High-activity plant lactobacillus KL100 for relieving anxiety and depression and application of high-activity plant lactobacillus KL100

Abstract

The invention belongs to the technical field of microorganisms, and relates to high-activity plant lactobacillus KL100 for relieving anxiety and depression and application of the high-activity plant lactobacillus KL100. The plant lactobacillus provided by the invention is KL100, is preserved in the China General Microbiological Culture Collection Center (CGMCC), has a preservation number of CGMCC No.27192, and is classified and named as the plant lactobacillus. The plant lactobacillus is KL100, has a good gastrointestinal passing rate, shows a breeding characteristic in the stomach after being embedded, is improved in cholate-resistant survival rate, and can be used for preparing the plant lactobacillus. According to the application disclosed by the invention, the expression of IL-1beta and TNF-alpha in a mouse body can be effectively reduced, the expression of IL-10 can be improved, and the secretion of 5-hydroxytryptamine by chromaffin cells in intestinal tracts can be promoted, so that anxiety and depression can be relieved, the strain resource is expanded, the development of the biological medicine field is promoted, and a new direction is provided for treating anxiety and depression.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a highly active Lactobacillus plantarum KL100 for alleviating anxiety and depression and a use thereof. Background Art

[0002] Depression is one of the most common mental illnesses in the world, with high morbidity, high disability and high mortality. Currently, more than 320 million people suffer from depression worldwide. With the development of society, the incidence of depression has gradually increased and is gradually tending to be younger. Nearly three-quarters of adult depression patients have their first symptoms in childhood or adolescence. Therefore, adolescent depressive disorder has become a major public mental health issue that needs to be addressed urgently.

[0003] Currently, the treatment of depression is still mainly based on drugs, but about one-third of depression patients do not improve their clinical symptoms after antidepressant treatment. As researchers delve deeper into the relationship between human microorganisms and depression, related intervention or treatment methods have emerged, such as probiotics and fecal transplantation, which show good application prospects.

[0004] Probiotics help restore the disordered intestinal flora of patients with depression, help improve depression, and provide a new entry point for the treatment of depression. Probiotics can treat and relieve depression from the neuroendocrine, vagus nerve, and immune pathways. The Chinese patent application with publication number CN 118240718 A discloses the use of a Lactobacillus rhamnosus TM08 and its products in improving depression. Through animal experiments, it is proved that the strain has the effect of relieving depression and nerve damage in mice; the Chinese patent application with publication number CN 117379464 A discloses the use of a Lactobacillus helveticus LH05 in the preparation of antidepressant drugs. Through experiments, it is proved that Lactobacillus helveticus LH05 can significantly increase the activity level of zebrafish and the concentration of 5-HT in brain tissue, that is, it has an antidepressant effect; the Chinese patent application with publication number CN 117887646 A discloses a composite probiotic for anti-anxiety and depression and its application, using Bacillus coagulans BC99 strain and Bifidobacterium bifidum bifidum) BBi32 strain. Compared with a single strain, the combination of the two bacteria has significantly improved the effect of anti-anxiety and depression. However, my country's probiotics for the treatment of depression are heavily dependent on imports. Although the problem of lack of proprietary strains for the treatment of depression is gradually improving, there is an extreme lack of strain production and supporting technologies, and the strains cannot be commercialized with high efficiency and high activity. Summary of the invention

[0005] The present invention aims to provide a highly active Lactobacillus plantarum KL100 for relieving anxiety and depression and its use. The present invention screens a strain of Lactobacillus plantarum KL100 that can promote the secretion of 5-HT by intestinal chromaffin cells (EC) from the human intestine, combines it with a high-density fermented, tryptophan-rich protein peptide, and realizes peptide segment self-assembly embedding to obtain a Lactobacillus plantarum KL100 preparation with good gastric acid and bile salt resistance and high antidepressant activity. The preparation can effectively relieve depression and anxiety symptoms.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a highly active Lactobacillus plantarum KL100 for relieving anxiety and depression. The strain is screened from the human intestine and was deposited in the General Microbiology Center of the China Culture Collection Administration on April 23, 2023. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, the deposit number is CGMCC No. 27192, and the classification name is Lactobacillus plantarum.

[0008] The invention discloses plant lactobacillus KL100 with a preservation number of CGMCC No. 27192. The bacterium presents milky white, round and regular colonies on an MRS solid culture medium.

[0009] The present invention provides use of the above-mentioned highly active Lactobacillus plantarum KL100 for relieving anxiety and depression in the preparation of a drug for relieving anxiety and depression.

[0010] The term "alleviation" herein refers to all actions including making the disease better or improved by administering the Lactobacillus plantarum KL100.

[0011] The present invention provides a highly active Lactobacillus plantarum KL100 embedding composition for relieving anxiety and depression, comprising the above-mentioned Lactobacillus plantarum KL100, skim milk, protein peptide and polysaccharide.

[0012] Preferably, the protein peptide is whey protein peptide.

[0013] Preferably, the polysaccharide is maltodextrin.

[0014] In addition, the present invention also provides a highly active Lactobacillus plantarum KL100 preparation for relieving anxiety and depression. The active ingredients of the Lactobacillus plantarum KL100 preparation include the above-mentioned Lactobacillus plantarum KL100 embedding composition, fruit powder, protein peptide and compound vitamin and mineral.

[0015] Preferably, the fruit powder is strawberry fruit powder.

[0016] Preferably, 1 g of the composite vitamin mineral contains 1.092-1.638 mg vitamin A, 1.04-1.56 mg vitamin B1, 1.04-1.56 mg vitamin B2, 1.04-1.56 mg vitamin B6, 0.1306-0.1958 mg vitamin B12, 130.6-195.8 mg vitamin C, 130.6-195.8 mg magnesium, and 8.40-12.60 mg zinc.

[0017] Preferably, the dosage form of the Lactobacillus plantarum KL100 preparation includes one or more of a solution, a powder, a gel, a granule, an emulsion, a suspension and a lyophilized agent.

[0018] In addition, the present invention also provides a method for preparing a highly active Lactobacillus plantarum KL100 preparation for relieving anxiety and depression, comprising:

[0019] Step S1, taking the Lactobacillus plantarum KL100 seed liquid as described above, inoculating it into a fermentation medium for high-density fermentation to obtain a bacterial liquid of Lactobacillus plantarum KL100 high-density fermentation, centrifuging to obtain bacterial mud, mixing the bacterial mud with skim milk, protein peptides, and polysaccharides, and freeze-drying to obtain a Lactobacillus plantarum KL100 embedding composition;

[0020] Step S2, mixing milk powder, Lactobacillus plantarum KL100 embedding composition, fruit powder, whey protein peptide and compound vitamin and mineral to obtain Lactobacillus plantarum KL100 preparation.

[0021] Preferably, in step S1, the high-density fermentation conditions are pH 6.5, fermentation temperature 35°C, inoculation amount 20 mL / L, dissolved oxygen content 0-100 mL / L, stirring speed 50 r / min, tank pressure 0.03 MPa, and fermentation time 20-24 h.

[0022] Preferably, in step S1, the mass ratio of the bacterial mud to the skim milk, protein peptide and polysaccharide is 1:1:4:4.

[0023] Preferably, in step S1, the freeze-drying operation is as follows: the temperature is -35°C in the 0th to 1st hour, the temperature is increased to -20°C in the 1st to 6th hour, the temperature is increased to -15°C in the 6th to 11th hour, the temperature is increased to -15°C in the 16th to 21st hour, the temperature is increased to -10°C in the 21st to 26th hour, the temperature is increased to 0°C in the 26th to 31st hour, the temperature is increased to 10°C in the 31st to 41st hour, the temperature is increased to 20°C in the 41st to 58th hour, the temperature is maintained at 20°C in the 58th to 58.5th hour, the temperature is increased to 25°C in the 58.5th to 60.5th hour, and the temperature is maintained at 25°C in the 60.5th to 62.5th hour; the freeze-drying vacuum degree is 0.22 mbar.

[0024] Preferably, in step S2, the mass ratio of the Lactobacillus plantarum KL100 embedding composition, milk powder, fruit powder, protein peptide and composite vitamin and mineral is 20-30:30-60:10-50:10-20:1-2.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) The present invention is based on intestinal chromaffin cells (EC) culture and uses high-throughput cell screening technology to screen a strain of Lactobacillus plantarum KL100 that has the ability to promote EC cells to secrete 5-hydroxytryptamine (5-HT) from the human intestine. The strain is preserved in the General Microbiology Center of the China National Microbiological Culture Collection Administration and obtains a strain with its own intellectual property rights.

[0027] (2) The present invention adopts high-density fermentation to obtain highly active probiotics, and adopts freeze-drying technology to maintain the high-activity probiotic activity. High-density fermentation technology can significantly increase the biomass of probiotics in a unit volume of fermentation liquid, thereby obtaining a large amount of probiotic products in a relatively short period of time to meet the large-scale market demand. In the present invention, the fermentation density of Lactobacillus plantarum KL100 in a 10 L fermentation tank can reach 5.62×10 12 CFU / mL, achieving high-density fermentation, making the subsequent application and production of Lactobacillus plantarum products possible.

[0028] (3) The present invention compounds the probiotics with a protective agent consisting of protein peptides, skim milk and polysaccharides, realizes peptide chain self-assembly during the freeze-drying process, forms a peptide chain cross-linking structure, and forms a protective film on the surface of the probiotics, thereby reducing the "solute damage" caused by excessive dehydration and severe shrinkage of the cells before drying, and the freeze-drying damage that causes cell membrane rupture, thereby increasing the gastrointestinal passage rate of the probiotics, obtaining a highly active probiotic preparation, and achieving a highly active Lactobacillus plantarum KL100 preparation that effectively relieves anxiety and depression, promotes the increase of 5-HT, and reduces the level of inflammation, thereby having a significant effect on improving depression.

[0029] Description of biological deposit:

[0030] A highly active Lactobacillus plantarum KL100 that relieves anxiety and depression was deposited in the General Microbiology Center of the China Culture Collection Administration on April 23, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, the deposit number is CGMCC No. 27192, and the classification name is Lactobacillus plantarum. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1This is a colony image of Lactobacillus plantarum KL100.

[0032] Figure 2 This is a comparison chart of the gastrointestinal passage rate test of Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation, A is the gastric passage rate of Lactobacillus plantarum KL100 powder; B is the gastric passage rate of Lactobacillus plantarum KL100 preparation; C is the duodenum passage rate of Lactobacillus plantarum KL100 powder; D is the duodenum passage rate of Lactobacillus plantarum KL100 preparation.

[0033] Figure 3 This is a graph showing the test results of the open field experiment on mice intervened by the normal group, model group, fluoxetine group, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation.

[0034] Figure 4 These are the test results of the tail suspension experiment on mice in the normal group, model group, fluoxetine group, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation. A is the immobility time of mice in each group during the experiment; B is the number of immobility times of mice in each group during the experiment; C is the percentage of the immobility time of mice in each group in the total experimental time.

[0035] Figure 5 This is a graph showing the weight measurement results of mice in the normal group, model group, fluoxetine group, Lactobacillus plantarum KL100 powder, and Lactobacillus plantarum KL100 preparation intervention during the experimental period.

[0036] Figure 6 The graph shows the results of IL-1β, TNF-α and IL-10 determination in the serum of mice in the normal group, model group, fluoxetine group, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation intervention groups.

[0037] Figure 7 The graph shows the test results of 5-HT concentration in the serum of mice in the normal group, model group, fluoxetine group, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation intervention groups. DETAILED DESCRIPTION

[0038] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are not intended to limit the protection scope of the present invention.

[0039] Unless otherwise specified, the reagents and equipment involved in the following examples were purchased from commercial channels.

[0040] The strains, reagents and culture media involved in the embodiment are:

[0041] The Lactobacillus plantarum KL100 involved in the embodiment is obtained from the intestine by screening using high-throughput cell screening technology based on intestinal EC cell culture.

[0042] Fermentation medium: 25 g / L maltose and glucose mixture (mass ratio of 1:1), 10 g / L beef extract and yeast powder mixture (mass ratio of 1:1), 2 g / L dipotassium hydrogen phosphate, 2 g / L triammine citrate, 5 g / L sodium acetate, 0.58 g / L magnesium sulfate, 0.07 g / L manganese sulfate, 1 mL / L Tween 80, pH of the culture medium was 6.5.

[0043] MRS liquid culture medium: peptone 10 g / L, beef extract 5 g / L, yeast extract 4 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 mL / L, make up to 1000 mL with distilled water, sterilize at 121°C for 15 min, the pH of the culture medium is 6.5.

[0044] MRS solid culture medium: Add 20 g / L agar powder to MRS liquid culture medium and sterilize at 121℃ for 15 min for viable bacteria counting. The pH of the culture medium is 6.5.

[0045] SGF electrolyte stock solution: KCl 6.9 mmol / L, KH 2 PO 4 0.9 mmol / L, NaHCO 3 25 mmol / L, NaCl47.2mmol / L, MgCl 2 (H 2 O) 6 0.1 mmol / L, (NH 4 ) 2 CO 3 0.5 mmol / L.

[0046] SIF electrolyte stock solution: KCl 6.8 mmol / L, KH 2 PO 4 0.8 mmol / L, NaHCO 3 85 mmol / L, NaCl38.4mmol / L, MgCl 2 (H 2 O) 6 0.33 mmol / L.

[0047] Strawberry fruit powder was purchased from Tuofeng Health Technology (Nantong) Co., Ltd.

[0048] Compound vitamins and minerals were purchased from Wuhu Meno Nutrition Technology Co., Ltd. Among them, 1 g of compound vitamins and minerals contains 1.092~1.638 mg vitamin A, 1.04~1.56 mg vitamin B1, 1.04~1.56 mg vitamin B2, 1.04~1.56 mg vitamin B6, 0.1306~0.1958 mg vitamin B12, 130.6~195.8 mg vitamin C, 130.6~195.8 mg magnesium, 8.40~12.60 mg zinc, and the rest is excipients.

[0049] Example 1 Cultivation of Lactobacillus plantarum KL100 and preparation of seed solution

[0050] Take 100 μL of Lactobacillus plantarum KL100 preserved in glycerol, inoculate it into 10 mL of MRS liquid culture medium, and culture it in a constant temperature incubator at 37°C for 20 h to obtain activated Lactobacillus plantarum KL100.

[0051] Take the activated Lactobacillus plantarum KL100, streak three lines on MRS solid culture medium, culture at 37°C for 48 h, and obtain a single colony of Lactobacillus plantarum KL100.

[0052] Pick a single colony of Lactobacillus plantarum KL100, put it into 10 mL of MRS liquid culture medium, and culture it at 37°C for 20 h to obtain Lactobacillus plantarum KL100 bacterial liquid; take 6 mL of Lactobacillus plantarum KL100 bacterial liquid and inoculate it into 200 mL of MRS liquid culture medium to obtain Lactobacillus plantarum KL100 seed liquid.

[0053] Example 2 Preparation of Lactobacillus plantarum KL100 preparation

[0054] Step S1, take 20 mL of plant lactobacillus KL100 seed liquid, inoculate it into 1 L of fermentation medium, and carry out high-density fermentation for 24 h at 35°C, with a dissolved oxygen content of 0 mL / L, a stirring speed of 40 r / min, and a tank pressure of 0.03 MPa to obtain a bacterial solution of plant lactobacillus KL100 high-density fermentation; the bacterial solution is centrifuged at a speed of 3000 r / min to obtain bacterial mud, 5 g of bacterial mud is mixed with 20 g of skim milk, 5 g of whey protein peptides, and 20 g of maltodextrin, and placed in a freeze dryer with a vacuum degree of 0.22 mbar for drying, at 0-1 h, the temperature is -35°C, at 1-6 h, the temperature is raised to -20°C, at 6-11 h, the temperature is raised to -15°C, at 16-21 h, the temperature is raised to -15°C, at 21-26 h, the temperature is raised to -10°C, at 26-31 h, the temperature is raised to 0°C, and at 31-41 h, the temperature is raised to 0°C. h, the temperature was raised to 10°C, from 41 to 58 h, the temperature was raised to 20°C, from 58 to 58.5 h, the temperature was maintained at 20°C, from 58.5 to 60.5 h, the temperature was raised to 25°C, from 60.5 to 62.5 h, the temperature was maintained at 25°C, and finally a Lactobacillus plantarum KL100 embedding composition was obtained.

[0055] Step S2, weigh 60 g of milk powder, 20 g of Lactobacillus plantarum KL100 embedding composition, 30 g of strawberry fruit powder, 10 g of whey protein peptide and 1 g of compound vitamin and mineral, and mix them to obtain a Lactobacillus plantarum KL100 preparation.

[0056] Example 3 Gastrointestinal Transit Rate Test of Lactobacillus plantarum KL100 Powder and Lactobacillus plantarum KL100 Preparation

[0057] Experimental steps:

[0058] (1) The bacterial sludge obtained in step S1 of Example 2 was directly freeze-dried to obtain Lactobacillus plantarum KL100 powder, and the Lactobacillus plantarum KL100 powder and the Lactobacillus plantarum KL100 preparation were dissolved in sterile water, respectively, and gradient dilution was performed to take 10 -7 CFU / mL, 10 -8 CFU / mL, 10 -9 The bacterial liquids with three CFU / mL gradients were spread on MRS solid medium, cultured for 24 h, and counted.

[0059] (2) Gastric passage rate: 22.5 mL of SGF electrolyte stock solution, 15 μL of 0.3 mol / L CaCl were added to 30 mL of a mixture of Lactobacillus plantarum KL100 powder and gastric digestive juice (powder: gastric digestive juice volume ratio was 1:1). 2And 4.8 mL of pepsin solution dissolved from SGF electrolyte stock solution, supplemented with distilled water to 60 mL. Digestion was performed at 37°C and pH 2, 3, and 4 for 0 h, 1 h, 2 h, and 3 h. The mixed solution at different time points was taken for gradient dilution, and the dilution factor was 10 -7 CFU / mL, 10 -8 CFU / mL, 10 -9 100 μL of the mixed solution with CFU / mL was spread, and the colonies were counted after incubation at 37°C for 24 h to calculate the gastric pass rate of Lactobacillus plantarum KL100 powder; the gastric pass rate of Lactobacillus plantarum KL100 preparation was calculated in the same way as above.

[0060] (3) Intestinal transit rate: 16.5 mL of SIF electrolyte stock solution, 7.5 mL of pancreatic enzyme solution dissolved in SIF electrolyte stock solution, 3.75 mL of bile salts of different concentrations (0.1%, 0.3%, 0.5%), and 60 μL of 0.3 mol / L CaCl were added to 30 mL of a mixture of Lactobacillus plantarum KL100 powder and gastric digestive juice (the volume ratio of powder to small intestinal digestive juice was 1:1). 2 , add distilled water to the system to reach 60 mL, shake in a 37℃ water bath for 0 h, 1 h, 2 h and 3 h, take the mixed solution at different time points for gradient dilution, and take the dilution factor of 10 -9 CFU / mL, 10 -10 CFU / mL, 10 -11 100 μL of the mixed solution with CFU / mL was applied, and the colonies were counted after incubation at 37°C for 24 h to calculate the intestinal permeability rate of Lactobacillus plantarum KL100 powder; the gastric permeability rate of Lactobacillus plantarum KL100 preparation was calculated in the same way as above, except that the mixed solution at different time points was diluted stepwise after shaking in a 37°C water bath for 0 h, 1 h, 2 h and 3 h, and the dilution factor of 10 was taken respectively. -8 CFU / mL, 10 -9 CFU / mL, 10 -10 100 μL of the mixed solution with CFU / mL was spread and cultured at 37°C for 24 h before colony counting to calculate the intestinal passage rate of Lactobacillus plantarum KL100 preparation.

[0061] like Figure 2As shown, A is the gastric rate of Lactobacillus plantarum KL100 powder, which is about 40%, and B is the gastric rate of Lactobacillus plantarum KL100 preparation, which is about 100%. Compared with Lactobacillus plantarum KL100 powder, the gastric rate of Lactobacillus plantarum KL100 preparation is increased by 2.5 times. C is the duodenum rate of Lactobacillus plantarum KL100 powder, which is about 0.2%, and D is the duodenum rate of Lactobacillus plantarum KL100 preparation, which is about 20%. Compared with Lactobacillus plantarum KL100 powder, the duodenum rate of Lactobacillus plantarum KL100 preparation is increased by 100 times. The experimental results show that Lactobacillus plantarum KL100 preparation has a good gastric survival rate and exhibits reproduction characteristics in the stomach.

[0062] Example 4 Animal Experiment

[0063] 1. Establishing the Animal Model

[0064] The experiment started after 1 week of adaptive feeding, and the mice were randomly divided into a normal group, a model group, a positive drug group (fluoxetine group), Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation, with 10 mice in each group. The treatment methods are shown in Table 1.

[0065] Table 1 Experimental mouse groups and treatment methods

[0066]

[0067] The experimental period was 7 weeks. Mouse modeling and drug administration were carried out simultaneously. The drug administration period was 5 weeks. The mice were weighed every 7 days during the experiment, and the gavage volume was adjusted according to the body weight. Mouse behavioral tests began in the 6th week.

[0068] At the end of the 7th week, fresh feces were collected from mice. All mice were fasted but not watered for 12 h, and their eyeballs were removed for blood collection. Serum was collected by centrifugation at 4°C and 3000 r / min for 10 min. The mice were killed after blood collection, and the hippocampus, cerebral cortex and colon tissues were dissected out.

[0069] CUMS depression modeling method: The first to fifth weeks of the experiment are the modeling period. 10 different stimuli are applied to the mice, and one stimulus is randomly given every day. The same stimulus cannot be applied for 3 consecutive days to ensure that the animals are not expected. Normal mice have smooth and shiny fur and good mental state. Depressed mice have yellow and dull fur, are listless, and their cleanliness is significantly reduced.

[0070] The 10 different stimuli are:

[0071] ① Tilted cage: raise one end of the cage and tilt it 45° for 24 hours;

[0072] ②Fasting: remove the mouse food for 24 h;

[0073] ③ Water deprivation + empty bottle stimulation: pour out the water in the bottle and put it back in place for 24 hours;

[0074] ④ Bedding: Take out the bedding, 8 hours;

[0075] ⑤Continuous light, 24 h;

[0076] ⑥ Crowding stimulation: Place partitions in the cage to reduce the activity area of ​​the mice for 24 hours;

[0077] ⑦ Clamp the tail: Use hemostatic forceps to clamp the tail 1 cm away from the tip for 60 seconds;

[0078] ⑧ Day and night reversal: Adjust the day and night cycle, cover the mouse cage with black cloth during the day, remove the black cloth at night, turn on the lights, and create an environment that is dark during the day and bright at night;

[0079] ⑨ Restraint: confined in a 50 mL plastic syringe, 2 h;

[0080] ⑩ Wet bedding: Sprinkle 200 mL of water into the bedding for 8 h.

[0081] 2. Open field experiment: The experimental room should be quiet and softly lit. The open field box is a black box with a size of 40 cm×40 cm×40 cm. The mice are placed in the central area at the bottom of the open field box and allowed to move freely. After placement, the experimenter stays away from the activity box and uses the camera equipment supporting the experimental device to take pictures. The filming time is 13 minutes. The experiment calculates the time the mice stay in the center of the open field and the total distance they travel in the last 10 minutes. After each mouse experiment, wipe the inner wall and bottom of the open field box with 75% alcohol to remove animal excrement and odor. The experimental results are based on Figure 3 As shown in the figure, the total distance, number of grid crossings and percentage of time spent in the middle grid in the open field in the model group were significantly lower than those in the other groups (p < 0.05). The total distance, number of grid crossings and percentage of time spent in the middle grid in the open field in the mice treated with fluoxetine, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation were significantly increased. Figure 3 As shown in the results, CUMS induced anxiety in mice, and their activity level decreased. After intervention with fluoxetine, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation, there was a significant improvement, and the activity level of mice tended to be normal. The regulatory effect of Lactobacillus plantarum KL100 preparation was better than that of Lactobacillus plantarum KL100 powder. The experimental results show that Lactobacillus plantarum KL100 preparation can effectively improve the anxiety of mice and significantly restore the activity of mice.

[0082] 3. Tail suspension test: A black tail suspension box was used for the experiment. The mouse's tail was fixed on a hook in the center of the top plate, with the head about 5 cm from the bottom of the box. The behavioral changes of the mouse were recorded for 6 minutes, and the cumulative immobility time in the last 4 minutes was analyzed. The criterion for the immobility of the mouse was that the mouse stopped struggling and its body was vertically suspended and motionless.

[0083] Figure 4 The figure shows the results of the tail suspension test in mice. The tail suspension test can reflect the despair of animals, and the immobility time is positively correlated with their despair. A is the immobility time of mice in each group during the experiment. Compared with the normal group mice, the immobility time of mice in the model group increased significantly (p < 0.05), while after intervention with fluoxetine, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation, the immobility time of mice decreased significantly (p < 0.05), among which the Lactobacillus plantarum KL100 preparation was more significant than the Lactobacillus plantarum KL100 powder, and there was no significant difference with the fluoxetine group (p > 0.05). B is the number of immobility times of mice in each group during the experiment. Compared with the mice in the normal group, the number of immobility times of mice in the model group increased significantly (p < 0.05). After intervention with fluoxetine, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation, the number of immobility times of mice decreased (p < 0.05). The intervention effect of Lactobacillus plantarum KL100 preparation was better than that of Lactobacillus plantarum KL100 powder, and there was no significant difference with the fluoxetine group (p > 0.05). C is the percentage of immobility time of mice in each group during the experiment in the total experimental time. Compared with the mice in the normal group, the time that mice in the model group gave up struggling and remained immobile during the experiment accounted for the highest proportion of the total time. After intervention with fluoxetine, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation, especially after intervention with Lactobacillus plantarum KL100 preparation, the proportion of time that mice gave up struggling and remained immobile was significantly reduced (p < 0.05).

[0084] The experimental results showed that the mice in the model group had strong feelings of despair, and the intervention with fluoxetine, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation could effectively improve this situation, among which the effect of Lactobacillus plantarum KL100 preparation was better than that of Lactobacillus plantarum KL100 powder.

[0085] 4. Weight of experimental mice: Emotions can affect appetite, which generally leads to decreased appetite, which in turn affects body weight. Changes in body weight reflect the depression of CUMS mice to a certain extent. Figure 5As shown in the figure, there was no significant difference in the weight of mice in each group before the experiment. After CUMS intervention, the appetite of mice in the model group was affected and their weight decreased significantly. Compared with the model group, the situation of mice intervened with Lactobacillus plantarum KL100 powder was improved, while there was no significant difference between the Lactobacillus plantarum KL100 preparation and fluoxetine groups and the normal group. The experimental results show that Lactobacillus plantarum KL100 preparation can significantly alleviate the weight loss of mice with depression induced by CUMS.

[0086] Example 5 Mouse cytokine assay

[0087] The experimental steps were carried out according to the kit instructions. IL-1β, TNF-α and IL-10 are important anti-inflammatory cytokines and are closely related to depression and anxiety. Figure 6 As shown in the results, the serum IL-1β and TNF-α levels of CUMS-induced depressed mice were significantly increased (p < 0.05), and the IL-10 level was significantly decreased (p < 0.05). Figure 6 As shown in the data, the IL-1β and TNF-α in the serum of mice intervened with the fluoxetine group, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation decreased, and IL-10 increased. Among them, the effect of Lactobacillus plantarum KL100 preparation intervention was equivalent to that of the fluoxetine group. The experiment proved that Lactobacillus plantarum KL100 preparation can significantly improve the level of serum inflammatory factors in mice.

[0088] Example 6 Detection of 5-HT in mouse serum

[0089] The experimental steps were carried out according to the instructions of the kit. 5-HT is a neurotransmitter that can guide the brain to produce pleasant emotions. The reduction of 5-HT is directly related to changes in psychology and emotions, thus leading to the occurrence of depression. The higher the 5-HT concentration, the lower the degree of depression. Figure 7 As shown, the 5-HT level of mice in the model group was the lowest, which was significantly different from that of other groups (p < 0.05); the 5-HT concentration in the serum of mice after intervention with fluoxetine, Lactobacillus plantarum KL100 powder and Lactobacillus plantarum KL100 preparation was significantly increased (p < 0.05), among which the concentration of Lactobacillus plantarum KL100 preparation was higher than that of Lactobacillus plantarum KL100 powder, and there was no significant difference with the fluoxetine group (p > 0.05). The experimental results show that Lactobacillus plantarum KL100 preparation can effectively promote the secretion of 5-HT neurotransmitter, thereby improving depression in mice.

[0090] 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 technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A highly active Lactobacillus plantarum KL100 for relieving anxiety and depression, characterized in that: The plant lactobacillus KL100 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee with a deposit number of CGMCC No.27192 and is classified and named as plant lactobacillus ( Lactiplantibacillus plantarum ).

2. Use of the highly active Lactobacillus plantarum KL100 for relieving anxiety and depression as claimed in claim 1 in the preparation of a preparation for relieving anxiety and depression.

3. A highly active Lactobacillus plantarum KL100 embedding composition for relieving anxiety and depression, characterized in that: The invention comprises the plant lactobacillus KL100 as claimed in claim 1, skim milk, protein peptide and polysaccharide.

4. The highly active Lactobacillus plantarum KL100 embedding composition for relieving anxiety and depression according to claim 3, wherein the protein peptide is whey protein peptide; and the polysaccharide is maltodextrin.

5. A highly active Lactobacillus plantarum KL100 preparation for relieving anxiety and depression, characterized in that: The effective ingredients of the Lactobacillus plantarum KL100 preparation include the Lactobacillus plantarum KL100 embedding composition according to claim 3 or 4.

6. A highly active Lactobacillus plantarum KL100 preparation for relieving anxiety and depression according to claim 5, characterized in that: The Lactobacillus plantarum KL100 preparation also includes fruit powder, whey protein peptides and compound vitamins and minerals; the dosage form of the Lactobacillus plantarum KL100 preparation includes one or more of a solution, a powder, a gel, a granule, an emulsion, a suspension and a lyophilized agent.

7. A highly active Lactobacillus plantarum KL100 preparation for relieving anxiety and depression according to claim 6, characterized in that: The fruit powder is strawberry fruit powder; 1 gram of compound vitamin and mineral contains 1.092-1.638 mg vitamin A, 1.04-1.56 mg vitamin B1, 1.04-1.56 mg vitamin B2, 1.04-1.56 mg vitamin B6, 0.1306-0.1958 mg vitamin B12, 130.6-195.8 mg vitamin C, 130.6-195.8 mg magnesium, and 8.40-12.60 mg zinc.

8. A method for preparing a highly active Lactobacillus plantarum KL100 preparation for relieving anxiety and depression, characterized in that: include: Step S1, taking the Lactobacillus plantarum KL100 seed liquid of claim 1, inoculating it into a fermentation medium for high-density fermentation to obtain a high-density fermentation bacterial liquid of Lactobacillus plantarum KL100, centrifuging the bacterial liquid to obtain bacterial mud, mixing the bacterial mud with skim milk, protein peptides, and polysaccharides, and freeze-drying to obtain a Lactobacillus plantarum KL100 embedding composition; Step S2, mixing milk powder, Lactobacillus plantarum KL100 embedding composition, fruit powder, whey protein peptide and compound vitamin and mineral to obtain Lactobacillus plantarum KL100 preparation.

9. The method for preparing a highly active Lactobacillus plantarum KL100 preparation for relieving anxiety and depression according to claim 8, characterized in that: In the step S1, the high-density fermentation conditions are as follows: pH 6.5, fermentation temperature 35°C, inoculation volume 20 mL / L, dissolved oxygen content 0-100 mL / L, stirring speed 50 r / min, tank pressure 0.03 MPa, and fermentation time 20-24 h; the mass ratio of the bacterial sludge to skim milk, protein peptide, and polysaccharide is 1:1:4:4; the freeze-drying operation is as follows: from 0 to 1 h, the temperature is -35°C; from 1 to 6 h, the temperature is increased to -20°C; from 6 to 11 h, the temperature is increased to -15°C; from 16 to 21 h, the temperature is increased to -15°C; from 21 to 26 h, the temperature is increased to -10°C; from 26 to 31 h, the temperature is increased to 0°C; from 31 to 41 h, the temperature is increased to 10°C; from 41 to 58 h, the temperature is increased to 20°C; from 58 to 58.5 h, the temperature is maintained at 20°C; from 58.5 to 60 ... h, the temperature was raised to 25°C, and from 60.5 to 62.5 h, the temperature was maintained at 25°C; the freeze-drying vacuum degree was 0.22 mbar.

10. The method for preparing a highly active Lactobacillus plantarum KL100 preparation for relieving anxiety and depression according to claim 8, characterized in that: In the step S2, the mass ratio of the Lactobacillus plantarum KL100 embedding composition, milk powder, fruit powder, protein peptide and composite vitamin and mineral is 20-30:30-60:10-50:10-20:1-2.

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