Probiotic composition for relieving anxiety and application

Abstract

The invention relates to the technical field of microorganisms, in particular to a probiotic composition for relieving anxiety and application, and the probiotic composition comprises lactobacillus rhamnosus LGD-0817, lactobacillus rhamnosus LDL-1708, lactobacillus plantarum LEP-1208 and lactobacillus plantarum MCA-3066. The probiotic composition is used for fermenting the traditional Chinese medicine composition, the obtained traditional Chinese medicine fermentation composition contains probiotics and traditional Chinese medicine fermentation liquor, the probiotics and the traditional Chinese medicine fermentation liquor can effectively achieve a synergistic effect, and the medicine effect of improving and treating depression is achieved; the traditional Chinese medicine fermentation composition plays a role through a plurality of ways, on one hand, the CRF content of hypothalamus and the ACTH content secreted by pituitary are reduced, so that the level of serum corticosterone is regulated and controlled, and the anti-depression and anti-anxiety effects are achieved; on the other hand, the 5-HT level of the central nervous system can be increased, the anti-depression effect is achieved, and it is prompted that the compound has the prospect of developing new anti-depression and anti-anxiety drugs and has clinical practical value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a probiotic composition for relieving anxiety and its application. Background Art

[0002] Benzodiazepines are currently the most widely used anti-anxiety medications in clinical practice, with common examples including diazepam, alprazolam, lorazepam, and oxazepam. However, some patients refuse to use these medications due to fear of drug tolerance and side effects, limiting their clinical application. For example, common side effects of diazepam include drowsiness, dizziness, and fatigue; high doses may cause ataxia and tremors. It can also cause blurred vision, affecting driving and fine motor skills. Due to its long half-life, patients may experience a hangover effect the day after taking the medication, manifesting as persistent weakness and fatigue. Long-term use may lead to drug dependence and addiction, and cause cognitive impairment and altered consciousness. Furthermore, diazepam may cause cardiovascular abnormalities (such as decreased blood pressure), respiratory depression (especially in the elderly or patients with respiratory insufficiency), as well as side effects such as headache, rash, leukopenia, and liver and kidney damage.

[0003] Compared with Western medicine, the active ingredients of traditional Chinese medicine (TCM) can exert unique therapeutic effects, characterized by significant efficacy and fewer side effects. TCM primarily works through relevant signaling pathways in the intervention of anxiety disorders, employing methods such as the use of active ingredients and compound preparations of TCM. However, its efficacy still needs further improvement.

[0004] Fermented traditional Chinese medicine (TCM) products have achieved five revolutionary breakthroughs and accomplishments: enhanced efficacy, improved safety, better patient compliance, improved microecological environment and enzyme balance in the human body, and reduced dosage of medicinal materials. Fermented TCM involves using one or more strains of selected probiotics from a carefully selected intestinal flora as inoculum extracts of natural medicines (TCM). Utilizing microecological and biomimetic methods, through biological grafting, the in vitro environment of the human gut and the digestive and decomposition process of TCM components are simulated. This process biologically transforms the extracted effective components of TCM, converting large molecules into small molecules that can be directly absorbed by the human gut, making TCM a new type of drug with rapid absorption and quantitative therapeutic effects. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a probiotic composition for relieving anxiety, and studies its bioactivity and medicinal uses.

[0006] In a first aspect, the present invention provides a probiotic composition for relieving anxiety, comprising Lactobacillus rhamnosus LGD-0817, Lactobacillus rhamnosus LDL-1708, Lactobacillus plantarum LEP-1208 and Lactobacillus plantarum MCA-3066; The preservation number of Lactobacillus rhamnosus LGD-0817 is CGMCC No. 23632; The Lactobacillus rhamnosus LDL-1708 has the accession number CGMCC No. 23960; The preservation number of the Lactobacillus plantarum LEP-1208 is CGMCC No. 24012; The preservation number of the Lactobacillus plantarum MCA-3066 is CGMCC No. 24489.

[0007] In this invention, the Lactobacillus rhamnosus LGD-0817 is classified and named as Lactobacillus rhamnosus. The depository institution is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 23632, and the deposit date is October 20, 2021. The Lactobacillus rhamnosus LDL-1708, classified and named Lactobacillus rhamnosus, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 23960, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on November 24, 2021. The Lactobacillus plantarum LEP-1208, classified and named Lactobacillus Plantarum, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24012 and deposit date of December 1, 2021. The Lactobacillus plantarum MCA-3066 is classified and named Lactobacillus Plantarum. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 24489 and deposit date of March 9, 2022.

[0008] Furthermore, the total live bacteria count in the probiotic composition is 10e. 3 -10e 12 CFU / mL, or 10e 3 -10e 9 CFU / mL.

[0009] Secondly, the present invention provides the use of probiotic compositions in the preparation of medicaments for relieving anxiety or depression.

[0010] Furthermore, the probiotic composition is formulated into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0011] Furthermore, the excipients include one or more of solvents, disintegrants, flavoring agents, preservatives, colorants, binders, lubricants, diluents, and drug carriers.

[0012] Furthermore, the dosage form is an oral dosage form or an injectable dosage form, and may also be a controlled-release or sustained-release dosage form known in the modern pharmaceutical industry.

[0013] Furthermore, a probiotic composition is added to the Chinese herbal extract for fermentation to obtain a Chinese herbal ferment that can relieve anxiety or depression. The Chinese herbal extract is obtained by extracting the Chinese herbal composition, which consists of jujube seed, longan pulp, lily bulb, poria cocos, and lotus seed.

[0014] Further, by mass parts, the traditional Chinese medicine composition consists of the following raw materials: 10-30 parts of jujube seed, 5-25 parts of longan pulp, 10-30 parts of lily bulb, 15-35 parts of poria cocos, and 10-30 parts of lotus seed.

[0015] Thirdly, the present invention provides a traditional Chinese medicine fermentation composition, which is obtained by fermenting a traditional Chinese medicine extract with a probiotic composition. The probiotic composition consists of the following probiotic strains: Lactobacillus rhamnosus LGD-0817 with accession number CGMCC No23632, Lactobacillus rhamnosus LDL-1708 with accession number CGMCC No23960, Lactobacillus plantarum LEP-1208 with accession number CGMCC No24012, and Lactobacillus plantarum MCA-3066 with accession number CGMCC No24489. The traditional Chinese medicine extract is obtained by extracting the traditional Chinese medicine composition, which consists of the following raw materials: 10-30 parts of jujube seed, 5-25 parts of longan pulp, 10-30 parts of lily bulb, 15-35 parts of Poria cocos, and 10-30 parts of lotus seed.

[0016] In this instruction manual, the "parts by weight" for each drug component indicates the dosage ratio between the components, not the actual unit of mass. Depending on the actual situation, a unit of weight can be any mass, such as 1 part by weight can be 1g, 500g, or 1kg, or even 15g, 30g, etc.

[0017] Fourthly, the present invention provides a method for preparing a traditional Chinese medicine fermentation composition, comprising the following steps: S1. Crush and disperse each Chinese herbal raw material in water, enzymatically hydrolyze it, and then heat to extract the aqueous extract. S2. Add the probiotic composition to the initial fermentation material and carry out a first fermentation for 24-48 hours until the live bacteria count reaches 10e. 3 -10e9 ; S3. After fermentation is complete, add the secondary fermentation material to the primary fermentation broth, then add the extracts of various Chinese herbal medicines from S1, and ferment for 48-72 hours until the viable cell count reaches 10e. 3 -10e 12 ; S4. After the secondary fermentation is completed, the fermentation liquid is sterilized at high temperature to obtain the traditional Chinese medicine fermentation composition.

[0018] Furthermore, in S1, the mass ratio of the Chinese herbal medicine raw material to water is 1:(5-15). Further, the traditional Chinese medicine composition in S1 comprises the following raw materials: 10-30 parts of jujube seed, 5-25 parts of longan pulp, 10-30 parts of lily bulb, 15-35 parts of poria cocos, and 10-30 parts of lotus seed; and / or, The probiotic composition in S2 consists of the following probiotic strains: Lactobacillus rhamnosus LGD-0817 with accession number CGMCC No23632, Lactobacillus rhamnosus LDL-1708 with accession number CGMCC No23960, Lactobacillus plantarum LEP-1208 with accession number CGMCC No24012, and Lactobacillus plantarum MCA-3066 with accession number CGMCC No24489; Furthermore, in S2, the preliminary fermentation material includes wort, oat liquor, fructooligosaccharides, and inulin; Furthermore, in step S3, the secondary fermentation material is blueberry pulp and banana puree, and the amount added is 5-10% (w / v) of the primary fermentation liquid; the total amount of the traditional Chinese medicine extract added is 10-20% (v / v) of the primary fermentation liquid. Fifthly, the present invention provides the application of the above-mentioned fermented Chinese medicine composition and the fermented Chinese medicine composition prepared by the above-mentioned preparation method in the preparation of drugs for relieving anxiety or depression.

[0019] Furthermore, the drug is either a gastrointestinal or non-gastrointestinal dosage form.

[0020] Specifically, the non-gastrointestinal route of administration is selected from injection, inhalation, skin, mucosal, or cavity administration.

[0021] Non-gastrointestinal drug delivery preparations are selected from injections, sprays, aerosols, patches, etc.

[0022] Specifically, the gastrointestinal administration formulation is selected from tablets, capsules, powders, granules, pills, solutions, emulsions, or syrups.

[0023] Furthermore, the dosage form is an oral dosage form or an injectable dosage form, and may also be a controlled-release or sustained-release dosage form known in the modern pharmaceutical industry.

[0024] Furthermore, the dosage form uses a traditional Chinese medicine fermentation composition as the active ingredient and is made with pharmaceutically acceptable excipients.

[0025] Those skilled in the art will fully understand that excipients (e.g., pharmaceutical excipients) include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0026] The diluent can be one or more of mannitol, sucrose, lactose, sorbitol, xylitol, polyethylene glycol, propylene glycol, vegetable oil, and mineral oil; the disintegrant can be one or more of croscarmellose sodium, colloidal silica, and citric acid; the binder can be one or more of starch paste, ethanol, water, and povidone alcohol solution; the preservative can be one or more of ethylparaben, propylparaben, sorbic acid, potassium sorbate, calcium propionate, sodium dehydroacetate, sodium diacetate, and sodium lactate; and the antioxidant can be ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, butylated hydroxytoluene, glycine, inositol, and other antioxidants. The flavoring agent may be one or more of the following: citric acid, sodium ascorbate, lecithin, malic acid, hydroquinone, citric acid, succinic acid, and sodium metabisulfite; the flavoring agent may be one or more of the following: aspartame, sucrose, xylitol, steviol glycosides, cyclamate, sorbitol, cocoa, pure vanilla, vanillin, ethyl vanillin, chocolate, malt, and mint; the suspending agent may be one or more of the following: xanthan gum, polyvinylpyrrolidone, sodium alginate, aluminum stearate, and hydrogenated vegetable oil; the emulsifier may be one or more of the following: alkyl sulfate, soap, dodecylbenzene sulfonate, lactate, sulfosuccinate, monoglyceride sulfonate, phosphate ester, siloxane, and taurine.

[0027] The dosage of this invention depends on many factors, such as the sex, age, weight, and individual response of the patient or animal, the route of administration, and the frequency of administration. The dosage can be administered as a single dose or in several doses, such as two, three, or four doses. The dosage level must be selected based on the specific route of administration, the severity of the condition being treated, and the patient's condition and medical history. However, it is the practice in the art to start with a dosage below the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0028] However, it should be recognized that the total daily dosage of this invention must be determined by the attending physician within the bounds of reliable medical judgment. For any specific patient, the specific effective therapeutic dose level must be determined based on a variety of factors, including the disorder being treated and its severity; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate; the duration of treatment; any drugs used in combination or concurrently; and similar factors known in the medical field. For example, it is practiced in the art to start with a dose below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0029] Generally speaking, the effective ingredient of the present invention can be calculated for use in mammals, especially humans, at a dose between 1 and 1000 mg / kg body weight / day, for example, between 1 and 500 mg / kg body weight / day, for example, between 50 and 500 mg / kg body weight / day, or 100 and 500 mg / kg, or 150 and 500 mg / kg, or 200 and 500 mg / kg, or 250 and 500 mg / kg.

[0030] Compared with the prior art, the present invention has the following beneficial effects: (1) The probiotic composition provided by the present invention can be used to ferment Chinese medicine liquid. During the fermentation process, the probiotic species have a good synergistic effect, which can effectively improve the fermentation effect of the probiotic composition on Chinese medicinal materials and improve the efficacy of Chinese medicinal materials.

[0031] (2) The traditional Chinese medicine fermentation composition obtained by the present invention contains probiotics and traditional Chinese medicine fermentation liquid. The two can work synergistically to promote the efficacy of traditional Chinese medicine fermentation products in improving and treating depression. (3) Through animal experiments and clinical verification, the fermented Chinese medicine composition obtained by the present invention exerts its effects in the following two ways: on the one hand, it regulates the level of serum corticosterone by downregulating the CRF content of the hypothalamus and the ACTH content secreted by the pituitary gland, thereby exerting antidepressant and anti-anxiety effects; on the other hand, it can increase the level of 5-HT in the central nervous system and exert antidepressant effects, suggesting that it has the prospect of developing new antidepressant and anti-anxiety drugs and has clinical practical value. Attached Figure Description

[0032] Figure 1 This invention relates to the effect of the anxiety-like phenotype. Among them, 1a is the experimental result diagram of forced swimming; 1b is the experimental result diagram of tail suspension; 1c is the experimental result diagram of open field; 1d is the experimental result diagram of elevated cross maze; * indicates that P≤0.05 compared with the model group; △ indicates that P≤0.05 compared with Example 6; △* indicates that P≤0.05 compared with the model group and Example 6.

[0033] Figure 2 This invention relates to the effect of HPA axis hypertrophy; Among them, 2a is a graph showing changes in hypothalamic CRF levels; 2b is a graph showing changes in serum basal ACTH levels. 2c shows the changes in serum basal corticosterone levels; * indicates P≤0.05 compared to the model group; △ indicates P≤0.05 compared to Example 1; △* indicates P≤0.05 compared to the model group and Example 6.

[0034] Figure 3 This invention relates to the effect of 5-HT in brain tissue; Wherein, 3a represents the effect of 5-HT in the prefrontal cortex; 3b represents the effect of 5-HT in the brainstem; * indicates that compared with the model group, P≤0.05; △ indicates that compared with Example 6, P≤0.05; △* indicates that compared with the model group and Example 6, P≤0.05; Figure 4 This is a flowchart illustrating the preparation process of the traditional Chinese medicine fermentation composition of this invention. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the test materials used in the following embodiments were purchased from conventional biochemical reagent stores. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0036] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0037] The "microbiota-gut-brain axis" theory primarily relies on the vagus nerve, immune system, and neuroendocrine system to influence the interaction between the gut and brain. This invention, based on the "microbiota-gut-brain axis" theory, aims to improve disease symptoms by modulating the gut microbiota to influence the function of the central nervous system.

[0038] Probiotics and certain traditional Chinese medicines are beneficial to the human gut microbiota, intestinal barrier, and immune system, exhibiting synergistic effects in many aspects.

[0039] Based on the present invention, Lactobacillus rhamnosus LGD-0817, Lactobacillus rhamnosus LDL-1708, Lactobacillus plantarum LEP-1208 and Lactobacillus plantarum MCA-3066 are selected and compounded to form a probiotic composition.

[0040] In some specific embodiments, one or more of the following can be added for compounding: Bifidobacterium longum, Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacillus fermentum, Bacillus coagulans, Kluyveromyces marxi, Lactobacillus paracasei, Lactobacillus casei, and Lactobacillus plantarum.

[0041] Based on this invention, a traditional Chinese medicine composition is made by combining jujube seed, longan pulp, lily bulb, poria cocos, and lotus seed, which are both food and medicine.

[0042] In some specific embodiments, one or more of the following ingredients may be added for compounding: Angelica sinensis, Atractylodes macrocephala, Gastrodia elata, Astragalus membranaceus, Lycium barbarum, Siraitia grosvenorii, Citrus medica, and Platycladus orientalis.

[0043] The probiotic composition biologically transforms the effective components of traditional Chinese medicine, converting the macromolecules of traditional Chinese medicine into small molecules that can be directly absorbed by the human intestine through microbial transformation, thereby improving the therapeutic effect, expanding the treatment scope, altering or decomposing toxic components, reducing adverse reactions, and increasing the extraction rate of effective components.

[0044] Based on the present invention, the total live bacteria count of the probiotic composition is 10e. 3 -10e 12 CFU / mL or 10e 3 -10e 12 CFU / g, for example, 1×10e 3 CFU / mL (CFU / g), 2×10e 4 CFU / mL (CFU / g), 3×10e 5 CFU / mL (CFU / g), 4×10e 6 CFU / mL (CFU / g), 5×10e 7 CFU / mL (CFU / g), 6×10e 8 CFU / mL (CFU / g), 7×10e 9 CFU / mL (CFU / g), 8×10e 10 CFU / mL (CFU / g), 9×10e 11 CFU / mL (CFU / g), 1×10e 12CFU / mL (CFU / g), etc. Other specific point values ​​within this range can be selected, which will not be elaborated here.

[0045] Please see Figure 4 The traditional Chinese medicine fermentation composition is prepared by the following steps: S1. Crush and disperse each Chinese herbal raw material in water, enzymatically hydrolyze it, and then heat to extract the aqueous extract. S2. Add the probiotic composition to the initial fermentation material and carry out a first fermentation for 24-48 hours to obtain a live bacteria count of 10e. 3 -10e 9 The primary fermentation broth; S3. After fermentation is complete, add the secondary fermentation material to the primary fermentation broth, then add the extracts of various Chinese herbal medicines from S1, and ferment for 24-48 hours to obtain a bacterial count in the range of 10e. 3 -10e 12 The secondary fermentation broth; S4. After the secondary fermentation is completed, the fermentation liquid can be centrifuged or not, and then sterilized at high temperature to obtain the traditional Chinese medicine fermentation composition.

[0046] The synergistic effect of two fermentations significantly improves the product's quality and functionality. The first fermentation, through high-density cultivation, creates favorable conditions for the growth of probiotics and the generation of their basic metabolites. The second fermentation adds fruit pulp and medicinal / edible extracts to the first fermentation broth, further enriching the product's flavor and functional components. This staged fermentation not only achieves efficient resource utilization and reduces production costs but also ensures the stability and continuity of the bacterial strain. It also avoids the risk of contamination caused by excessively long fermentation times and reduces the inhibitory effect of medicinal / edible components (such as phenols and tannins) on probiotics.

[0047] In some specific embodiments, the mass ratio of Chinese herbal raw materials to water is 1:(5-15), for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0048] In some specific embodiments, the fermentation time is 24-48h, for example, 24h, 26h, 32h, 36h, 42h, 48h, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0049] In some specific embodiments, enzymatic hydrolysis uses a complex enzyme of cellulase and pectinase with a mass ratio of 1:(0.1-10), such as 1:0.1, 1:0.5, 1:1, 1:3, 1:5, 1:7, 1:9, 1:10, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0050] In some specific embodiments, the mass of the compound enzyme is 1-5% of the volume of the water; the enzymatic hydrolysis temperature is 55-60℃, the enzymatic hydrolysis pH is 5.0-7.5, and the enzymatic hydrolysis time is 2-4h.

[0051] In some specific embodiments, the initial fermentation feed includes wort, oat liquor, fructooligosaccharides, and inulin.

[0052] In some specific embodiments, the secondary fermentation material is blueberry pulp and banana puree, and the amount added is 5-10% (w / v) of the primary fermentation liquid; for example, 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), 10% (w / v), and other specific values ​​within this range can be selected, which will not be elaborated here.

[0053] In some specific embodiments, the total amount of the herbal extract added is 10-20% (v / v) of the supernatant; for example, 10% (v / v), 12% (v / v), 14% (v / v), 16% (v / v), 18% (v / v), 20% (v / v), etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0054] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0055] Example 1 Probiotic Composition Take 3 parts by weight of *Lactobacillus rhamnosus* LGD-0817, 2 parts by weight of *Lactobacillus rhamnosus* LDL-1708, 2 parts by weight of *Lactobacillus plantarum* LEP-1208, and 2 parts by weight of *Lactobacillus plantarum* MCA-3066, and inoculate them into MRS liquid medium. Incubate at 37°C for 18-24 hours, repeating 2-3 times for activation. Inoculate the activated strains into fresh MRS liquid medium at a 2-3% inoculation rate and incubate at 37°C until the logarithmic growth phase, preparing a 10⁻⁶ concentration. 8 -10 9 CFU / mL bacterial suspension.

[0056] Example 2 Probiotic Composition Take 2 parts by weight of *Lactobacillus rhamnosus* LGD-0817, 2 parts by weight of *Lactobacillus rhamnosus* LDL-1708, 1 part by weight of *Lactobacillus plantarum* LEP-1208, and 1 part by weight of *Lactobacillus plantarum* MCA-3066, and inoculate them into MRS liquid medium. Incubate at 37°C for 18-24 hours, repeating 2-3 times for activation. Inoculate the activated strains into fresh MRS liquid medium at a 2-3% inoculation rate and incubate at 37°C until the logarithmic growth phase, preparing a 10⁻⁶ concentration. 8 -10 9 CFU / mL bacterial suspension.

[0057] Example 3 Probiotic Composition Take 1 part by weight of Lactobacillus rhamnosus LGD-0817, 1 part by weight of Lactobacillus rhamnosus LDL-1708, 1 part by weight of Lactobacillus plantarum LEP-1208, and 1 part by weight of Lactobacillus plantarum MCA-3066, and inoculate them into MRS liquid medium. Incubate at 37°C for 18-24 hours, repeating 2-3 times for activation. Inoculate the activated strains into fresh MRS liquid medium at a 2-3% inoculation rate and incubate at 37°C until the logarithmic growth phase, preparing a 10⁻⁶ concentration. 8 -10 9 CFU / mL bacterial suspension.

[0058] Example 4: Fermented Chinese Medicine Composition Preparation of Traditional Chinese Medicine Extracts: Weigh the following raw materials: 10 parts by weight of jujube seed, 25 parts by weight of longan pulp, 30 parts by weight of lily bulb, 20 parts by weight of poria cocos, and 10 parts by weight of lotus seed. Crush the jujube seed and disperse it in water at a ratio of 1:10 to 1:12. Add 1% by weight of a compound enzyme (a 1:1 ratio of cellulase and pectinase). Enzymatically hydrolyze the extract at 55℃ and pH 6.0 for 2 hours. After hydrolysis, extract in boiling water at 100℃ for 30-40 minutes. Filter and sterilize at 121℃ for 15 minutes to obtain the jujube seed aqueous extract. Crush the longan pulp and disperse it in water at a ratio of 1:8 to 1:10. Add 1% by weight of a compound enzyme (a 1:1 ratio of cellulase and pectinase). The compound enzyme (1:1 ratio of cellulase and pectinase) was used for enzymatic hydrolysis at 55℃ and pH 6.0 for 2 hours. After hydrolysis, it was extracted in boiling water at 100℃ for 20-30 minutes, filtered, and sterilized at 115℃ for 20 minutes to obtain a longan pulp aqueous extract. Lily bulbs were pulverized and dispersed in water at a ratio of 1:10 to 1:12, and 1% of the water mass of the compound enzyme (1:1 ratio of cellulase and pectinase) was added. Enzymatic hydrolysis was carried out at 55℃ and pH 6.0 for 2 hours. After hydrolysis, it was extracted in boiling water at 100℃ for 20-30 minutes, filtered, and sterilized at 121℃ for 15 minutes to obtain a lily bulb aqueous extract. Poria cocos was pulverized and dispersed in water at a ratio of 1:10... - Add 1% (by weight) of a compound enzyme (1:1 ratio of cellulase and pectinase) to a 1:15 volume of water. Enzymatically hydrolyze the mixture at 55°C and pH 6.0 for 2 hours. After hydrolysis, extract the extract in boiling water at 100°C for 30-40 minutes. Filter and sterilize at 121°C for 15 minutes to obtain a Poria cocos aqueous extract. Remove the core from lotus seeds, crush them, and disperse them in a 1:10-1:12 volume of water. Add 1% (by weight) of a compound enzyme (1:1 ratio of cellulase and pectinase) to a 1:12 volume of water. Enzymatically hydrolyze the mixture at 55°C and pH 6.0 for 2 hours. After hydrolysis, extract the extract in boiling water at 100°C for 30-40 minutes. Filter and sterilize at 121°C for 15 minutes to obtain a lotus seed aqueous extract. Mix the extracts for later use. Primary fermentation: The bacterial culture obtained in Example 1 was inoculated into the culture medium (wheat and oat liquor in a 1:1 mass ratio) at a rate of 5-8%, and 5-6% (w / v) of fructooligosaccharides (by volume of the culture medium) was added simultaneously. The fermentation temperature was controlled at 37°C for constant temperature culture, and the initial pH was controlled at 5.5-6.0. The pH was adjusted using a sodium bicarbonate solution (5-10%). The mixture was stirred at 150-200 rpm in an anaerobic environment for 24 hours. Secondary fermentation: After the primary fermentation is completed, add 5-10% (w / v) of blueberry pulp and banana puree to the primary fermentation liquid, and then add 10-20% (v / v) of the traditional Chinese medicine extract to the supernatant. Control the secondary fermentation temperature at 35℃, the initial pH value at 5.5-6.0, and stir at 100-150 rpm. In an anaerobic environment, the fermentation time is 48 hours. After the secondary fermentation is completed, sterilize the fermentation liquid at 95-100℃ for 10-15 minutes to obtain the traditional Chinese medicine fermentation composition.

[0059] Example 5 The preparation method is the same as in Example 4, except that the traditional Chinese medicine composition is composed of the following raw materials: 30 parts by weight of jujube seed, 5 parts by weight of longan pulp, 10 parts by weight of lily bulb, 15 parts by weight of poria cocos, and 20 parts by weight of lotus seed. The bacterial solution used in the first fermentation was the one from Example 2.

[0060] Example 6 The preparation method is the same as in Example 4, except that the traditional Chinese medicine composition is composed of the following raw materials: 20 parts by weight of jujube seed, 15 parts by weight of longan pulp, 20 parts by weight of lily bulb, 25 parts by weight of poria cocos, and 20 parts by weight of lotus seed. The bacterial solution used in the first fermentation was the one from Example 2.

[0061] Example 7 The preparation method is the same as in Example 4, except that the traditional Chinese medicine composition is composed of the following raw materials: 25 parts by weight of jujube seed, 10 parts by weight of longan pulp, 15 parts by weight of lily bulb, 35 parts by weight of poria cocos, and 30 parts by weight of lotus seed. The bacterial solution used in the first fermentation was the one from Example 3.

[0062] Comparative Example 1 The preparation method is the same as in Example 6, except that only one fermentation is performed and no Chinese herbal extract is added.

[0063] Comparative Example 2 The preparation method is the same as in Example 6, except that Lactobacillus rhamnosus LGD-0817 and Lactobacillus rhamnosus LDL-1708 are removed from the probiotic composition.

[0064] Comparative Example 3 The preparation method is the same as in Example 6, except that the raw materials of each Chinese medicine are not crushed, and are enzymatically hydrolyzed by adding hemicellulase and protease in a 1:1 ratio.

[0065] The efficacy of fermented Chinese medicine compositions was verified.

[0066] Experimental animals: C57BL / 6J male rats, 5 weeks old, weighing 180~220 g; Animal grouping and administration: Mice were randomly divided into four groups of eight: a healthy control group, an anxiety model group, Example 5, Example 6, Comparative group 1, Comparative group 2, and Comparative group 3. After one week of acclimatization (temperature 23±2℃, humidity 50±10%, 12-hour diurnal cycle), the other five groups (except the control group) underwent CUMS modeling for four weeks. Mice in the Example and Comparative groups received an administration dose of 20 mg / kg / day. The healthy control and anxiety model groups were administered an equal volume of saline via gavage as controls. Mice had free access to food and water during this period. After five weeks, behavioral experiments were conducted. After the behavioral experiments, the mice were fasted and deprived of water for 12 hours, then sacrificed and their tissues were collected. Whole blood was centrifuged for 15 minutes (3500 r / min), and the supernatant serum was collected. Tissues such as the prefrontal cortex and brainstem were collected on ice and promptly removed. Pre-cooled in liquid nitrogen and stored at -80℃ for later use.

[0067] Data were analyzed and plotted using Graphpad Prism 9.4.0 software. Results are expressed as mean ± standard error (mean ± SEM). The student's t-test was used for comparing data from two or more groups, and one-way ANOVA was also used for comparing data from two or more groups.

[0068] (1) Investigate the effect of drug administration on anxiety-like phenotype. Mice underwent forced swimming, tail suspension, elevated cross maze, open field, and elevated cross maze tests, respectively. The results are shown in Table 1 and 2. Figure 1 As shown.

[0069] Forced swimming (FST): Mice were subjected to forced swimming stimulation. During the experiment, mice were forced to swim in a confined space. They initially swam frantically in an attempt to escape, then remained in a floating, immobile state, with only their nostrils exposed for breathing. Their limbs occasionally paddled to prevent them from sinking. This state, known as the immobile state, essentially represents the animal giving up hope of escape. Each stimulation lasted 10 minutes from the time the mouse entered the water until it left the pool (6 minutes for adaptation, 4 minutes for sampling and measurement).

[0070] Figure 1 As can be seen, after treatment with the drug in the example group and the comparative group, the time of forced swimming stillness in each group of mice was significantly shorter than that in the model group, and the degree of behavioral despair was reduced. However, the example group could significantly shorten the time of forced swimming stillness in mice, indicating that the antidepressant effect of the present invention is significant. Tail Suspension Test (TST): The tail suspension test (TST) is a classic and rapid method for evaluating antidepressant drugs. Its principle is based on the fact that mice, after being suspended by their tails, attempt to escape but are unable to, thus giving up the struggle and entering a specific state of depressive immobility. During the experiment, the duration of immobility is recorded to reflect the depressive state. Antidepressants and stimulants can significantly shorten or alter this state. In the experiment, the mouse's tail is clamped and suspended. The suspended mouse struggles to overcome the abnormal position, but after a certain period of activity, it becomes intermittently immobile, showing a willingness to give up the adjustment. Each stimulus, from the time the mouse's tail is clamped and suspended to the time it is released and rests, lasts for 10 minutes (6 minutes for adaptation, 4 minutes for sampling and measurement).

[0071] Figure 1 b. As can be seen, after treatment with the drug in the example group and the comparative group, it was found that the drug administration could shorten the immobility time of the mouse tail suspension. However, the example group could significantly shorten the immobility time of the mouse tail suspension, indicating that the present invention can enhance the desire of mice with suspended tails to struggle and escape.

[0072] Open Field Test (OFT): This test assesses an animal's spontaneous activity and exploratory behavior in a novel opening environment, evaluating the degree of its interest. Before the experiment, mice acclimatize for 1 hour in a quiet room with soft lighting. The open field box consists of four 50cm-high white acrylic panels arranged in a 50cm x 50cm square. The base is also made of white acrylic. A camera connected to a computer tracking system is positioned directly above the box. Each mouse is placed in the center of the open field, and its behavior is recorded for 3 minutes. The following parameters are analyzed using the computer tracking system: total walking distance and number of times the animal stands up (the number of times both front paws leave the ground simultaneously). A decrease in total walking distance and number of times the animal stands up indicates a decrease in its interest in the novel environment.

[0073] Figure 1 c indicates that chronic stress in the model group reduced the autonomous exploration behavior of experimental mice in new environments, mainly reflected in a significant reduction in the time spent in the central area of ​​the open field (P<0.05). After treatment with the drug in the example group and the control group, the total walking distance of mice in each group was increased compared with that in the model group, but the example group could significantly increase the total walking distance of mice, indicating that the drug administration of the present invention can increase the interest of depressed model animals in novel environments.

[0074] The Elevated Cross Maze (EPM) utilizes the contradictory behavior of animals—their exploratory nature towards novel environments and their fear of open, suspended arms—to examine their anxiety levels. The EPM has one pair of open arms and one pair of closed arms. Rodents, due to their preference for darkness, tend to move within the closed arms, but out of curiosity and exploration, they also move within the open arms. When faced with novel stimuli, animals simultaneously experience the impulse to explore and the fear, creating a conflict between exploration and avoidance, thus generating anxiety. Anti-anxiety medications can significantly increase the frequency and duration of entry into the open arms.

[0075] like Figure 1 As shown in d, continuous stress significantly reduced the time mice spent in the open arm of the elevated cruciate maze (P<0.05). Both the example group and the comparative group showed that administration of the drug increased the time mice spent in the open arm, but the example group significantly increased the time OB mice spent entering the open arm. This indicates that the present invention can enhance the exploratory characteristics of depressed mice in novel environments and has a good effect on combating anxiety in animals.

[0076] Table 1

[0077] As shown in the table above, the compound probiotics of the present invention perform secondary fermentation on a specific food-medicine homology composition, and the two can work synergistically to promote the therapeutic effect on depression. Comparative Examples 1-3 differ from Examples 5-6 in the active fermentation components and fermentation methods; although they have some antidepressant effects, their therapeutic effect is inferior to that of the present invention.

[0078] (2) Investigate the effect of drug administration on HPA axis hyperactivity. Protein expression levels were determined using enzyme-linked immunosorbent assay (ELISA): mouse hypothalamic CRF, serum ACTH, and serum corticosterone were measured using an ELISA kit. The detailed experimental procedures were performed according to the corresponding instruction manuals. The results are shown in Table 2.

[0079] Table 2

[0080] The HPA axis is a major biological system mediating central nervous system stress, and existing research indicates that abnormal hyperactivity of the HPA axis is an important factor in the course of depression. Studies have shown that basal corticosterone levels in elderly patients with depression are significantly higher than in healthy individuals in the morning, afternoon, and evening. Results are as follows... Figure 2 As shown in Table 2, under chronic stress, the model group exhibited hyperexcitability of the HPA axis and elevated CRF in the hypothalamus. CRF directly acts on the pituitary gland, leading to an upregulation of ACTH secreted by the pituitary. Simultaneously, ACTH, through blood circulation, acts on the adrenal glands, resulting in excessive corticosterone secretion. The CRF and ACTH levels in the example group and the control group showed varying degrees of downregulation, with the example group showing a significantly greater downregulation of serum corticosterone levels compared to the control group, indicating a more pronounced antidepressant effect.

[0081] (3) Investigate the effects of drug administration on the synthesis and metabolism of 5-HT in the prefrontal cortex and brainstem. The level of 5-HT in animal brain tissue was determined using a high-performance liquid chromatography-fluorescence detection system. The determination conditions were as follows: Column: Shimadzu Inertsil ODS-3 (5 μm 4.6 mm × 250 mm). Mobile phase A was 0.1 mol / L NaAc (containing 0.1 mmol / L EDTA-2Na), adjusted to pH 5.1 with HAc. Mobile phase B was methanol. Both mobile phases A and B were filtered through a 0.45 μm microporous membrane and degassed by sonication for 30 min. The mobile phase ratio was A:B = 85:15, the flow rate was 1.0 mL / min, the excitation wavelength was 290 nm, the emission wavelength was 330 nm, and the injection volume was 20 μL. The results are shown in Table 3.

[0082] Table 3

[0083] 5-HT, as an important brain-gut peptide, plays a crucial role in the regulation of mental disorders. The classic theory of depression is that low levels of 5-HT in the brain are the cause. Figure 3 As shown in Table 3, the model group exhibited a significant decrease in 5-HT levels in the prefrontal cortex and brainstem under stress. Both the example and comparative groups were able to reverse these changes, and the example group significantly increased brain 5-HT levels compared to the comparative group, demonstrating a more pronounced antidepressant effect.

[0084] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. A probiotic composition for relieving anxiety, characterized in that, Including Lactobacillus rhamnosus LGD-0817, Lactobacillus rhamnosus LDL-1708, Lactobacillus plantarum LEP-1208 and Lactobacillus plantarum MCA-3066; The preservation number of Lactobacillus rhamnosus LGD-0817 is CGMCC No. 23632; The Lactobacillus rhamnosus LDL-1708 has the accession number CGMCC No. 23960; The preservation number of the Lactobacillus plantarum LEP-1208 is CGMCC No. 24012; The preservation number of the Lactobacillus plantarum MCA-3066 is CGMCC No. 24489.

2. The probiotic composition according to claim 1, characterized in that, The total live bacteria count of the probiotic composition is 10e. 3 -10e 12 CFU / mL, or 10e 3 -10e 9 CFU / mL.

3. The use of the probiotic composition according to claim 1 or 2 in the preparation of a medicament for relieving anxiety or depression.

4. The application according to claim 3, characterized in that, The extract is made by adding a probiotic composition to a traditional Chinese medicine extract and then fermenting it. The traditional Chinese medicine extract is obtained by extracting the traditional Chinese medicine composition, which consists of jujube seed, longan pulp, lily bulb, poria cocos, and lotus seed.

5. The application according to claim 4, characterized in that, The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 10-30 parts of jujube seed, 5-25 parts of longan pulp, 10-30 parts of lily bulb, 15-35 parts of poria cocos, and 10-30 parts of lotus seed.

6. A fermented traditional Chinese medicine composition, characterized in that, The herbal extract is prepared by fermenting a traditional Chinese medicine extract with a probiotic composition. The probiotic composition consists of the following probiotic strains: Lactobacillus rhamnosus LGD-0817 (CGMCC No. 23632), Lactobacillus rhamnosus LDL-1708 (CGMCC No. 23960), Lactobacillus plantarum LEP-1208 (CGMCC No. 24012), and Lactobacillus plantarum MCA-3066 (CGMCC No. 24489). The traditional Chinese medicine extract is prepared by extracting the herbal extract from the herbal extract. The herbal extract consists of the following raw materials: 10-30 parts of jujube seed, 5-25 parts of longan pulp, 10-30 parts of lily bulb, 15-35 parts of Poria cocos, and 10-30 parts of lotus seed.

7. A method for preparing a fermented composition of traditional Chinese medicine, characterized in that, Includes the following steps: S1. Crush and disperse each Chinese herbal raw material in water, enzymatically hydrolyze it, and then heat to extract the aqueous extract. S2. Add the probiotic composition to the initial fermentation material and carry out a first fermentation for 24-48 hours until the live bacteria count reaches 10e. 3 -10e 9 CFU / mL; S3. After fermentation is complete, add the secondary fermentation material to the primary fermentation broth, then add the extracts of various Chinese herbal medicines from S1, and ferment for 48-72 hours until the viable cell count reaches 10e. 3 -10e 12 CFU / mL; S4. After the secondary fermentation is completed, the mixture is centrifuged or not, and then sterilized at high temperature to obtain the fermented Chinese medicine composition.

8. The preparation method according to claim 7, characterized in that, The traditional Chinese medicine raw materials mentioned in S1 consist of the following ingredients: 10-30 parts of jujube seed, 5-25 parts of longan pulp, 10-30 parts of lily bulb, 15-35 parts of poria cocos, and 10-30 parts of lotus seed; and / or, The probiotic composition in S2 consists of the following probiotic strains: Lactobacillus rhamnosus LGD-0817 with accession number CGMCC No23632, Lactobacillus rhamnosus LDL-1708 with accession number CGMCC No23960, Lactobacillus plantarum LEP-1208 with accession number CGMCC No24012, and Lactobacillus plantarum MCA-3066 with accession number CGMCC No24489.

9. The use of the traditional Chinese medicine fermentation composition as described in claim 6 in the preparation of a drug for relieving anxiety or depression.

10. The application according to claim 9, wherein the drug is one of a gastrointestinal dosage form or a non-gastrointestinal dosage form.

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