Application of compound probiotic preparation and metagen thereof in reducing neurodegenerative diseases, resisting oxidation and preventing Alzheimer's disease

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a composite probiotic preparation and application of a metagen thereof in reducing neurodegenerative diseases, resisting oxidation and preventing Alzheimer's disease. The invention provides a mixed probiotic preparation which is prepared from lactobacillus rhamnosus NKU ML1-2, bifidobacterium breve NKU BB1-2 and bifidobacterium longum subsp.infantis NKU FB 3-14 according to a specific proportion, and further studies the afterbiogenic form of the mixed probiotic preparation. Through in-vitro antioxidant screening, in-vitro AD cell model verification, metabonomics and molecular docking analysis, the efficacy and potential mechanism of the mixed probiotic preparation and the metabiotics thereof in the aspect of AD prevention are systematically evaluated, and a safe and efficient microbial solution with a definite action target is provided for Alzheimer's disease prevention.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of compound probiotic preparations and their postbiotics in reducing neurodegenerative diseases, anti-oxidation, and preventing Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by senile plaques formed by extracellular β-amyloid (Aβ) deposition, neurofibrillary tangles formed by intracellular hyperphosphorylated tau protein, and accompanying synaptic loss, neuronal death, and chronic neuroinflammation. Currently, the number of AD patients worldwide continues to rise, placing a heavy burden on society and individuals; however, effective curative drugs remain scarce.

[0003] In recent years, the gut-brain axis ( Gut-Brain Axis The theory provides a new perspective for the prevention and treatment of Alzheimer's disease (AD). The gut microbiota communicates bidirectionally with the central nervous system through multiple pathways, including the immune system, neuroendocrine system, and vagus nerve. Studies have found that AD patients often have gut microbiota dysbiosis, which may exacerbate neuroinflammation, oxidative stress, and Aβ deposition. Therefore, regulating the gut microbiota through probiotics, prebiotics, or post-biotics has become a potential strategy for the prevention and intervention of AD.

[0004] Probiotics are live microorganisms that are beneficial to the health of the host when ingested in sufficient quantities. However, live bacteria preparations have issues such as stability, tolerance to gastric acid and bile, and potential infection risks (in certain populations).

[0005] Post-natal Yuan ( Postbiotics Metabiotics refer to preparations of non-living microorganisms and / or their components that are beneficial to the health of the host, including cell components (such as peptidoglycans and surface proteins) and metabolites (such as short-chain fatty acids, bacteriocins, and vitamins). Metabiotics have advantages such as high safety, good stability, and well-defined targets, and there is no need to consider the survival of the microorganisms.

[0006] Currently, research on probiotics or metabiotics for Alzheimer's disease (AD) mainly focuses on single strains or simple mixtures. There is a lack of research on the optimization of synergistic strain combinations targeting multiple pathological mechanisms of AD, and there are also few systematic comparisons of the differences in neuroprotective efficacy between different forms of metabiotics (such as supernatant and bacterial cells).

[0007] Therefore, there is an urgent need to develop a safe, efficient, and targeted microbial solution for the prevention of Alzheimer's disease, which maximizes the antioxidant and beneficial metabolite production capabilities of probiotic systems by optimizing culture conditions and formulations. Summary of the Invention

[0008] The purpose of this invention is to provide a mixed probiotic preparation and its postbiotics in reducing risk factors for neurodegenerative diseases or preventing Alzheimer's disease. The mixed probiotic preparation provided by this invention is not only safe and efficient, but also has a clear target of action, and can effectively prevent Alzheimer's disease.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a mixed probiotic preparation comprising Lactobacillus rhamnosus NKUML1-2, Bifidobacterium longum subsp. infantis NKU FB3-14, and Bifidobacterium breve NKU BB1-2; The preservation number of Lactobacillus rhamnosus NKU ML1-2 is GDMCC No. 66837; the preservation number of Bifidobacterium breve NKIBB1-2 is GDMCC No. 65956; and the preservation number of Bifidobacterium longum subsp. infantis NKU FB 3-14 is CGMCC No. 25762.

[0010] Preferably, the total number of live bacteria in the mixed probiotic preparation is not less than 1×10⁻⁶. 8 CFU / mL; The mixed probiotic preparation contains Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKUFB 3-14, and Bifidobacterium breve NKU BB1-2 in a ratio of 1:3:1.

[0011] This invention also provides a method for preparing the above-mentioned mixed probiotic preparation, specifically including the following steps: S1. Inoculate Lactobacillus rhamnosus NKU ML1-2 into MRS liquid medium, Bifidobacterium longum subsp. infantis NKUFB 3-14 into BS liquid medium, and Bifidobacterium breve NKU BB1-2 into BBL liquid medium, and culture at 37℃ under anaerobic conditions for 20-24 h respectively. S2. After centrifuging the cultured Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB3-14 and Bifidobacterium breve NKU BB1-2, discard the supernatant, mix them in proportion and inoculate them into BBL medium, add growth aids and culture in an anaerobic environment for 24-48 h to obtain a mixed probiotic preparation.

[0012] Preferably, in step S1, Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB 3-14, and Bifidobacterium breve NKU BB1-2 are all inoculated into the culture medium at a volume fraction of 5%.

[0013] Preferably, the mixed inoculation ratio of Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB 3-14 and Bifidobacterium breve NKU BB1-2 in step S2 is 1:3:1.

[0014] Preferably, the concentration of the growth aid in step S2 in the BBL medium is not less than 1%; the growth aid is one or more of glucose, fructooligosaccharides, galactooligosaccharides and inulin.

[0015] The present invention also provides a mixed probiotic postbiotic, which is prepared by culturing, heat inactivation and drying of the above-mentioned mixed probiotic preparation or the mixed probiotic preparation obtained by the above-mentioned preparation method.

[0016] The present invention also provides the application of the above-mentioned mixed probiotic preparation or the mixed probiotic preparation obtained by the above-mentioned preparation method or the above-mentioned mixed probiotic postbiotic in the preparation of antioxidant products.

[0017] This invention also provides the application of the above-mentioned mixed probiotic preparation or the mixed probiotic preparation prepared by the above-mentioned preparation method or the above-mentioned mixed probiotic postbiotic in inhibiting the inflammatory response of nerve cells or inhibiting the activity of BACE1, a key enzyme in the production of β-amyloid protein.

[0018] The present invention also provides the application of the above-mentioned mixed probiotic preparation or the mixed probiotic preparation prepared by the above-mentioned preparation method or the above-mentioned mixed probiotic postbiotic in the preparation of products for the prevention of Alzheimer's disease.

[0019] Preferably, the mixed probiotic preparation or mixed probiotic postbiotic can produce differential metabolites with high binding affinity to key targets of Alzheimer's disease; The key targets are glycogen synthase kinase-3β and β-secretase; The metabolites include one or more of acetyl strychnine, manzamin F, senoside A, taurcholic acid, and tryptamine.

[0020] The beneficial effects of this invention are: The mixed probiotic preparation prepared by this invention and the postbiotic obtained by heat inactivation treatment can safely and efficiently reduce key risk factors of Alzheimer's disease such as oxidative stress, neuroinflammation, and β-amyloid protein production, and further prevent neurodegenerative diseases such as Alzheimer's disease. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Statistical chart of DPPH free radical scavenging capacity of different ratio groups and single strain groups of mixed probiotic preparations; Figure 2 Statistical chart of hydroxyl radical scavenging capacity of different ratio groups and single strain groups of mixed probiotic preparations; Figure 3 A statistical chart showing the total number of viable bacteria in 24-hour culture for different ratios of mixed probiotic preparations and single bacterial groups; Figure 4 A statistical chart showing the comprehensive index scores of different ratio groups and single strain groups of mixed probiotic preparations; Figure 5 Radar chart for comprehensive index evaluation of different ratio groups and single strain groups of mixed probiotic preparations; Figure 6 A is a statistical chart of the total number of viable bacteria in a 24-hour culture of a probiotic preparation containing different growth accelerators; B is a statistical chart of the DPPH scavenging rate of a probiotic preparation containing different growth accelerators; C is a statistical chart of the hydroxyl radical scavenging rate of a probiotic preparation containing different growth accelerators (where *: P<0.05; **: P<0.01). Figure 7 A shows the total number of viable bacteria in 24 hours of culture for different ratios of mixed probiotic preparations under 1% inulin growth adjuvant; B shows the DPPH scavenging rate of different ratios of mixed probiotic preparations under 1% inulin growth adjuvant; C shows the hydroxyl radical scavenging rate of different ratios of mixed probiotic preparations under 1% inulin growth adjuvant (where, **: P<0.01). Figure 8 A is a statistical chart of the total number of viable bacteria in the mixed probiotic preparation under different culture times and different oxygen environments; B is a statistical chart of the DPPH scavenging rate of the mixed probiotic preparation under different culture times and different oxygen environments; C is a statistical chart of the hydroxyl radical scavenging rate of the mixed probiotic preparation under different culture times and different oxygen environments (where *: P<0.05; **: P<0.01). Figure 9Principal component analysis (PCA) plots of non-target metabolites in different ratios of mixed probiotic preparations (where A is the PCA plot for the ratios of ML1-2:FB 3-14:BB1-2 of 1:1:1 and 1:3:1; B is the PCA plot for the ratios of ML1-2:FB 3-14:BB1-2 of 3:1:1 and 1:3:1; C is the PCA plot for the ratios of ML1-2:FB 3-14:BB1-2 of 1:1:3 and 1:3:1). Figure 10 This is a peak area diagram of differentially metabolites in a mixed probiotic preparation in different formulations; where A is acetyl strychnine ( Acetylspartioidine B is manzamin F (); Manzamine F C is tansine amide A (); Sesbanimide A D is taurine ( Taurocholic acid E is tryptophan (); Tryptamine (Where *: P<0.05; **: P<0.01); Figure 11 This is a diagram showing the docking results between the GSK-3β protein target molecule and its differential metabolites; where A represents acetylstigmine ( Acetylspartioidine B is manzamin F (); Manzamine F C is tansine amide A (); Sesbanimide A); D is taurine ( Taurocholic acid E is tryptophan (); Tryptamine ); Figure 12 This is a diagram showing the docking results between β-secretase target molecules and differentially metabolites; where A represents acetylstigmine ( Acetylspartioidine B is manzamin F (); Manzamine F C is tansine amide A (); Sesbanimide A D is taurine ( Taurocholic acid E is tryptophan (); Tryptamine ); Figure 13 A statistical chart showing the hydroxyl radical scavenging rates of different components in a mixed probiotic preparation (where **: P<0.01). Figure 14 Statistical chart of DPPH free radical scavenging rate of different components of mixed probiotic preparation (where, **: P<0.01). Figure 15 Figure showing the effect of okadaic acid on the viability of HT-22 cells; Figure 16Statistical graph showing the effect of different forms of postbiotics in mixed probiotic preparations on HT-22 cell viability; among them, the semi-quantitative analysis results of HT-22 cell viability are Mean±SD; compared with the Con group, ##: P<0.01; compared with the OA group, *: P<0.05; **: P<0.01; Figure 17 The statistical graph shows the effect of different forms of postbiotics in mixed probiotic preparations on MDA content in HT-22 cells; among them, compared with the Con group, ##: P<0.01; compared with the OA group, **: P<0.01 in each postbiotic intervention group. Figure 18 Statistical chart showing the effects of different forms of postbiotics in mixed probiotic preparations on inflammatory factors in HT-22 cells; among them, compared with the Con group, ##: P<0.01; compared with the OA group, **: P<0.01 in each postbiotic intervention group; Figure 19 The statistical graph shows the effect of different forms of postbiotics in mixed probiotic preparations on BACE1 enzyme activity in HT-22 cells; among them, compared with the Con group, ##: P<0.01; compared with the OA group, **: P<0.01 in each postbiotic intervention group. Detailed Implementation

[0023] This invention provides a mixed probiotic preparation and a postbiotic obtained by heat inactivation treatment. The mixed probiotic preparation is composed of Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB 3-14 and Bifidobacterium breve NKIBB1-2 in a ratio of 1:3:1.

[0024] The Lactobacillus rhamnosus NKU ML1-2 described in this invention has the accession number GDMCC No. 66837 (disclosed in patent application No. 202511982720.7); the Bifidobacterium breve NKU BB1-2 has the accession number GDMCC No. 65956 and is deposited at the Guangdong Provincial Center for Microbial Culture Collection (disclosed in patent application No. 202511573503.2); the Bifidobacterium longum subsp. infantis NKU FB 3-14 has the accession number CGMCC No. 25762 (disclosed in patent application No. 202410977294.7).

[0025] Through screening for optimal proportions and optimization of culture components and culture conditions, this invention has determined that the ratio of viable bacteria of NKU ML1-2, NKU FB3-14, and NKU BB1-2 strains in the mixed probiotic preparation is 1:3:1. Furthermore, the optimal culture conditions for the mixed probiotic preparation have been determined to be anaerobic, 24 hours, and the addition of 1% inulin.

[0026] This invention, through molecular-level studies and examples, demonstrates that five differentially expressed metabolites (acetyl stigmine, manzamin F, stigmine A, taurine, and tryptophan) in a mixed probiotic formulation can bind with high affinity to the key AD targets GSK-3β and BACE1. Manzamin F exhibits the strongest binding affinity to both targets and is the most promising lead compound. These metabolites, by occupying the active site, forming key hydrogen bonds, and engaging in hydrophobic interactions, can directly inhibit the kinase activity of GSK-3β (thus reducing tau protein hyperphosphorylation) and the protease activity of BACE1 (thus reducing Aβ production).

[0027] On the other hand, in vitro antioxidant experiments in the embodiments of the present invention showed that all three metabiotic forms possessed certain free radical scavenging capabilities. The DPPH and hydroxyl radical scavenging rates of the heat-inactivated supernatant were significantly higher than those of the heat-inactivated bacterial cells and the heat-inactivated supernatant + bacterial cell group. This indicates that the soluble metabolites produced by the mixed probiotics during fermentation and present in the supernatant are its main antioxidant active components, and heat inactivation did not significantly destroy the activity of these substances.

[0028] On the other hand, in the HT-22 cell AD model, all three metabiotic forms of the mixed probiotics improved OA-induced cytotoxicity and reduced oxidative stress damage (lowering MDA). The heat-inactivated supernatant (S) showed the most significant effect, followed by heat-inactivated supernatant + bacterial cells (H), while heat-inactivated bacterial cells (J) had a relatively weaker effect. This suggests that soluble metabolites (such as short-chain fatty acids, bacteriocins, and enzymes) in the probiotic fermentation supernatant may play a central role in neuroprotection.

[0029] On the other hand, in the OA-induced AD model of HT-22 cells, the mixed probiotic postbiotics could alleviate neuroinflammation by inhibiting pro-inflammatory factors and promoting the secretion of anti-inflammatory factors, and could also inhibit the activity of BACE1, a key enzyme in Aβ production. Among all the tested postbiotic forms, heat-inactivated supernatant (S) showed the strongest anti-inflammatory and Aβ-inhibition potential, followed by heat-inactivated supernatant + bacterial cells (H), while heat-inactivated bacterial cells (J) had relatively limited effects. This further confirms that the soluble metabolites produced by probiotics during fermentation are the main active ingredients exerting neuroprotective, anti-inflammatory, and anti-Aβ effects.

[0030] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0032] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0033] Example 1 Lactobacillus rhamnosus NKU ML1-2 was inoculated into MRS liquid medium at a volume fraction of 5%, and then cultured in a constant temperature incubator at 37℃ for 20-24 h for subsequent mixed culture.

[0034] Bifidobacterium longum infantis subspecies NKU FB 3-14 was inoculated into BS liquid medium at a volume fraction of 5%, and then anaerobically cultured at 37℃ for 20-24 h for subsequent mixed culture.

[0035] Bifidobacterium breve NKU BB1-2 was inoculated into BBL liquid medium at a volume fraction of 5%, and then anaerobically cultured at 37℃ for 20-24 h for subsequent mixed culture.

[0036] MRS liquid medium: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 mL; BS liquid medium: peptone 10.0 g / L, liver extract 5.0 g / L, beef extract 3.0 g / L, yeast extract 5.0 g / L, casein peptone 8.0 g / L, soluble starch 0.5 g / L, sodium chloride 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1 g / L, glucose 10 g / L, ferric sulfate heptahydrate 0.01 g / L, manganese sulfate 0.005 g / L, L-cysteine ​​0.5 g / L; BBL liquid medium: peptone 15.0 g / L, glucose 20.0 g / L, yeast extract 2.0 g / L, soluble starch 0.5 g / L, sodium chloride 5.0 g / L, L-cysteine ​​0.5 g / L, tomato extract 5.0 g / L, liver extract 2.0 g / L, Tween 80 1.0 mL.

[0037] The suspensions of *Lactobacillus rhamnosus* NKU ML1-2, *Bifidobacterium longum* subsp. *infantii* NKU FB 3-14, and *Bifidobacterium breve* NKIBB1-2 were centrifuged (8000 g, 15 min, 4℃), and the supernatant was discarded. The bacterial concentrations of the three bacteria were adjusted to 1-5 × 10⁻⁶ using BBL liquid medium. 9 CFU / mL. The three bacteria were inoculated into BBL liquid medium at a volume ratio of 4% and cultured in an anaerobic environment for 24 h to obtain a mixed probiotic preparation.

[0038] Example 2: Screening of the optimal ratio for a mixed probiotic preparation Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB 3-14, and Bifidobacterium breve NKIBB1-2 were cultured individually for 20-24 h in an aerobic environment of MRS liquid medium, an anaerobic environment of BS liquid medium, and an anaerobic environment of BBL liquid medium, respectively.

[0039] The suspensions of *Lactobacillus rhamnosus* NKU ML1-2, *Bifidobacterium longum* subsp. *infantii* NKU FB 3-14, and *Bifidobacterium breve* NKIBB1-2 were centrifuged (8000 g, 15 min, 4℃), and the supernatant was discarded. The bacterial concentrations of the three bacteria were adjusted to 1-5 × 10⁻⁶ using BBL liquid medium. 9 CFU / mL. The three bacteria were inoculated at a 4% (v / v) ratio into BBL liquid medium and cultured anaerobicly for 24 h for subsequent determination of total viable count and antioxidant capacity. Different ratios of viable counts of *Lactobacillus rhamnosus* NKU ML1-2, *Bifidobacterium longum* subsp. infantis NKU FB 3-14, and *Bifidobacterium breve* NKU BB1-2 were set up for comparison with single-bacterial groups. The total bacterial count in each group was 1.85 × 10⁻⁶ CFU / mL. 8 CFU / mL, see Table 1 below for details.

[0040] Table 1. Mixed probiotic formulation ratios and single-strain concentrations

[0041] like Figure 1As shown, the DPPH scavenging rates of the mixed groups with different ratios were significantly higher than those of any single bacterial group, indicating a synergistic effect among the strains. Among them, the mixed group with a ratio of 1:3:1 (ML1-2:FB3-14:BB1-2) had the highest DPPH scavenging rate, which was significantly better than other mixed ratios and single bacterial groups (P<0.05).

[0042] like Figure 2 As shown, the 1:3:1 ratio group exhibited the strongest hydroxyl radical scavenging ability, further demonstrating that the antioxidant activity produced synergistically by the strains under this ratio was the highest.

[0043] like Figure 3 As shown, after 24 hours of anaerobic mixed culture, the 1:3:1 ratio group had the highest total number of viable bacteria, indicating that this ratio is most conducive to the proliferation and survival of bacteria in the coexisting environment, forming a stable symbiotic system.

[0044] like Figure 4-5 As shown, the entropy weight method was used to comprehensively evaluate three indicators: DPPH scavenging rate, hydroxyl radical scavenging rate, and total viable bacteria count. The analysis revealed that when the ratio of *Lactobacillus rhamnosus* NKU ML1-2, *Bifidobacterium longum* subsp. infantis NKU FB 3-14, and *Bifidobacterium breve* NKU BB1-2 in the mixed probiotic preparation was 1:3:1, the mixed probiotic preparation exhibited the strongest antioxidant activity and the highest total viable bacteria count.

[0045] Therefore, based on a comprehensive evaluation of antioxidant activity and bacterial proliferation capacity, the optimal live bacteria ratio of Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB 3-14, and Bifidobacterium breve NKU BB1-2 in the mixed probiotic preparation was determined to be 1:3:1.

[0046] Example 3: Screening of culture components and conditions for mixed probiotic preparations Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB 3-14, and Bifidobacterium breve NKIBB1-2 were cultured individually for 20-24 h in an aerobic environment of MRS liquid medium, an anaerobic environment of BS liquid medium, and an anaerobic environment of BBL liquid medium, respectively.

[0047] The suspensions of *Lactobacillus rhamnosus* NKU ML1-2, *Bifidobacterium longum* subsp. *infantii* NKU FB 3-14, and *Bifidobacterium breve* NKIBB1-2 were centrifuged (8000 g, 15 min, 4℃), and the supernatant was discarded. The bacterial concentrations of the three bacteria were adjusted to 1-5 × 10⁻⁶ using BBL liquid medium. 9CFU / mL. The three bacteria were inoculated into BBL liquid medium at a volume ratio of 4%. The viable count ratio of Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB 3-14 and Bifidobacterium breve NKU BB1-2 was 1:3:1.

[0048] Different growth aid groups were set up: 1% inulin, fructooligosaccharide, and galactooligosaccharide were added to the mixed culture system, and the mixture was cultured in an anaerobic environment for 24 h. The differences in antioxidant index and total viable count index between the different growth aid groups and the control group (pure BBL liquid medium) were compared.

[0049] Depend on Figure 6 (AC) It was found that the culture system with 1% inulin had significantly higher 24-hour total viable bacteria count, DPPH scavenging rate, and hydroxyl radical scavenging rate than the system with other prebiotics or the control group (P<0.05). As a high-quality prebiotic, inulin can be efficiently utilized by Bifidobacteria and Lactobacillus, significantly promoting their growth and metabolism, thereby producing more metabolites with antioxidant activity.

[0050] Therefore, in the subsequent experiments, 1% inulin was selected as the growth promoter for the mixed probiotics.

[0051] Furthermore, the antioxidant properties and total viable bacteria count of mixed probiotic preparations with different formulations containing 1% inulin were compared. The results are as follows: Figure 7 As shown in (AC), under culture conditions with 1% inulin, the ratio of *Lactobacillus rhamnosus* NKU ML1-2, *Bifidobacterium longum* subsp. infantis NKU FB 3-14, and *Bifidobacterium breve* NKU BB1-2 of 1:3:1 remained the ratio with the strongest antioxidant activity and the highest total viable count. Therefore, ML1-2:FB3-14:BB1-2 = 1:3:1 was selected as the optimal ratio for the mixed probiotic preparation, and 1% inulin was added as a growth promoter in the culture system.

[0052] Depend on Figure 8 (AC) results showed that under anaerobic conditions and 24-hour culture, the total viable count and antioxidant activity of the mixed probiotic preparation (1:3:1 ratio, containing 1% inulin) reached their peak values. Extending the culture time to 48 hours resulted in a slight decrease in the viable count, but no significant increase in antioxidant activity.

[0053] Therefore, anaerobic conditions, 24 hours, and the addition of 1% inulin were determined to be the optimal culture conditions for the subsequent mixed probiotic preparation.

[0054] Example 4: Non-target metabolomics analysis and molecular docking with AD targets of mixed probiotic preparations By comparing the differences in metabolites among different ratios of mixed probiotic preparations (ML1-2:FB3-14:BB1-2=1:3:1, 1:1:1, 3:1:1, and 1:1:3), we further explored the differentially expressed metabolites associated with Alzheimer's disease in the selected mixed probiotic preparation (ML1-2:FB3-14:BB1-2=1:3:1) and screened out the differentially expressed metabolites that were specifically enriched or significantly more abundant in the 1:3:1 group.

[0055] Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB3-14, and Bifidobacterium breve NKIBB1-2 were cultured individually for 20-24 h in an aerobic environment of MRS liquid medium, an anaerobic environment of BS liquid medium, and an anaerobic environment of BBL liquid medium, respectively.

[0056] The suspensions of *Lactobacillus rhamnosus* NKU ML1-2, *Bifidobacterium longum* subsp. *infantii* NKU FB3-14, and *Bifidobacterium breve* NKIBB1-2 were centrifuged (8000 g, 15 min, 4℃), and the supernatant was discarded. The bacterial concentrations of the three bacteria were adjusted to 1–5 × 10⁻⁶ using BBL liquid medium. 9 CFU / mL. The three bacteria were inoculated into BBL liquid medium (containing 1% inulin) at a volume ratio of 4% and cultured in an anaerobic environment for 24 h for non-targeted metabolomics assays.

[0057] PCA score chart as follows Figure 9 (AC) showed that the selected mixed probiotic preparation (ML1-2:FB 3-14:BB1-2 =1:3:1) was clearly separated from the other groups and located in a separate region of the PCA plot, indicating that its metabolite profile was unique.

[0058] like Figure 10 As shown in (AE), in the selected mixed probiotic formulation (ML1-2:FB 3-14:BB1-2=1:3:1), acetyl strychnine ( Acetylspartioidine ), Manzamin F ( Manzamine F ), stigmine A ( Sesbanimide A The relative peak areas of these three metabolites were significantly higher than those of other ratio groups, with taurine ( Taurocholic acid ), tryptamine ( Tryptamine The relative peak areas of these two metabolites were significantly lower than those in other ratios. Manzamin alkaloids possess anti-inflammatory and neuroprotective activities; taurine is involved in lipid metabolism and signal transduction; tryptamine is a precursor to serotonin and is associated with mood and cognitive regulation. The enrichment of these metabolites suggests that the 1:3:1 ratio may have produced more substances with potential neuroactive activity.

[0059] Therefore, further molecular docking was performed on the differentially expressed metabolites in the selected mixed probiotic preparation (ML1-2:FB 3-14:BB1-2 = 1:3:1) with Alzheimer's disease-related protein targets. The five differentially expressed metabolites were molecularly docked with two key target proteins of AD—glycogen synthase kinase-3β (GSK-3β, PDB: 4AFJ) and β-secretase (BACE1, PDB: 1FKN). PDB ID 1FKN represents human β-secretase (…). Beta-Secretase The crystal structure of which binds to its inhibitor.

[0060] Molecular docking utilizes the MEMAPSIN 2 protein (A chain) in the structure, removing water and inhibitor molecules, and selecting the inhibitor binding site (green cavity) as the docking site. PDB ID 4AFJ is a human glycogen synthase kinase-3β (GSK-II). Glycogen Synthase Kinase-3 beta The structure of a crystal complex of GSK-3β bound to a high-performance inhibitor (5-aryl-4-carboxamide-1,3-oxazole compound) was described. This docking method utilizes the GLYCOGEN SYNTHASEKINASE-3 BETA protein (chains A and B) from the structure, removing water and the inhibitor molecule, and selecting the binding site of one of the inhibitors (the green cavity) as the docking site.

[0061] The docking results are shown in Table 2. All five metabolites exhibited good binding affinity to both target sites (Vina score ≤ -5.5 kcal / mol). Among them, manzamin F ( Manzamine F The binding scores for GSK-3β and BACE1 were as high as -10.289 and -8.192 kcal / mol, respectively, indicating strong binding potential. Sesantamine A and acetyl stigmine also showed strong binding ability. This computational simulation predicts that specific metabolites produced by the 1:3:1 mixed probiotic preparation may intervene in the pathological process of Alzheimer's disease by directly acting on GSK-3β (associated with tau protein phosphorylation) and BACE1 (associated with Aβ production).

[0062] Table 2. Comparison of molecular docking binding affinity between differentially metabolites and Alzheimer's-related protein targets.

[0063] Figure 11 (AE) and Figure 12(AE) presents the molecular docking results of five key differentially expressed metabolites in a mixed probiotic formulation (ML1-2:FB3-14:BB1-2=1:3:1) with two core pathological targets of Alzheimer's disease (AD): glycogen synthase kinase-3β (GSK-3β) and β-secretase (BACE1). This analysis reveals the possible mechanisms of action of these metabolites in three dimensions, providing a structural biology basis for their potential application in AD prevention and treatment.

[0064] Figure 11 (AE) represents the molecular docking results of GSK-3β protein with differential metabolites.

[0065] GSK-3β is a key kinase involved in the abnormal phosphorylation of tau protein. Its overactivation promotes the formation of neurofibrillary tangles and is a significant pathogenic mechanism of Alzheimer's disease (AD). Figure 11 As shown in (AE), all five metabolites can effectively enter and stably bind to the ATP-binding pocket (active center) or adjacent regulatory region of GSK-3β, and interact with key amino acid residues.

[0066] Manzamin F ( Manzamine F )like Figure 11 (B) exhibits the strongest binding ability (docking score -10.289 kcal / mol). Its complex polycyclic structure is precisely embedded in a hydrophobic pocket composed of hydrophobic residues (such as Val70, Ala83, Tyr134), forming strong hydrophobic interactions and van der Waals forces. Simultaneously, the nitrogen atom in its molecule may form crucial hydrogen bonds with the carbonyl group of the main chain or the side chain (such as Asp133), thereby efficiently and competitively inhibiting ATP binding and suppressing the kinase activity of GSK-3β. Acetyl strychnine ( Acetylspartioidine )like Figure 11 (A) shows the relationship between guaramide A ( SesbanimideA )like Figure 11 (C) also shows strong binding affinity (scores of -7.664 and -7.720 kcal / mol, respectively). Both bind to the hydrophobic region of the active pocket through their molecular backbone, and may form hydrogen bond networks with polar residues (such as Lys85, Arg141) at the edge of the active site through their respective polar groups (such as ester bonds, hydroxyl groups), stabilizing the binding conformation and interfering with substrate recognition. Taurocholic acid ( Taurocholic acid )like Figure 11 (D) shows the relationship with tryptamine ( Tryptamine )like Figure 11(E) indicates that the binding site may be an allosteric regulatory site or channel entrance near the active pocket. The sulfate group of taurocholic acid and the indole ring of tryptophan may interact with positively charged residues (such as Arg96) or aromatic residues (such as Phe67) through electrostatic or π-π stacking interactions, thereby inducing conformational changes and indirectly inhibiting GSK-3β function.

[0067] Figure 12 (AE) Analysis of molecular docking results between β-secretase (BACE1) and differentially expressed metabolites: BACE1 is a key rate-limiting enzyme catalyzing the cleavage of amyloid precursor protein (APP) to produce Aβ, and is a core target for inhibiting Aβ production. For example... Figure 12 As shown in (AE), each metabolite can occupy the substrate-binding cleft of BACE1.

[0068] Manzamin F Figure 12 (B) again demonstrates excellent binding potential (docking score -8.192 kcal / mol). Its large, rigid structure may span across the bis-aspartic acid catalytic site (Asp32 / Asp228), interacting with the catalytic center and multiple substrate binding pockets through multiple hydrophobic arms (such as Tyr71, Phe108, Trp115) and potential hydrogen bond donors / acceptors, thereby generating extremely strong competitive inhibition. (Selenamide A, for example...) Figure 12 (C) and acetyl strychnine Figure 12 (A) shows that binding occurs through the "flap" regions of BACE1 (e.g., Gly230-Thr232) or S2 / S3 sub-pockets (e.g., Leu30, Tyr198). The epoxy structure of guaramide A and the acetoxy group of acetyl stigmine may act as hydrogen bond acceptors, forming hydrogen bonds with key amino acids (e.g., Arg235, Ser325) to fix the open conformation of the "flap" or hinder the correct orientation of the substrate APP. Taurocholic acid, such as... Figure 12 (D) shows that its taurine side chain extends into the solvent region through its steroid backbone and hydrophobic channels of BACE1, potentially acting as an anchor and increasing solubility. Tryptamines, for example... Figure 12 (E) shows that the indole ring is inserted into a hydrophobic pocket composed of aromatic residues (such as Tyr198, Phe108), and its ethylamine side chain may mimic the substrate P1 site and interact weakly with catalytic aspartic acid.

[0069] In conclusion, molecular docking results, based on three-dimensional structural analysis, confirmed that the five differentially expressed metabolites of the mixed probiotic formulation (1:3:1) all bind with high affinity to the key AD targets GSK-3β and BACE1. Manzamin F exhibited the strongest binding affinity to both targets, suggesting it may be the most promising lead compound. These metabolites, by occupying the active site, forming key hydrogen bonds, and engaging in hydrophobic interactions, are expected to directly inhibit the kinase activity of GSK-3β (thereby reducing tau protein hyperphosphorylation) and the protease activity of BACE1 (thereby reducing Aβ production). This provides an important molecular mechanism hypothesis and structural basis for explaining the intervention of the mixed probiotic formulation and its postbiotics in the core pathological process of AD through the "microbial metabolites-gut-brain axis" pathway.

[0070] Example 5: Preparation and antioxidant properties of different forms of biogenics after mixing probiotic formulations Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB 3-14, and Bifidobacterium breve NKIBB1-2 were cultured individually for 20-24 h in an aerobic environment of MRS liquid medium, an anaerobic environment of BS liquid medium, and an anaerobic environment of BBL liquid medium, respectively.

[0071] The suspensions of *Lactobacillus rhamnosus* NKU ML1-2, *Bifidobacterium longum* subsp. *infantii* NKU FB 3-14, and *Bifidobacterium breve* NKIBB1-2 were centrifuged (8000 g, 15 min, 4℃), and the supernatant was discarded. The bacterial concentrations of the three bacteria were adjusted to 1-5 × 10⁻⁶ using BBL liquid medium. 9 CFU / mL. The three bacteria were inoculated into BBL liquid medium (containing 1% inulin) at a volume ratio of 4% and cultured in an anaerobic environment for 24 h to prepare the mixed probiotic postbiotic.

[0072] The culture medium was centrifuged at 8000 g for 5 min to separate the supernatant and bacterial precipitate. The culture medium, supernatant, and bacterial cells were heated in a water bath at 70℃-80℃ for 25-35 min and then freeze-dried under vacuum to obtain three postbiotic forms of mixed probiotic preparations: heat-inactivated bacterial cells + supernatant, heat-inactivated supernatant, and heat-inactivated bacterial cells.

[0073] In vitro antioxidant experiments such as Figure 13-14 This indicates that all three metabiotic forms possess a certain degree of free radical scavenging ability. However, the DPPH and hydroxyl radical scavenging rates of the heat-inactivated supernatant (S) were significantly higher than those of the heat-inactivated bacterial cells (J) and the heat-inactivated supernatant + bacterial cells (H) group (P<0.05). This suggests that the soluble metabolites produced by the mixed probiotics during fermentation and present in the supernatant are their main antioxidant active components, and heat inactivation did not significantly destroy the activity of these substances.

[0074] Example 6: Improvement of cell viability and oxidative stress in AD cell models by different postbiotic forms of mixed probiotic preparations. 6.1 Cell Culture HT-22 cells were cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution in a 37°C, 5% CO2 incubator using conventional methods.

[0075] (1) Recovery: Take out the frozen cells from -80℃, thaw them quickly in a 37℃ water bath, transfer them into a centrifuge tube containing 5 mL of complete culture medium, centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend them in fresh culture medium, inoculate them into 100 mm culture dishes (containing 8-10 mL of culture medium), mix well and place them in an incubator.

[0076] (2) Subculture: When the cells adhere to the wall and grow to 80-90% confluence, wash three times with 1×PBS, digest with 0.25% trypsin, add complete culture medium to stop the culture, centrifuge and discard the supernatant, resuspend and dispense into new culture dishes in an appropriate ratio for continued culture.

[0077] (3) Cryopreservation: Fetal bovine serum containing 10% DMSO was used as the cryopreservation solution. Cells in good condition were digested, centrifuged, resuspended in the cryopreservation solution and aliquoted into cryovials. They were first placed in a -20℃ freezer for 30 min and then transferred to -80℃ for long-term storage.

[0078] (4) Counting: Dilute the cell suspension 20 times with complete culture medium, add 10 μL to a cell counting chamber, and count the total number of cells in the four squares according to the formula (total number / 4 × 20 × 10). 4 ) Calculate the cell concentration in the original suspension and use this as a basis for subsequent plate laying experiments.

[0079] 6.2 MTT assay to screen okadaic acid for half-maximal inhibitory concentration (IC50) in HT-22 cells. Using Okada acid ( Okadaic Acid An in vitro AD model was constructed by inducing the mouse hippocampal neuronal cell line HT-22 with OA. HT-22 cells were treated with different concentrations of OA (20, 40, 60, 80, 100 nmol / L), and cell viability was detected by MTT assay.

[0080] Cell viability (%) = (Experimental group absorbance - Zeroing well absorbance) / (Control group absorbance - Zeroing well absorbance) × 100%.

[0081] Through the MTT method ( Figure 15 The half-maximal inhibitory concentration (IC50) of OA on HT-22 cells was determined to be approximately 80 nmol / L, and this concentration was used for subsequent experiments to establish the model.

[0082] 6.3 Experimental Grouping The experiment consisted of six groups: a control group (Con group), a model group (OA group), a heat-inactivated supernatant + bacterial cell intervention group (OA+H group), a heat-inactivated supernatant intervention group (OA+S group), a heat-inactivated bacterial cell intervention group (OA+J group), and a positive control group (donepezil hydrochloride group) (OA+DP group). The control group was treated with complete culture medium, the model group was treated with 80 nmol / L okadaic acid (OA), and each intervention group received different forms of postbiotic preparations in addition to OA. The positive control group received donepezil hydrochloride in addition to OA.

[0083] The preparation methods for each group of drugs are as follows: (1) Postbiotic preparations: Take 200-300 mg of heat-inactivated supernatant (S), heat-inactivated supernatant + bacterial cells (H), and heat-inactivated bacterial cells (J) lyophilized powder (all derived from a total bacterial concentration of approximately 1.5 × 10⁻⁶ before inactivation). 10 A mixed probiotic culture (CFU / mL) was diluted 200 times with DMEM complete medium to obtain the working solution.

[0084] (2) OA stock solution and working solution: Weigh 50 µg of OA powder and dissolve it in 310 µL of DMSO to prepare a 200 µmol / L stock solution. Aliquot the stock solution and store at -80℃. When using, dilute it 2500 times with DMEM complete medium to obtain an 80 nmol / L OA working solution.

[0085] (3) DP stock solution and working solution: Weigh 5 mg donepezil hydrochloride (DP), dissolve it in 102 µL DMSO to prepare a 0.1 mol / L stock solution, and aliquot and store at -80℃. When using, dilute 2000 times with DMEM complete medium to obtain a 50 µmol / L DP working solution.

[0086] 6.4 Detection of HT-22 cell viability using the CCK-8 assay HT-22 cell viability was detected using the CCK-8 assay. The specific steps are as follows: (1) Cells were spaced at 1.5 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 µL in 96-well plates, with 6 replicates per group. 100 µL of cell suspension was added to each well and the cells were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0087] (2) After the cells adhere to the culture medium, replace the culture medium with the corresponding drugs (OA and different post-genetics or positive control drugs) according to the above grouping, and continue to culture for 12-24 hours.

[0088] (3) After the intervention, add 10 µL of CCK-8 reagent mixture (volume ratio 10:1) to each well, shake gently to mix, and then incubate in a 37℃, 5% CO2 incubator for 2 hours in the dark.

[0089] (4) After incubation, the optical density (OD) value of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0090] The cell viability calculation formula is: (OD value of experimental group - OD value of blank well) / (OD value of control group - OD value of blank well) × 100%.

[0091] Figure 16 The results showed that cell viability was significantly decreased in the OA model group compared with the Con group (P<0.001). Compared with the OA group, all intervention groups improved cell viability to varying degrees. Among them, the heat-inactivated supernatant (S) group showed the most significant effect, restoring cell viability to 85.4% of that in the Con group, which was better than the heat-inactivated supernatant + bacterial cells (H) group and the heat-inactivated bacterial cells (J) group, and the effect was close to that of the positive control drug donepezil (DP).

[0092] 6.5 TBA method for detecting MDA levels Prepare the required solutions according to the kit instructions. Perform experimental grouping and drug preparation for each group as described in section 6.3, and incubate in a boiling water bath for 15 min. Cool to room temperature and centrifuge at 1000×g for 10 min. Add 200 μL of supernatant to each 96-well plate and measure the absorbance of each well at 532 nm using a microplate reader.

[0093] MDA is the end product of lipid peroxidation. For example... Figure 17 As shown, the MDA content in the OA model group was significantly higher than that in the Con group (P<0.001). All postbiotic intervention groups reduced MDA levels, with group S showing the best effect, significantly superior to groups H and J. This confirms that mixed probiotic postbiotics, especially their supernatant, can effectively alleviate OA-induced oxidative stress damage in nerve cells.

[0094] In the HT-22 cell AD model, all three metabiotic forms of the mixed probiotics improved OA-induced cytotoxicity and reduced oxidative stress damage (lowering MDA). Their protective effects differed: heat-inactivated supernatant (S) showed the most significant effect, followed by heat-inactivated supernatant + bacterial cells (H), while heat-inactivated bacterial cells (J) had a relatively weaker effect. This indicates that soluble metabolites (such as short-chain fatty acids, bacteriocins, and enzymes) in the probiotic fermentation supernatant play a central role in neuroprotection.

[0095] Example 7: Effects of different postbiotic forms of mixed probiotic preparations on inflammation levels and β-amyloid (Aβ) production in an AD cell model. 7.1 ELISA method for detecting inflammatory factors HT-22 cells were divided into four groups: control group (Con group), model group (OA group), heat-inactivated supernatant + bacterial cell group (OA+H group), heat-inactivated supernatant group (OA+S group), heat-inactivated bacterial cell group (OA+J group), and donepezil hydrochloride positive control group (OA+DP group). After culturing in the appropriate conditioned medium for 24 h, the cell culture supernatant from each group was collected (centrifuged at 4000 r / min, 4℃ for 20 min) to remove cell particles and polymers. The supernatant was stored below -20℃ to avoid repeated freeze-thaw cycles. The levels of inflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-10) in the cell samples of each group were analyzed using an ELISA kit. Samples and biotin antigen were added, and the mixture was incubated at 37℃ for 0.5 h; the plate was washed 5 times; avidin HRP was added, and the mixture was incubated at 37℃ for 0.5 h, followed by 5 washes. Indicators A and B were added sequentially, and the mixture was incubated at 37℃ for 10 min. Add the stop solution, then measure the OD value using a microplate reader. Each sample was repeated three times. The concentration of inflammatory factors in each group was calculated using a standard curve, in ng / L. All procedures were strictly performed according to the kit instructions, and standard wells and blank wells were prepared.

[0096] The results are as follows Figure 18 As shown, OA stimulation significantly promoted the secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α, while inhibiting the expression of the anti-inflammatory factor IL-10. All three post-biotic interventions exhibited anti-inflammatory effects: they significantly inhibited the release of pro-inflammatory factors and promoted IL-10 expression. Similarly, the heat-inactivated supernatant (S) group showed the most significant effects in inhibiting IL-1β, IL-6, and TNF-α, and also exhibited the strongest upregulation of IL-10; its overall anti-inflammatory effect was significantly superior to that of the H and J groups.

[0097] 7.2 FRET method for detecting BACE1 enzyme activity (1) Cell processing and lysis: HT-22 cells were seeded in 6-well plates (2×10⁻⁶ cells / wells). 5 / well), and treated in groups for 24 h. Discard the supernatant, wash twice with pre-cooled PBS, add 200 μL of RIPA lysis buffer containing protease inhibitor to each well, and lyse on ice for 30 min. Centrifuge at 12000×g for 15 min at 4℃, collect the supernatant, determine the protein concentration by BCA method, and adjust to 1 μg / μL.

[0098] (2) Enzyme activity assay: In a black 96-well plate, add 50 μL of cell lysis buffer (containing 50 μg of total protein) and 50 μL of reaction buffer (pH 4.5) containing FRET substrate (final concentration 10 μmol / L) to each well. After gently mixing, incubate at 37°C for 60 min in the dark.

[0099] (3) Fluorescence measurement and calculation: A multi-functional microplate reader was used, with the excitation wavelength set to 325 nm and the emission wavelength to 393 nm, to measure the fluorescence value (F1). Blank wells (reaction buffer + substrate only) and model group (OA group) controls were set up.

[0100] BACE1 relative enzyme activity (%) = [(Experimental group F1 - Blank group F1) / (OA group F1 - Blank group F1)] × 100%.

[0101] β-secretase (BACE1) is a key rate-limiting enzyme catalyzing the production of Aβ from amyloid precursor protein (APP). BACE1 activity was significantly increased in the OA model group. Figure 19 As shown, all postbiotic intervention groups inhibited BACE1 activity. Group S showed the highest inhibition rate of BACE1 activity, comparable to the positive control group DP, and significantly superior to group J. This suggests that the mixed probiotic postbiotics, especially the active substances in their supernatant, reduce Aβ production by inhibiting BACE1, thereby intervening in the core pathological process of AD.

[0102] In an OA-induced AD model of HT-22 cells, the mixed probiotic postbiotics alleviated neuroinflammation by inhibiting pro-inflammatory factors and promoting the secretion of anti-inflammatory factors, and also inhibited the activity of BACE1, a key enzyme in Aβ production. Among all tested postbiotic forms, heat-inactivated supernatant (S) showed the strongest anti-inflammatory and Aβ-inhibition potential, followed by heat-inactivated supernatant + bacterial cells (H), while heat-inactivated bacterial cells (J) had relatively limited effects. This further confirms that the soluble metabolites produced by probiotics during fermentation are the main active components exerting neuroprotective, anti-inflammatory, and anti-Aβ effects.

[0103] This study systematically compared the protective effects of three different postbiotic forms of mixed probiotic preparations (heat-inactivated bacterial cells J, heat-inactivated supernatant + bacterial cells H, and heat-inactivated supernatant S) on an okadaic acid (OA)-induced HT-22 cell Alzheimer's disease (AD) model. The results showed that all forms improved cell viability, reduced oxidative stress and neuroinflammation, and inhibited the Aβ production pathway. However, a clear gradient of efficacy was observed: heat-inactivated supernatant (S) > heat-inactivated supernatant + bacterial cells (H) > heat-inactivated bacterial cells (J).

[0104] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a mixed probiotic preparation, characterized in that, Includes the following steps: S1. Inoculate Lactobacillus rhamnosus NKU ML1-2 into MRS liquid medium, Bifidobacterium longum subsp. infantis NKU FB3-14 into BS liquid medium, and Bifidobacterium breve NKU BB1-2 into BBL liquid medium, and culture at 37℃ under anaerobic conditions for 20-24 h respectively. S2. After centrifuging the cultured Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB3-14 and Bifidobacterium breve NKU BB1-2, discard the supernatant, mix them in proportion and inoculate them into BBL medium, add growth aids and culture in an anaerobic environment for 24-48 h to obtain a mixed probiotic preparation. The total number of live bacteria in the mixed probiotic preparation is not less than 1×10⁻⁶. 8 CFU / mL.

2. The preparation method according to claim 1, characterized in that, In step S1, Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB 3-14, and Bifidobacterium breve NKU BB1-2 were inoculated into the culture medium at a volume fraction of 5%.

3. The preparation method according to claim 1, characterized in that, The mixed inoculation ratio of Lactobacillus rhamnosus NKU ML1-2, Bifidobacterium longum subsp. infantis NKU FB 3-14 and Bifidobacterium breve NKU BB1-2 in step S2 is 1:3:

1.

4. The preparation method according to claim 1, characterized in that, The concentration of the growth aid in step S2 in the BBL medium is not less than 1%; the growth aid is one or more of glucose, fructooligosaccharide, galactooligosaccharide and inulin.

5. A mixed probiotic postbiotic, characterized in that, The mixed probiotic postbiotic is prepared by culturing, heat inactivation and drying of a mixed probiotic preparation obtained by any one of the preparation methods described in claims 1-4.

6. The application of the mixed probiotic preparation obtained by the preparation method according to any one of claims 1-4 or the mixed probiotic postbiotic according to claim 5 in the preparation of antioxidant products.

7. The use of the mixed probiotic preparation obtained by the preparation method according to any one of claims 1-4 or the mixed probiotic postbiotic according to claim 5 in the preparation of products for inhibiting the inflammatory response of nerve cells or inhibiting the activity of BACE1, a key enzyme in the production of β-amyloid protein.

8. The use of the mixed probiotic preparation obtained by the preparation method according to any one of claims 1-4 or the mixed probiotic postbiotic according to claim 5 in the preparation of products for the prevention of Alzheimer's disease.

9. The application according to claim 8, characterized in that, The mixed probiotic preparation or mixed probiotic postbiotic can produce differential metabolites with high binding affinity to key targets of Alzheimer's disease. The key targets are glycogen synthase kinase-3β and β-secretase; The metabolites include one or more of acetyl strychnine, manzamin F, senoside A, taurcholic acid, and tryptamine.

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