Probiotic preparation for preventing and treating Alzheimer's disease and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a probiotic preparation for preventing and treating Alzheimer's disease and a preparation method thereof. The probiotic powder comprises the following raw materials in parts by weight: 5 to 6 parts of probiotic freeze-dried powder, 5 to 7 parts of whey protein powder, 8 to 10 parts of pullulan, 2.5 to 3.5 parts of quaternary ammonium salt modified chitosan, 35 to 40 parts of trehalose, 10 to 12 parts of maltodextrin, 10 to 12 parts of prebiotics, 3 to 3.5 parts of glycine, 1.3 to 1.8 parts of glycerol, 4.5 to 6 parts of sodium alginate, 1.2 to 1.8 parts of L-glutamine, 3 to 3.5 parts of fructo-oligosaccharide and 1 to 1.2 parts of lecithin. The probiotic preparation provided by the invention contains lactobacillus rhamnosus, bifidobacterium longum and lactobacillus plantarum, and the inner layer is a vitrification freeze-drying protection matrix of trehalose / protein, so that freeze-drying stability and rehydration activity are ensured; the outer layer is a sodium alginate / quaternized chitosan electrostatic self-assembly delivery system, so that gastric acid protection and small intestine targeted release are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a probiotic preparation for the prevention and treatment of Alzheimer's disease and its preparation method. Background Technology

[0002] Alzheimer's disease is a progressive neurodegenerative disease. Current medications primarily focus on improving neurotransmitters or managing symptoms, making it difficult to fundamentally alter the disease's progression. In recent years, the link between the gut microbiota and the central nervous system has become increasingly clear, and the role of the gut-brain axis in cognitive function, inflammatory responses, and metabolic homeostasis has attracted attention. Nutritional interventions centered on the gut microbiota are gradually becoming a potential direction for cognitive impairment management; however, a significant gap remains between basic research and commercially viable formulations.

[0003] Probiotic preparations are widely used in the food and health care industries, but existing products are mostly positioned for digestive relief and general immune regulation, and the selection and compatibility principles for strains targeting cognitive impairment are still imperfect. Different strains have varying tolerance characteristics to processing and storage conditions and the gastrointestinal environment. Without targeted protection and delivery design, the activity of the preparation is prone to decline during storage, reconstitution, and passage through the stomach, thus affecting its efficacy after reaching the small intestine. Existing freeze-dried probiotics mostly use single or small amounts of protective agents (such as sucrose, trehalose, skim milk powder, etc.), and the stress control at each stage of freezing nucleation, sublimation drying, and rehydration is relatively crude. Common problems include: difficulty in stably controlling the size and distribution of ice crystals during pre-freezing; insufficient solid network support during sublimation leading to an increased risk of structural collapse; and large changes in osmotic gradient during rehydration, which can easily induce cellular stress. These factors combined reduce the number of viable bacteria and functional retention after rehydration. On the other hand, single carbohydrate protection systems have limited ability to synergistically protect the interface and membrane structure, making it difficult to meet the consistent protection needs of multiple strains. Therefore, given the aforementioned shortcomings, it is extremely necessary to develop a stable, high-survival-rate, slow-release, and highly compatible probiotic preparation for the prevention and treatment of Alzheimer's disease. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a probiotic preparation for the prevention and treatment of Alzheimer's disease and its preparation method. To solve the problems of poor live bacteria stability and low colonization efficiency after administration in existing probiotic preparations for Alzheimer's disease intervention, this invention proposes a technical solution that uses *Lactobacillus rhamnosus*, *Bifidobacterium longum*, and *Lactobacillus plantarum* as core bacteria, employing a multi-bacterial synergistic mechanism to act on the gut-brain axis, thereby achieving multi-pathway regulation of cognitive impairment in Alzheimer's disease. By introducing a vitrification protective agent system mainly composed of trehalose, combined with a ternary synergistic system of whey protein and glycine, the probiotic cell membrane forms a glass-like stable structure during freeze-drying, avoiding cell membrane rupture and protein denaturation caused by the mechanical stress of ice crystals. This achieves the technical effects of improving the freeze-dried survival rate of probiotics and enhancing their storage stability at room temperature.

[0005] The technical effects described in this invention are achieved through the following technical solution: a probiotic preparation for preventing and treating Alzheimer's disease, comprising the following raw materials: 5-6 parts of probiotic freeze-dried powder, 5-7 parts of whey protein powder, 8-10 parts of pullulan, 2.5-3.5 parts of quaternary ammonium salt modified chitosan, 35-40 parts of trehalose, 10-12 parts of maltodextrin, 10-12 parts of prebiotics, 3-3.5 parts of glycine, 1.3-1.8 parts of glycerol, 4.5-6 parts of sodium alginate, 1.2-1.8 parts of L-glutamine, 3-3.5 parts of fructooligosaccharides, and 1-1.2 parts of lecithin.

[0006] Preferably, the probiotic freeze-dried powder is prepared from Lactobacillus rhamnosus, Bifidobacterium longum and Lactobacillus plantarum in a mass ratio of 5:3:2; Preferably, the activity of the *Lactobacillus rhamnosus* is 0.5–1 × 10⁻⁶. 10 CFU / g; the activity of the Bifidobacterium longum is 1–5 × 10⁻⁶. 9 CFU / g; the activity of the *Lactobacillus plantarum* is 1–3 × 10⁻⁶. 9 CFU / g; Preferably, the prebiotic is any one of fructooligosaccharides, resistant dextrin, and inulin; more preferably, it is resistant dextrin. Preferably, the preparation steps of the quaternary ammonium salt modified chitosan are as follows: S1: Dissolve chitosan in 0.5% glacial acetic acid, stir at 400 rpm for 1-2 hours, then heat to 45-50℃, adjust the pH to 8-8.5 with 1M NaOH solution, stir at constant temperature for 30 minutes, slowly add glycidyl trimethylammonium chloride, and continue stirring for 6-8 hours. S2: After the reaction in step S1 is completed, cool to room temperature, adjust pH to 6.5-7, add an equal volume of 85wt% ethanol, let stand for 30-60 min, centrifuge, wash repeatedly with ethanol, resuspend in pure water and dialyze for 24 h, dry in stages to obtain quaternary ammonium salt modified chitosan. Preferably, in step S1, the ratio of chitosan, glacial acetic acid, and glycidyltrimethylammonium chloride is 0.5-0.8 g: 20 mL: 2-3 mL; Preferably, in step S1, the glycidyl trimethylammonium chloride is added dropwise over 60 minutes; Preferably, in step S2, the molecular weight cutoff for dialysis is 3-5 kDa; Preferably, in step S2, the staged drying operation is as follows: under vacuum conditions, pre-freezing at -40℃ for 2-3 hours, drying at -35℃ for 12 hours, and drying at 20℃ for 6-8 hours; Preferably, another aspect of the present invention provides a method for preparing a probiotic preparation for preventing and treating Alzheimer's disease, comprising the following steps: S101: Disperse whey protein powder in pure water and stir at 300 rpm for 20-30 min. Then add pullulan and continue stirring for 20-30 min. Adjust the pH to neutral with 0.5 M NaOH solution. Under nitrogen protection, heat to 55-65℃ and stir for 2-3 h. Cool to room temperature and pre-cool at 4℃ for 2 h. Control the total solid content to 10% and set aside. S102: Add trehalose, maltodextrin, and prebiotics to pure water in sequence, stir and mix until dissolved; then add the solution from step S101, stir and mix until dissolved, then add glycine and glycerol in sequence, adjust the pH to 6.8-7.2 with 0.5M NaOH solution and 0.5M HCl solution, pre-cool at 4℃ for 2 hours, degas under vacuum for 10-15 minutes, control the total solid content to 20-30%, and set aside. S103: Slowly cool the solution from step S102 to -40℃, keep it at that temperature for 2-3 hours, cool it to -35--40℃ and vacuum dry it for 2-4 hours, then heat it to 20-25℃ and vacuum dry it for 6-10 hours, and then pass it through an 80-180 mesh sieve to obtain vitrified matrix powder. S104: Dissolve 85% of the total weight of sodium alginate in pure water, adjust the pH to neutral, and then add L-glutamine, fructooligosaccharides and lecithin in sequence. Pre-emulsify under ice bath conditions at high speed of 5000-7000 rpm for 3-5 min to obtain an emulsion. S105: Slowly add the emulsion from step S104 dropwise to a 30 mM CaCl2 solution, stir at 100–200 rpm for 10–15 min, centrifuge, filter, and resuspend the wet particles in a 0.1 wt% quaternary ammonium salt modified chitosan solution. Adjust the pH to 5.8–6.2 with 0.1 M HCl solution, stir gently at 200 rpm for 10–15 min, centrifuge, filter, and resuspend the wet particles in a 0.1 wt% sodium alginate aqueous solution. Stir gently at 200 rpm for 10–15 min, sonicate under ice bath, centrifuge, filter, and obtain LbL self-assembled microparticles; S106: Freeze-dry the LbL self-assembled microparticles from step S105 to obtain synergistic layer powder; under a nitrogen atmosphere, add the synergistic layer powder and the vitrified matrix powder from step S103 into a mixer, premix at 10-15 rpm for 8-10 min, add the probiotic freeze-dried powder and mix again for 5-8 min to obtain the probiotic preparation. Preferably, in step S103, the cooling parameter is: cooling at a rate of 0.5 to 1 °C / min; the heating parameter is: heating at a rate of 0.25 to 0.35 °C / min. Preferably, in step S105, the emulsion is added at a rate of 0.5–2 mL / min and at a height of 2–5 cm. Preferably, in step S105, the quaternary ammonium salt modified chitosan solution is prepared by dissolving quaternary ammonium salt modified chitosan in a 0.5% aqueous acetic acid solution; Preferably, in step S105, the sodium alginate aqueous solution is prepared by dissolving the remaining parts by weight of sodium alginate in pure water; Preferably, in step S105, the ultrasonic-assisted processing parameters are: power 100-200W, frequency 20kHz, 2s on / 2s off, and time 60-120s. Preferably, in step S106, the freeze-drying process is as follows: add 5% trehalose, cool to -40℃ at a rate of 0.8-1.2℃ / min for 2 hours to pre-freeze, cool to -35℃ for 4-6 hours to vacuum dry, and heat to 20℃ at a rate of 0.25-0.35℃ / min for 6-8 hours to vacuum dry.

[0007] The beneficial effects of this invention are as follows: This invention utilizes *Lactobacillus rhamnosus*, *Bifidobacterium longum*, and *Lactobacillus plantarum* to construct a composite probiotic strain group. *Lactobacillus rhamnosus*, as the core strain, exhibits significant advantages in regulating neurotransmitter balance and mediating brain-gut signal feedback. *Bifidobacterium longum* helps maintain intestinal mucosal structural stability and blocks the invasion of peripheral inflammatory factors into the central nervous system. *Lactobacillus plantarum*, with its broad-spectrum metabolic capacity, promotes the production of short-chain fatty acids, providing stable metabolic support for the enteric nervous system. The three strains complement each other in metabolic pathways, ecological adaptability, and colonization sites, improving the survival ability of the formulation in the complex intestinal environment and enhancing the overall biological effect, overcoming the functional limitations commonly found in single-strain applications.

[0008] This invention uses an inner vitrified matrix as the main freeze-drying protective structure, with trehalose providing the vitrified matrix. Whey protein and pullulan, after mild hygrothermal glycosylation, form an interfacial protective layer. Low-concentration glycerol and glycine are used as permeation and membrane stabilizing agents. By controlling the pre-freezing cooling rate and secondary drying conditions, the impact of freezing and drying processes on the temperature and osmotic pressure of probiotics is reduced, minimizing the risk of inactivation caused by ice crystals and drying stress. Rehydration facilitates the formation of a stable hydration environment, thereby improving freeze-drying stability and activity retention after rehydration. Regarding outer layer protection and in vivo release, sodium alginate and calcium ions first form a gel core, followed by electrostatic layer-by-layer self-assembly of chitosan with quaternized sodium alginate to obtain a composite barrier structure. This structure maintains good integrity under acidic conditions, providing protection in the stomach. Upon entering the small intestine, with changes in pH and ionic strength, as well as calcium ion exchange, the gel and composite layer gradually swell and partially depolymerize, promoting release and delivery in the small intestine. The quaternized cationic properties of chitosan allow for gentle electrostatic interactions with anionic groups on the intestinal mucosa surface, improving local retention and contact efficiency. Introducing a small amount of lecithin into the carrier phase to form lipid microdomains enhances compatibility and synergistic effects after rehydration, gently encapsulating compatible small molecule components (such as glutamine and fructooligosaccharides). After rehydration, these microdomains, along with the outer composite network, influence the diffusion pathway, achieving synergistic release that matches the release of live bacteria, thus balancing prebiotic supply and local microecological support. In terms of processing, the vitrified matrix powder and the layered self-assembled synergistic layers are independently freeze-dried. The final layer is then dry-blended with the freeze-dried probiotic powder under low-shear, low-humidity, and low-oxygen conditions. This helps avoid compatibility conflicts when processing multi-layered structures in the same container and reduces the chance of live bacteria being directly exposed to freeze-drying, acidic / alkaline, and high-ionic-strength environments. After oral administration, the formula undergoes hydration, swelling, and ion exchange processes in sequence according to the characteristics of its materials. This manifests as a continuous synergy of freeze-drying protection, gastric tolerance, and small intestinal release, thereby improving the survival, delivery, and early-stage efficacy of probiotics in vivo from both material and process perspectives. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 These are graphs showing the rehydration test results of the probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-3 of this invention; Figure 2 This is a graph showing the changes in CFU activity in the stability test of probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-3 of this invention; Figure 3 This is a graph showing the change in moisture content during stability testing of probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-3 of this invention; Figure 4 This is a graph showing the results of gastric segment simulated tolerance tests on probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-3 of this invention; Figure 5 The graph shows the results of intestinal segment simulated tolerance tests of probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-3 of this invention. Detailed Implementation

[0011] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It should be noted that, unless otherwise specified, the raw materials involved in the present invention are all purchased through conventional commercial channels.

[0012] Example 1: A probiotic preparation for preventing and treating Alzheimer's disease, comprising the following ingredients: 5.7 parts of lyophilized probiotic powder, 6.5 parts of whey protein powder, 9 parts of pullulan, 3 parts of quaternary ammonium salt modified chitosan, 38 parts of trehalose, 11 parts of maltodextrin, 10.5 parts of prebiotics, 3.2 parts of glycine, 1.6 parts of glycerol, 5.2 parts of sodium alginate, 1.5 parts of L-glutamine, 3.2 parts of fructooligosaccharides, and 1.1 parts of lecithin.

[0013] The probiotic freeze-dried powder was prepared by Lactobacillus rhamnosus, Bifidobacterium longum and Lactobacillus plantarum in a mass ratio of 5:3:2. The activity of the Lactobacillus rhamnosus was 0.8 × 10⁻⁶. 10 CFU / g; the activity of the Bifidobacterium longum is 3×10⁻⁶. 9 CFU / g; the activity of the *Lactobacillus plantarum* was 2 × 10⁻⁶.9 CFU / g; The preparation steps of the quaternary ammonium salt modified chitosan are as follows: S1: Dissolve 3.5g of chitosan in 100mL of 0.5% glacial acetic acid, stir at 400rpm for 1.5h, then heat to 48℃, adjust the pH to 8.3 with 1M NaOH solution, stir at constant temperature for 30min, slowly add 13mL of glycidyltrimethylammonium chloride over 60min, and continue stirring for 7h. S2: After the reaction in step S1 is completed, cool to room temperature, adjust pH to 6.7, add an equal volume of 85wt% ethanol, let stand for 50 min, centrifuge, wash repeatedly with ethanol, resuspend in pure water, dialyze for 24 h with a molecular weight cutoff of 3.5 kDa, pre-freeze at -40℃ for 2.5 h under vacuum, dry at -35℃ for 12 h, and dry at 20℃ for 7 h to obtain quaternary ammonium salt modified chitosan. The preparation of probiotic formulations for preventing and treating Alzheimer's disease includes the following steps: S101: Disperse whey protein powder in pure water, stir at 300 rpm for 25 min, then add pullulan and continue stirring for 25 min. Adjust the pH to neutral with 0.5 M NaOH solution. Under nitrogen protection, heat to 60℃ and stir for 2.5 h. Cool to room temperature, pre-cool at 4℃ for 2 h, and control the total solid content to 10%. S102: Add trehalose, maltodextrin and resistant dextrin to pure water in sequence, stir and mix until dissolved; then add the solution from step S101, stir and mix until dissolved, then add glycine and glycerol in sequence, adjust the pH to 7 with 0.5M NaOH solution and 0.5M HCl solution, pre-cool at 4℃ for 2 hours, degas under vacuum for 12 minutes, control the total solid content to 25%, and set aside. S103: The solution from step S102 is cooled to -40℃ at a rate of 0.8℃ / min, kept at this temperature for 2.5h, and then vacuum dried at -40℃ for 2h. After that, the temperature is increased to 25℃ at a rate of 0.3℃ / min and vacuum dried for 6h. The solution is then sieved through a 180-mesh screen to obtain the vitrified matrix powder. S104: Dissolve 85% of the total weight of sodium alginate in pure water, adjust the pH to neutral, and then add L-glutamine, fructooligosaccharides and lecithin in sequence. Pre-emulsify by high-speed shearing at 6000 rpm for 4 minutes in an ice bath to obtain an emulsion. S105: The emulsion from step S104 was slowly added dropwise to 4 times its volume of 30 mM CaCl2 solution at a drop height of 4 cm and a speed of 1 mL / min. The mixture was stirred at 150 rpm for 12 min, centrifuged, filtered, and the wet particles were resuspended in 0.1 wt% quaternary ammonium salt modified chitosan solution. The pH was adjusted to 6 with 0.1 M HCl solution, and the mixture was gently stirred at 200 rpm for 12 min. The mixture was centrifuged, filtered, and the wet particles were resuspended in 0.1 wt% sodium alginate aqueous solution. The mixture was gently stirred at 200 rpm for 12 min, and then sonicated under ice bath conditions at a power of 150 W, a frequency of 20 kHz, a 2 s on / 2 s off cycle, and a time of 100 s. The mixture was centrifuged and filtered to obtain LbL self-assembled microparticles. S106: The LbL self-assembled microparticles from step S105 are freeze-dried, 5% trehalose is added, and the mixture is pre-frozen at -40℃ for 2 hours at a rate of 1℃ / min, then pre-dried under vacuum at -35℃ for 5 hours, and then vacuum-dried at 20℃ for 7 hours at a rate of 0.3℃ / min to obtain the synergistic layer powder. Under a nitrogen atmosphere, the synergistic layer powder and the vitrified matrix powder from step S103 are added to a mixer and pre-mixed at 12 rpm for 9 minutes. After adding the probiotic freeze-dried powder, the mixture is mixed again for 7 minutes to obtain the probiotic preparation.

[0014] Example 2: A probiotic preparation for preventing and treating Alzheimer's disease, comprising the following ingredients: 5 parts probiotic freeze-dried powder, 5 parts whey protein powder, 8 parts pullulan, 2.5 parts quaternary ammonium salt modified chitosan, 35 parts trehalose, 10 parts maltodextrin, 10 parts prebiotics, 3 parts glycine, 1.3 parts glycerol, 4.5 parts sodium alginate, 1.2 parts L-glutamine, 3 parts fructooligosaccharides, and 1 part lecithin.

[0015] The probiotic freeze-dried powder was prepared by Lactobacillus rhamnosus, Bifidobacterium longum and Lactobacillus plantarum in a mass ratio of 5:3:2. The activity of the Lactobacillus rhamnosus was 0.5 × 10⁻⁶. 10 CFU / g; the activity of the Bifidobacterium longum is 1×10⁻⁶. 9 CFU / g; the activity of the *Lactobacillus plantarum* was 1×10⁻⁶. 9 CFU / g; The preparation steps of the quaternary ammonium salt modified chitosan are as follows: S1: Dissolve 2.5g of chitosan in 100mL of 0.5% glacial acetic acid, stir at 400rpm for 1h, then heat to 45℃, adjust the pH to 8.5 with 1M NaOH solution, stir at constant temperature for 30min, slowly add 10mL of glycidyl trimethylammonium chloride over 60min, and continue stirring for 8h. S2: After the reaction in step S1 is completed, cool to room temperature, adjust pH to 6.5, add an equal volume of 85wt% ethanol, let stand for 30 min, centrifuge, wash repeatedly with ethanol, resuspend in pure water, dialyze with a molecular weight cutoff of 5kDa for 24 h, pre-freeze at -40℃ for 2 h under vacuum, dry at -35℃ for 12 h, and dry at 20℃ for 6 h to obtain quaternary ammonium salt modified chitosan. The preparation of probiotic formulations for preventing and treating Alzheimer's disease includes the following steps: S101: Disperse whey protein powder in pure water, stir at 300 rpm for 20 min, then add pullulan and continue stirring for 20 min. Adjust the pH to neutral with 0.5 M NaOH solution. Under nitrogen protection, heat to 55℃, stir and react for 3 h, cool to room temperature, pre-cool at 4℃ for 2 h, and control the total solid content to 10%. S102: Add trehalose, maltodextrin and inulin to pure water in sequence, stir and mix until dissolved; then add the solution from step S101, stir and mix until dissolved, then add glycine and glycerol in sequence, adjust the pH to 6.8 with 0.5M NaOH solution and 0.5M HCl solution, pre-cool at 4℃ for 2 hours, degas under vacuum for 10 minutes, control the total solid content to 20%, and set aside. S103: The solution from step S102 is cooled to -40℃ at a rate of 0.5℃ / min, kept at that temperature for 2 hours, cooled to -35℃ and vacuum dried for 4 hours, then heated to 20℃ at a rate of 0.25℃ / min and vacuum dried for 10 hours, and then sieved through an 80-mesh screen to obtain vitrified matrix powder. S104: Dissolve 85% of the total weight of sodium alginate in pure water, adjust the pH to neutral, and then add L-glutamine, fructooligosaccharides and lecithin in sequence. Pre-emulsify by high-speed shearing at 5000 rpm for 5 min in an ice bath environment to obtain an emulsion. S105: The emulsion from step S104 was slowly added dropwise to 4 times its volume of 30 mM CaCl2 solution at a height of 2 cm and a speed of 0.5 mL / min. The mixture was stirred at 100 rpm for 15 min, centrifuged, filtered, and the wet particles were resuspended in 0.1 wt% quaternary ammonium salt modified chitosan solution. The pH was adjusted to 5.8 with 0.1 M HCl solution, and the mixture was gently stirred at 200 rpm for 10 min. The mixture was centrifuged, filtered, and the wet particles were resuspended in 0.1 wt% sodium alginate aqueous solution. The mixture was gently stirred at 200 rpm for 10 min, and the mixture was subjected to ultrasonic treatment under ice bath conditions at a power of 100 W, a frequency of 20 kHz, a 2 s on / 2 s off cycle, and a time of 120 s. The mixture was centrifuged and filtered to obtain LbL self-assembled microparticles. S106: The LbL self-assembled microparticles from step S105 are freeze-dried, 5% trehalose is added, and the mixture is pre-frozen at -40℃ for 2 hours at a rate of 0.8℃ / min, then vacuum-dried at -35℃ for 4 hours, and vacuum-dried at 20℃ for 8 hours at a rate of 0.25℃ / min to obtain the synergistic layer powder. Under a nitrogen atmosphere, the synergistic layer powder and the vitrified matrix powder from step S103 are added to a mixer and pre-mixed at 10 rpm for 10 minutes. After adding the probiotic freeze-dried powder, the mixture is mixed again for 5 minutes to obtain the probiotic preparation.

[0016] Example 3: A probiotic preparation for preventing and treating Alzheimer's disease, comprising the following ingredients: 6 parts of lyophilized probiotic powder, 7 parts of whey protein powder, 10 parts of pullulan, 3.5 parts of quaternary ammonium salt modified chitosan, 40 parts of trehalose, 12 parts of maltodextrin, 12 parts of prebiotics, 3.5 parts of glycine, 1.8 parts of glycerol, 6 parts of sodium alginate, 1.8 parts of L-glutamine, 3.5 parts of fructooligosaccharides, and 1.2 parts of lecithin.

[0017] The probiotic freeze-dried powder was prepared by Lactobacillus rhamnosus, Bifidobacterium longum and Lactobacillus plantarum in a mass ratio of 5:3:2. The activity of the Lactobacillus rhamnosus is 1×10⁻⁶. 10 CFU / g; the activity of the Bifidobacterium longum is 5×10⁻⁶. 9 CFU / g; the activity of the *Lactobacillus plantarum* was 3 × 10⁻⁶. 9 CFU / g; The preparation steps of the quaternary ammonium salt modified chitosan are as follows: S1: Dissolve 4g of chitosan in 100mL of 0.5% glacial acetic acid, stir at 400rpm for 2h, then heat to 50℃, adjust pH to 8 with 1M NaOH solution, stir at constant temperature for 30min, slowly add 15mL of glycidyltrimethylammonium chloride over 60min, and continue stirring for 6h. S2: After the reaction in step S1 is completed, cool to room temperature, adjust pH to 7, add an equal volume of 85wt% ethanol, let stand for 60 min, centrifuge, wash repeatedly with ethanol, resuspend in pure water, dialyze with a molecular weight cutoff of 3kDa for 24 h, pre-freeze at -40℃ for 3 h under vacuum, dry at -35℃ for 12 h, and dry at 20℃ for 8 h to obtain quaternary ammonium salt modified chitosan. The preparation of probiotic formulations for preventing and treating Alzheimer's disease includes the following steps: S101: Disperse whey protein powder in pure water, stir at 300 rpm for 30 min, then add pullulan and continue stirring for 30 min. Adjust the pH to neutral with 0.5 M NaOH solution. Under nitrogen protection, heat to 65℃, stir and react for 3 h, cool to room temperature, pre-cool at 4℃ for 2 h, and control the total solid content to 10%. S102: Add trehalose, maltodextrin and resistant dextrin to pure water in sequence, stir and mix until dissolved; then add the solution from step S101, stir and mix until dissolved, then add glycine and glycerol in sequence, adjust the pH to 7.2 with 0.5M NaOH solution and 0.5M HCl solution, pre-cool at 4℃ for 2 hours, degas under vacuum for 15 minutes, control the total solid content to 30%, and set aside. S103: The solution from step S102 is cooled to -40℃ at a rate of 1℃ / min, kept at that temperature for 3h, cooled to -38℃ and vacuum dried for 3h, then heated to 24℃ at a rate of 0.35℃ / min and vacuum dried for 8h, and then sieved through a 150-mesh screen to obtain vitrified matrix powder. S104: Dissolve 85% of the total weight of sodium alginate in pure water, adjust the pH to neutral, and then add L-glutamine, fructooligosaccharides and lecithin in sequence. Pre-emulsify by high-speed shearing at 7000 rpm for 3 minutes in an ice bath to obtain an emulsion. S105: The emulsion from step S104 was slowly added dropwise to 4 times its volume of 30 mM CaCl2 solution at a drop height of 5 cm and a speed of 2 mL / min. The mixture was stirred at 200 rpm for 10 min, centrifuged, filtered, and the wet particles were resuspended in 0.1 wt% quaternary ammonium salt modified chitosan solution. The pH was adjusted to 6.2 with 0.1 M HCl solution, and the mixture was gently stirred at 200 rpm for 15 min. The mixture was centrifuged, filtered, and the wet particles were resuspended in 0.1 wt% sodium alginate aqueous solution. The mixture was gently stirred at 200 rpm for 15 min, and then sonicated under ice bath conditions at a power of 200 W, a frequency of 20 kHz, a 2 s on / 2 s off cycle, and a time of 60 s. The mixture was centrifuged and filtered to obtain LbL self-assembled microparticles. S106: The LbL self-assembled microparticles from step S105 are freeze-dried, 5% trehalose is added, and the mixture is pre-frozen at -40℃ for 2 hours at a rate of 1.2℃ / min, then pre-dried under vacuum at -35℃ for 6 hours, and then vacuum-dried at 20℃ for 8 hours at a rate of 0.35℃ / min to obtain the synergistic layer powder. Under a nitrogen atmosphere, the synergistic layer powder and the vitrified matrix powder from step S103 are added to a mixer and pre-mixed at 15 rpm for 8 minutes. After adding the probiotic freeze-dried powder, the mixture is mixed again for 8 minutes to obtain the probiotic preparation.

[0018] Comparative Example 1: The operation process of Comparative Example 1 is basically the same as that of Example 1. The specific difference is that unmodified chitosan is used instead of quaternary ammonium salt modified chitosan in Comparative Example 1, while the rest of the operation process remains the same.

[0019] Comparative Example 2: The operation process of Comparative Example 2 is basically the same as that of Example 1. The specific difference is that unglycosylated whey protein and pullulan are used in Comparative Example 2. Specifically, the whey protein powder and pullulan in step S101 are directly added to step S102 for mixing, and the rest of the operation process remains the same.

[0020] Comparative Example 3: The operation process of Comparative Example 3 is basically the same as that of Example 1. The specific difference is that the lyophilization operation of the vitrification matrix powder in step S103 is cancelled in Comparative Example 3, and the solution in step S102 is kept for later use; the lyophilization treatment in step S106 is cancelled; the LbL self-assembled microparticles, the solution in step S102, and the probiotic lyophilized powder are directly mixed and then lyophilized. 5% trehalose is added, and the mixture is pre-frozen at -40°C for 2 hours at a rate of 1°C / min, then pre-dried under vacuum at -35°C for 5 hours, and then vacuum dried at 20°C at a rate of 0.3°C / min for 7 hours; the rest of the operation process remains the same.

[0021] Performance testing: Rehydration test: 100 mg of the probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-3 were placed in isotonic rehydration medium (1 g / L peptone + 8.5 g / L sodium chloride + distilled water to volume). After gently inverting and mixing for 1 min at room temperature, a 10-fold serial dilution was immediately performed and plate counting (initial equivalence calibration). The sample weight of each group was corrected to ensure that the starting CFU of different groups were consistent. A sample with equivalent CFU after initial equivalence calibration was taken, and isotonic rehydration medium was added to 10 mL. The sample was incubated at 25°C or shaken slowly at 60 rpm. 1 mL samples were taken at 0.5 min, 1 min, 5 min, and 10 min respectively (1 mL of fresh isotonic rehydration medium was added). The samples were spread on culture medium (MRS agar + 0.05% L-cysteine) and anaerobically cultured at 37°C for 48-72 h to count CFU. The CFU recovery rate at each time point was calculated as follows: CFU at sampling point / Equivalent CFU at the starting point × 100%. The results are as follows. Figure 1 As shown.

[0022] based on Figure 1The results showed that the probiotic preparations prepared in the embodiments of the present invention exhibited good rehydration kinetics and activity maintenance after rehydration in the rehydration test. Based on the results of Example 1 and Comparative Example 1, the activity rise rate and recovery activity of Comparative Example 1 were weaker than those of Example 1. This may be because unmodified chitosan is only fully positively charged under slightly acidic conditions, making it more prone to discontinuity or local thinning of the membrane layer during layer deposition. Uneven distribution of water entering the channels during rehydration resulted in a slightly lower curve in the early stage. Unmodified chitosan has strong self-polymerization ability and the membrane is more brittle, making it more difficult to coordinate with the inner matrix when the size changes during rehydration, resulting in incomplete release and recovery. Based on the results of Example 1 and Comparative Example 2, Comparative Example 2 showed a slow initial start-up and a lower recovery rate. In the later stage, it was significantly lower than that of Example 1 due to the influence of osmosis and interfacial stress. This may be due to the lack of mild glycosylation, which makes the protein... The stability and hydrophilicity of the white polysaccharide interface layer decreased, resulting in slower interface reconstruction during rehydration and a greater likelihood of local powder aggregation. Simultaneously, the synergistic effect with trehalose weakened, leading to a decrease in osmotic buffering capacity. Based on the results of Example 1 and Comparative Example 3, Comparative Example 3 exhibited limited water absorption and dispersion in the early stages, with a significantly slower curve rise than Example 1, and significantly lower recovery activity in the later stages. This may be due to placing the outer microparticles, inner solution, and probiotics in the same freeze-drying pan, leading to freezing competition and phase separation: multiple components iced together, resulting in uneven local solid concentration and discontinuous pores; concentrated drying stress caused proteins, sugars, salts, and colloids to sublimate in the same space, forming a denser or brittle dried layer, reducing specific surface area and interconnected pores; and uneven residual water and water activity, resulting in rapid initial rehydration followed by slower rehydration or aggregation, affecting overall recovery efficiency.

[0023] Stability test: The probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-3 were sealed in nitrogen-filled packaging and placed at 40℃ / 75%RH for 3 months. Samples were taken at 0, 1, 2 and 3 months to test the CFU retention activity (%) = CFU at the sampling point / CFU before the test × 100%, and the water content fluctuation. The results are as follows: Figure 2 and Figure 3 As shown.

[0024] based on Figure 2 and Figure 3The results analysis showed that the probiotic preparations prepared in the embodiments of the present invention exhibited high activity retention and small water content fluctuations in long-term accelerated testing, demonstrating excellent stability and long-term stable storage. Based on the results of Example 1 and Comparative Example 1, the CFU retention rate of Comparative Example 1 decreased, and water content fluctuations were amplified. This may be because unmodified chitosan requires a slightly acidic environment to fully charge, and during layer-by-layer assembly, it is more susceptible to small fluctuations in pH and ionic strength, leading to localized weak or discontinuous regions. Under high temperature and high humidity accelerated conditions, these weak regions are more sensitive to moisture, resulting in faster rehydration and subsequent activity decay. Furthermore, unmodified chitosan molecules have a strong tendency for self-aggregation, insufficient membrane flexibility, and poor reversibility of size changes during hygrothermal cycling, easily forming permeation channels over time, further reducing retention. Based on the results of Example 1 and Comparative Example 2, the CFU retention rate and water content stability of Comparative Example 2 decreased significantly. This may be due to the reduced compatibility and formation of proteins and polysaccharides when not glycosylated. The membrane properties decrease, and the glassy network is more prone to relaxation under humid heat. Under the same level of rehydration, the molecular migration rate increases, the activity decays faster, and the lack of interfacial stability brought about by glycosylation makes it difficult for the buffering effect of glycine / glycerol to be effectively transferred to the membrane surface. The cumulative damage from the rehydration-drying cycle stress is more obvious. The instability of the interfacial layer leads to uneven wetting of the particle surface and an increased probability of agglomeration. During storage, local moisture enrichment occurs, and water content and water activity increase faster. Based on the results of Example 1 and Comparative Example 3, the CFU retention rate and water content fluctuation of Comparative Example 3 are the most significant. This may be because the freeze-drying of sodium alginate-calcium, chitosan, lecithin and live bacteria in the same system results in uneven distribution of ice crystal paths and pores. Mass transfer of thick material is limited, and residual water is more difficult to reduce and is unevenly distributed. It is most prone to rehydration under accelerated conditions. The coexistence of calcium salts, counterions and acid residues will increase the hydrophilicity of the system and the water binding mode. The plasticizing effect is more obvious in the humid heat environment, which leads to faster matrix softening, faster decrease in CFU retention, and significant impact on long-term stability.

[0025] Tolerance test: 100 mg of the probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-3 were placed in centrifuge tubes, and then 5 mL of simulated gastric fluid (2 mL / L hydrochloric acid + 3 g / L pepsin + 8.5 g / L sodium chloride + distilled water) at 37°C was added. The tubes were placed in a 37°C water bath shaker at 100 rpm. 1 mL samples were taken at 0 min, 15 min, and 30 min (using parallel independent tubes). Immediately, 1 mL of 0.1 M NaHCO3 was added to neutralize the sample to neutral. After neutralization, the sample was rapidly diluted 10-fold (0.9% NaCl solution) and spread onto culture medium (MRS agar + 0.05% L-cysteine). The samples were anaerobically cultured at 37°C for 48-72 h, and CFU were counted. The gastric segment survival rate (%) was calculated as: (CFU at sampling point / CFU before test) × 100%. The results are shown below. Figure 4 As shown; After completing the 30-minute simulated gastric fluid test, samples from Examples 1-3 and Comparative Examples 1-3 were centrifuged at 3000g for 5 minutes. The supernatant was discarded, and the samples were resuspended in 5 mL of simulated intestinal fluid (trypsin 1 g / L + bile salts 3.6 g / L + sodium chloride 8.5 g / L + potassium hydrogen phosphate dihydrate 6.8 g / L + distilled water) at 37°C. The samples were then placed in a 37°C water bath at 100 rpm and 1 mL of sample was taken at 0 min, 30 min, 60 min, and 90 min (1 mL of fresh simulated intestinal fluid was added). After sampling, 9 times the volume of ice-cold isotonic diluent (0.1% PBS buffer) was added and mixed thoroughly. The samples were then spread on culture medium (MRS agar + 0.05% L-cysteine) and anaerobically cultured at 37°C for 48-72 h. The CFU count was then calculated, and the intestinal segment viability (%) was calculated as: CFU at sampling point / CFU before test × 100%. The results are shown below. Figure 5 As shown.

[0026] based on Figure 4 and Figure 5 Results analysis showed that the probiotic preparations prepared in the embodiments of the present invention exhibited excellent survival rates in simulated gastric juice tests. Based on the results of Example 1 and Comparative Example 1, the survival rate of the gastric segment in Comparative Example 1 was significantly reduced. This may be due to the narrow electrical window leading to membrane discontinuity. Unmodified chitosan is only fully protonated under slightly acidic conditions, and its layer-by-layer deposition is easily affected by small fluctuations in pH / ionic strength, resulting in poor membrane continuity and density. It is more prone to swelling-erosion in gastric acid, increasing the permeation rate of acid and enzymes. Unmodified chitosan has strong self-polymerization and is brittle. When the volume of the acid segment changes, it does not cooperate sufficiently with the inner matrix to form microchannels, further weakening the barrier. Based on the results of Example 1 and Comparative Example 2, the survival rate of the gastric segment in Comparative Example 2 decreased significantly. This may be because the outer barrier is still a layer-by-layer composite of quaternized chitosan / sodium alginate, so the early acid-enzyme diffusion is still strongly restricted, and the early survival rate is slightly reduced. However, the compatibility and film formation of unglycosylated whey protein and pullulan are good. The membrane exhibits poor stability, making it more prone to uneven wetting and aggregation upon exposure to acid, thus weakening its buffering capacity and membrane stability. Cumulative stress leads to additional losses. Furthermore, the buffering effect of glycine / glycerol is difficult to effectively transfer to the vicinity of the bacterial membrane in the absence of a stable interface layer, resulting in a significant decrease in survival rate under long-term exposure. Based on the results of Example 1 and Comparative Example 3, the gastric segment of Comparative Example 3 showed the most significant decrease in survival rate. This may be due to the uneven distribution of ice crystal paths and pores in the freeze-drying of sodium alginate-calcium, chitosan, lecithin, and live bacteria in the same container, causing the outer composite membrane to be locally too thin or discontinuous. Gastric acid and pepsin can rapidly enter the vicinity of the bacteria along these channels. The same container system is more likely to retain calcium salts, counterions, and acidic residues, creating a high ionic strength / low pH microenvironment upon exposure to acid, which has a greater impact on the cell membrane, resulting in a significant decrease in survival rate in the early stages. In addition, the swelling-rearrangement speed during rehydration of the freeze-dried composite is slow, making it difficult for the inner layer to form an effective buffer in time, leading to a significant decrease in overall survival rate.

[0027] Based on the results of Example 1 and Comparative Example 1, this may be because the charge density of unmodified chitosan decreased significantly under neutral conditions (pH 6.8), making the composite layer with sodium alginate more prone to loosening or local desorption. Bile salts and enzymes could penetrate more easily, leading to a faster and continuous decline in activity in the middle and later stages. The unmodified chitosan membrane was also more brittle, with poor reversibility of size changes. Microchannels formed under intestinal oscillation / ion exchange, resulting in a significant overall decrease in activity. Based on the results of Example 1 and Comparative Example 2, the intestinal survival rate of Comparative Example 2 decreased slightly in the early stage and significantly in the middle and later stages. This may be because the ammonium-modified chitosan / sodium alginate composite layer could still effectively limit the rapid diffusion of bile salts and enzymes. Unglycosylated whey protein had weak compatibility and film-forming properties with pullulan, making it more prone to instability under bile salt-protein interactions and enzymatic hydrolysis. In addition, The lack of a stable interface makes it difficult for the glycine / glycerol buffering effect to sustain the protection of the bacterial film, and the cumulative stress leads to a significant decrease in long-term survival rate. Based on the results of Example 1 and Comparative Example 3, the overall survival rate of the intestinal segment in Comparative Example 3 showed a significant decrease. The freeze-drying in the same container caused the outer and inner layers to form uneven channels and local salt / acid enrichment in the integrated matrix. When ion exchange and bile salts are present in the intestinal segment, the weak areas penetrate first and the whole becomes unstable faster. It is more difficult to completely remove calcium salts and counterions in the same container system. Phosphate and sodium ions in the intestinal fluid exchange further, accelerating the loosening of the alginate-calcium network. The interfacial disturbance of bile salt-lipid / protein is more difficult to buffer in this heterogeneous structure. The swelling / rearrangement of the single-freeze-dried complex in the intestinal fluid is slower and it is difficult to form a stable interface in time, resulting in the most significant decrease over a long period.

[0028] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A probiotic preparation for preventing and treating Alzheimer's disease, characterized in that, Its composition includes the following ingredients: 5-6 parts of probiotic freeze-dried powder, 5-7 parts of whey protein powder, 8-10 parts of pullulan, 2.5-3.5 parts of quaternary ammonium salt modified chitosan, 35-40 parts of trehalose, 10-12 parts of maltodextrin, 10-12 parts of prebiotics, 3-3.5 parts of glycine, 1.3-1.8 parts of glycerol, 4.5-6 parts of sodium alginate, 1.2-1.8 parts of L-glutamine, 3-3.5 parts of fructooligosaccharides, and 1-1.2 parts of lecithin.

2. The probiotic preparation for preventing and treating Alzheimer's disease according to claim 1, characterized in that, The probiotic freeze-dried powder was prepared from Lactobacillus rhamnosus, Bifidobacterium longum, and Lactobacillus plantarum in a mass ratio of 5:3:2; the Lactobacillus rhamnosus activity was 0.5–1 × 10⁻⁶. 10 CFU / g; the activity of the Bifidobacterium longum is 1–5 × 10⁻⁶. 9 CFU / g; the activity of the *Lactobacillus plantarum* is 1–3 × 10⁻⁶. 9 CFU / g.

3. The probiotic preparation for preventing and treating Alzheimer's disease according to claim 2, characterized in that, The prebiotic is any one of fructooligosaccharides, resistant dextrin, and inulin.

4. A probiotic preparation for preventing and treating Alzheimer's disease according to claim 3, characterized in that, The preparation steps of the quaternary ammonium salt modified chitosan are as follows: S1: Dissolve chitosan in glacial acetic acid, stir, then heat, adjust the pH with 1M NaOH solution, keep the temperature constant and stir, slowly add glycidyl trimethylammonium chloride, and continue stirring to react; S2: After the reaction in step S1 is completed, cool to room temperature, adjust the pH, add an equal volume of ethanol, let stand, centrifuge, wash repeatedly with ethanol, resuspend in pure water and dialyze, and dry in stages to obtain quaternary ammonium salt modified chitosan.

5. A probiotic preparation for preventing and treating Alzheimer's disease according to claim 4, characterized in that, In step S1, the ratio of chitosan, glacial acetic acid and glycidyltrimethylammonium chloride is 0.5-0.8 g: 20 mL: 2-3 mL; the glycidyltrimethylammonium chloride is added dropwise over 60 min.

6. A probiotic preparation for preventing and treating Alzheimer's disease according to claim 5, characterized in that, In step S2, the molecular weight cutoff for dialysis is 3-5 kDa; the staged drying operation is as follows: under vacuum conditions, pre-freeze at -40℃ for 2-3 hours, dry at -35℃ for 12 hours, and dry at 20℃ for 6-8 hours.

7. A method for preparing a probiotic preparation for preventing and treating Alzheimer's disease according to any one of claims 1-6, characterized in that, Includes the following steps: S101: Disperse whey protein powder in pure water, stir, then add pullulan and continue stirring. Adjust the pH with NaOH solution. Under nitrogen protection, heat and stir the reaction. Cool to room temperature, pre-cool, control the total solids content, and set aside. S102: Add trehalose, maltodextrin and prebiotics to pure water in sequence, stir and mix until dissolved; then add the solution from step S101, stir and mix until dissolved, then add glycine and glycerol in sequence, NaOH solution and HCl solution to adjust the pH, pre-cool, vacuum degas, control the total solid content, and set aside. S103: Slowly cool down the solution from step S102, keep it at that temperature, continue cooling, vacuum dry, then heat up, vacuum dry, and screen to obtain vitrified matrix powder. S104: Dissolve some sodium alginate in pure water, adjust the pH, and then add L-glutamine, fructooligosaccharides and lecithin in sequence. Under ice bath conditions, perform high-speed shear pre-emulsification to obtain an emulsion. S105: Slowly add the emulsion from step S104 to the CaCl2 solution, stir the reaction, centrifuge, filter, and resuspend the wet particles in the quaternary ammonium salt modified chitosan solution. Adjust the pH with HCl solution, stir gently, centrifuge, filter, and resuspend the wet particles in sodium alginate aqueous solution. Stir gently, treat with ultrasound under ice bath, centrifuge, filter, and obtain LbL self-assembled microparticles. S106: Freeze-dry the LbL self-assembled microparticles from step S105 to obtain synergistic layer powder; under a nitrogen atmosphere, add the synergistic layer powder and the vitrified matrix powder from step S103 into a mixer, premix, add the probiotic freeze-dried powder, and mix again to obtain a probiotic preparation.

8. A method for preparing a probiotic preparation for preventing and treating Alzheimer's disease according to claim 7, characterized in that, In step S103, the cooling parameter is: cooling at a rate of 0.5 to 1 °C / min; the heating parameter is: heating at a rate of 0.25 to 0.35 °C / min.

9. A method for preparing a probiotic preparation for preventing and treating Alzheimer's disease according to claim 8, characterized in that, In step S105, the emulsion dripping rate is 0.5-2 mL / min, and the dripping height is 2-5 cm; the ultrasonic-assisted processing parameters are: power 100-200 W, frequency 20 kHz, 2 s on / 2 s off, and time 60-120 s.

10. A method for preparing a probiotic preparation for preventing and treating Alzheimer's disease according to claim 9, characterized in that, In step S106, the freeze-drying process is as follows: add 5% trehalose, cool to -40℃ at a rate of 0.8-1.2℃ / min for 2 hours for pre-freezing, cool to -35℃ for vacuum initial drying for 4-6 hours, and heat to 20℃ at a rate of 0.25-0.35℃ / min for vacuum drying for 6-8 hours.

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