[0075] The mouse was placed in a corner of the open field, and the timing and recording were started immediately. The recording was stopped after 10 minutes of observation. When replacing the animal, in order to avoid affecting the next test result, the surface needs to be wiped with 75% alcohol to remove the residual information of the previous animal (such as urine, feces, odor, etc.). Smart 3.0 analysis software was used for real-time recording and analysis, and the time (s) that the mouse stayed in the center area of ​​the open field was counted.

[0076] The results are as follows Figure 5 As shown in the figure, compared with the normal group, the time for the mice in the CUMS model group to enter the center of the open field was significantly shortened (p < 0.01). Compared with the model group, the time for the mice in the probiotic composition preparation group to enter the center of the open field was significantly increased (p < 0.05). This shows that the probiotic composition preparation can effectively alleviate the anxiety behavior of mice accompanied by depression-like symptoms caused by CUMS.

[0077] 7. Elevated plus maze test

[0078] Place the mouse in the central area of ​​the elevated plus maze (the mouse faces the open arm), start timing and recording immediately, and stop recording after 5 minutes of observation. When replacing the animal, in order to avoid affecting the next test result, it is necessary to wipe the surface with 75% alcohol to remove the residual information of the previous animal (such as urine, feces, odor, etc.). Use Smart 3.0 analysis software for real-time recording and analysis, and count the number of times the mouse enters the open arm, and the percentage of time in the open arm and the closed arm (%).

[0079] The results are as follows Figure 6 As shown in the figure, compared with the normal group, the number of times the mice in the CUMS model group entered the open arm and the percentage of time in the open arm were significantly shortened (p < 0.05). Compared with the model group, the number of times the mice in the probiotic composition preparation group entered the open arm and the percentage of time in the open arm were significantly increased (p < 0.0001), and the time in the closed arm was significantly shortened (p < 0.0001), indicating that the probiotic composition preparation strain can effectively alleviate the anxiety behavior of mice accompanied by depression-like symptoms caused by CUMS.

[0080] 8. New object recognition experiment

[0081] The novel object recognition test (NOR) is mainly used to evaluate the cognitive function, especially memory, of rodents (such as mice and rats). The core of this test is the difference in the animals' exploration behavior towards new and old objects, which is used to evaluate their memory and cognitive abilities.

[0082] Before the formal test, the mice were trained for 2 days. On the first day, the mice were allowed to freely explore and adapt in the open field for 10 minutes. On the second day, two identical objects A were placed in diagonal positions, and the mice were allowed to explore freely for 5 minutes. One hour later, one of the objects A was replaced with another new object B, and the mice were allowed to explore freely for 5 minutes. It should be noted that each time the animal is replaced, in order to avoid affecting the results of the next test, it is necessary to wipe the open field and the surface of the object with 75% alcohol to remove the residual information of the previous animal (such as urine, feces, odor, etc.). In the formal experiment, object A was retained in the open field, and object B was replaced with object C. Each mouse was placed in the same direction and allowed to explore freely for 5 minutes. Smart 3.0 analysis software was used for real-time video recording and analysis, and the percentage (%) of time the mouse explored the new object C was counted.

[0083] The results are as follows Figure 7 As shown in the figure, compared with the normal group, the percentage of time spent exploring new objects in the CUMS model group was significantly shortened (p < 0.01). Compared with the model group, the percentage of time spent exploring new objects in the probiotic composition preparation group was significantly increased (p < 0.01), indicating that the probiotic composition preparation strain can effectively improve the cognitive behavior of CUMS depressed mice and improve the memory of mice.

[0084] Example 3

[0085] Detection of serum 5-HT and BDNF levels in mice

[0086] Three groups of mice were treated according to the method described in Example 2, and appropriate amounts of serum were taken from the three groups of mice, respectively, and the detection was performed according to the instructions of the mouse 5-HT ELISA kit (provided by Jianglai Bio) and the mouse BDNF ELISA kit (provided by Lianke Bio). The mouse hippocampus tissue was detected according to the instructions of the total RNA extraction kit (provided by Promeg Bio), the single-stranded synthesis kit (provided by Quanshijin), and the 2X SYBRGreen qPCRMiX (provided by Si Kejie). The primers used for qPCR are shown in Table 2 below.

[0087] Table 2 Primer sequences for qPCR detection

[0088]

[0089] The results of 5-HT level detection in mouse serum are as follows Figure 8As shown, compared with the normal group, the 5-HT level in the serum of the mice in the CUMS model group was significantly decreased (p<0.001). Compared with the model group, the 5-HT content in the mice in the probiotic composition preparation group was significantly increased (p<0.05).

[0090] The results of BDNF level detection in mouse serum are as follows Fig. 9 As shown, compared with the normal group, the BDNF level in the serum of the mice in the CUMS model group was significantly decreased (p<0.0001). Compared with the model group, the BDNF content in the mice in the probiotic composition preparation group was significantly increased (p<0.01).

[0091] The results of BDNF detection in mouse hippocampus are as follows Fig.10 As shown in the figure, compared with the normal group, the BDNF level in the hippocampus of the CUMS model group mice decreased significantly (p < 0.001). Compared with the model group, the BDNF content of the mice in the probiotic composition preparation group increased significantly (p < 0.01), increasing by about 7 times. It can be seen that the probiotic composition preparation may relieve the depressive-like behavior of mice by increasing the levels of 5-HT and BDNF, and can effectively improve the cognitive behavior of CUMS depressed mice and improve the memory of mice.

[0092] Example 4

[0093] The number of viable bacteria in different proportions of probiotic composition preparations

[0094] The strains A21196, A20001, A21337 and other strains A2104 (Lactobacillus reuteri) and A21077 (Lactobacillus casei) in the probiotic composition were streaked and activated on MRS plates, respectively. After 3 times of MRS liquid transfer, the obtained bacterial suspensions were mixed and cultured with different viable counts according to Table 3. After 16 hours, the corresponding bacterial suspensions were taken and diluted by multiple dilution (10 -6 , 10 -7 , 10 -8 ) method was used to count the number of viable bacteria in turn, and each dilution gradient was plated twice.

[0095] Table 3 Preparation methods of probiotic compositions in different schemes

[0096]

[0097]

[0098] According to the above-mentioned live bacteria count statistics, serial numbers 1 to 8 are all different proportions of the probiotic composition. The results show that when the probiotic composition is combined in a ratio of 1:1:1, the live bacteria count is the highest. A sufficient amount of live bacteria count may ensure that the probiotic composition is successfully colonized in the intestine and exerts a probiotic effect. If any two of the probiotic compositions are compounded with other strains, the optimal effect cannot be achieved.

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A probiotic composition, characterized in that: The invention comprises Limosilactobacillusfermentum strain A21196, Pediococcus pentosaceus strain A20001 and Lactiplantibacillusplantarum strain A21337 having a viable cell count ratio of (0.5-1.5):(0.5-1.5):(0.5-1.5); The deposit number of the fermented Lactobacillus mucilaginosus strain A21196 is GDMCC No: 62959; The deposit number of the Pediococcus pentosaceus strain A20001 is GDMCC No: 64674; The deposit number of the plant lactobacillus strain A21337 is GDMCC No: 64677.

2. The probiotic composition according to claim 1, characterized in that: The live cell count ratio of the fermentative Lactobacillus mucilaginosus strain A21196, the Pediococcus pentosaceus strain A20001 and the Lactobacillus plantarum strain A21337 is (0.8~1.2):(0.8~1.2).

3. The probiotic composition according to claim 1, characterized in that: The viable cell count ratio of the fermentative Lactobacillus mucilaginosus strain A21196, Pediococcus pentosaceus strain A20001 and Lactobacillus plantarum strain A21337 was 1:1:

1.

4. A probiotic, characterized in that: The active ingredient comprises the probiotic composition according to any one of claims 1 to 3.

5. The probiotic according to claim 4, characterized in that: The dosage form of the probiotics includes aqueous solution and / or powder.

6. The probiotic according to claim 4 or 5, characterized in that: The total live bacteria concentration in the probiotic is 1×10 10 CFU / mL~100×10 10 CFU / mL or 1×10 10 CFU / g~100×10 10 CFU / g.

7. A drug, characterized in that The invention comprises the probiotic composition according to any one of claims 1 to 3 or the probiotic agent according to any one of claims 4 to 6 and a pharmaceutically acceptable excipient.

8. The drug according to claim 7, characterized in that: The total live bacteria concentration in the drug is 1×10 10 CFU / mL~100×10 10 CFU / mL or 1×10 10 CFU / g~100×10 10 CFU / g.

9. Use of the probiotic composition according to any one of claims 1 to 3 or the probiotic agent according to any one of claims 4 to 6 in the preparation of a medicament for preventing and / or treating at least one of the following neurological diseases or indications: anxiety, depression and cognitive dysfunction.

10. The use according to claim 9, characterized in that: The dosage form of the drug includes at least one of the following: powder, tablet, oral solution, capsule and drops.

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