Compound probiotic preparation capable of being used for emotion regulation and preparation method of compound probiotic preparation

Abstract

The invention discloses a compound probiotic preparation capable of being used for emotion regulation and a preparation method of the compound probiotic preparation. The compound probiotic preparation comprises 15-22% of bifidobacterium longum R01755; 18 to 25 percent of lactobacillus helveticus R00522; 12%-20% of lactobacillus plantarum 299v; 8%-15% of lactobacillus rhamnosus GG; 5 to 10 percent of bifidobacterium breve; 15-25% of a lily and rehmannia soup polysaccharide extract; 5-10% of an auxiliary protective agent; and the balance of a carrier substrate; wherein the auxiliary protective agent comprises sodium alginate, chitosan and glycerol. According to the present invention, the emotion is synergistically regulated through multiple mechanisms; the probiotic combination regulates the tryptophan metabolic pathway, and improves the levels of 5-HT and GABA in the brain; the lactobacillus plantarum 299v can enhance the intestinal barrier and reduce neuroinflammation caused by LPS (Lipopolysaccharide) entering blood; the expression of IL-10 is up-regulated by lactobacillus rhamnosus GG, and immune activation mediated by TNF-alpha is inhibited; the bifidobacterium breve efficiently produces butyric acid and activates an intestinal pheochromophilic cell 5-HT synthesis pathway.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a compound probiotic preparation that can be used for mood regulation and its preparation method. Background Technology

[0002] Mood disorders such as anxiety and depression have become significant public health issues. Traditional treatments, such as drug therapy, often involve side effects, dependence, or unstable efficacy. In recent years, the development of the gut-brain axis theory has revealed the close connection between gut microbiota and the central nervous system. Probiotics have demonstrated their potential to regulate mood by modulating gut microbiota, influencing neurotransmitter synthesis, and reducing inflammatory responses.

[0003] Currently, several probiotic products for mood regulation have appeared on the market, but they still have the following shortcomings:

[0004] Single or arbitrary strains: Most products use a single strain or a simple mixture, lacking systematic screening and ratio optimization. The synergistic effect between strains is unclear, which affects the overall regulatory effect.

[0005] Lack of precise delivery system: Ordinary formulations cannot achieve targeted colonization of probiotics in specific areas of the intestine (such as the terminal ileum and Peyer's patches), affecting their persistence and effectiveness;

[0006] There is insufficient research on the combination of traditional Chinese medicine extracts and probiotics: classic prescriptions such as Baihe Dihuang Decoction have the effects of calming the mind and improving intelligence, but the synergistic effect and mechanism of their extracts and probiotics have not been fully explored and applied.

[0007] Therefore, developing a compound probiotic preparation with strong strain synergy, high survival rate, multi-mechanism synergy, and precise delivery capability has significant scientific research value and application prospects. Summary of the Invention

[0008] This application provides a compound probiotic preparation for mood regulation and its preparation method, aiming to solve the problems of existing technologies with single or arbitrary strains and a lack of precise delivery systems.

[0009] Firstly, a compound probiotic preparation for mood regulation, comprising:

[0010] Bifidobacterium longum R0175 15-22%; Lactobacillus helveticus R0052 18-25%; Lactobacillus plantarum 299v 12-20%; Lactobacillus rhamnosus GG 8-15%; Bifidobacterium breve 5-10%; Lily and Rehmannia glutinosa decoction polysaccharide extract 15-25%; adjuvant protectant 5-10%;

[0011] And the remaining carrier matrix; wherein the auxiliary protective agent comprises sodium alginate, chitosan and glycerol.

[0012] Optionally, the lily and rehmannia polysaccharide extract is prepared by the following steps: lily and rehmannia slices are mixed in a mass ratio, water is added for extraction, the filtrate is concentrated and precipitated with alcohol, the precipitate is collected, purified by column chromatography, and then dried to obtain the extract.

[0013] Optionally, in the auxiliary protective agent, the mass ratio of sodium alginate, chitosan, and glycerol is 3:2:1.

[0014] Optionally, the carrier matrix comprises microcrystalline cellulose and spray-dried lactose, and the compound probiotic preparation is in tablet form, wherein the total number of live bacteria is not less than 1.0 × 10⁻⁶. 9 CFU / film.

[0015] Optionally, the alcohol precipitation step controls the final ethanol concentration to 65%-75% and allows it to stand at 4±0.5℃.

[0016] Secondly, a method for preparing a compound probiotic preparation that can be used for mood regulation includes the following steps:

[0017] S1: Bifidobacterium longum R0175, Lactobacillus helveticus R0052, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG and Bifidobacterium breve were cultured separately, the bacterial cells were collected, and the probiotic freeze-dried powder was prepared by freeze drying.

[0018] S2: Preparation of polysaccharide extract from Lily and Rehmannia Decoction;

[0019] S3: The probiotic freeze-dried powder obtained in S1 is mixed with the auxiliary protective agent solution and then spray-dried to prepare probiotic microcapsules;

[0020] S4: The probiotic microcapsules prepared in S3, the polysaccharide extract of Lilium brownii and Rehmannia glutinosa prepared in S2, and the carrier matrix are mixed, granulated, dried, and compressed to obtain the compound probiotic preparation.

[0021] Optionally, in step S1, the culture is carried out under anaerobic conditions, and the dissolved oxygen content of the fermentation broth is controlled to be below 0.5% saturation; the freeze drying includes primary drying and secondary drying.

[0022] Optionally, in step S3, the inlet air temperature of the spray dryer is 125-135℃, the outlet air temperature is 70-80℃, and the atomization pressure is 0.28-0.32 MPa.

[0023] Optionally, the polysaccharide extract of Lily and Rehmannia Decoction satisfies a polysaccharide content of ≥60.0%.

[0024] Compared with the prior art, this application has at least the following beneficial effects:

[0025] This application utilizes a scientifically formulated ratio of Bifidobacterium longum R0175, Lactobacillus helveticus R0052, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG, and Bifidobacterium breve to form a metabolic synergistic network. Experiments show that R0175 can increase the GABA production efficiency of R0052 by 37.2%, significantly enhancing the neurotransmitter synthesis capacity.

[0026] This application regulates mood through multiple mechanisms: the probiotic combination regulates the tryptophan metabolism pathway and increases the levels of 5-HT and GABA in the brain; Lactobacillus plantarum 299v enhances the intestinal barrier and reduces neuroinflammation caused by LPS entering the bloodstream; Lactobacillus rhamnosus GG upregulates IL-10 expression and inhibits TNF-α-mediated immune activation; and Bifidobacterium breve efficiently produces butyrate, activating the 5-HT synthesis pathway in enterochromaffin cells.

[0027] This application utilizes the polysaccharide extract of Lilium and Rehmannia glutinosa as a targeted prebiotic, which not only promotes the proliferation of probiotics, but its metabolites can also affect the function of the central nervous system through the gut-brain axis signaling pathway, upregulate BDNF expression in the hippocampus, and enhance the effect of mood regulation.

[0028] This application uses a sodium alginate-chitosan-glycerol composite protectant for spray drying microencapsulation, which enables the survival rate of probiotics in artificial gastric fluid to reach 86.2% and achieves targeted delivery to the terminal ileum, significantly improving the colonization rate in the Peyer's patches area. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating a method for preparing a compound probiotic preparation for mood regulation, provided as an embodiment of this application. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0031] This application provides a compound probiotic preparation for mood regulation, comprising:

[0032] The compound probiotic preparation is precisely composed of the following components by weight percentage: *Bifidobacterium longum* R0175 18.5%, *Lactobacillus helveticus* R005 222.0%, *Lactobacillus plantarum* 299v 16.5%, *Lactobacillus rhamnosus* GG 12.0%, *Bifidobacterium breve* 7.5%, *Lily and Rehmannia glutinosa* polysaccharide extract 20.0%, excipient protectant 7.5%, and the balance being a carrier matrix. The sources and technical details of each component are as follows:

[0033] The core probiotic blend includes:

[0034] Bifidobacterium longum R0175 and Lactobacillus helveticus R0052 constitute a core pair for mood regulation, and their metabolic synergy was verified through in vitro co-culture: the metabolite of R0175 can increase the efficiency of γ-aminobutyric acid (GABA) production by R0052 by 37.2% (HPLC determination, n=6). This combination was demonstrated in a randomized double-blind clinical trial (NCT04876321) to significantly reduce cortisol levels (P<0.01) and regulate HPA axis overactivation.

[0035] Lactobacillus plantarum 299v (DSM 9843) exhibits high adhesion (≥18% adhesion to Caco-2 cells), strengthening intestinal barrier integrity and reducing neuroinflammation induced by LPS entry into the bloodstream; Lactobacillus rhamnosus GG (ATCC 53103) inhibits TNF-α-mediated neuroimmune activation by upregulating intestinal IL-10 expression; Bifidobacterium breve (JCM 11944) efficiently produces butyrate (≥12.5 mmol / L in 48h fermentation broth), activating the 5-HT synthesis pathway in enterochromaffin cells and providing a material basis for gut-brain signal transduction;

[0036] Note: The viable count of each strain's freeze-dried powder is ≥2.0×10⁻⁶. 11 CFU / g, bacterial species identification was performed using 16S rRNA gene sequencing (primers 27F / 1492R), and purity was verified using the API 50 CHL system;

[0037] The polysaccharide extract of Lilium brownii var. viridulum from Gansu Province and Rehmannia glutinosa from Jiaozuo, Henan Province, was obtained by mixing the two herbs at a mass ratio of 5:5. A three-factor, three-level orthogonal experiment (L9(3)) was conducted to determine the polysaccharide content. 4Optimization: Add 12 times the amount of water, dynamically extract at 105℃ for 90 min (pressure 0.08MPa), concentrate the filtrate to d=1.08 (60℃), control the ethanol flow rate at 15mL / min during alcohol precipitation, and maintain a settling temperature of 4±0.5℃. The resulting precipitate was subjected to DEAE-52 cellulose column chromatography (elution gradient: 0→0.3mol / L NaCl), and the main peak fraction was collected and spray-dried (air intake 160℃) to obtain a pale yellow powder. The polysaccharide content was determined to be 64.3±1.8% (n=3) by the phenol-sulfuric acid method. HPLC-ELSD analysis showed the presence of characteristic peaks of lily polysaccharides (retention time 18.7 min) and peaks of rehmannia glutinosa derivatives (24.3 min). This extract promoted the proliferation rate (OD) of R0175. 600 The efficacy reached 215%, and the in vitro blood-brain barrier model (hCMEC / D3 cells) confirmed that its metabolites could penetrate the barrier and upregulate BDNF mRNA expression in the hippocampus (qPCR, P<0.05).

[0038] The auxiliary protective agents were sodium alginate (low viscosity type, Mw≈50kDa, Qingdao Mingyue Seaweed Group), chitosan (degree of deacetylation ≥90%, Zhejiang Jinke Biotechnology), and glycerin (pharmaceutical grade) mixed in a 3:2:1 ratio. The particle size D of the microcapsules after spray drying was determined by a laser particle size analyzer. 50 =18.7±2.3μm, Zeta potential -28.5mV, encapsulation efficiency 89.4% (plate count method). In vitro simulated digestion experiments (referring to INFUGEST 2.0 protocol) showed that the survival rate after artificial gastric fluid (pH2.5, 2h) was 86.2%, and the cumulative release rate after artificial intestinal fluid (pH6.8, 4h) reached 92.7%, which was significantly better than the monolayer encapsulation system (P<0.01).

[0039] The carrier matrix was a 1:1 mixture of microcrystalline cellulose (PH-102, JRS Pharma, Germany) and spray-dried lactose (DCL-11, DFE Pharma), with a specific surface area of ​​1.85 m² / g determined by the BET method, ensuring uniform mixing (RSD < 3%). This combination maintained a formulation moisture content ≤ 4.5% and a stable disintegration time of 12-16 min in an accelerated testing at 40℃ / 75%RH (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0921).

[0040] This formulation achieves mood regulation through a triple pathway: ① The probiotic combination regulates intestinal tryptophan metabolism (Kynurenine / 5-HT pathway), increasing 5-HT and GABA levels in the brain (LC-MS / MS verification); ② Lily and Rehmannia polysaccharides act as targeted prebiotics, selectively enriching butyrate-producing bacteria and uploading signals via GLP-1 receptors in enteric neurons; ③ The microcapsule structure ensures targeted delivery of live bacteria to the terminal ileum (SITZMARK® marker verification), establishing colonization dominance in the Peyer's patches region. Animal experiments showed that, according to Shapley value analysis, the contribution of each component to the elevated cruciate maze OE% index under this ratio was: R0175 / R0052 accounted for 52.3%, polysaccharide extract accounted for 28.7%, and the remaining components accounted for 19.0%, confirming that it is not a simple additive effect.

[0041] In one embodiment, a method for preparing a compound probiotic preparation that can be used for mood regulation is provided, comprising the following steps:

[0042] S1: Standardized preparation of probiotic powders: Glycerol storage tubes (-80℃) containing *Bifidobacterium longum* R0175 (ATCC BAA-999), *Lactobacillus helveticus* R0052 (CNCM I-1728), *Lactobacillus plantarum* 299v (DSM 9843), *Lactobacillus rhamnosus* GG (ATCC 53103), and *Bifidobacterium breve* JCM 11944 were streaked onto modified MRS agar plates (supplemented with 0.05% L-cysteine ​​hydrochloride, 0.1% Tween 80, pH 6.2±0.1) and anaerobic incubated at 37℃ for 48 h (anaerobic workstation: 85% N2, 10% H2, 5% N2). CO2 (Whitley DG250). A single colony was picked and inoculated into 50 mL of seed culture (37℃, static incubation for 16 h), then transferred to a 5L fermenter (BioFlo 320, Eppendorf) at a 3% (v / v) inoculation rate. Control parameters: temperature 37±0.5℃, stirring speed 100 rpm, pressure 0.03 MPa, pH 6.0 (maintained by automatic addition of 2 mol / L NaOH). Cultured until OD... 600The growth rate reached 1.8-2.0 (approximately 18-22 hours). The fermentation broth was centrifuged using a disc centrifuge (Westfalia Separator, 8000 rpm, 15 min, 4℃) to collect the bacterial sludge. The sludge was washed three times with pre-cooled sterile physiological saline (0.85% NaCl, 4℃) (centrifugation conditions were the same each time) to obtain wet bacterial cells. Skim milk powder (Inner Mongolia Yili Industrial Group, protein content ≥34%) was added at 10% (w / w) of the wet bacterial cell mass. After mixing, the mixture was dispensed into freeze-drying trays (liquid layer thickness ≤1.5 cm), pre-frozen at -80℃ for 2 hours (cooling rate -1℃ / min), and then transferred to a freeze dryer for: first drying (-40℃, 20Pa, 12 hours), and second drying (25℃, 10Pa, 36 hours). The obtained lyophilized powder was aseptically pulverized (using a universal pulverizer, 304 stainless steel sieve, 80 mesh), vacuum-sealed in aluminum foil bags (residual oxygen ≤1%), and stored at -20℃ in the dark. The viable count of each strain of lyophilized powder was determined by plate counting (MRS agar, anaerobic incubation at 37℃ for 72 h), and all samples showed a viable count ≥2.5 × 10⁻⁶. 11 CFU / g, moisture content ≤3.0% (Karl Fischer method, GB / T 13531.1-2008).

[0043] (Note: Key control points during fermentation: Dissolved oxygen level should be maintained at <0.5% saturation; the strain should not be passaged more than 5 times to prevent degeneration; the freeze-drying protectant can be replaced with a trehalose / monosodium glutamate complex system (mass ratio 4:1).)

[0044] S2: Refining of the polysaccharide extract from Lilium brownii var. viridulum (Gansu Lanzhou) and Rehmannia glutinosa (Henan Jiaozuo) slices (500g each) conforming to the 2020 edition of the Chinese Pharmacopoeia were prepared. After identification (microscopic characteristics and TLC fingerprint comparison), the samples were pulverized to 20 mesh (0.85 mm pore size) using a pulverizer (FW-100, Tianjin Tester) and mixed evenly. The mixture was placed in a multi-functional extraction vessel (TQ-2.0, Shanghai Minjie), and 25 times (w / v) of purified water (conductivity ≤15μS / cm) was added. The mixture was steam-heated in a jacket to 100±2℃ and dynamically extracted for 90 min (stirring speed 30 rpm). The mixture was then filtered through a 200-mesh (74μm pore size) cotton filter at normal pressure. The residue was extracted with 20 times the amount of water using the same method for 60 min, and the filtrates were combined. The filtrate was concentrated under reduced pressure (vacuum degree -0.085 MPa) at 60±2℃ using a double-effect concentrator (ZN-100, Wenzhou Jinbang) to a relative density of 1.08 (60℃, measured by a DHZ-3 hydrometer). The concentrate was pumped into an alcohol precipitation tank (316L stainless steel), and 95% ethanol (pharmaceutical grade, Shandong Xinhua Pharmaceutical) was added dropwise at a flow rate of 15 mL / min with stirring (60 rpm), and the alcohol concentration was monitored in real time (ALM-155 alcohol meter) until a final concentration of 70% (v / v) was reached; the solution was then allowed to stand in a cold storage at 4±0.5℃ for 24 hours. The precipitate was collected by centrifugation at 4000 rpm for 20 min using a tubular centrifuge (GQ105, Shanghai Pudong), washed twice with 70% ethanol (each time the volume of precipitate was 3 times), and dried in a vacuum drying oven (DZF-6050, Shanghai Yiheng) at 50±2℃ for 12 h (vacuum degree -0.09 MPa). The powder was then pulverized through an 80-mesh sieve (pore size 0.18 mm) using a universal pulverizer to obtain a pale yellow powder. The polysaccharide content was determined according to the phenol-sulfuric acid method in the "Technical Specifications for Inspection and Evaluation of Health Foods" (2003 edition): 10.0 mg of sample was accurately weighed, and distilled water was added to a final volume of 10 mL. 1.0 mL of this solution was reacted with 1.0 mL of 5% phenol solution and 5.0 mL of concentrated sulfuric acid. The absorbance was measured at 490 nm. Using glucose as a standard, the polysaccharide content of this product was calculated to be 63.8±1.2% (n=3). HPLC-ELSD chromatograms (Agilent 1260, XBridge BEH Amide 2.5 μm 4.6×150 mm; mobile phase: acetonitrile-water gradient elution) showed characteristic peak retention times of 18.7 min (lily polysaccharide) and 24.3 min (rehmannia glutinosa derivative).

[0045] (Note: Key parameters for the alcohol precipitation process: the ethanol drop rate affects the uniformity of the precipitate particles; the ethanol in the supernatant after centrifugation can be recovered and reused; confirmation of the polysaccharide structure requires HPGPC-MALLS coupled analysis (data stored in the applicant's laboratory archives, number POLY-2023-087).)

[0046] S3: Targeted encapsulation of probiotic microcapsules. The above-mentioned freeze-dried bacterial powders were accurately weighed according to the following mass ratios (Bifidobacterium longum R0175: 25.0%, Lactobacillus helveticus R0052: 25.0%, Lactobacillus plantarum 299v: 20.0%, Lactobacillus rhamnosus GG: 15.0%, Bifidobacterium breve: 15.0%), and mixed in a three-dimensional motion mixer (SYH-100, Changzhou Yibu) for 20 min (15 rpm). Separately, low-viscosity sodium alginate (Mw≈50 kDa, Qingdao Mingyue Seaweed Group), chitosan (degree of deacetylation ≥90%, Zhejiang Jinke Biotechnology), and pharmaceutical-grade glycerin (Sinopharm Group) were mixed in a 3:2:1 ratio, and purified water was added to prepare a 15% solution. The solution was stirred in a 40℃ water bath until completely dissolved. The bacterial powder and the protectant solution were added to a high-speed shear emulsifier (IKA T25, 10000 rpm, 5 min) at a mass ratio of 85:15 to prepare a 20% homogeneous suspension (viscosity 85±5 mPa·s, Brookfield DV2T measurement). The suspension was fed into a spray drying tower (B-290 Mini Spray Dryer, Büchi) at a flow rate of 8 mL / min using a peristaltic pump (BT100-2J, Baoding Lange). The process parameters were: inlet air temperature 130±2℃ (real-time monitoring with PT100 thermocouple), outlet air temperature 75±2℃, two-fluid nozzle atomization pressure 0.30±0.02 MPa, and negative pressure inside the tower -150 Pa. The microcapsule powder was collected from the bottom of the tower and the cyclone separator, and sealed in an aluminum foil bag filled with nitrogen. Encapsulation efficiency determination: Take 0.1g of microcapsules, add 10mL of 0.1% Tween 80 physiological saline, shake at 37℃ for 10min to wash off surface bacteria, centrifuge, resuspend the precipitate, and count on plates. Encapsulation efficiency = (total viable bacteria - surface bacteria) / total viable bacteria × 100%. This process achieved 88.5±2.1% (n=3). The microcapsule D[4,3] was measured to be 18.7±1.5μm and the Zeta potential was -28.5±1.2mV (Malvern Zetasizer Nano ZS) using a laser particle size analyzer (Mastersizer 3000).

[0047] (Note: Key controls for spray drying: maintaining feed liquid temperature at 25±2℃ to prevent bacterial inactivation; achieving a tower wall adhesion rate of <5% by optimizing inlet air temperature and atomization pressure; validating microcapsule enteric coagulation using USP) <711> Dissolution testing device, simulating intestinal fluid (pH 6.8), showed a cumulative release rate of >90% over 4 hours.

[0048] S4: Formulation and quality control of the final formulation product: Take 55.0g of microcapsule bacterial powder, 20.0g of Lilium brownii polysaccharide extract, 12.5g of microcrystalline cellulose (PH-102, JRS Pharma), and 12.5g of spray-dried lactose (DCL-11, DFE Pharma), and mix them in a V-type mixer (HY-10, Changzhou Yibu) for 30min (20rpm). Add 3% hydroxypropyl methylcellulose (HPMC E5, Shin-Etsu) 70% ethanol solution as a binder (the amount should be such that it "can be formed into a ball by hand and crumbles easily when lightly pressed"). Mix the mixture in a trough mixer for 5min (25rpm), and granulate it using a gyratory granulator (KZL-180, Changzhou Yibu) with a 16-mesh sieve (1.18mm aperture). The wet granules were dried in a fluidized bed dryer (FL-5, Changzhou Yibu) at 40±2℃ until the moisture content was ≤4.5% (halogen moisture analyzer, Mettler Toledo HR83), and then sieved through a 16-mesh sieve. The granules were then compressed into tablets using a tablet press (ZP-35D, Shanghai Tianxiang) (die: 8.5mm shallow concave punch), with a tablet weight of 0.25g±5% and a hardness of 4.5-6.0kg (YPY-200, Shanghai Huanghai). The finished product was tested according to the General Rules of Part IV of the 2020 edition of the Chinese Pharmacopoeia: disintegration time 12-16min (0921), viable bacteria count ≥1.0×10⁻⁶. 9 CFU / tablet (selective culture medium: MRS agar for Lactobacillus, modified Bifidobacterium agar for Bifidobacterium, anaerobic culture at 37℃ for 72h), moisture ≤5.0% (0832), microbial limits meet the requirements (1105, 1106).

[0049] In one embodiment, a validation test system for the formulation is provided, including in vitro cell models and animal behavioral tests, to evaluate the effect of the formulation on mood regulation through multidimensional indicators;

[0050] The in vitro mechanism verification experiments include:

[0051] A study on the regulation of key neurotransmitters in the gut-brain axis was conducted using a Caco-2 / HT-29 co-culture gut model and a SH-SY5Y neuronal cell transmembrane co-culture system. The cells were divided into four groups: (1) blank control; (2) single strain R0175; (3) single strain R0052; and (4) complete compound preparation (10...). 8 (CFU / mL). After 48 h of culture, the concentrations of GABA, 5-HT, and BDNF in the culture supernatant were detected by LC-MS / MS.

[0052] The following results were obtained:

[0053] The amount of GABA produced in the compound preparation group was (125.7±8.3) μg / mL, which was significantly higher than that in the R0175 monotherapy group (78.2±5.1) μg / mL and the R0052 monotherapy group (82.4±6.7) μg / mL (P<0.01);

[0054] 5-HT levels increased 2.8 times, and the conversion rate of tryptophan metabolism to the 5-HT pathway increased by 37.5%;

[0055] Neuronal BDNF expression increased 3.1-fold, and TrkB phosphorylation level increased 2.7-fold;

[0056] The following conclusions were drawn: there is a synergistic metabolic effect among the composite strains; the metabolites of R0175 promote the GABA synthase activity of R0052, verifying that "the metabolites of R0175 can increase the GABA production efficiency of R0052 by 37.2%";

[0057] To verify the intestinal barrier function and inflammation regulation, a TNF-α (10 ng / mL)-induced Caco-2 cell inflammation model was established, and transmembrane resistance (TEER), tight junction protein (ZO-1, occludin) expression and LPS permeability were detected.

[0058] The following results were obtained:

[0059] After 24 hours of treatment with the compound formulation, the TEER value recovered to (385±27) Ω·cm. 2 Approaching normal level (412±18) Ω·cm 2 ;

[0060] LPS permeability decreased by 62.3%, and serum LBP concentration decreased by 41.7%;

[0061] IL-10 expression was upregulated 3.8-fold, and TNF-α expression decreased by 76.2%.

[0062] The following conclusions were drawn: Lactobacillus plantarum 299v reduces LPS entry into the bloodstream by enhancing intestinal barrier function; Lactobacillus rhamnosus GG upregulates IL-10 and inhibits neuroinflammation, verifying the mechanisms of "reducing LPS entry into the bloodstream to induce neuroinflammation" and "inhibiting TNF-α-mediated neuroimmune activation".

[0063] Animal experiments included:

[0064] Chronic unpredictable stress (CUMS) mouse models, specifically including:

[0065] Eighty C57BL / 6J mice were randomly divided into four groups: (1) normal control group; (2) CUMS + placebo; (3) CUMS + low-dose combination preparation (1×10^9 CFU / kg); (4) CUMS + high-dose combination preparation (5×10^9 CFU / kg). The mice were administered the drugs for four consecutive weeks, with behavioral indicators assessed weekly. After the treatment, changes in hippocampal neurotransmitters, HPA axis activity, and gut microbiota were measured.

[0066] The behavioral results are as follows:

[0067] The rate of preference for sucrose in the high-dose group (78.3±4.2)% was significantly higher than that in the placebo group (52.1±3.8)% (P<0.001);

[0068] The time spent immobile during forced swimming decreased by 53.7% (118.4±12.3s vs 255.6±18.7s);

[0069] The percentage of open arm exploration time (OE%) in the elevated cross maze was (46.8±3.5)%, close to the normal level (51.2±2.8)%, while the placebo group was only (24.3±2.1)%.

[0070] The following biochemical indicators were obtained:

[0071] The serum cortisol level in the high-dose group was (152.7±18.3) ng / mL, which was significantly lower than that in the placebo group (328.6±25.4) ng / mL (P<0.001);

[0072] The concentration of 5-HT in the hippocampus increased by 2.3 times, and the concentration of GABA increased by 1.9 times.

[0073] The butyric acid content in short-chain fatty acids in feces increased by 3.1 times;

[0074] The results of gut microbiota analysis were as follows: 16S rRNA sequencing showed that the abundance of Akkermansia and Faecalibacterium increased by 4.7-fold and 3.2-fold, respectively, in the high-dose group, and the genera positively related to mood were significantly enriched.

[0075] Validation of the gut-brain signal transduction pathway, using fluorescently labeled probiotics and SITZMARK® capsule tracking technology, confirmed that the microcapsule structure successfully targeted and delivered live bacteria to the terminal ileum, increasing colony colonization in the Peyer's patches area by 4.8 times. A vagus nerve transection experiment revealed that the formulation reduced the improvement in anxiety behavior by 76.3%, demonstrating that the vagus nerve is a key signal transduction pathway.

[0076] In conclusion, the following conclusions can be drawn:

[0077] Shapley value analysis confirmed that "the contribution of each component to the OE% index of the elevated cross maze is: R0175 / R0052 accounts for 52.3%, polysaccharide extract accounts for 28.7%, and other components account for 19.0%", revealing a non-simple additive effect. The polysaccharide extract of Lily and Rehmannia Decoction significantly enhances probiotic colonization and activity.

[0078] Microencapsulation technology resulted in a survival rate of 86.2% after exposure to an acidic gastric environment (artificial gastric juice pH 2.5, 2h), which was significantly better than the unencapsulated group (23.7%, P<0.001), ensuring that the effective bacterial count reached the intestinal target.

[0079] The triple mechanism of action was confirmed: (1) the probiotic combination directly regulates neurotransmitters; (2) the polysaccharide extract selectively enriches beneficial bacteria as a targeted prebiotic; (3) the microcapsule structure enables precise delivery, and the three work together to regulate mood through the gut-brain axis.

[0080] This formulation has shown significant efficacy in patients with mild to moderate mood disorders, and is particularly suitable for those who are intolerant to conventional antidepressants or wish to avoid drug side effects. Preliminary clinical studies have shown that significant improvements in mood indicators can be observed after 8 weeks of continuous use, approaching the early efficacy of SSRIs, with superior safety.

[0081] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A compound probiotic preparation for mood regulation, characterized in that, include: Bifidobacterium longum R0175 15-22%; Lactobacillus helveticus R0052 18-25%; Lactobacillus plantarum 299v 12-20%; Lactobacillus rhamnosus GG 8-15%; Bifidobacterium breve 5-10%; Lily and Rehmannia glutinosa decoction polysaccharide extract 15-25%; adjuvant protectant 5-10%; And the remaining carrier matrix; wherein the auxiliary protective agent comprises sodium alginate, chitosan and glycerol.

2. The compound probiotic preparation for mood regulation according to claim 1, characterized in that, The polysaccharide extract of Lily and Rehmannia Decoction is prepared by the following steps: Lily and Rehmannia slices are mixed in a mass ratio, water is added for extraction, the filtrate is concentrated and precipitated with alcohol, the precipitate is collected, purified by column chromatography and dried to obtain the extract.

3. The compound probiotic preparation for mood regulation according to claim 1, characterized in that, In the auxiliary protective agent, the mass ratio of sodium alginate, chitosan, and glycerol is 3:2:

1.

4. The compound probiotic preparation for mood regulation according to claim 1, characterized in that, The carrier matrix comprises microcrystalline cellulose and spray-dried lactose, and the compound probiotic preparation is in tablet form, wherein the total number of live bacteria is not less than 1.0 × 10⁻⁶. 9 CFU / film.

5. The compound probiotic preparation for mood regulation according to claim 2, characterized in that, The alcohol precipitation step controls the final ethanol concentration to be 65%-75% and allows it to stand at 4±0.5℃.

6. A method for preparing a compound probiotic preparation for mood regulation as described in any one of claims 1-5, characterized in that, Includes the following steps: S1: Bifidobacterium longum R0175, Lactobacillus helveticus R0052, Lactobacillus plantarum 299v, Lactobacillus rhamnosus GG and Bifidobacterium breve were cultured separately, the bacterial cells were collected, and the probiotic freeze-dried powder was prepared by freeze drying. S2: Preparation of polysaccharide extract from Lily and Rehmannia Decoction; S3: The probiotic freeze-dried powder obtained in S1 is mixed with the auxiliary protective agent solution and then spray-dried to prepare probiotic microcapsules; S4: The probiotic microcapsules prepared in S3, the polysaccharide extract of Lilium brownii and Rehmannia glutinosa prepared in S2, and the carrier matrix are mixed, granulated, dried, and compressed to obtain the compound probiotic preparation.

7. The method for preparing the compound probiotic preparation for mood regulation according to claim 6, characterized in that, In step S1, the culture is carried out under anaerobic conditions, and the dissolved oxygen content of the fermentation broth is controlled to be below 0.5% saturation; the freeze drying includes primary drying and secondary drying.

8. The method for preparing the compound probiotic preparation for mood regulation according to claim 6, characterized in that, In step S3, the inlet air temperature of the spray dryer is 125-135℃, the outlet air temperature is 70-80℃, and the atomization pressure is 0.28-0.32MPa.

9. The method for preparing the compound probiotic preparation for mood regulation according to claim 6, characterized in that, The polysaccharide extract of Lily and Rehmannia Decoction meets the requirement of polysaccharide content ≥60.0%.

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