A probiotic complex for improving memory and its preparation method

Abstract

This invention discloses a probiotic complex for improving memory and its preparation method. In preparation, each bacterial strain is first activated and expanded to obtain a seed culture, which is then inoculated into a fermentation medium for fermentation. After fermentation, the bacterial sludge is collected by centrifugation. The bacterial sludge is mixed with a solution containing a complex protective agent to prepare a bacterial suspension, which is then pre-frozen, freeze-dried, pulverized, and sieved to obtain a lyophilized powder. This powder is then mixed with a filler to prepare a dosage form. This invention, through specific strain combinations and process optimization, solves the problems of limited memory improvement effects in existing technologies due to a lack of synergistic effects, poor stability, and neglect of the neuroregulatory metabolic environment.

Description

Technical Field

[0001] This invention belongs to the field of probiotic preparation technology, and relates to a probiotic complex for improving memory and its preparation method. Background Technology

[0002] With the deepening understanding of the "gut-brain axis" theory, the bidirectional regulatory relationship between gut microbiota and brain function has become a hot topic in the interdisciplinary research of neuroscience and microbiology. Numerous studies have shown that gut microbiota influences the host's cognitive, emotional, and memory functions through multiple pathways, including neural, endocrine, and immune pathways. Probiotics, as an important means of regulating gut microecological balance, have shown broad application prospects in improving cognitive impairment and promoting memory. Specific strains of *Lactobacillus* and *Bifidobacterium* have been shown to effectively improve learning and memory abilities by regulating gut microbiota structure, promoting the production of neuroactive metabolites (such as γ-aminobutyric acid and glycerophospholipids), and regulating neuroinflammation and the activity of the hypothalamic-pituitary-adrenal (HPA) axis. These findings provide a theoretical basis for developing probiotic-based cognitive function intervention strategies and have promoted the research and development of functional foods and neurohealth products. However, although some probiotic products have entered the market, their actual effectiveness in improving memory remains limited by technological bottlenecks. Current research largely focuses on the mechanisms of action of single strains, lacking a systematic exploration and utilization of the synergistic effects of multiple strains. This results in limited functional targets and thus limited memory-enhancing effects. Furthermore, probiotics are highly susceptible to factors such as temperature, humidity, and oxygen during formulation processing (e.g., freeze-drying, tableting) and long-term storage, leading to bacterial inactivation, significantly reducing the viable cell count and bioavailability of the product, thereby affecting its colonization ability and function in the gut. In addition, current formulation designs often neglect the metabolic environment required for probiotics to exert their neuroregulatory effects, failing to specifically add prebiotics or other auxiliary ingredients to promote the synthesis of key neuroactive substances, thus weakening the transmission efficiency of gut-brain axis signals. More critically, existing manufacturing processes have not been specifically optimized around the core function of "improving memory," making it difficult to maintain the physicochemical stability of the formulation while ensuring high strain activity. These issues collectively result in current probiotic products failing to meet the demand for highly effective, stable, and targeted memory-enhancing formulations in clinical healthcare. Therefore, there is an urgent need to develop a novel probiotic complex preparation technology. This technology involves scientifically screening memory-improving strains with synergistic effects, optimizing formulations to enhance bacterial stability and intestinal colonization, and combining this with function-oriented process design to integrate cofactors such as prebiotics. This would enable multi-dimensional and highly efficient memory intervention, break through current technological bottlenecks, and promote the in-depth application of probiotics in the field of neurohealth. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides a probiotic complex for improving memory and its preparation method, thereby solving the technical problems that existing probiotic preparations suffer from limited memory improvement effects and difficulty in meeting the requirements for efficient and stable application due to the lack of multi-strain synergistic effects, poor formulation stability, neglect of the neuroregulatory metabolic environment, and lack of optimization of the preparation process for the "memory improvement" function.

[0004] This invention is achieved through the following technical solution: A method for preparing a probiotic complex for improving memory includes the following steps: S1: Inoculate the compound strains into their respective activation media and culture them until the OD of each strain reaches its maximum. 600 The value reaches 1.0~2.0 to obtain an activated strain; then the activated strain is inoculated into the corresponding seed culture medium and cultured until the strain is in the logarithmic growth phase to obtain the strain seed liquid; the compound strain consists of Lactobacillus apis BB1, Limosilactobacillus fermentum A2.8, Lactobacillus paracasei PS23 and Bifidobacterium bifidum BGN4.

[0005] S2: Inoculate the seed culture of each strain into the fermentation medium and culture until the total viable count in the fermentation broth reaches 1×10⁻⁶. 10 ~8×10 10 CFU / mL; then the fermentation broth was centrifuged, the cell precipitate was collected, and the cell precipitate was washed several times with sterile physiological saline to obtain pure bacterial sludge; S3: Dissolve the composite protectant in deionized water, sterilize and cool to obtain a composite protectant solution; then stir and mix the composite protectant solution with pure bacterial mud to obtain a bacterial suspension. S4: After pre-freezing, freeze-drying, pulverizing and sieving the bacterial suspension, freeze-dried powder is obtained. Then, filler is added to obtain mixed powder. Finally, the mixed powder is prepared into a dosage form to obtain the probiotic complex that improves memory.

[0006] Preferably, during cultivation, Lactobacillus apis BB1, Limosilactobacillus fermentum A2.8, and Lactobacillus paracasei PS23 are cultured on MRS medium under anaerobic conditions for 18-24 h; while Bifidobacterium bifidum BGN4 is cultured on BHI medium under CO2 conditions with a volume concentration of 5%-8% for 20-26 h.

[0007] Preferably, in step S2, the seed culture of each strain is inoculated into the fermentation medium according to the CFU ratio of the four strains as (1~5):(1~5):(1~5):(1~5).

[0008] Preferably, in step S2, the seed culture of each strain is inoculated into the fermentation medium according to the CFU ratio of the four strains being 3:2:2:2.

[0009] Preferably, the seed culture of each strain is inoculated into the fermentation medium and cultured in two stages. The first stage is cultured for 0-12 hours, maintaining a pH of 5.0 and a temperature of 36-38℃. The second stage is cultured for 12-24 hours, controlling the pH to rise linearly to 6.5 and maintaining a temperature of 36-38℃.

[0010] Preferably, in step S3, the composite protective agent is composed of maltodextrin, fructooligosaccharides, monosodium glutamate and sodium ascorbate; the ratio of maltodextrin, fructooligosaccharides, monosodium glutamate and sodium ascorbate by mass is (3~8):(1.5~4):(0.6~1.5):(0.3~0.8).

[0011] Preferably, in step S3, the composite protective agent is composed of maltodextrin, fructooligosaccharides, monosodium glutamate, sodium ascorbate, and β-cyclodextrin, and the ratio of maltodextrin, fructooligosaccharides, monosodium glutamate, sodium ascorbate, and β-cyclodextrin is 5:2:1:0.6:1.2 by mass.

[0012] Preferably, in step S3, the ratio of the pure bacterial mud to the composite protective agent solution is 1:(1.8~2.2) by mass.

[0013] Preferably, in step S4, the pre-freezing process is carried out at a temperature of -25 to -45°C for 2 to 3 hours; more preferably, the temperature is carried out at -39 to -42°C for 2.5 hours. In the freeze-drying process, the vacuum degree is 15 to 30 Pa, the shelf temperature is 48 to -52°C, and the time is 24 to 30 hours.

[0014] A probiotic complex for improving memory was prepared by the method described above.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects: This invention discloses a method for preparing a probiotic complex that improves memory. The method involves the combined screening and simultaneous activation culture of four specific strains (BB1, A2.8, PS23, and BGN4) in step S1, constructing a biological basis with synergistic effects from the source, effectively solving the technical defects of single strains having limited function and combinations lacking synergy. Step S2 ensures high purity and activity of the bacteria by controlling the viable cell count at the fermentation endpoint and the centrifugation washing process, guaranteeing the stability and colonization ability of the formulation. Step S3, the introduction of a compound protectant not only maintains bacterial activity during freeze-drying but also indirectly optimizes the metabolic environment for neural regulation through the metabolic regulation of the protectant components. Step S4, through the control of process parameters throughout the pre-freezing, freeze-drying, and dosage form processing, achieves functional-oriented optimization of the preparation process. Ultimately, the resulting probiotic complex, while ensuring high stability, fully leverages the synergistic effects of multiple strains to improve memory function, thus overcoming the technical bottlenecks of limited memory improvement and application difficulties caused by the lack of synergistic effects, poor stability, neglect of the metabolic environment, and non-specific processes in existing technologies. Detailed Implementation

[0016] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0017] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0018] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0019] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0020] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0021] This invention provides a method for preparing a probiotic complex that improves memory, comprising the following steps: S1: Inoculate the compound strains into their respective activation media and culture them until the OD of each strain reaches its maximum. 600 When the value reaches 1.0~2.0, an activated strain is obtained; then the activated strain is inoculated into the corresponding seed culture medium and cultured until the strain is in the logarithmic growth phase to obtain the strain seed liquid; Specifically: Inoculate the compound strains into their respective activation media and incubate them at 30-40℃ until the OD of the strains reaches the target value. 600 When the OD value reaches 1.0~2.0, activated strains are obtained. Then, each activated strain is inoculated into its corresponding seed culture medium at an inoculation rate of 2%~8% (v / v), and the above culture environment is maintained until the strains reach the logarithmic growth phase, thus obtaining the seed culture of each strain. The "OD" in this invention... 600 The "value" refers to the optical density value at a wavelength of 600 nm.

[0022] More specifically: By weight, 0.5–3 parts of the compound strain were inoculated into 50–100 parts of activation medium and cultured at 30–40°C in a specific gas environment until the strain reached its OD value. 600 The value reaches 1.0~2.0; then, each activated strain is inoculated into the corresponding seed culture medium at an inoculation rate of 2%~8% (v / v), and the amount of seed culture medium is 1.5~2 times that of the activation culture medium. The above culture environment is maintained until the strain is in the logarithmic growth phase to obtain the seed liquid of each strain.

[0023] The composite strain consisted of Lactobacillus apis BB1, Limosilactobacillus fermentum A2.8, Lactobacillus paracasei PS23, and Bifidobacterium bifidum BGN4, all of which were purchased from the German Microbial Culture Collection (DSMZ).

[0024] During cultivation, Lactobacillus apis BB1, Limosilactobacillus fermentum A2.8, and Lactobacillus paracasei PS23 were cultured on MRS medium under anaerobic conditions for 18–24 h; Bifidobacterium bifidum BGN4 was cultured on BHI medium under CO2 conditions with a volume concentration of 5%–8% for 20–26 h. In this invention, the MRS medium is the Moli-Rogosa-Sharp medium, which is specifically used for the cultivation of lactic acid bacteria; the BHI medium is brain heart extract medium, which is used for the cultivation of bifidobacteria. The colony morphology of the activated strains must meet the following requirements: Lactobacillus apis BB1 is a round, milky-white colony with neat edges; Limosilactobacillus fermentum A2.8 is a round, pale yellow colony with a smooth surface; Lactobacillus paracasei PS23 is a round, milky-white colony with a viscous texture; and Bifidobacterium bifidum BGN4 is a round, milky-white colony with serrated edges. S2: Inoculate the seed culture of each strain into the fermentation medium and culture until the total viable count in the fermentation broth reaches 1×10⁻⁶. 10 ~8×10 10 CFU / mL; then the fermentation broth was centrifuged, the cell precipitate was collected, and the cell precipitate was washed several times with sterile physiological saline to obtain pure bacterial sludge; In one specific embodiment, the steps are as follows: First, the seed culture of each strain is inoculated into the fermentation medium according to a certain colony forming unit (CFU) ratio; for Lactobacillus strains, nitrogen gas is introduced into the fermentation medium before use; for Bifidobacterium bifidum BGN4, CO2 gas with a volume concentration of 5% is introduced into the fermentation medium before use to remove oxygen; the nitrogen or CO2 gas introduction time is 30-60 minutes. Second, under controlled culture conditions, the culture is carried out until the total viable count in the fermentation broth reaches 1×10⁻⁶. 10 ~8×10 10CFU / mL; Next, the fermentation broth was centrifuged under certain conditions. During centrifugation, the temperature was 3~15℃, the centrifugal force was 7000~9000×g, and the time was 10~25min; the bacterial precipitate was collected; Finally, the precipitate was washed 2~3 times with sterile physiological saline. During the washing process, the volume ratio of the sterile physiological saline to the bacterial precipitate was (2~7):1. The stirring speed was controlled at 50~200r / min during the washing process. After each washing, the precipitate was collected under the same centrifugation conditions to remove the culture medium residue and obtain pure bacterial sludge.

[0025] Preferably, the CFU ratio of the four strains is (1~5):(1~5):(1~5):(1~5), and more preferably, the CFU ratio of the four strains is 3:2:2:2. This invention uses a Box-Behnken experimental design to systematically optimize the CFU ratio of four strains (Lactobacillus apisBB1, Limosilactobacillus fermentum A2.8, Lactobacillus paracasei PS23, and Bifidobacterium bifidum BGN4). The experiment uses the ratio of the four strains as the independent variable (encoding range 1-5:1-5:1-5:1-5), and the viable cell count, GABA yield, and LPA (14:0) yield in the fermentation broth as response values, constructing a quadratic multinomial regression model.

[0026] In this invention, GABA production refers to Gamma-Aminobutyric Acid (γ-aminobutyric acid) production, which is the total amount of GABA accumulated in the fermentation broth at the end of fermentation. LPA (14:0) refers to Myristoyl LPA, a saturated fatty acid chain containing 14 carbon atoms. LPA (16:0) refers to Palmitoyl LPA, a saturated fatty acid chain containing 16 carbon atoms (palmitoic acid). LPA (14:0) production refers to the total amount of LPA in the fermentation broth at the end of fermentation. In the Box-Behnken experimental design of this invention, these two indicators are the core standards for measuring the "functional activity" of probiotics: GABA production represents the ability of the bacterial community to regulate the balance of nerve inhibition / excitation. LPA (14:0) production represents the ability of the bacterial community to promote nerve growth and synaptic plasticity. By optimizing the ratio of the four strains, the aim is to maximize the production of these two functional metabolites, thereby achieving the best memory improvement effect.

[0027] The results showed that the interaction coefficient was optimal when the CFU ratio of the four strains was 3:2:2:2 (the interaction coefficient between Lactobacillus apis BB1 and Limosilactobacillus fermentum A2.8 was 0.87, with a confidence level of p<0.01), at which point the total viable count in the fermentation broth reached 4.2 × 10⁻⁶. 10 The CFU / mL concentration was increased by 23% compared to the previous concentration; GABA production reached 185 mg / L, and LPA (14:0) production reached 22.3 μg / L, which were 2.1 times and 1.8 times that of single-strain culture, respectively. Validation experiments showed that at this ratio, the time spent in the target quadrant of the Morris water maze in SAMP8 mice was prolonged by 48%, which was significantly higher than other ratio combinations (p<0.05).

[0028] The interaction coefficient is a key parameter in the Box-Behnken response surface methodology (BSA) mathematical model. This coefficient represents the strength and direction of the synergistic effect between strains BB1 and A2.8. A positive value indicates a synergistic effect between the two strains, while a negative value indicates an antagonistic effect. In this invention, the interaction coefficient is 0.87, meaning that when BB1 and A2.8 are present simultaneously, they promote each other, producing an effect greater than the sum of their individual effects. The confidence level p is used to determine whether the aforementioned "synergistic effect" is genuine and credible, rather than accidental. In statistics, the p-value represents the probability that "the two are assumed to be unrelated, but the result shows a relationship," i.e., the false positive probability. Generally, statistics consider p < 0.05 to be significant. In this application, p < 0.01 indicates a genuine and strong synergistic effect between strains BB1 and A2.8, a highly reliable result not due to experimental error.

[0029] Seed cultures of each strain were inoculated into fermentation medium. During cultivation, the temperature was 36–38°C, the pH was 5.0–7.0, and the cultivation time was 24–30 hours. More specifically, this step employed a phased, dynamic pH control strategy. The first phase was the cell growth phase, during which the pH was maintained at 5.0 for 0–12 hours. This phase corresponds to the logarithmic growth phase of *Lactobacillus apis BB1*, and the goal was to rapidly increase the cell density to reach OD. 600=1.8, which promotes the growth of Lactobacillus apis BB1; the second stage is the product synthesis period, that is, the pH is linearly increased to 6.5 for 12-24 hours to induce secondary metabolism, that is, to initiate the synthesis pathway of GABA and LPA. The increase in pH induces the expression of GAD gene (glutamate decarboxylase), while inhibiting GABA transaminase (the enzyme that breaks down GABA), thus promoting GABA synthesis. At the same time, the increase in pH increases the activity of phosphodiesteryltransferase, increases the precursor phosphatidic acid, and promotes LPA synthesis. Metabolomics analysis showed that after the pH was increased, the expression level of GAD gene (glutamate decarboxylase) was significantly increased by 2.1 times, and the expression level of GABA transaminase gene was decreased by 35%, resulting in a 1.7-fold increase in GABA synthesis flux. At the same time, the activity of phosphodiesteryltransferase (an important LPA synthase) increased by 42%, and the content of phosphatidic acid, an intermediate product of the LPA synthesis pathway, increased by 68%. This strategy resulted in a GABA to LPA molar ratio of 8.3:1 in the fermentation broth, a ratio that has been shown to maximize gut-brain axis signal transduction efficiency.

[0030] S3: Dissolve the composite protectant in deionized water, sterilize and cool to obtain a composite protectant solution; then stir and mix the composite protectant solution with pure bacterial mud to obtain a bacterial suspension. In one specific embodiment, the above process is as follows: weigh each protective agent according to a certain proportion, add deionized water, stir until completely dissolved, and prepare a composite protective agent solution with a certain mass concentration; secondly, sterilize the composite protective agent solution at 115~121℃ for 10~30min, and cool for later use; finally, mix the pure bacterial mud and the composite protective agent solution according to a certain proportion, and stir at 3~5℃ and 100~150r / min for 20~30min to form a uniform bacterial suspension.

[0031] Further preferred method: Mix 0.3-8 parts of the composite protectant with 100-200 parts of deionized water, stir under certain conditions until completely dissolved, to prepare a composite protectant solution with a mass concentration of 15%-20%; next, sterilize the composite protectant solution at 115-121℃ for 12-18 minutes, and cool to 3-5℃ for later use; finally, add pure bacterial mud to the composite protectant solution in proportion, and stir at 3-5℃ and 100-150r / min for 20-30 minutes to form a uniform bacterial suspension.

[0032] The composite protective agent is composed of maltodextrin, fructooligosaccharides, monosodium glutamate and sodium ascorbate; by mass parts, the ratio of maltodextrin, fructooligosaccharides, monosodium glutamate and sodium ascorbate is (3~8):(1.5~4):(0.6~1.5):(0.3~0.8); The maltodextrin has a glucose equivalent (DE) value of 8-12 and a degree of polymerization of fructooligosaccharides of 2-8. The composite protective agent is composed of maltodextrin, fructooligosaccharides, monosodium glutamate, sodium ascorbate, and β-cyclodextrin. The ratio of maltodextrin, fructooligosaccharides, monosodium glutamate, sodium ascorbate, and β-cyclodextrin is 5:2:1:0.6:1.2 by mass.

[0033] The ratio of the pure bacterial sludge to the composite protective agent solution is 1:(1.8~2.2) by mass.

[0034] S4: After pre-freezing, freeze-drying, pulverizing and sieving the bacterial suspension, freeze-dried powder is obtained. Then, filler is added to obtain mixed powder. Finally, the mixed powder is prepared into a dosage form to obtain the probiotic complex that improves memory.

[0035] In one specific embodiment, the process is as follows: First, the bacterial suspension is dispensed into freeze-drying trays and pre-frozen at -25~-45℃ for 2~3 hours to ensure complete freezing. Second, the pre-frozen bacterial suspension is transferred to a vacuum freeze dryer and freeze-dried under controlled conditions until the sample moisture content is ≤5%, obtaining probiotic freeze-dried powder. Next, the freeze-dried powder is pulverized under sterile conditions and passed through a 70~90 mesh sieve to remove clumps. Finally, according to the formulation requirements, fillers are added, and the freeze-dried powder and fillers are mixed evenly in a certain proportion. Finally, the mixed powder is made into probiotic complex capsules, probiotic complex powder, granules, or chewable tablets, thus obtaining a probiotic complex for improving memory.

[0036] During the pre-freezing process, the temperature is -25~-45℃ and the time is 2~3 hours; more preferably, the temperature is -39~-42℃ and the time is 2.5 hours. During the freeze-drying process, the vacuum degree is 15~30Pa, the shelf temperature is -48~-52℃, and the time is 24~30h; in this invention, the shelf is the freeze-drying tray, and controlling the shelf temperature is controlling the temperature of the cold source. The filler is one or more of microcrystalline cellulose, maltodextrin, mannitol and lactose; The ratio of the freeze-dried powder to the filler is (5~14):1 by mass parts; After the mixed powder is prepared into a dosage form, it needs to be sealed and packaged. The packaging material is an oxygen-barrier and moisture-barrier aluminum-plastic composite film.

[0037] In another preferred embodiment, during the preparation of the mixed powder into a dosage form, a synergistic protective system of microencapsulation and three-layer oxygen barrier packaging is employed. During the microencapsulation process, sodium alginate-chitosan microcapsules (core material:wall material = 1:3, calcium chloride concentration 2%) are prepared using a sharp-pore coagulation bath method. The average microcapsule particle size is 520 μm, and the encapsulation rate is 91.3%. Specifically, the mixed powder is added to sodium alginate to form a suspension, which is then subjected to a sharp-pore coagulation bath.

[0038] The three-layer oxygen barrier packaging consists of an inner layer of polyethylene (50 μm thick), a middle layer of aluminum foil (9 μm thick), and an outer layer of polyethylene terephthalate (12 μm thick), achieving an oxygen permeability as low as 0.08 cm. 3 / (m 2 •d·atm). Accelerated stability testing (40℃ / 75%RH) showed that this synergistic system enabled the probiotics to retain 92.1% of their live bacteria after 3 months of storage, significantly higher than the single microencapsulation group (76.5%) and the single oxygen-barrier packaging group (68.3%). Furthermore, the microcapsules achieved a 90.5% survival rate in simulated gastric fluid after 2 hours and a 95.2% release rate in simulated intestinal fluid after 4 hours, achieving targeted drug delivery.

[0039] In summary, this invention discloses a method for preparing a probiotic complex that improves memory. The method involves activating and culturing the complex strains, then transferring them to a seed culture medium to obtain a seed culture in the logarithmic growth phase, laying the foundation for subsequent high-activity culture. Subsequently, the culture medium is inoculated in a specific ratio to achieve a high viable count in the fermentation broth. Low-temperature centrifugation and washing with physiological saline effectively remove impurities and improve bacterial purity. Next, a protective agent is added, sterilized, and then mixed with the bacterial sludge at low temperature to reduce bacterial damage and maintain activity. Finally, optimized freeze-drying conditions control the sample moisture content to ensure the stability of the freeze-dried powder. Fillers are added to suit capsule and powder formulations, and oxygen- and moisture-barrier packaging extends the shelf life. The synergistic effect of each step ensures both the activity and stability of the probiotics and the suitability of the formulation for storage and use, ultimately enabling the complex to stably perform its memory-improving function with long-lasting activity and convenient use.

[0040] The strains selected in this invention, Lactobacillus apis BB1, promote glycerophospholipid production; Limosilactobacillus fermentum synthesizes γ-aminobutyric acid (GABA); Lactobacillus paracaseiPS23 regulates neuroinflammation and antioxidant enzyme activity; and Bifidobacterium bifidum BGN4 improves intestinal flora balance. These strains work synergistically at different targets, retaining the aforementioned anti-inflammatory, intestinal barrier repair, and original metabolic effects, while also activating the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. The serum BDNF content in the combined group increased by 15%–20% compared to the dual-strain combination group and by 40%–45% compared to the single-strain group. BDNF has the effect of regulating synaptic plasticity, increasing the time spent in the target quadrant of the Morris water maze in SAMP8 mice by 45%–48% (far exceeding the 30%–35% increase achieved by the single-strain combination).

[0041] In this invention, the composite protectant forms a glassy matrix that encapsulates the bacteria, maintaining osmotic pressure and inhibiting oxidative damage. Combined with freeze-drying, this results in a viable count retention rate of over 85% after 12 months of storage at 4°C, a 30%–40% improvement compared to traditional freeze-dried formulations. Simultaneously, the protectant enhances the hydrophobicity of the bacterial surface, increasing intestinal colonization by 30%–40%. The targeted addition of monosodium glutamate (MSG) serves as a precursor for GABA synthesis in probiotics, strengthening gut-brain axis signal transduction. The selected bacterial strains are derived from natural foods or normal human gut flora, and have been validated through acute toxicity tests. The suitable population includes adolescents, adults, and the elderly. The entire preparation process is mild, with simple and controllable steps. Both fermentation and freeze-drying equipment are conventional probiotic production equipment, reducing costs by 20%–30% compared to formulations containing special carriers. This allows for ton-scale production, meeting industrial application requirements. Furthermore, this invention adds β-cyclodextrin (1.2% by mass) to the composite protectant, and its encapsulation effect on lipid-soluble metabolites is determined by HPLC-MS. Chromatographic conditions: An Agilent ZORBAX SB-C18 column (2.1 × 150 mm, 3.5 μm) was used; mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile, with gradient elution (0–5 min 30% B, 5–15 min 30%–90% B, 15–20 min 90% B); flow rate was 0.3 mL / min; column temperature was 40℃. Mass spectrometry conditions: Electrospray ionization (ESI+) mode, scan range m / z 100–1000, capillary voltage 3.5 kV, and drying gas temperature 350℃. Results showed that β-cyclodextrin achieved an encapsulation rate of 89.2% for LPA (14:0) and 85.7% for LPA (16:0), significantly improving the intestinal absorption efficiency of lipid-soluble metabolites. Differential scanning calorimetry analysis of the encapsulated complex showed that its phase transition temperature increased by 8.5 °C, indicating that the encapsulation structure enhanced the thermal stability of the metabolite.

[0042] For the probiotic complex for improving memory in this invention, the viable bacteria retention rate was ≥85% after storage at 4°C for 12 months using the plate count method, which is 30%~40% higher than that of traditional freeze-dried preparations. The synergistic effect of the compound protectant effectively inhibited bacterial oxidative damage and activity loss. A mouse gavage experiment (1×10⁻⁶) was conducted. 9 CFU / animal, for 7 consecutive days), fecal samples were collected on day 14 after gavage to detect the number of target strains, with a colonization rate ≥1×10⁻⁶. 6 CFU / g feces increased the colonization rate by 30%~40% compared to the sum of single strains, and the protectant significantly enhanced the hydrophobicity of the bacterial surface; using a SAMP8 mouse model (16 weeks old, 15 mice per group), 1×10 CFU / g feces was administered by gavage daily. 9CFU / animal, for 12 consecutive weeks, was tested using the Morris water maze test: the time spent in the target quadrant was ≥45% longer than the control group, and the escape latency was ≥35% shorter than the control group; the serum brain-derived neurotrophic factor (BDNF) content was ≥40% higher than the control group and ≥15% higher than the dual-strain combination group; the serum LPA (14:0) content was ≥35% higher than the control group, the GABA content was ≥25% higher than the control group, and the serum inflammatory factor TNF-α content was ≥20% lower than the control group.

[0043] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0044] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0045] Example 1 1) Activation culture of compound probiotic strains First, one sample of *Lactobacillus apis* BB1 and one sample of *Limosilactobacillus fermentum* A2.8 were selected and inoculated into 50 portions of MRS medium, respectively. The *Lactobacillus* spp. were then cultured anaerobically at 37°C for 20 hours until the OD (Organic Demand) reached its maximum. 600 =1.3; then, 75 MRS seed culture media were inoculated at a 3% (v / v) inoculation rate, maintained at 37℃ in an anaerobic environment, and cultured for 14 h to obtain seed liquid.

[0046] 2) Large-scale culture and cell collection of compound probiotics First, 200 portions of fermentation medium were inoculated at a CFU ratio of 3:2, incubated at 37°C and pH 6.2 (with 1 mol / L lactic acid added dropwise), under nitrogen purging for 40 min, and cultured for 26 h. The total viable count in the fermentation broth was 2.5 × 10⁻⁶. 10 CFU / mL; then, centrifuge at 4℃, 8000×g for 15 min to collect the precipitate; finally, wash with 100 parts of sterile physiological saline, the sludge has a water content of 75%.

[0047] 3) Preparation of compound protectant and mixing with probiotics First, add 100 parts deionized water to a mixture of 5 parts maltodextrin, 3 parts fructooligosaccharides, 1 part monosodium glutamate, and 0.5 parts sodium ascorbate, and stir at 35°C to dissolve, preparing an 18% protective agent solution; sterilize at 121°C for 15 minutes, and cool to 4°C; mix the bacterial sludge and protective agent at a ratio of 1:2, and stir at 4°C and 120 rpm for 25 minutes, resulting in a bacterial suspension with a viable count of 7 × 10⁻⁶. 9 CFU / mL.

[0048] 4) Freeze-drying The bacterial suspension was dispensed into freeze-drying trays and pre-frozen at -39℃ for 2.5 hours until completely frozen. The trays were then transferred to a vacuum freeze dryer, and the vacuum was controlled at 15 Pa and the shelf temperature at -50℃. The samples were freeze-dried for 26 hours until the water content was 2.5%, thus obtaining probiotic freeze-dried powder. The freeze-dried powder was then placed in a sterile environment and pulverized for 2 minutes using a high-speed grinder (9000 r / min). The powder was then passed through an 80-mesh sterile sieve to remove any clumped particles.

[0049] 5) Formulation The freeze-dried bacterial powder and sterilized filler were added to a three-dimensional motion mixer at a mass ratio of 10:1 and mixed at 60 rpm for 18 minutes. A fully automatic capsule filling machine was used, selecting No. 1 empty capsules and setting the filling amount to 0.4 g / capsule. The filled capsules were then placed in a polishing machine and polished at 350 rpm for 6 minutes. The qualified capsules were then packed into aluminum-plastic blister packs (12 capsules per pack), with a blister forming temperature of 90℃ and a sealing pressure of 0.4 MPa. The blister packs were then placed into aluminum-plastic composite bags with 8% silica gel desiccant inside, vacuumed (vacuum degree 0.07 MPa), and heat-sealed at 130℃ for 3 seconds to complete the packaging.

[0050] The probiotic complex capsules prepared in this example retained 85% of their live bacteria count after 12 months of storage at 4°C; the colonization rate in mouse feces was 1.2 × 10⁻⁶. 6 CFU / g; SAMP8 mice showed a 32% longer time spent in the target quadrant and a 26% shorter escape latency compared to the control group; serum LPA (14:0) increased by 32%, GABA increased by 22%, and serum BDNF levels increased by 28% compared to the control group.

[0051] Example 2 1) Activation culture of compound probiotic strains 1.5 portions of *Lactobacillus paracasei* PS23 and 1 portion of *Bifidobacterium bifidum* BGN4 were inoculated into 80 portions of MRS / BHI medium and cultured at 38°C for 22 hours (OD200). 600 =1.4), Bifidobacterium bifidum BGN45%CO224h (OD600 =1.3); 120 seed culture media were inoculated at a 4% inoculum rate, Lactobacillus was cultured for 13 h, and Bifidobacterium bifidum BGN4 was cultured for 17 h, with a viable count of 4 × 10⁻⁶ in the seed culture. 9 CFU / mL.

[0052] 2) Large-scale culture and cell collection of compound probiotics Inoculate 300 portions of fermentation medium at a CFU ratio of 2:1. Incubate at 38℃, pH 6.4 (with 1 mol / L sodium hydroxide added), purge with nitrogen for 50 min for Lactobacillus, and incubate with Bifidobacterium bifidum BGN at 45% CO2 for 28 h. The total viable count in the fermentation broth is 4 × 10⁻⁶. 10 CFU / mL; centrifuged at 4℃ and 8500×g for 15 min, washed with 150 portions of sterile physiological saline, the bacterial sludge had a water content of 72%.

[0053] 3) Preparation of compound protectant and mixing with probiotics Mix 4 parts maltodextrin, 4 parts fructooligosaccharides, 1.2 parts monosodium glutamate, and 0.6 parts sodium ascorbate with 150 parts deionized water and stir at 38°C to prepare a 16% protective agent solution; sterilize at 118°C for 16 minutes and cool to 3°C; mix the bacterial sludge and protective agent in a ratio of 1:2.1, stir at 3°C ​​and 140 rpm for 22 minutes, and the viable count of the bacterial suspension is 8 × 10⁻⁶. 9 CFU / mL.

[0054] 4) Freeze-drying The bacterial suspension was dispensed into freeze-drying trays and pre-frozen at -40℃ for 3 hours until completely frozen. It was then transferred to a vacuum freeze dryer, with the vacuum level controlled at 12Pa and the shelf temperature at -51℃, and freeze-dried for 28 hours until the sample moisture content was 2.2%, thus obtaining probiotic freeze-dried powder. The freeze-dried powder was then placed in a sterile environment and pulverized for 3 minutes using a high-speed pulverizer (10000r / min). The powder was then passed through a 70-mesh sterile sieve to ensure that the powder particle size was ≤150μm.

[0055] 5) Formulation The freeze-dried bacterial powder and filler were added to a double cone mixer at a mass ratio of 12:1 and stirred at 50 rpm for 28 minutes. A fully automatic powder filling machine was used, with oxygen-barrier aluminum foil bags (oxygen barrier rate ≥99.5%) selected. The filling amount was set to 2g / bag, and the ambient temperature was controlled at 20℃ and the relative humidity at 38%. Nitrogen gas (purity ≥99.9%) was introduced during the filling process for protection to ensure that the number of viable bacteria per gram of powder was 9.8 × 10⁸. 9 CFU; Place the qualified aluminum foil bag into the outer packaging box, and put the product instruction manual and silica gel desiccant (6% of the powder) inside the box.

[0056] The probiotic complex powder prepared in this embodiment retained 89% of its live bacteria count after 12 months of storage at 4°C; the colonization rate in mouse feces was 1.3 × 10⁻⁶. 6 CFU / g; SAMP8 mice showed a 35% longer time spent in the target quadrant and a 28% shorter escape latency compared to the control group; serum LPA (14:0) increased by 30%, GABA increased by 24%, and serum inflammatory factor TNF-α levels decreased by 20% compared to the control group.

[0057] Example 3 1) Activation culture of compound probiotic strains Three samples of Lactobacillus apis BB1 and one sample of Bifidobacterium bifidum BGN4 were selected and inoculated into 100 samples of MRS / BHI medium and cultured at 36°C for 18 hours in anaerobic conditions (OD). 600 =1.2), Bifidobacterium bifidum BGN45%CO220h (OD 600 =1.2); 150 seed culture media were inoculated at a 2% inoculum rate, Lactobacillus genus cultured for 16 h, and Bifidobacterium bifidum BGN4 cultured for 20 h to obtain seed liquid.

[0058] 2) Large-scale culture and cell collection of compound probiotics Inoculate 500 portions of fermentation medium at a CFU ratio of 3:1, incubate at 36°C and pH 6.0 (adjusted with 0.8 mol / L lactic acid), purge with nitrogen for 60 min for Lactobacillus, and incubate with Bifidobacterium bifidum BGN at 45% CO2 for 24 h. The total viable count in the fermentation broth is 2 × 10⁻⁶. 10 CFU / mL; centrifuged at 5℃ and 7500×g for 15 min, washed with 200 portions of sterile physiological saline, the bacterial sludge had a water content of 78%.

[0059] 3) Preparation of compound protectant and mixing with probiotics A 20% protectant solution was prepared by mixing 6 parts maltodextrin, 2 parts fructooligosaccharides, 0.8 parts monosodium glutamate, and 0.4 parts sodium ascorbate with 200 parts deionized water and stirring at 32°C. The solution was then sterilized at 121°C for 12 minutes and cooled to 5°C. The mixture of mycelial sludge and protectant was then mixed at a ratio of 1:1.8 and stirred at 100 rpm for 30 minutes at 5°C. The resulting bacterial suspension had a viable count of 6 × 10⁻⁶. 9 CFU / mL.

[0060] 4) Freeze-drying The bacterial suspension was dispensed into freeze-drying trays and pre-frozen at -42℃ for 2 hours until completely frozen. It was then transferred to a vacuum freeze dryer, and the vacuum degree was controlled at 20Pa and the shelf temperature at -48℃. The mixture was freeze-dried for 24 hours until the sample moisture content was 2.8%, thus obtaining probiotic freeze-dried powder. The freeze-dried powder was then placed in a sterile environment and pulverized for 2.5 minutes using a high-speed pulverizer (8500r / min). The powder was then passed through a 90-mesh sterile sieve to remove any agglomerated particles.

[0061] 5) Formulation Freeze-dried bacterial powder and filler were mixed at a mass ratio of 14:1, and 8% of 75% anhydrous ethanol was added as a wetting agent. The mixture was stirred at 30 rpm for 12 minutes in a sterile kneader to form a soft mass. The soft mass was then fed into a wet granulation machine, granulated at 60 rpm using an 18-mesh sieve, with the ambient temperature controlled at 20℃ and relative humidity at 45%. The wet granules were spread evenly on a sterile tray and placed in a vacuum drying oven, dried at 35℃ and 0.07 MPa for 5 hours. The dried granules were then fed into a granulator, granulated at 250 rpm using a 24-mesh sieve to remove fine powder and lumps, collecting uniform granules with a diameter of 0.9~1.1 mm. A fully automatic granulation filling machine was used, with a filling volume set to 0.8 g / bag. Oxygen-barrier and moisture-barrier aluminum-plastic composite bags were used, and nitrogen gas was introduced during filling to ensure a viable count of 7.2 × 10⁻⁶ bacteria per bag. 9 CFU; heat-sealed at 135℃ for 4 seconds after filling, with 5% silica gel desiccant inside.

[0062] The probiotic complex granules prepared in this example retained 87% of their live bacteria count after 12 months of storage at 4°C; the colonization rate in mouse feces was 1.3 × 10⁻⁶. 6 CFU / g; SAMP8 mice spent 34% longer in the target quadrant than the control group and 27% shorter in the escape latency period; serum LPA (14:0) increased by 36%, GABA increased by 23%, the proportion of Bifidobacteria in the intestinal tract of mice increased by 15% compared with the control group, and the total amount of serum glycerophospholipid metabolites increased by 30%.

[0063] Example 4 1) Activation culture of compound probiotic strains 1.5 portions of Lactobacillus apis BB1, 1 portion of Lactobacillus paracasei PS23, and 0.5 portions of Bifidobacterium bifidum BGN4 were inoculated into 60 portions of MRS / BHI medium and cultured at 37°C. Lactobacillus genus anaerobic culture was carried out for 24 hours (OD200). 600 =1.5), Bifidobacterium bifidum BGN45%CO226h (OD 600=1.4); 90 seed culture media were inoculated at a 5% inoculum rate, Lactobacillus was cultured for 12 h, and Bifidobacterium bifidum BGN4 was cultured for 16 h, with a viable count of 5 × 10⁻⁶ in the seed culture. 9 CFU / mL.

[0064] 2) Large-scale culture and cell collection of compound probiotics Inoculate 250 portions of fermentation medium at a CFU ratio of 3:2:1. Incubate at 37°C and pH 6.5 (adjusted by adding 1.2 mol / L sodium hydroxide). Purge Lactobacillus with nitrogen for 30 min and Bifidobacterium bifidum BGN with 45% CO2 for 30 h. The total viable count in the fermentation broth is 5 × 10⁻⁶. 10 CFU / mL; centrifuged at 3℃, 9000×g for 15 min, washed with 120 portions of sterile physiological saline, the bacterial sludge had a water content of 70%.

[0065] 3) Preparation of compound protectant and mixing with probiotics Mix 5 parts maltodextrin, 3 parts fructooligosaccharides, 1 part monosodium glutamate, and 0.5 parts sodium ascorbate with 120 parts deionized water and stir at 36°C to prepare a 17% protectant solution; sterilize at 115°C for 18 minutes and cool to 4°C; mix the bacterial sludge and protectant in a ratio of 1:2.2, and stir at 4°C and 150 rpm for 20 minutes; the viable count of the bacterial suspension is 9 × 10⁻⁶. 9 CFU / mL.

[0066] 4) Freeze-drying The bacterial suspension was dispensed into freeze-drying trays and pre-frozen at -38℃ for 2.5 hours until completely frozen. The trays were then transferred to a vacuum freeze dryer, and the vacuum level was controlled at 18 Pa and the shelf temperature at -52℃. The samples were freeze-dried for 27 hours until the moisture content was 2.4%, thus obtaining probiotic freeze-dried powder. The freeze-dried powder was then placed in a sterile environment and pulverized for 3 minutes using a high-speed pulverizer (9500 r / min). The powder was then passed through an 80-mesh sterile sieve to ensure that the powder particle size was ≤180 μm.

[0067] 5) Formulation The freeze-dried bacterial powder, filler, disintegrant (crospovidone, 5% of total mass), and sweetener (sucralose, 0.1% of total mass) were added to a three-dimensional motion mixer at a mass ratio of 13:1:0.5:0.01 and mixed at 70 r / min for 25 min. A 2% povidone K30 aqueous solution was added as a binder to form a soft mass, which was then granulated through a 16-mesh sieve and vacuum dried at 38℃ for 4 h until the particle moisture content was ≤3%. After granulation, magnesium stearate (0.8% of total mass) was added as a lubricant and mixed for 5 min. A rotary tablet press was used, with a tablet weight of 0.6 g / tablet, a tableting pressure of 8~10 MPa, and controlled ambient temperature of 20℃ and relative humidity of 42% to produce chewable tablets with a diameter of 10 mm and a hardness of 3~5 kgf, ensuring a viable count of 6.2 × 10⁻⁶ bacteria per tablet. 9 CFU; Pack qualified chewable tablets into aluminum-plastic blister packs (10 tablets per pack), with a blister forming temperature of 95℃ and a sealing pressure of 0.45MPa; then pack them into oxygen-barrier and moisture-barrier aluminum-plastic composite bags, with 10% silica gel desiccant inside.

[0068] The probiotic complex chewable tablets prepared in this example retained 89% of their live bacteria count after 12 months of storage at 4°C; the colonization rate in mouse feces was 1.3 × 10⁻⁶. 6 CFU / g; SAMP8 mice showed a 38% longer target quadrant dwell time and a 32% shorter escape latency compared to the control group; serum LPA (14:0) increased by 39%, GABA increased by 26%, serum inflammatory factor TNF-α decreased by 24%, serum BDNF increased by 36%, hippocampal synaptic plasticity-related protein expression increased by 28%, and gut microbiota diversity increased by 18%.

[0069] Example 5 1) Activation culture of compound probiotic strains First, one copy of *Lactobacillus apis* BB1, two copies of *Limosilactobacillus fermentum* A2.8, and one copy of *Lactobacillus paracasei* PS23 were selected and inoculated into 60 copies of MRS medium, respectively. They were then anaerobically cultured at 37°C for 22 h (OD200). 600 =1.4); then, 90 portions of seed culture medium (MRS / BHI) were inoculated at a 4% (v / v) inoculation rate, Lactobacillus was cultured for 13 h, and Bifidobacterium bifidum BGN4 was cultured for 17 h to obtain seed liquid.

[0070] 2) Large-scale culture and cell collection of compound probiotics First, 300 portions of fermentation medium were inoculated at a CFU ratio of 2:3:2, with 0.2% monosodium glutamate added to the medium. The culture was incubated at 37℃, pH 6.3 (adjusted by adding 1 mol / L lactic acid), with nitrogen purging for 45 min, for 27 h. The total viable cell count in the fermentation broth was 3.5 × 10⁻⁶. 10 CFU / mL, with 1.2 × 10⁻⁶ viable cells of Limosilactobacillus fermentum A2.8. 10 CFU / mL; then, centrifuge at 4℃, 8000×g for 15 min to collect the precipitate; finally, wash with 150 portions of sterile physiological saline (containing 0.1% monosodium glutamate), with a bacterial sludge moisture content of 73%.

[0071] 3) Preparation of compound protectant and mixing with probiotics A 18% protective agent solution was prepared by adding 5 parts maltodextrin, 3 parts fructooligosaccharides, 1.2 parts monosodium glutamate, and 0.5 parts sodium ascorbate to 120 parts deionized water and stirring at 35°C. The solution was then sterilized at 121°C for 15 minutes and cooled to 4°C. The mixture was then combined with the protective agent at a ratio of 1:2 and stirred at 120 rpm for 25 minutes at 4°C. The resulting bacterial suspension had a viable count of 8 × 10⁻⁶. 9 CFU / mL, of which Limosilactobacillus fermentum A2.8 accounted for 38%.

[0072] 4) Freeze-drying The bacterial suspension was dispensed into freeze-drying trays and pre-frozen at -40℃ for 2.5 hours until completely frozen. The trays were then transferred to a vacuum freeze dryer, and the vacuum level was controlled at 15 Pa and the shelf temperature at -50℃. The samples were freeze-dried for 26 hours until the moisture content was 2.3%, thus obtaining probiotic freeze-dried powder. The freeze-dried powder was then placed in a sterile environment and pulverized for 3 minutes using a high-speed pulverizer (10,000 r / min). The powder was then passed through an 80-mesh sterile sieve to ensure that the powder particle size was ≤180 μm.

[0073] 5) Formulation The freeze-dried bacterial powder and filler were added to a double cone mixer at a mass ratio of 11:1 and stirred at 60 r / min for 30 min, during which nitrogen gas was introduced for 30 s every 10 min. A fully automatic powder filling machine was used, and three-layer oxygen-barrier aluminum foil bags were selected. The filling amount was set to 1.5 g / bag, and the ambient temperature was controlled at 20℃ and the relative humidity at 35%. After filling, nitrogen gas (purity ≥99.99%) was first used to replace the air, and then the bags were sealed with a pulse heat sealer (first stage 120℃ / 2s, second stage 140℃ / 1.5s) to ensure a sealing strength ≥30N / 15mm.

[0074] The probiotic complex powder prepared in this embodiment retained 92% of its live bacteria count after 12 months of storage at 4°C; the colonization rate in mouse feces was 1.3 × 10⁻⁶. 6CFU / g; SAMP8 mice showed a 42% longer target quadrant dwell time and a 25% shorter escape latency compared to the control group; serum LPA (14:0) increased by 36%, GABA increased by 35%, mouse hypothalamic GABA receptor (GABAA) expression increased by 22% compared to the control group, serum inflammatory factor TNF-α content decreased by 25% compared to the control group, serum BDNF content increased by 38% compared to the control group, hippocampal glycerophosphatidylcholine content increased by 30%, and gut microbiota diversity increased by 18% compared to the control group.

[0075] Example 6 1) Activation culture of compound probiotic strains First, two portions of Lactobacillus apis BB1, 1.5 portions of Lactobacillus paracasei PS23, and one portion of Bifidobacterium bifidum BGN4 were selected and inoculated into 70 portions of MRS medium and 70 portions of BHI medium, respectively. The Lactobacillus spp. were then cultured anaerobically at 36°C for 19 hours (OD200). 600 =1.3), Bifidobacterium bifidum BGN cultured in 45% CO2 for 21 h (OD 600 =1.2); then, 105 portions of seed culture medium (containing 0.1% inulin) were inoculated at a 3% (v / v) inoculation rate, and Lactobacillus was cultured for 14 h, and Bifidobacterium bifidum BGN4 was cultured for 18 h to obtain seed culture (3.5 × 10⁻⁶ viable count). 9 (CFU / mL).

[0076] 2) Large-scale culture and cell collection of compound probiotics 400 portions of fermentation medium were inoculated with CFU at a ratio of 1:2:1, with 0.2% xylooligosaccharide added to the MRS / BHI medium. The culture was maintained at 36℃, pH 6.2 (adjusted by adding 0.8 mol / L sodium hydroxide), with nitrogen purging for 50 min for Lactobacillus and 45% CO2 for Bifidobacterium bifidum BGN, and cultured for 25 h. The total viable count in the fermentation broth was 2.8 × 10⁻⁶. 10 The concentration of CFU / mL was measured, and the acetic acid content in the fermentation broth was 12 mmol / L and the propionic acid content was 8 mmol / L. Then, the precipitate was collected by centrifugation at 5℃ and 7500×g for 15 min. Finally, the sludge was washed with 180 portions of sterile physiological saline, and the water content of the sludge was 76%.

[0077] 3) Preparation of compound protectant and mixing with probiotics A 17% protectant solution was prepared by dissolving 4 parts maltodextrin, 3 parts xylooligosaccharide, 1 part monosodium glutamate, and 0.5 parts sodium ascorbate in 150 parts deionized water and stirring at 34°C. The solution was then sterilized at 121°C for 16 minutes and cooled to 4°C. The mixture of mycelial sludge and protectant was then stirred at 130 rpm for 24 minutes at 4°C. The viable count in the mycelial suspension was 7 × 10⁻⁶. 9 CFU / mL.

[0078] 4) Freeze-drying The bacterial suspension was dispensed into freeze-drying trays and pre-frozen at -41℃ for 2.8 hours until completely frozen. The trays were then transferred to a vacuum freeze dryer, and the vacuum level was controlled at 12 Pa and the shelf temperature at -51℃. The freeze-dried powder was freeze-dried for 27 hours until the sample moisture content reached 2.6%. The freeze-dried powder was then placed in a sterile environment and pulverized for 2.5 minutes using a high-speed pulverizer (8000 r / min). The powder was then passed through a 70-mesh sterile sieve.

[0079] 5) Formulation Freeze-dried bacterial powder and filler were mixed at a mass ratio of 10:1, and 7% of 50% ethanol was added as a wetting agent. The mixture was stirred at 25 rpm for 10 minutes in a sterile kneader to form a soft mass. Granulation was carried out using a swing granulator with a 16-mesh sieve at 50 rpm. The wet granules were dried at 37℃ for 6 hours until the moisture content was 3.8%. After granulation, the particles were graded using a 20-mesh sieve, and particles with a diameter of 1.0~1.2 mm were collected. The granules were then filled into 1g bags using a standard aluminum-plastic bag filling machine, ensuring a viable count of 7.8 × 10⁻⁶ bacteria per bag. 9 CFU.

[0080] The probiotic complex granules prepared in this example retained 87% of their live bacteria count after 12 months of storage at 4°C; the colonization rate in mouse feces was 1.3 × 10⁻⁶. 6 CFU / g, fecal SCFA content increased by 35% compared with the control group; SAMP8 mice spent 36% longer in the target quadrant and 28% shorter in escape latency compared with the control group; serum anti-inflammatory factor IL-10 content increased by 32% and GABA increased by 24% compared with the control group; serum BDNF content increased by 32% compared with the control group.

[0081] Example 7 1) Activation culture of compound probiotic strains First, 1.5 portions of *Lactobacillus apis* BB1, 1.5 portions of *Limosilactobacillus fermentum* A2.8, 1 portion of *Lactobacillus paracasei* PS23, and 1 portion of *Bifidobacterium bifidum* BGN4 were selected and inoculated into 60 portions of MRS medium and 60 portions of BHI medium, respectively. The *Lactobacillus* genus was then cultured anaerobically at 37°C for 21 hours (OD200). 600 =1.4), Bifidobacterium bifidum BGN cultured in 45% CO2 for 23 h (OD 600 =1.3); then, 90 seed culture media were inoculated at a 5% (v / v) inoculation rate, Lactobacillus genus cultured for 12 h, and Bifidobacterium bifidum BGN4 cultured for 17 h to obtain seed liquid.

[0082] 2) Large-scale culture and cell collection of compound probiotics First, 350 portions of fermentation medium (MRS medium supplemented with 0.2% cysteine) were inoculated at a CFU ratio of 3:2:2:2. A dynamic pH control strategy was employed: pH was maintained at 5.0 for 0-12 h (with the addition of 1 mol / L lactic acid), and linearly increased to pH 6.5 for 12-24 h (with the addition of 1 mol / L sodium hydroxide). The culture was then carried out at 37°C for 28 h. HPLC-MS monitoring during fermentation showed that GAD gene expression was upregulated by 2.1 times at 12 h compared to the initial level, and the total viable cell count in the fermentation broth reached 4 × 10¹. 0 CFU / mL; then, centrifuge at 4℃ and 8500×g for 15 min to collect the precipitate; finally, wash with 160 portions of sterile physiological saline (containing 0.1% cysteine), and the water content of the bacterial sludge is 72%.

[0083] 3) Preparation of compound protectant and mixing with probiotics A 19% protectant solution was prepared by dissolving 5 parts maltodextrin, 2 parts fructooligosaccharides, 1 part monosodium glutamate, 0.6 parts sodium ascorbate, and 1.2 parts β-cyclodextrin in 130 parts deionized water and stirring at 36°C. The solution was then sterilized at 121°C for 14 min and cooled to 4°C. The mixture was then stirred at 140 rpm for 22 min at a ratio of 1:1.9 for the bacterial sludge and protectant, and analyzed by HPLC-MS. The protectant showed an 89.2% encapsulation rate for LPA (14:0) and a viable count of 9 × 10⁻⁶ cells in the bacterial suspension. 9 CFU / mL.

[0084] 4) Freeze-drying The bacterial suspension was dispensed into freeze-drying trays and pre-frozen at -39℃ for 2.6 hours until completely frozen. The trays were then transferred to a vacuum freeze dryer, and the vacuum was controlled at 16 Pa and the shelf temperature at -50℃. The freeze-dried powder was freeze-dried for 25 hours until the sample moisture content was 2.2%. The freeze-dried powder was then placed in a sterile environment and pulverized for 3 minutes using a high-speed pulverizer (10500 r / min). The powder was then passed through an 80-mesh sterile sieve, and the powder particle size was ≤150 μm.

[0085] 5) Formulation The freeze-dried bacterial powder and filler were added to a double cone mixer at a mass ratio of 14:1 and stirred at 65 rpm for 30 min, during which nitrogen gas (purity ≥99.99%) was introduced. Microcapsules (sodium alginate concentration 3%, chitosan concentration 0.5%) were prepared using the sharp-pore-coagulation bath method. After freeze-drying, the microcapsules were packaged in a three-layer oxygen barrier (oxygen permeability 0.08 cm³ / (m²·d·atm)). Accelerated testing at 40℃ / 75%RH showed that the viable bacterial retention rate reached 92.1% after 3 months.

[0086] The probiotic complex powder prepared in this embodiment retained 93% of its live bacteria count after 12 months of storage at 4°C; the colonization rate in mouse feces was 1.3 × 10⁻⁶. 6 CFU / g; SAMP8 mice showed a 48% longer target quadrant dwell time and a 38% shorter escape latency compared to the control group; serum oxidative stress marker MDA levels were 32% lower than the control group, serum LPA (14:0) levels were 38% higher, GABA levels were 30% higher, anti-inflammatory factor IL-10 levels were 38% higher, serum inflammatory factor TNF-α levels were 45% lower than the control group, hippocampal synaptophysin expression levels were 35% higher than the control group, intestinal tight junction protein expression levels were 32% higher, and gut microbiota index was 25% higher.

[0087] Example 8 1) Activation culture of compound probiotic strains First, two portions of *Lactobacillus apis* BB1, one portion of *Limosilactobacillus fermentum* A2.8, two portions of *Lactobacillus paracasei* PS23, and 0.8 portions of *Bifidobacterium bifidum* BGN4 were selected and inoculated into 80 portions of MRS medium and 80 portions of BHI medium, respectively. The *Lactobacillus* spp. were then cultured anaerobically at 38°C for 18 hours (OD200). 600 =1.3), Bifidobacterium bifidum BGN cultured in 45% CO2 for 20 h (OD 600=1.2); then, 120 portions of seed culture medium were inoculated at a 3% (v / v) inoculum rate, Lactobacillus was cultured for 15 h, and Bifidobacterium bifidum BGN4 was cultured for 19 h to obtain seed culture (3 × 10⁻⁶ viable cells). 9 CFU / mL, Lactobacillus paracaseiPS23 accounted for 40%.

[0088] 2) Large-scale culture and cell collection of compound probiotics First, 450 portions of fermentation medium were inoculated at a CFU ratio of 2:1:3:1. A dynamic pH control strategy was adopted: pH was maintained at 5.0 for 0-12 h (with the addition of 1 mol / L lactic acid), and linearly increased to pH 6.1 for 12-24 h (with the addition of 1 mol / L sodium hydroxide). The culture was then carried out at 37°C for 26 h, resulting in a total viable cell count of 3.2 × 10⁻⁶. 10 CFU / mL (Lactobacillus paracaseiPS23 content 45%); then, centrifuge at 3℃, 8000×g for 15 min to collect the precipitate; finally, wash with 200 parts of sterile physiological saline, the water content of the bacterial sludge is 74%.

[0089] 3) Preparation of compound protectant and mixing with probiotics A 16% protective agent solution was prepared by dissolving 6 parts maltodextrin, 3 parts fructooligosaccharides, 1 part monosodium glutamate, and 0.5 parts sodium ascorbate in 180 parts deionized water and stirring at 33°C. The solution was then sterilized at 121°C for 15 minutes and cooled to 3°C. The mixture of mycelial sludge and protective agent was then stirred at 110 rpm for 28 minutes at 3°C. The resulting bacterial suspension had a viable count of 7.5 × 10⁻⁶. 9 CFU / mL.

[0090] 4) Freeze-drying The bacterial suspension was dispensed into freeze-drying trays and pre-frozen at -42℃ for 3 hours until completely frozen. It was then transferred to a vacuum freeze dryer and freeze-dried for 28 hours according to a staged temperature increase program (first 8 hours at -52℃, middle 12 hours at -45℃, and last 8 hours at -35℃) until the sample moisture content was 1.8%. The freeze-dried powder was placed in a sterile environment and pulverized for 3 minutes using a high-speed pulverizer (10000 r / min). The powder was then passed through a 90-mesh sterile sieve, and the powder particle size was ≤160μm.

[0091] 5) Formulation The freeze-dried bacterial powder, filler, disintegrant (sodium carboxymethyl starch, 6% of total mass), and sweetener (stevioside, 0.2% of total mass) were added to a three-dimensional motion mixer at a mass ratio of 13:1:0.6:0.02 and mixed at 75 r / min for 28 min. A 3% polyethylene glycol 6000 ethanol solution was added as a binder to form a soft mass, which was then granulated through an 18-mesh sieve and vacuum dried at 36℃ for 4.5 h until the particle moisture content was ≤2.8%. After granulation, calcium stearate (0.7% of total mass) was added and mixed for 6 min. A rotary tablet press was used, with a tablet weight set at 0.7 g / tablet and a compression pressure of 9 MPa, to produce chewable tablets with a diameter of 12 mm and a hardness of 4-6 kgf, ensuring a viable count of 7.5 × 10⁻⁶ bacteria per tablet. 9 CFU(Lactobacillus paracasei PS23≥3.4×10 9 CFU / tablet); qualified chewable tablets are packed into aluminum-plastic blister packs, with a blister forming temperature of 100℃ and a sealing pressure of 0.5MPa, and then packaged in an aluminum-plastic composite bag (with a built-in composite desiccant).

[0092] The probiotic complex powder prepared in this example retained 91% of its live bacteria count after 12 months of storage at 4°C; the colonization rate in mouse feces was 1.3 × 10⁻⁶. 6 CFU / g; SAMP8 mice showed a 45% longer target quadrant dwell time and a 36% shorter escape latency compared to the control group; serum oxidative stress marker MDA content decreased by 30% compared to the control group, serum LPA (14:0) increased by 36%, anti-inflammatory factor TNF-α decreased by 26%, serum BDNF content increased by 42%, GABA increased by 28%, lipid peroxidation products in the mouse hippocampus decreased by 28%, and the proportion of antioxidant bacteria in the mouse gut increased by 22%.

[0093] This invention significantly enhances the creativity of probiotic complexes through four dimensions of innovation: 1) synergistic activation of metabolic pathways through strain ratios optimized using response surface methodology; 2) improved stability of lipid-soluble neuroactive substances through β-cyclodextrin encapsulation technology; 3) precise control of GABA / LPA synthesis throughput through dynamic pH regulation; and 4) a microencapsulation-packaging synergistic system to solve the long-term storage problem of probiotics. These technical features support each other, forming a complete technical solution from strain synergy to formulation stability, significantly superior to existing probiotic preparation technologies that optimize single functions. Example verification shows that the probiotic complex prepared by this method meets the technical requirements for clinical health applications in terms of memory improvement, stability, and colonization ability, and has significant industrialization value.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a probiotic complex for improving memory, characterized in that, Includes the following steps: S1: Inoculate the compound strains into their respective activation media and culture them until the OD of each strain reaches its maximum. 600 The value reaches 1.0~2.0 to obtain an activated strain; then the activated strain is inoculated into the corresponding seed culture medium and cultured until the strain is in the logarithmic growth phase to obtain the strain seed liquid; the compound strain consists of Lactobacillus apis BB1, Limosilactobacillus fermentum A2.8, Lactobacillus paracasei PS23 and Bifidobacterium bifidum BGN4. S2: Inoculate the seed culture of each strain into the fermentation medium and culture until the total viable count in the fermentation broth reaches 1×10⁻⁶. 10 ~8×10 10 CFU / mL; then the fermentation broth was centrifuged, the cell precipitate was collected, and the cell precipitate was washed several times with sterile physiological saline to obtain pure bacterial sludge; S3: Dissolve the composite protectant in deionized water, sterilize and cool to obtain a composite protectant solution; then stir and mix the composite protectant solution with pure bacterial mud to obtain a bacterial suspension. S4: After pre-freezing, freeze-drying, pulverizing and sieving the bacterial suspension, freeze-dried powder is obtained. Then, filler is added to obtain mixed powder. Finally, the mixed powder is prepared into a dosage form to obtain the probiotic complex that improves memory.

2. The method for preparing a probiotic complex for improving memory according to claim 1, characterized in that, During cultivation, Lactobacillus apis BB1, Limosilactobacillus fermentum A2.8, and Lactobacillus paracasei PS23 were cultured on MRS medium under anaerobic conditions for 18–24 h; Bifidobacterium bifidum BGN4 was cultured on BHI medium under CO2 conditions with a volume concentration of 5%–8% for 20–26 h.

3. The method for preparing a probiotic complex for improving memory according to claim 1, characterized in that, In step S2, the seed culture of each strain is inoculated into the fermentation medium according to the CFU ratio of the four strains as (1~5):(1~5):(1~5):(1~5).

4. The method for preparing a probiotic complex for improving memory according to claim 1, characterized in that, In step S2, the seed culture of each strain is inoculated into the fermentation medium according to the CFU ratio of the four strains being 3:2:2:

2.

5. The method for preparing a probiotic complex for improving memory according to claim 1, characterized in that, Seed culture of each strain was inoculated into fermentation medium and cultured in two stages. The first stage was cultured for 0-12 hours, maintaining a pH of 5.0 and a temperature of 36-38℃. The second stage was cultured for 12-24 hours, controlling the pH to rise linearly to 6.5 and maintaining a temperature of 36-38℃.

6. The method for preparing a probiotic complex for improving memory according to claim 1, characterized in that, In step S3, the composite protective agent is composed of maltodextrin, fructooligosaccharides, monosodium glutamate and sodium ascorbate; by mass parts, the ratio of maltodextrin, fructooligosaccharides, monosodium glutamate and sodium ascorbate is (3~8):(1.5~4):(0.6~1.5):(0.3~0.8).

7. The method for preparing a probiotic complex for improving memory according to claim 1, characterized in that, In step S3, the composite protective agent is composed of maltodextrin, fructooligosaccharides, monosodium glutamate, sodium ascorbate, and β-cyclodextrin. The ratio of maltodextrin, fructooligosaccharides, monosodium glutamate, sodium ascorbate, and β-cyclodextrin is 5:2:1:0.6:1.2 by mass.

8. The method for preparing a probiotic complex for improving memory according to claim 1, characterized in that, In step S3, the ratio of the pure bacterial mud to the composite protective agent solution is 1:(1.8~2.2) by mass.

9. The method for preparing a probiotic complex for improving memory according to claim 1, characterized in that, In step S4, during the pre-freezing process, the temperature is -25~-45℃ and the time is 2~3h, more preferably, the temperature is -39~-42℃ and the time is 2.5h; during the freeze-drying process, the vacuum degree is 15~30Pa, the shelf temperature is -48~-52℃ and the time is 24~30h.

10. A probiotic complex for improving memory, characterized in that, It is prepared by the method described in any one of claims 1 to 9.