A composition for improving cognitive function and its preparation method, product and application

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a composition for improving cognitive function, a preparation method, products and applications thereof. The composition is prepared from soybean meal powder, whey protein peptide powder and bovine collagen peptide powder in a mass ratio of 1:3:2 as a peptide source raw material, in combination with AMY001, Lactobacillus plantarum LP-90 and Clostridium butyricum (3:1:1) composite strains, through Bacillus amyloliquefaciens enzyme production self-digestion, peptide-bacteria anaerobic synergistic fermentation and sodium alginate-HPMC-CPP composite carrier embedding. The composition can work synergistically through multiple mechanisms such as clearing hippocampal Aβ1-42 protein, increasing BDNF content and inhibiting IL-6 expression, has high memory improvement rate and Aβ1-42 clearance rate, and can significantly repair cognitive impairment. The composition has high stability and bioavailability, the preparation process is controllable, and is suitable for the development of drugs for preventing or assisting in improving cognitive impairment related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composition for improving cognitive function, its preparation method, product, and application. Background Technology

[0002] Cognitive impairment-related diseases (such as Alzheimer's disease) have become a major health challenge in the context of global population aging. Their pathogenesis is complex, and the core pathological features are abnormal deposition of β-amyloid protein (Aβ) in the brain to form toxic oligomers, insufficient expression of neurotrophic factors (such as brain-derived neurotrophic factor BDNF) leading to neuronal damage, and excessive activation of neuroinflammatory response exacerbating neurodegenerative diseases. Ultimately, this leads to irreversible cognitive impairment such as memory decline and spatial cognitive ability loss, which seriously affects the quality of life of patients and places a heavy burden on families and society.

[0003] Currently, products related to improving cognitive function are mainly divided into three categories: single active peptide preparations, probiotic preparations, and simple compound products. However, all of them have significant technical bottlenecks: single active peptide preparations mostly rely on exogenous protease hydrolysis for preparation. The hydrolysis process makes it difficult to precisely control the proportion of target peptides, resulting in low bioavailability of active peptides and difficulty in penetrating the blood-brain barrier to exert targeted neuroprotective effects. Probiotic preparations mostly use simple compounding of conventional neuroprotective strains, lacking compatibility design with active ingredients. The colonization rate of strains in the intestine is limited, and the regulation of the gut-brain axis is not targeted enough, making it unable to effectively intervene in the pathological process of the central nervous system. Simple compound products only achieve physical mixing of components, without solving key problems such as low peptide-bacteria interaction efficiency and easy degradation and inactivation of active ingredients in the gastrointestinal environment. As a result, the cognitive function improvement effect of the products is not good and cannot meet the needs of clinical application.

[0004] Therefore, developing a composition based on multi-component synergistic design and multi-mechanism combined intervention, which has high bioavailability, strong environmental stability and clear neuroprotective effects, can not only effectively fill the technological gaps of existing products in terms of synergistic action mechanism and targeted intervention capability, but also provide new solutions for the prevention and improvement of cognitive dysfunction-related diseases. It has important clinical value, social significance and broad application prospects. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention provides a composition for improving cognitive function. It is prepared using whey protein peptide powder, bovine bone collagen peptide powder, and soybean meal powder as raw materials, combined with functional strains such as Akk bacteria (AMY001), Lactobacillus plantarum LP-90, and Clostridium butyricum. The preparation process involves enzymatic self-degradation by Bacillus amyloliquefaciens, peptide-bacterial anaerobic synergistic fermentation, and encapsulation with a composite carrier. This composition can efficiently remove Aβ1-42 protein from hippocampal tissue, increase BDNF levels, and inhibit neuroinflammation, significantly repairing cognitive function. Furthermore, it exhibits high stability and bioavailability, and the related preparation method is controllable, making it suitable for the development of related pharmaceuticals.

[0006] The technical solution of this invention is as follows: On one hand, the present invention provides a method for preparing a composition that improves cognitive function, comprising the following steps: (1) Raw material pretreatment and mixing: After crushing and sieving soybean meal powder, mix it with whey protein peptide powder and bovine bone collagen peptide powder, add deionized water, stir to form a homogeneous substrate, sterilize and cool for later use; (2) Activation and inoculation of enzyme-producing strains: Bacillus amyloliquefaciens was activated and inoculated into the above substrate for enzyme fermentation; (3) Enzyme production fermentation and peptide purification: After fermentation, the supernatant is collected by centrifugation and filtered to remove impurities to obtain active peptide solution; (4) Activation and mixing of functional strains: AMY001, Lactobacillus plantarum LP-90 and Clostridium butyricum were activated separately and mixed in proportion to form a mixed bacterial solution; (5) Anaerobic co-fermentation: The mixed bacterial culture is inoculated into the active peptide liquid and co-fermented under anaerobic conditions to obtain the fermentation product; (6) Preparation of carrier solution: Sodium alginate, HPMC and CPP are mixed and dissolved in deionized water to prepare carrier solution; (7) Encapsulation and drying: The fermentation product is mixed with the carrier solution, and after solidification, rinsing and drying, the final product of the composition is obtained.

[0007] Specifically, in step (1), the mass ratio of soybean meal powder: whey protein peptide powder: bovine bone collagen peptide powder is 1:3:2.

[0008] Specifically, the inoculation amount of Bacillus amyloliquefaciens in step (2) can be 8%-12% v / v; the viable count of the activated Bacillus amyloliquefaciens is approximately 2 × 10⁻⁶. 9 The inoculum size of *Bacillus amyloliquefaciens* was 8%-12% v / v; the fermentation conditions for enzyme production were: temperature 35-40℃, stirring speed 60-100 r / min, and aeration rate 0.8-1.2 L / (L). Fermentation time: 36-44 h (min), pH 7.0-8.0.

[0009] Preferably, the inoculum size of *Bacillus amyloliquefaciens* is 10% v / v; the inoculum size of *Bacillus amyloliquefaciens* is 8%-12% v / v; the enzyme-producing fermentation conditions are: temperature 38℃, stirring speed 80 r / min, and aeration rate 1.0 L / (L). Fermentation time: 40 h (min), pH 7.0-8.0.

[0010] Specifically, in step (3), the centrifugation speed is 7000-9000 r / min and the centrifugation time is 15 min; the filtration uses a 0.22 μm microporous membrane.

[0011] Preferably, in step (3), the centrifugation speed is 8000 r / min and the centrifugation time is 12-18 min; the filtration uses a 0.20-0.25 μm microporous filter membrane.

[0012] Specifically, in step (4), the volume ratio of AMY001, Lactobacillus plantarum LP-90, and Clostridium butyricum is 3:1:1; the viable count of the 3:1:1 ratio is ≥3×10⁻⁶. 10 CFU / mL, viable count of Lactobacillus plantarum LP-90 ≥1×10⁻⁶ 10 CFU / mL, viable count of Clostridium butyricum ≥1×10⁻⁶ 10 CFU / mL; Total viable count of mixed bacterial culture ≥8×10 10 CFU / mL.

[0013] Specifically, the inoculation amount of the mixed bacterial solution in step (5) can be 8%-12% v / v; the conditions for the anaerobic co-fermentation can be: fermentation for 36-44 hours in an anaerobic environment at 30-35℃ and a stirring speed of 40-60 r / min.

[0014] Preferably, the inoculation amount of the mixed bacterial solution in step (5) can be 10% v / v; the conditions for the anaerobic co-fermentation can be: fermentation for 40 h in an anaerobic environment at 33°C and a stirring speed of 50 r / min.

[0015] Specifically, the carrier solution in step (6) includes 2.0-3.0g of sodium alginate, 0.8-1.2g of HPMC and 0.4-0.8g of CPP.

[0016] Preferably, the carrier solution in step (6) comprises 2.5g sodium alginate, 1.0g HPMC and 0.6g CPP.

[0017] Specifically, in step (7), the fermentation product and the carrier solution are mixed at a mass ratio of 1:2-3.

[0018] Preferably, the fermentation product and the carrier solution in step (7) are mixed at a mass ratio of 1:2.5.

[0019] In another aspect, the present invention provides a composition prepared by the aforementioned preparation method.

[0020] In another aspect, the present invention provides the use of the aforementioned composition in the preparation of products that help improve memory.

[0021] Specifically, the product in question is a medicine or health supplement.

[0022] In another aspect, the present invention provides a medicament comprising the aforementioned composition.

[0023] Specifically, the drug also includes pharmaceutically acceptable excipients.

[0024] More specifically, the pharmaceutically acceptable excipients are selected from one or more of the following: fillers, binders, disintegrants, lubricants, flow aids, flavoring agents, preservatives, stabilizers, solubilizers, suspending agents, and coating materials.

[0025] Preferably, the filler is selected from one or more of lactose, mannitol, microcrystalline cellulose, starch, and pregelatinized starch.

[0026] Preferably, the adhesive is selected from one or more of hydroxypropyl methylcellulose, povidone, sodium carboxymethyl cellulose, starch paste, and gelatin.

[0027] Preferably, the disintegrant is selected from one or more of crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and sodium crospovidone carboxymethyl cellulose.

[0028] Preferably, the lubricant is selected from one or more of magnesium stearate, sodium stearate fumarate, talc, and polyethylene glycol.

[0029] Preferably, the flow aid is selected from one or two of silica and micronized silica gel.

[0030] Preferably, the flavoring agent is selected from one or more of steviol glycosides, sucralose, lemon flavoring, and menthol.

[0031] Preferably, the preservative is selected from one or more of sodium benzoate, potassium sorbate, and parabens.

[0032] Preferably, the stabilizer is selected from one or more of vitamin C, disodium EDTA, citric acid, and tartaric acid.

[0033] Preferably, the solubilizer is selected from one or more of polysorbate 80 and poloxamer 188.

[0034] Preferably, the suspending agent is selected from one or more of sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and xanthan gum.

[0035] Preferably, the coating material is selected from one or more of hydroxypropyl methylcellulose, acrylic resin, and ethylcellulose.

[0036] Specifically, the dosage form of the drug is selected from oral preparations, including one of capsules, tablets, granules, powders, suspensions, and oral liquids.

[0037] The beneficial effects of this invention are as follows: (1) The composition of the present invention can completely repair the memory function of Alzheimer's disease model mice. In the Morris water maze test, the average escape latency was not significantly different from that of the normal control group. The time spent in the target quadrant and the number of times the platform was crossed were close to the normal physiological level. The memory improvement rate was high and it could effectively reverse cognitive impairment.

[0038] (2) The composition of the present invention achieves neuroprotection through multiple synergistic mechanisms. On the one hand, it can efficiently clear pathogenic Aβ1-42 protein in hippocampal tissue; on the other hand, it can significantly increase the content of neurotrophic factor BDNF (significantly higher than the normal control group) and at the same time significantly reduce the level of pro-inflammatory factor IL-6.

[0039] (3) The technical solution of the present invention achieves the technical effect of "1+1+1>3". The proportion of target peptides generated by Bacillus amyloliquefaciens self-decomposition is more suitable, and the bioavailability is much higher than that of exogenous protease hydrolysis products; the anaerobic synergistic fermentation constructs a bidirectional empowerment system of "peptide protecting bacteria and bacteria promoting peptides", which has a higher colonization rate and stronger synergistic effect than simple physical mixing.

[0040] (4) The present invention uses sodium alginate, HPMC and CPP composite carrier encapsulation technology to prepare microspheres, which can effectively protect the stability of active peptides and probiotics, avoid their inactivation in the gastrointestinal environment, and improve the targeted release efficiency of active ingredients, ensuring that the composition plays a highly effective role after entering the body. Detailed Implementation

[0041] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0042] The raw materials and strains of this invention are shown in Table 1: Table 1. Sources of raw materials and strains

[0043] Example 1 1. Raw material pretreatment and mixing Take 100g of soybean meal powder and grind it for 2 minutes using a high-speed grinder (20000r / min). Then pass it through an 80-mesh sieve. Add 300g of whey protein peptide powder and 200g of bovine bone collagen peptide powder directly. After mixing evenly, add 1200mL of deionized water to adjust the water content of the liquid to 58%. Mix at 1000r / min for 10 minutes to form a homogeneous substrate.

[0044] Transfer the substrate to a 5L fermenter, autoclave at 121℃ for 20 minutes, and cool to 38℃ for later use.

[0045] 2. Activation and inoculation of enzyme-producing strains Activation of Bacillus amyloliquefaciens: Inoculate 0.2g of bacterial powder into 100mL of culture medium (3g beef extract + 10g HiVeg peptone + 5g NaCl + 1L deionized water, pH 7.5, sterilized at 121℃ for 20min), and incubate at 37℃ and 180r / min on a shaker for 18h until the bacterial culture reaches OD. 600 Reaching 1.2-1.5 (live bacteria count ≈ 2 × 10⁻⁵) 9 CFU / mL). Inoculate 300 mL of the activated bacterial solution (10% v / v) into the cooled substrate. Set the fermenter parameters as follows: temperature 38℃, stirring speed 80 r / min, aeration rate 1.0 L / (L). 10% malic acid solution was added via an automatic feeding system (flow rate 0.5 mL / min) to maintain pH 7.2-7.8.

[0046] 3. Enzyme-producing fermentation and peptide purification After fermentation for 40 hours, the fermentation broth was centrifuged at 8000 r / min for 15 min, and the supernatant (containing active peptides) was collected. The supernatant was filtered through a 0.22 μm microporous membrane to remove impurities, and the active peptide solution was obtained.

[0047] 4. Activation and mixing of functional strains AMY001 activation: Inoculate 0.1g of yeast extract into 100mL of culture medium (10g yeast extract + 5g tryptone + 5g glucose + 0.5g L-cysteine ​​+ 5g NaCl + 1L deionized water, pH 7.0, sterilized at 121℃ for 20min), and anaerobic culture at 37℃. The number of viable bacteria after activation should be ≥3×10⁻⁶. 10 CFU / mL.

[0048] Activation of Lactobacillus plantarum LP-90: Inoculate 0.5g of bacterial powder into 100mL of modified MRS medium (casein peptone 10g + beef extract 8g + yeast extract 4g + glucose 20g + sodium acetate 5g + Tween-80 1mL + MgSO4). 7H₂O 0.2g + MnSO₄ 0.05 g of 4H₂O + 1 L of deionized water (pH 6.2), incubated at 37°C, resulted in a viable bacterial count ≥ 1 × 10⁻⁵ after activation. 10 CFU / mL.

[0049] Clostridium butyricum activation: Inoculate 5 mL of liquid bacterial agent into 500 mL of meat culture medium (10 g beef extract + 5 g peptone + 5 g soluble starch + 3 g yeast extract + 0.5 g L-cysteine ​​+ 1 L deionized water, pH 7.2), and anaerobic culture at 37℃. The number of viable bacteria after activation is ≥1×10⁻⁶. 10 CFU / mL.

[0050] The activated bacterial solution was prepared by mixing Akk bacteria, Lactobacillus plantarum, and Clostridium butyricum in a volume ratio of 3:1:1, with a total viable count ≥8×10⁻⁶. 10 CFU / mL.

[0051] 5. Anaerobic co-fermentation Nitrogen gas was introduced into the active peptide solution obtained in step 3 for 30 minutes (the oxygen concentration was monitored by the online DO electrode of the fermenter and dropped to below 0.3%), and then 300 mL of the above mixed bacterial solution was added (inoculation amount 10% v / v). Set the fermenter parameters as follows: temperature 33℃, anaerobic environment (no aeration), stirring speed 50r / min, fermentation time 40h, and obtain the fermentation product.

[0052] 6. Preparation of carrier solution Take 2.5g of sodium alginate, 1.0g of HPMC, and 0.6g of CPP, add 100mL of deionized water, stir in a 60℃ constant temperature water bath for 30min (stirring speed 800r / min) until completely dissolved, and cool to room temperature for later use.

[0053] 7. Embedding and Drying The fermentation product from step 5 was mixed with the carrier solution at a mass ratio of 1:2.5. After stirring evenly, the mixture was transferred to a syringe with a 2mm needle and 2% CaCl2 solution was dripped in at a rate of 1mL / min (temperature 25℃). The mixture was allowed to stand and solidify for 30min to form 1.5-2.5mm microspheres. The microspheres were rinsed twice with deionized water and then dried in a vacuum freeze dryer (-50℃, 0.1MPa) for 24 hours to obtain the final product composition.

[0054] Comparative Example A comparative example was set up with reference to Example 1. The differences between the comparative example and Example 1 are shown in Table 2: Table 2

[0055] Example of effect 1.1 Experimental Design and Grouping Experimental animals: SPF-grade C57BL / 6 mice, male, 8 months old, weighing 25-30g (Beijing Vital River Laboratory Animal Technology Co., Ltd.); Alzheimer's disease model was established by bilateral hippocampal injection of Aβ1-42 oligomer (5μg / μL, 2μL per side). After injection, mice were fed for 14 days, and mice with a successful escape latency of >90s were screened by Morris water maze preliminary test.

[0056] Grouping is shown in Table 3: Table 3 Grouping Scheme

[0057] 1.2 Detection Indicators 1.2.1 Memory function test (Morris water maze test) (1) Experimental equipment The Morris water maze system includes a 120cm diameter circular pool (30cm deep, 22±1℃), a 10cm diameter transparent platform (located in the center of the 4th quadrant, with the top of the platform 1cm above the water surface and not visible underwater), and video tracking and analysis software (recording swimming path, speed, and time).

[0058] (2) Detection steps Positioning and navigation training (days 36-40, 5 days in total): Mice were placed in the water from four different entry points (the edge of the pool, corresponding to the four quadrants) each day. The time it took for the mice to find the platform (i.e., the escape latency) was recorded. If the mice did not find the platform within 60 seconds, they were guided to the platform by the experimenter and stayed there for 10 seconds. The latency was recorded as 60 seconds. The training was conducted four times a day, with a 30-minute interval between each session. The average value of the five training days was taken as the positioning and navigation result for the sixth week.

[0059] Space Exploration Test (Day 41): Remove the platform from the pool and place the mouse into the water from the entry point in quadrant 1. Let it swim freely for 2 minutes. The video software automatically records the time the mouse spends in the quadrant where the original platform is located (quadrant 4) and the number of times it crosses the original platform position. Calculate the proportion of the time spent in the target quadrant to the total swimming time.

[0060] (3) Data statistics Memory improvement rate (%) = (mean escape latency of the model control group - mean escape latency of the experimental group) / mean escape latency of the model control group × 100%.

[0061] 1.2.2 Pathological Improvement Detection (Hippocampal Tissue Indicators) (1) Preparation of hippocampal tissue samples After fasting for 12 hours, the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the brains were quickly decapitated and removed. The hippocampal tissue was then dissected in pre-cooled physiological saline (4°C), and the surface moisture was absorbed with filter paper before weighing.

[0062] Add RIPA lysis buffer containing protease inhibitor (1% PMSF) to hippocampal tissue at a ratio of 1:10 (g:mL). Homogenize for 3 min using a tissue homogenizer (3000 rpm) under ice bath conditions. Centrifuge at 12000 rpm for 30 min at 4°C. Collect the supernatant (i.e., hippocampal tissue protein extract) and determine the protein concentration using a BCA protein quantification kit (Lambolid, B5001). Adjust the protein concentration of all samples to be consistent (2 μg / μL) for later use.

[0063] (2) Detection of Aβ1-42 content in the hippocampus (ELISA method) Detection method: Mouse Aβ1-42 ELISA kit (Kanglang Biotechnology, KL-E11823).

[0064] Clearance rate calculation: Aβ1-42 clearance rate (%) = (Aβ1-42 content in the model control group - Aβ1-42 content in the experimental group) / Aβ1-42 content in the model control group × 100%.

[0065] (3) Detection of BDNF and IL-6 levels in the hippocampus (ELISA method) Detection methods: Mouse BDNF ELISA kit (YLKbio, E2775-YLK), Mouse IL-6 ELISA kit (Xitang Bio, F10830).

[0066] 1.3 Results and Analysis 1.3.1 Memory function test (Morris water maze test) The results are shown in Table 4. The average escape latency of the Example 1 group was not significantly different from that of the normal control group (P>0.05). The time spent in the target quadrant and the number of times the platform was crossed were close to the normal physiological level. The memory improvement rate reached 78.2%, indicating that the composition of the present invention can completely repair the memory function of Alzheimer's disease model mice.

[0067] Although each comparative group showed some improvement in memory function (memory improvement rate 26.4%-41.6%), the effect was significantly weaker than that of Example 1 group: The escape latency of Comparative Example 1 (exogenous alkaline protease hydrolysis) was 44.0 s longer than that of Example 1, and the memory improvement rate was 44.7 percentage points lower. This is because exogenous enzymatic hydrolysis could not generate the target peptide ratio that matched the "Bacillus amyloliquefaciens enzyme self-hydrolysis", resulting in a decrease in the bioavailability of the active peptide. The number of platform crossings in Comparative Example 2 (peptide-bacteria physical mixing) was only 32.8% of that in Example 1, with the lowest memory improvement rate, confirming that "anaerobic synergistic fermentation" is the key to enhancing the synergistic effect of peptide-bacteria, and physical mixing cannot achieve the bidirectional empowerment of "peptide protecting bacteria and bacteria promoting peptide". The average escape latency of the three comparative groups (excluding Lactobacillus plantarum LP-90) was 57.5±5.2s, which was 36.0s longer than that of the first example group; the time spent in the target quadrant was 43.0±3.4s, which was only 62.3% of that of the first example group; the number of platform crossings was 2.9±0.3 times, which was less than 50% of that of the first example group; and the memory improvement rate was 41.6%, which was 36.6 percentage points lower than that of the first example group, confirming that the change in strain species weakened the restorative effect of the composition on memory function.

[0068] Table 4 Results of the Morris water maze experiment

[0069] Note: *** P < 0.001 (highly significant difference compared with the normal control group), ** P < 0.01 (significant difference compared with the normal control group).

[0070] 1.3.2 Pathological Improvement Detection (Hippocampal Tissue Indicators) The results are shown in Table 5. The hippocampal Aβ1-42 clearance rate in the Example 1 group reached 85.5%, the hippocampal Aβ1-42 content was close to that of the normal control group, while the BDNF content was significantly higher than that of the normal control group, and the IL-6 content was significantly reduced. This indicates that the composition of the present invention can intervene in the neuropathological process through multiple dimensions such as clearing pathogenic proteins, activating neurotrophic factors and inhibiting neuroinflammation.

[0071] The pathological improvement effects of each comparative group were not as good as those of Example 1 group: The Aβ1-42 clearance rate of Comparative Example 1 was only 38.3%, and the BDNF content was only 38.5% of that of Example 1, confirming that the peptides generated by Bacillus amyloliquefaciens enzyme self-degradation are more likely to penetrate the blood-brain barrier and can synergistically enhance the Aβ clearance ability with functional strains. The IL-6 content of Comparative Example 2 was 2.6 times that of Example 1. Due to the lack of a synergistic fermentation step, the colonization rate of the strain was low and it could not effectively inhibit neuroinflammation. The hippocampal Aβ1-42 clearance rate in Comparative Example 3 was 53.2%, only 62.2% of that in Example 1. The hippocampal Aβ1-42 content (1.10±0.11 ng / mg protein) was significantly higher than that in Example 1. The BDNF content was 20.5±1.7 ng / mg protein, only 51.9% of that in Example 1. The IL-6 content was 20.2±1.5 pg / mg protein, 1.58 times that of Example 1. This indicates that after removing Lactobacillus plantarum LP-90, the multi-dimensional intervention effects of the composition in clearing pathogenic proteins, activating neurotrophic factors, and inhibiting neuroinflammation were significantly reduced.

[0072] Table 5 Results of hippocampal tissue index testing

[0073] Note: *** P < 0.001 (highly significant difference compared with the normal control group), ** P < 0.01 (significant difference compared with the normal control group).

[0074] In summary, however, Example 1, through its innovative design of Bacillus amyloliquefaciens enzyme self-decomposition, peptide-bacterial anaerobic synergistic fermentation, and the combination of functional strains of Lactobacillus plantarum LP-90 and AMY001, achieved a breakthrough improvement in cognitive function repair and neuropathological intervention, with various indicators approaching normal physiological states. This result confirms that the technical solution of this invention is not a simple addition of components or process improvement, but rather achieves a "1+1+1>3" technical effect through synergistic innovation across multiple technical aspects.

[0075] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method for preparing a composition for improving cognitive function, characterized in that, Includes the following steps: (1) Raw material pretreatment and mixing: After crushing and sieving soybean meal powder, mix it with whey protein peptide powder and bovine bone collagen peptide powder, add deionized water, stir to form a homogeneous substrate, sterilize and cool for later use; (2) Activation and inoculation of enzyme-producing strains: Bacillus amyloliquefaciens was activated and inoculated into the above substrate for enzyme fermentation; (3) Enzyme production fermentation and peptide purification: After fermentation, the supernatant is collected by centrifugation and filtered to remove impurities to obtain active peptide solution; (4) Activation and mixing of functional strains: Ackermania glutinis AMY001, Lactobacillus plantarum LP-90 and Clostridium butyricum were activated separately and mixed in proportion to form a mixed bacterial solution; (5) Anaerobic co-fermentation: The mixed bacterial culture is inoculated into the active peptide liquid and co-fermented under anaerobic conditions to obtain the fermentation product; (6) Preparation of carrier solution: Sodium alginate, hydroxypropyl methylcellulose and casein phosphopeptide are mixed and dissolved in deionized water to prepare carrier solution; (7) Encapsulation and drying: The fermentation product is mixed with the carrier solution, and after solidification, rinsing and drying, the final product of the composition is obtained; In step (1), the mass ratio of soybean meal powder: whey protein peptide powder: bovine bone collagen peptide powder is 1:3:

2. The number of viable Bacillus amyloliquefaciens after activation in step (2) is approximately 2 × 10⁻⁶. 9 The inoculum size of *Bacillus amyloliquefaciens* was 8%-12% v / v; the fermentation conditions for enzyme production were: temperature 35-40℃, stirring speed 60-100 r / min, and aeration rate 0.8-1.2 L / (L). (min), pH 7.0-8.0, fermentation time 36-44h; In step (4), the volume ratio of AMY001, Lactobacillus plantarum LP-90, and Clostridium butyricum is 3:1:1; the viable count of AMY001 is ≥3×10⁻⁶. 10 CFU / mL, viable count of Lactobacillus plantarum LP-90 ≥1×10⁻⁶ 10 CFU / mL, viable count of Clostridium butyricum ≥1×10⁻⁶ 10 CFU / mL; Total viable count of mixed bacterial culture ≥8×10 10 CFU / mL; The inoculation amount of the mixed bacterial solution in step (5) is 8%-12% v / v; the conditions for the anaerobic co-fermentation are: fermentation for 36-44 hours in an anaerobic environment at 30-35℃ and a stirring speed of 40-60 r / min. The carrier solution in step (6) comprises 2.0-3.0g sodium alginate, 0.8-1.2g hydroxypropyl methylcellulose, and 0.4-0.8g casein phosphopeptide; In step (7), the fermentation product and the carrier solution are mixed at a mass ratio of 1:2-3.

2. The composition prepared by the method of claim 1.

3. The use of the composition of claim 2 in the preparation of products that help improve memory.

4. The application according to claim 3, characterized in that, The product in question is a medicine or health supplement.

5. A drug, characterized in that, The drug comprises the composition of claim 2.

6. The drug according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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