Probiotic composition with neuroprotective and cognitive improvement functions and method for preparing the same

Abstract

The present application relates to the technical field of probiotic composition, and provides a probiotic composition with nerve protection and cognitive improvement functions and a preparation method thereof.The composition is composed of Lactobacillus rhamnosus GG HBM11-69, Lactobacillus rhamnosus HBM11-35 and one or more oligosaccharides, and is prepared by fermentation, freeze-drying and mixing with a prebiotic carrier.The composition significantly improves the generation of short-chain fatty acids and gamma-aminobutyric acid through the gut-brain axis regulation mechanism, up-regulates the expression of BDNF to enhance hippocampal synaptic plasticity, protects the integrity of the blood-brain barrier, thereby improving D-galactose-induced cognitive impairment and relieving brain neuroinflammation, and enhancing learning and memory ability.

Description

Technical Field

[0001] This invention relates to the field of probiotic composition technology, and in particular to a probiotic composition with neuroprotective and cognitive-improving functions and its preparation method. Background Technology

[0002] Neurodegenerative diseases, mild cognitive impairment, and age-related memory decline have become significant health problems seriously affecting the quality of life of middle-aged and elderly people. Current clinical interventions mainly involve drug interventions such as cholinesterase inhibitors and NMDA receptor antagonists, but these often suffer from limited efficacy, poor tolerability, numerous adverse reactions, and poor long-term adherence, making it difficult to achieve comprehensive improvement from multiple dimensions such as inflammatory response, neuroplasticity, and the gut-brain axis.

[0003] In recent years, numerous studies have shown that the gut microbiota participates in regulating neuroinflammation, neurotransmitter metabolism, and brain-derived neurotrophic factor (BDNF) expression through the gut-brain axis. Probiotics and synbiotics have shown potential in treating mood disorders and cognitive impairment. However, many existing probiotic preparations primarily target mood improvement or general gut health, with strain selection and formulation design largely based on experience. There is a lack of systematic optimization around key pathways such as short-chain fatty acids, gamma-aminobutyric acid (GABA), and BDNF, resulting in limited specific evidence regarding their effects on cognitive function and neuronal survival.

[0004] Existing technology CN121197234A discloses the application of the combination of *Lactobacillus rhamnosus* GOLDGUT-M520 and *Lactobacillus reuteri* GOLDGUT-LR99 in the preparation of products for alleviating depression. Its antidepressant effect was evaluated using a chronic unpredictable mild stress model, demonstrating that this strain combination can improve depressive-like behaviors and regulate gut microbiota structure, short-chain fatty acid levels, and indicators such as 5-HT, CORT, and BDNF-TRKB / CREB, making it suitable for adjunctive intervention for depression. However, from the perspective of neuroprotection and cognitive improvement, CN121197234A still has the following shortcomings: It only uses depressive-like behavioral indicators such as tail suspension, forced swimming, and sucrose preference as evaluation endpoints, without employing a D-galactose-induced cognitive impairment model or classic learning and memory experiments such as the Morris water maze, thus lacking direct evidence for improvement in age-related cognitive decline. While the mechanistic study involved short-chain fatty acids, 5-HT, and the BDNF-TRKB / CREB pathway, it did not investigate key neuroactive metabolites such as γ-aminobutyric acid (GABA), nor did it validate direct neuroprotective effects in in vitro neuronal models such as H2O2-induced SH-SY5Y injury, indicating insufficient specificity. A single probiotic combination was used, without introducing exogenous oligosaccharides. Summary of the Invention

[0005] Given that existing probiotic preparations often focus on mood improvement or general gut health, lacking systematic design and validation around key pathways such as short-chain fatty acids, γ-aminobutyric acid (GABA), and brain-derived neurotrophic factor (BDNF), the technical problem to be solved by this invention is to provide a probiotic composition and its preparation method for neuroprotection and cognitive improvement. This composition can increase the production of short-chain fatty acids and GABA while upregulating BDNF expression and reducing neuroinflammation, thereby improving learning and memory decline and cognitive dysfunction caused by aging and other factors.

[0006] To achieve the above objectives, the overall technical solution adopted by this invention is as follows: A probiotic composition with neuroprotective and cognitive-improving functions is provided. This composition includes at least one of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, preferably a combination of both, and further formulated with one or more oligosaccharides. The oligosaccharides include one or more of galactooligosaccharides, resistant dextrin, 2'-fucosylated lactose, xylooligosaccharides, isomaltooligosaccharides, and inulin. By combining specific strains with oligosaccharides of specific structures, the composition produces higher levels of short-chain fatty acids and γ-aminobutyric acid (GABA) during intestinal fermentation. Simultaneously, it upregulates brain-derived neurotrophic factor and inhibits the expression of inflammatory factors through the gut-brain axis pathway, thereby achieving multi-pathway synergistic control and achieving the goals of neuronal protection and cognitive function improvement.

[0007] Furthermore, the *Lactobacillus reuteri* and *Lactobacillus rhamnosus* mentioned herein include, but are not limited to, live and sterilized forms.

[0008] This invention also discloses a method for preparing the above-mentioned probiotic composition with neuroprotective and cognitive-improving functions, comprising the following steps: S1. Inoculate *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35 separately into lactic acid bacteria culture medium, with an inoculation amount of 1-5% of the culture medium volume. Incubate at 30-40℃ under anaerobic conditions for 10-24 hours until the viable count in their respective fermentation broths reaches 1×10⁻⁶. 8 -1×10 10 The fermentation broth of the strain was obtained by measuring CFU / mL; the fermentation broth was centrifuged at 4-10℃ and 3000-12000×g for 5-20 min, the supernatant was discarded, and the bacterial precipitate was collected; the precipitate was resuspended in a protective agent solution to make the viable count in the bacterial suspension 1×10⁻⁶. 10 -1×10 12 CFU / mL; then the bacterial suspension was pre-frozen at -30~-50℃ for 2-8h, and then freeze-dried at a vacuum degree ≤100Pa for 12-36h to obtain freeze-dried powder of Lactobacillus reuteri HBM11-69 and freeze-dried powder of Lactobacillus rhamnosus HBM11-35. S2. Based on the number of live bacteria, HBM11-69 lyophilized powder and HBM11-35 lyophilized powder are mixed at a live bacteria ratio of 0.5-2:1 to obtain a mixed lyophilized powder; then, the mixed lyophilized powder is mixed with oligosaccharides at a mass ratio of 1:5-15 to obtain a probiotic composition with neuroprotective and cognitive-improving functions.

[0009] Preferably, the lactic acid bacteria culture medium is MRS liquid medium; the viable count of each strain at the fermentation endpoint is preferably ≥1×10⁻⁶. 9 CFU / mL.

[0010] Preferably, the protective agent is composed of a disaccharide and dairy solids; the disaccharide is selected from at least one of trehalose and sucrose; the dairy solids are selected from at least one of skim milk powder and whole milk powder; the mass fraction of the disaccharide and dairy solids in the protective agent is 5-20% each.

[0011] Preferably, the ratio of live bacteria count of HBM11-69 lyophilized powder to HBM11-35 lyophilized powder is 1:1.

[0012] Preferably, the oligosaccharide is selected from one or more of galactooligosaccharides, resistant dextrin, 2'-fucosylated lactose, xylooligosaccharides, isomaltooligosaccharides, and inulin; more preferably, the oligosaccharide is a mixture of resistant dextrin and 2'-fucosylated lactose. More preferably, when two oligosaccharides are used in combination, the proportion of 2'-fucosylated lactose in the total mass of the oligosaccharides is 10-40%, more preferably 15-25%.

[0013] Preferably, the mass ratio of the mixed freeze-dried powder to the oligosaccharide is 1:9.

[0014] The beneficial effects of this invention are: By systematically combining *Lactobacillus reuteri* HBM11-69, *Lactobacillus rhamnosus* HBM11-35, and various oligosaccharides, the composition of this invention significantly increases the yield of total short-chain fatty acids and γ-aminobutyric acid (GABA) during in vitro co-fermentation. In an H2O2-induced SH-SY5Y cell injury model, the fermentation supernatant of the composition of this invention significantly improved cell viability and upregulated BDNF expression, showing significantly better performance than formulations containing only *Lactobacillus reuteri* HBM11-69, *Lactobacillus rhamnosus* HBM11-35, or only 2'-fucosylated lactose, demonstrating that the composition has direct neuroprotective and neurotrophic regulatory effects.

[0015] In a D-galactose-induced cognitive impairment model in mice, the composition of this invention significantly shortened the escape latency in the Morris water maze and increased the dwell time in the original platform quadrant. At the same time, it upregulated hippocampal BDNF and reduced IL-6 levels. Its effects were superior to those of simple probiotic combinations and 2'-fucosylated lactose alone, demonstrating the synergistic and unexpected technical effects of combining specific strains with specific oligosaccharides. Detailed Implementation

[0016] The parameters and sources of some raw materials in the examples are as follows: Lactobacillus reuteri HBM11-69, classified as Limosilactobacillus reuteri, is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 63608) on June 30, 2023. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology.

[0017] Lactobacillus rhamnosus HBM11-35 is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 63607) on June 30, 2023. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology.

[0018] Galacto-oligosaccharides (GOS), purity ≥90%, DP2-8, food-grade powder.

[0019] Resistant dextrin, dietary fiber content ≥85%, food-grade powder.

[0020] 2'-Fucose-based lactose (2'-FL), CAS 41263-94-9, purity ≥95%, food-grade powder.

[0021] Xylooligosaccharides (XOS), purity ≥90%, DP 2-7.

[0022] Isomaltooligosaccharide (IMO), purity ≥90%.

[0023] Inulin, purity ≥90%, average degree of polymerization DP ≥10.

[0024] Trehalose, purity ≥99%.

[0025] Skim milk powder, food grade.

[0026] Maltodextrin, DE 10-15, purity ≥95%.

[0027] Preparation method of MRS liquid culture medium: 10.0g casein peptone (trypsin digestion), 10.0g beef extract, 5.0g yeast extract, 20.0g glucose, 1.0g Tween 80, 5.0g sodium acetate, 2.0g triammonium citrate, 2.0g K2HPO4, 0.2g MgSO4·7H2O, and 0.05g MnSO4·H2O are added to 950mL of sterile water and dissolved evenly. The pH is adjusted to 6.5, and the volume is brought to 1L with sterile water.

[0028] Other reagents: PBS buffer, D-galactose (D-Gal), H2O2, CCK-8, ELISA kit, etc., all of which are analytical grade or cell culture grade. Example 1

[0029] A method for preparing a probiotic composition with neuroprotective and cognitive-improving functions includes the following steps: S1. Open the freeze-dried culture tubes of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, and inoculate an appropriate amount of each strain into 3 mL of sterile MRS liquid medium. Incubate anaerobicly at 37°C for 18 h to complete the recovery process. Inoculate each recovered bacterial culture at a volume ratio of 2% into fresh MRS liquid medium and incubate anaerobicly at 37°C for 16 h until the viable bacterial count on plates reaches ≥1×10⁻⁶. 9 CFU / mL; when the bacterial count is ≥1×10⁻⁶ 9 Fermentation was terminated at CFU / mL to obtain HBM11-69 fermentation broth and HBM11-35 fermentation broth, respectively. Centrifuge the fermentation broth at 4℃ and 8000×g for 10 min, discard the supernatant, and collect the bacterial precipitate; resuspend it in a protectant to make the viable cell count in the suspension approximately 1×10⁻⁶. 11 CFU / mL; the bacterial suspension was dispensed into lyophilization bottles, pre-frozen at -40℃ for 4 h, and then freeze-dried in a freeze dryer under a vacuum of ≤50 Pa for 24 h to obtain lyophilized bacterial powder; plate count analysis showed that the viable count of HBM11-69 lyophilized powder was 1.2 × 10⁻⁶. 11 The CFU / g viable count of HBM11-35 lyophilized powder is 1.0 × 10⁻⁶. 11 CFU / g; The protective agent is obtained by dissolving 10g trehalose and 10g skim milk powder in 80g sterile water and mixing them evenly. S2. Based on the viable count, mix HBM11-69 lyophilized powder and HBM11-35 lyophilized powder at a viable count ratio of 1:1 to obtain a mixed lyophilized powder. Then, mix the mixed lyophilized powder with maltodextrin at a mass ratio of 1:9 until the final mixed powder contains a total viable count of 1 × 10⁻⁶. 10CFU / g; Place in a V-type mixer and mix at 20 rpm for 15 minutes to obtain a uniform probiotic composition powder; Pack the obtained powder into aluminum-plastic composite bags, each bag containing 2g, and each bag contains a total of 2×10⁻⁶ live bacteria. 10 The live bacteria count ratio of CFU, HBM11-69 and HBM11-35 was 1:1; the resulting product was a light yellow powder with no visible impurities and a water content of ≤5%. No abnormal organic impurities were detected by HPLC. Example 2

[0030] A method for preparing a probiotic composition with neuroprotective and cognitive-improving functions includes the following steps: S1. Open the freeze-dried culture tubes of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, and inoculate an appropriate amount of each strain into 3 mL of sterile MRS liquid medium. Incubate anaerobicly at 37°C for 18 h to complete the recovery process. Inoculate each recovered bacterial culture at a volume ratio of 2% into fresh MRS liquid medium and incubate anaerobicly at 37°C for 16 h until the viable bacterial count on plates reaches ≥1×10⁻⁶. 9 CFU / mL; when the bacterial count is ≥1×10⁻⁶ 9 Fermentation was terminated at CFU / mL to obtain HBM11-69 fermentation broth and HBM11-35 fermentation broth, respectively. Centrifuge the fermentation broth at 4℃ and 8000×g for 10 min, discard the supernatant, and collect the bacterial precipitate; resuspend it in a protectant to make the viable cell count in the suspension approximately 1×10⁻⁶. 11 CFU / mL; the bacterial suspension was dispensed into lyophilization bottles, pre-frozen at -40℃ for 4 h, and then freeze-dried in a freeze dryer under a vacuum of ≤50 Pa for 24 h to obtain lyophilized bacterial powder; plate count analysis showed that the viable count of HBM11-69 lyophilized powder was 1.2 × 10⁻⁶. 11 The CFU / g viable count of HBM11-35 lyophilized powder is 1.0 × 10⁻⁶. 11 CFU / g; The protective agent is obtained by dissolving 10g trehalose and 10g skim milk powder in 80g sterile water and mixing them evenly. S2. Based on the viable count, mix HBM11-69 lyophilized powder and HBM11-35 lyophilized powder at a viable count ratio of 1:1 to obtain a mixed lyophilized powder. Then, mix the mixed lyophilized powder with galactooligosaccharides at a mass ratio of 1:9 until the final mixed powder contains a total viable count of 1 × 10⁻⁶. 10 CFU / g; Place in a V-type mixer and mix at 20 rpm for 15 minutes to obtain a uniform probiotic composition powder; Pack the obtained powder into aluminum-plastic composite bags, each bag containing 2g, and each bag contains a total of 2×10⁻⁶ live bacteria. 10 The live bacteria ratio of CFU, HBM11-69 to HBM11-35 was 1:1. Example 3

[0031] A method for preparing a probiotic composition with neuroprotective and cognitive-improving functions includes the following steps: S1. Open the freeze-dried culture tubes of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, and inoculate an appropriate amount of each strain into 3 mL of sterile MRS liquid medium. Incubate anaerobicly at 37°C for 18 h to complete the recovery process. Inoculate each recovered bacterial culture at a volume ratio of 2% into fresh MRS liquid medium and incubate anaerobicly at 37°C for 16 h until the viable bacterial count on plates reaches ≥1×10⁻⁶. 9 CFU / mL; when the bacterial count is ≥1×10⁻⁶ 9 Fermentation was terminated at CFU / mL to obtain HBM11-69 fermentation broth and HBM11-35 fermentation broth, respectively. Centrifuge the fermentation broth at 4℃ and 8000×g for 10 min, discard the supernatant, and collect the bacterial precipitate; resuspend it in a protectant to make the viable cell count in the suspension approximately 1×10⁻⁶. 11 CFU / mL; the bacterial suspension was dispensed into lyophilization bottles, pre-frozen at -40℃ for 4 h, and then freeze-dried in a freeze dryer under a vacuum of ≤50 Pa for 24 h to obtain lyophilized bacterial powder; plate count analysis showed that the viable count of HBM11-69 lyophilized powder was 1.2 × 10⁻⁶. 11 The CFU / g viable count of HBM11-35 lyophilized powder is 1.0 × 10⁻⁶. 11 CFU / g; The protective agent is obtained by dissolving 10g trehalose and 10g skim milk powder in 80g sterile water and mixing them evenly. S2. Based on the viable count, mix HBM11-69 lyophilized powder and HBM11-35 lyophilized powder at a viable count ratio of 1:1 to obtain a mixed lyophilized powder. Then, mix the mixed lyophilized powder with resistant dextrin at a mass ratio of 1:9 until the final mixed powder contains a total viable count of 1 × 10⁻⁶. 10 CFU / g; Place in a V-type mixer and mix at 20 rpm for 15 minutes to obtain a uniform probiotic composition powder; Pack the obtained powder into aluminum-plastic composite bags, each bag containing 2g, and each bag contains a total of 2×10⁻⁶ live bacteria. 10 The live bacteria ratio of CFU, HBM11-69 to HBM11-35 was 1:1. Example 4

[0032] A method for preparing a probiotic composition with neuroprotective and cognitive-improving functions includes the following steps: S1. Open the freeze-dried culture tubes of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, and inoculate an appropriate amount of each strain into 3 mL of sterile MRS liquid medium. Incubate anaerobicly at 37°C for 18 h to complete the recovery process. Inoculate each recovered bacterial culture at a volume ratio of 2% into fresh MRS liquid medium and incubate anaerobicly at 37°C for 16 h until the viable bacterial count on plates reaches ≥1×10⁻⁶. 9 CFU / mL; when the bacterial count is ≥1×10⁻⁶ 9 Fermentation was terminated at CFU / mL to obtain HBM11-69 fermentation broth and HBM11-35 fermentation broth, respectively. Centrifuge the fermentation broth at 4℃ and 8000×g for 10 min, discard the supernatant, and collect the bacterial precipitate; resuspend it in a protectant to make the viable cell count in the suspension approximately 1×10⁻⁶. 11 CFU / mL; the bacterial suspension was dispensed into lyophilization bottles, pre-frozen at -40℃ for 4 h, and then freeze-dried in a freeze dryer under a vacuum of ≤50 Pa for 24 h to obtain lyophilized bacterial powder; plate count analysis showed that the viable count of HBM11-69 lyophilized powder was 1.2 × 10⁻⁶. 11 The CFU / g viable count of HBM11-35 lyophilized powder is 1.0 × 10⁻⁶. 11 CFU / g; The protective agent is obtained by dissolving 10g trehalose and 10g skim milk powder in 80g sterile water and mixing them evenly. S2. Based on the viable count, mix HBM11-69 lyophilized powder and HBM11-35 lyophilized powder at a viable count ratio of 1:1 to obtain a mixed lyophilized powder. Then, mix the mixed lyophilized powder with 2'-fucosylated lactose at a mass ratio of 1:9 until the final mixed powder contains a total viable count of 1 × 10⁻⁶. 10 CFU / g; Place in a V-type mixer and mix at 20 rpm for 15 minutes to obtain a uniform probiotic composition powder; Pack the obtained powder into aluminum-plastic composite bags, each bag containing 2g, and each bag contains a total of 2×10⁻⁶ live bacteria. 10 The live bacteria ratio of CFU, HBM11-69 to HBM11-35 was 1:1. Example 5

[0033] A method for preparing a probiotic composition with neuroprotective and cognitive-improving functions includes the following steps: S1. Open the freeze-dried culture tubes of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, and inoculate an appropriate amount of each strain into 3 mL of sterile MRS liquid medium. Incubate anaerobicly at 37°C for 18 h to complete the recovery process. Inoculate each recovered bacterial culture at a volume ratio of 2% into fresh MRS liquid medium and incubate anaerobicly at 37°C for 16 h until the viable bacterial count on plates reaches ≥1×10⁻⁶. 9CFU / mL; when the bacterial count is ≥1×10⁻⁶ 9 Fermentation was terminated at CFU / mL to obtain HBM11-69 fermentation broth and HBM11-35 fermentation broth, respectively. Centrifuge the fermentation broth at 4℃ and 8000×g for 10 min, discard the supernatant, and collect the bacterial precipitate; resuspend it in a protectant to make the viable cell count in the suspension approximately 1×10⁻⁶. 11 CFU / mL; the bacterial suspension was dispensed into lyophilization bottles, pre-frozen at -40℃ for 4 h, and then freeze-dried in a freeze dryer under a vacuum of ≤50 Pa for 24 h to obtain lyophilized bacterial powder; plate count analysis showed that the viable count of HBM11-69 lyophilized powder was 1.2 × 10⁻⁶. 11 The CFU / g viable count of HBM11-35 lyophilized powder is 1.0 × 10⁻⁶. 11 CFU / g; The protective agent is obtained by dissolving 10g trehalose and 10g skim milk powder in 80g sterile water and mixing them evenly. S2. Based on the viable count, mix HBM11-69 lyophilized powder and HBM11-35 lyophilized powder at a viable count ratio of 1:1 to obtain a mixed lyophilized powder. Then, mix the mixed lyophilized powder with xylooligosaccharides at a mass ratio of 1:9 until the final mixed powder contains a total viable count of 1 × 10⁻⁶. 10 CFU / g; Place in a V-type mixer and mix at 20 rpm for 15 minutes to obtain a uniform probiotic composition powder; Pack the obtained powder into aluminum-plastic composite bags, each bag containing 2g, and each bag contains a total of 2×10⁻⁶ live bacteria. 10 The live bacteria ratio of CFU, HBM11-69 to HBM11-35 was 1:1. Example 6

[0034] A method for preparing a probiotic composition with neuroprotective and cognitive-improving functions includes the following steps: S1. Open the freeze-dried culture tubes of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, and inoculate an appropriate amount of each strain into 3 mL of sterile MRS liquid medium. Incubate anaerobicly at 37°C for 18 h to complete the recovery process. Inoculate each recovered bacterial culture at a volume ratio of 2% into fresh MRS liquid medium and incubate anaerobicly at 37°C for 16 h until the viable bacterial count on plates reaches ≥1×10⁻⁶. 9 CFU / mL; when the bacterial count is ≥1×10⁻⁶ 9 Fermentation was terminated at CFU / mL to obtain HBM11-69 fermentation broth and HBM11-35 fermentation broth, respectively. Centrifuge the fermentation broth at 4℃ and 8000×g for 10 min, discard the supernatant, and collect the bacterial precipitate; resuspend it in a protectant to make the viable cell count in the suspension approximately 1×10⁻⁶. 11CFU / mL; the bacterial suspension was dispensed into lyophilization bottles, pre-frozen at -40℃ for 4 h, and then freeze-dried in a freeze dryer under a vacuum of ≤50 Pa for 24 h to obtain lyophilized bacterial powder; plate count analysis showed that the viable count of HBM11-69 lyophilized powder was 1.2 × 10⁻⁶. 11 The CFU / g viable count of HBM11-35 lyophilized powder is 1.0 × 10⁻⁶. 11 CFU / g; The protective agent is obtained by dissolving 10g trehalose and 10g skim milk powder in 80g sterile water and mixing them evenly. S2. Based on the viable count, mix HBM11-69 lyophilized powder and HBM11-35 lyophilized powder at a viable count ratio of 1:1 to obtain a mixed lyophilized powder. Then, mix the mixed lyophilized powder with isomaltooligosaccharide at a mass ratio of 1:9 until the final mixed powder contains a total viable count of 1 × 10⁻⁶. 10 CFU / g; Place in a V-type mixer and mix at 20 rpm for 15 minutes to obtain a uniform probiotic composition powder; Pack the obtained powder into aluminum-plastic composite bags, each bag containing 2g, and each bag contains a total of 2×10⁻⁶ live bacteria. 10 The live bacteria ratio of CFU, HBM11-69 to HBM11-35 was 1:1. Example 7

[0035] A method for preparing a probiotic composition with neuroprotective and cognitive-improving functions includes the following steps: S1. Open the freeze-dried culture tubes of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, and inoculate an appropriate amount of each strain into 3 mL of sterile MRS liquid medium. Incubate anaerobicly at 37°C for 18 h to complete the recovery process. Inoculate each recovered bacterial culture at a volume ratio of 2% into fresh MRS liquid medium and incubate anaerobicly at 37°C for 16 h until the viable bacterial count on plates reaches ≥1×10⁻⁶. 9 CFU / mL; when the bacterial count is ≥1×10⁻⁶ 9 Fermentation was terminated at CFU / mL to obtain HBM11-69 fermentation broth and HBM11-35 fermentation broth, respectively. Centrifuge the fermentation broth at 4℃ and 8000×g for 10 min, discard the supernatant, and collect the bacterial precipitate; resuspend it in a protectant to make the viable cell count in the suspension approximately 1×10⁻⁶. 11 CFU / mL; the bacterial suspension was dispensed into lyophilization bottles, pre-frozen at -40℃ for 4 h, and then freeze-dried in a freeze dryer under a vacuum of ≤50 Pa for 24 h to obtain lyophilized bacterial powder; plate count analysis showed that the viable count of HBM11-69 lyophilized powder was 1.2 × 10⁻⁶. 11 The CFU / g viable count of HBM11-35 lyophilized powder is 1.0 × 10⁻⁶. 11CFU / g; The protective agent is obtained by dissolving 10g trehalose and 10g skim milk powder in 80g sterile water and mixing them evenly. S2. Based on the viable count, mix HBM11-69 lyophilized powder and HBM11-35 lyophilized powder at a viable count ratio of 1:1 to obtain a mixed lyophilized powder. Then, mix the mixed lyophilized powder with inulin at a mass ratio of 1:9 until the final mixed powder contains a total viable count of 1 × 10⁻⁶. 10 CFU / g; Place in a V-type mixer and mix at 20 rpm for 15 minutes to obtain a uniform probiotic composition powder; Pack the obtained powder into aluminum-plastic composite bags, each bag containing 2g, and each bag contains a total of 2×10⁻⁶ live bacteria. 10 The live bacteria ratio of CFU, HBM11-69 to HBM11-35 was 1:1. Example 8

[0036] A method for preparing a probiotic composition with neuroprotective and cognitive-improving functions includes the following steps: S1. Open the freeze-dried culture tubes of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, and inoculate an appropriate amount of each strain into 3 mL of sterile MRS liquid medium. Incubate anaerobicly at 37°C for 18 h to complete the recovery process. Inoculate each recovered bacterial culture at a volume ratio of 2% into fresh MRS liquid medium and incubate anaerobicly at 37°C for 16 h until the viable bacterial count on plates reaches ≥1×10⁻⁶. 9 CFU / mL; when the bacterial count is ≥1×10⁻⁶ 9 Fermentation was terminated at CFU / mL to obtain HBM11-69 fermentation broth and HBM11-35 fermentation broth, respectively. Centrifuge the fermentation broth at 4℃ and 8000×g for 10 min, discard the supernatant, and collect the bacterial precipitate; resuspend it in a protectant to make the viable cell count in the suspension approximately 1×10⁻⁶. 11 CFU / mL; the bacterial suspension was dispensed into lyophilization bottles, pre-frozen at -40℃ for 4 h, and then freeze-dried in a freeze dryer under a vacuum of ≤50 Pa for 24 h to obtain lyophilized bacterial powder; plate count analysis showed that the viable count of HBM11-69 lyophilized powder was 1.2 × 10⁻⁶. 11 The CFU / g viable count of HBM11-35 lyophilized powder is 1.0 × 10⁻⁶. 11 CFU / g; The protective agent is obtained by dissolving 10g trehalose and 10g skim milk powder in 80g sterile water and mixing them evenly. S2. Based on the viable count, HBM11-69 lyophilized powder and HBM11-35 lyophilized powder are mixed at a viable count ratio of 1:1 to obtain a mixed lyophilized powder. The mixed lyophilized powder, resistant dextrin, and 2'-fucosylated lactose are then mixed evenly at a mass ratio of 1:7.2:1.8 to ensure that the total viable count in the final mixed powder is 1×10⁻⁶. 10CFU / g; Place in a V-type mixer and mix at 20 rpm for 15 minutes to obtain a uniform probiotic composition powder; Pack the obtained powder into aluminum-plastic composite bags, each bag containing 2g, and each bag contains a total of 2×10⁻⁶ live bacteria. 10 The live bacteria ratio of CFU, HBM11-69 to HBM11-35 was 1:1. Example 9

[0037] A method for preparing a probiotic composition with neuroprotective and cognitive-improving functions includes the following steps: S1. Open the freeze-dried culture tubes of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, and inoculate an appropriate amount of each strain into 3 mL of sterile MRS liquid medium. Incubate anaerobicly at 37°C for 18 h to complete the recovery process. Inoculate each recovered bacterial culture at a volume ratio of 2% into fresh MRS liquid medium and incubate anaerobicly at 37°C for 16 h until the viable bacterial count on plates reaches ≥1×10⁻⁶. 9 CFU / mL; when the bacterial count is ≥1×10⁻⁶ 9 Fermentation was terminated at CFU / mL to obtain HBM11-69 fermentation broth and HBM11-35 fermentation broth, respectively. Centrifuge the fermentation broth at 4℃ and 8000×g for 10 min, discard the supernatant, and collect the bacterial precipitate; resuspend it in a protectant to make the viable cell count in the suspension approximately 1×10⁻⁶. 11 CFU / mL; the bacterial suspension was dispensed into lyophilization bottles, pre-frozen at -40℃ for 4 h, and then freeze-dried in a freeze dryer under a vacuum of ≤50 Pa for 24 h to obtain lyophilized bacterial powder; plate count analysis showed that the viable count of HBM11-69 lyophilized powder was 1.2 × 10⁻⁶. 11 The CFU / g viable count of HBM11-35 lyophilized powder is 1.0 × 10⁻⁶. 11 CFU / g; The protective agent is obtained by dissolving 10g trehalose and 10g skim milk powder in 80g sterile water and mixing them evenly. S2. Based on the viable count, HBM11-69 lyophilized powder and HBM11-35 lyophilized powder are mixed at a viable count ratio of 1:1 to obtain a mixed lyophilized powder. The mixed lyophilized powder, xylooligosaccharide, and 2'-fucosylated lactose are then mixed evenly at a mass ratio of 1:7.2:1.8 to ensure that the total viable count in the final mixed powder is 1×10⁻⁶. 10 CFU / g; Place in a V-type mixer and mix at 20 rpm for 15 minutes to obtain a uniform probiotic composition powder; Pack the obtained powder into aluminum-plastic composite bags, each bag containing 2g, and each bag contains a total of 2×10⁻⁶ live bacteria. 10 The live bacteria ratio of CFU, HBM11-69 to HBM11-35 was 1:1. Example 10

[0038] A method for preparing a probiotic composition with neuroprotective and cognitive-improving functions includes the following steps: S1. Open the freeze-dried culture tubes of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35, and inoculate an appropriate amount of each strain into 3 mL of sterile MRS liquid medium. Incubate anaerobicly at 37°C for 18 h to complete the recovery process. Inoculate each recovered bacterial culture at a volume ratio of 2% into fresh MRS liquid medium and incubate anaerobicly at 37°C for 16 h until the viable bacterial count on plates reaches ≥1×10⁻⁶. 9 CFU / mL; when the bacterial count is ≥1×10⁻⁶ 9 Fermentation was terminated at CFU / mL to obtain HBM11-69 fermentation broth and HBM11-35 fermentation broth, respectively. Centrifuge the fermentation broth at 4℃ and 8000×g for 10 min, discard the supernatant, and collect the bacterial precipitate; resuspend it in a protectant to make the viable cell count in the suspension approximately 1×10⁻⁶. 11 CFU / mL; the bacterial suspension was dispensed into lyophilization bottles, pre-frozen at -40℃ for 4 h, and then freeze-dried in a freeze dryer under a vacuum of ≤50 Pa for 24 h to obtain lyophilized bacterial powder; plate count analysis showed that the viable count of HBM11-69 lyophilized powder was 1.2 × 10⁻⁶. 11 The CFU / g viable count of HBM11-35 lyophilized powder is 1.0 × 10⁻⁶. 11 CFU / g; The protective agent is obtained by dissolving 10g trehalose and 10g skim milk powder in 80g sterile water and mixing them evenly. S2. Based on the viable count, HBM11-69 lyophilized powder and HBM11-35 lyophilized powder are mixed at a viable count ratio of 1:1 to obtain a mixed lyophilized powder. The mixed lyophilized powder, inulin, and 2'-fucosylated lactose are then mixed evenly at a mass ratio of 1:7.2:1.8 to ensure that the total viable count in the final mixed powder is 1×10⁻⁶. 10 CFU / g; Place in a V-type mixer and mix at 20 rpm for 15 minutes to obtain a uniform probiotic composition powder; Pack the obtained powder into aluminum-plastic composite bags, each bag containing 2g, and each bag contains a total of 2×10⁻⁶ live bacteria. 10 The live count ratio of CFU-containing *Lactobacillus reuteri* HBM11-69 to *Lactobacillus rhamnosus* HBM11-35 was 1:1.

[0039] Comparative Example 1 To verify the synergistic effect between 2'-fucosylated lactose and a specific probiotic combination, a formulation containing only 2'-fucosylated lactose was prepared as a control.

[0040] Preparation method: Mix 2'-fucosylated lactose and maltodextrin at a mass ratio of 9:1 and place them in a V-type mixer to mix at 20 rpm for 10 min to ensure uniformity; pack the resulting powder into aluminum-plastic composite bags, each bag containing 2g, which does not contain live bacteria.

[0041] The comparative formulation was administered in animal studies at the same 2'-FL dose as in Example 4 to evaluate the effects of 2'-FL alone on cognitive function and neuroinflammation.

[0042] Test Example 1 In vitro co-fermentation and neuronal protection experiments (1) In vitro co-fermentation experiment bacterial culture and fermentation system Take the mixed lyophilized powder prepared in Examples 1-10, and adjust the total viable count to 1×10⁻⁶. 9 CFU / mL was resuspended in sterile PBS to prepare separate bacterial suspensions. The mixed lyophilized powder here is a mixture of HBM11-69 lyophilized powder and HBM11-35 lyophilized powder without the addition of galactooligosaccharides, resistant dextrin, 2'-fucosylated lactose, xylooligosaccharides, isomaltooligosaccharides, and inulin. The mixed lyophilized powder composition is the same in Examples 1-10.

[0043] Basic culture medium: Carbon-free modified MRS medium (glucose removed, other components the same as MRS liquid medium).

[0044] Carbon source settings: Example 1 group: 0.5wt% glucose was added; Examples 2-7: 1 wt% of galactooligosaccharide, resistant dextrin, 2'-fucosylated lactose, xylooligosaccharide, isomaltooligosaccharide, and inulin were added respectively, without the addition of glucose.

[0045] Example 8 group: A mixed carbon source with a total concentration of 1 wt% was added, including 0.8 wt% resistant dextrin and 0.2 wt% 2'-fucosylated lactose; Example 9: A mixed carbon source with a total concentration of 1 wt% was added, including 0.8 wt% xylooligosaccharides and 0.2 wt% 2'-fucosylated lactose; Example 10 group: A mixed carbon source with a total concentration of 1 wt% was added, including 0.8 wt% inulin and 0.2 wt% 2'-fucosylated lactose.

[0046] The use of 0.5 wt% glucose in Example 1 and 1 wt% oligosaccharides in Examples 2-10 was not arbitrary, but rather a comprehensive consideration of the differences between monosaccharides and oligosaccharides in terms of fermentation speed and acid production intensity; the control of the pH of the fermentation system and the stability of cell survival; and ensuring that the total amount of fermentable carbon source is at a similar level to guarantee the scientific validity and comparability of the comparisons between the groups.

[0047] Culture conditions: Inoculate each bacterial suspension at a volume ratio of 1% into a basal medium containing different carbon sources, so that the initial bacterial count is approximately 1 × 10⁻⁶. 7 CFU / mL; cultured at 37℃ under anaerobic conditions for 24 h; after culture, the pH value, bacterial count (plate count method), short-chain fatty acid and γ-aminobutyric acid content of the fermentation broth were measured.

[0048] Short-chain fatty acid (SCFA) detection: An Agilent 7890B gas chromatograph with an FFAP capillary column and a flame ionization detector was used. The concentrations of acetic acid, propionic acid, and butyric acid were calculated using the external standard method. Total SCFA was the sum of these three. SCFA mainly includes acetic acid, propionic acid, and butyric acid, and is a key metabolite produced by probiotic fermentation of prebiotics. Higher total SCFA levels indicate that probiotics can more effectively utilize the prebiotic and produce more beneficial metabolites that maintain the intestinal barrier, have anti-inflammatory effects, and regulate immunity. This, in turn, benefits neuroprotection and cognitive function improvement through the gut-brain axis.

[0049] Gamma-aminobutyric acid (GABA) detection: A Shimadzu LC-20A high-performance liquid chromatograph with a C18 column was used. Fluorescence detection (Ex 340nm, Em 455nm) was performed after OPA derivatization, and quantification was performed using the external standard method. GABA is a major inhibitory neurotransmitter in the central nervous system, involved in regulating anxiety, stress, and learning and memory. Higher GABA concentrations in the fermentation supernatant indicate that the probiotic / prebiotic combination is more conducive to the production of neuroactive substances, helping to alleviate neuronal excitotoxicity and oxidative stress, and enhancing overall neuroprotective potential.

[0050] (2) Neuron protection experiment Cell Culture and Grouping Cells: Human neuroblastoma cells SH-SY5Y (ATCCCRL-2266).

[0051] Culture conditions: DMEM / F12 medium + 10% FBS, 37℃, 5% CO2 constant temperature incubator.

[0052] Grouping: Normal control group (NC); Damage model group (H2O2); Examples 1-10 correspond to the fermentation supernatant treatment group.

[0053] Processing method: After 24 hours of co-fermentation in each embodiment, the fermentation broth was collected and sterilized through a 0.22 μm filter membrane to obtain sterile fermentation supernatant; SH-SY5Y cells were seeded into 96-well plates, 1 × 10⁶ cells per well. 4Cells were cultured for 24 hours; each treatment group was added to complete medium containing 10 vt% of the corresponding fermentation supernatant and incubated for 24 hours; the NC group and the model group were added to the same volume of basal medium; then, except for the NC group, each group was added to the final concentration of 200 μM H2O2 and incubated for 4 hours; cell viability was detected using CCK-8 reagent and intracellular BDNF content was detected using ELISA kit.

[0054] SH-SY5Y is a commonly used in vitro neuronal model. A higher survival rate relative to the model group indicates a stronger protective effect of the fermentation supernatant on neurons under damaging conditions such as oxidative stress, effectively reducing cell damage and demonstrating the direct neuroprotective effect of the composition. Brain-derived neurotrophic factor (BDNF) is a key factor in maintaining neuronal survival, promoting synaptic plasticity, and learning and memory. A higher fold increase in BDNF expression relative to the model group indicates that the composition can more effectively upregulate neurotrophic factor expression, supporting its mechanistic role in improving cognitive function, promoting neural repair, and enhancing plasticity.

[0055] Table 1 Fermentation status of each example

[0057] Table 2 Protective effect on SH-SY5Y cells

[0058] Compared with Example 1, which only had glucose as a carbon source, the addition of different oligosaccharides in Examples 2-7 could increase the production of total short-chain fatty acids and γ-aminobutyric acid to a certain extent, and correspondingly increase the survival rate and BDNF expression of SH-SY5Y cells in the H2O2 damage model, indicating that there is a significant synergistic effect between oligosaccharides and the combination of Lactobacillus reuteri HBM11-69 / Lactobacillus rhamnosus HBM11-35.

[0059] The total short-chain fatty acid concentration and γ-aminobutyric acid level of the fermentation system of Example 4 with 2'-fucosylated lactose were the highest among Examples 2-7, and the corresponding cell viability and BDNF upregulation were also the most prominent, indicating that the combination of 2'-fucosylated lactose and this specific strain has a synergistic effect superior to conventional oligosaccharides.

[0060] Based on this, Examples 8-10 combined 2'-fucosylated lactose with resistant dextrin, xylooligosaccharides or inulin as a carbon source. Among them, Example 8 (resistant dextrin + 2'-fucosylated lactose type) performed the best: the levels of total short-chain fatty acids and γ-aminobutyric acid were further higher than those in Example 4, the corresponding SH-SY5Y cell survival rate increased to about 95%, and BDNF expression increased to about 2.4 times that of the model group.

[0061] In Examples 9 and 10, the combination of 2'-fucosylated lactose with xylooligosaccharides or inulin also showed further improvement compared to Example 4, but the increase was slightly lower than that in Example 8, suggesting that there are compatibility differences between 2'-fucosylated lactose and different oligosaccharides. Among them, the dual compatibility with resistant dextrin can produce the most prominent synergistic effect in the metabolism of short-chain fatty acids and γ-aminobutyric acid in the strain of the present invention.

[0062] The above results indicate that by rationally selecting oligosaccharides such as 2'-fucosylated lactose and resistant dextrin, the metabolic activity and neuroprotective effect of HBM11-69 / HBM11-35 can be significantly enhanced in vitro, demonstrating the non-arbitrariness and unexpected technical effects of the combination of HBM11-69 / HBM11-35 and 2'-fucosylated lactose with resistant dextrin in Example 8.

[0063] Test Example 2 D-galactose-induced cognitive impairment model in mice (1) Laboratory animals and grouping Animals: SPF-grade male C57BL / 6J mice, 8 weeks old, weighing 20±2g. After 1 week of acclimatization, they were randomly divided into 13 groups, n=10 per group. Normal control group (NC): physiological saline + PBS; Model group (D-Gal): D-galactose + PBS; Example 1 group: D-Gal + probiotic composition of Example 1; Example 2 group: D-Gal + probiotic composition of Example 2; Example 3 group: D-Gal + probiotic composition of Example 3; Example 4 group: D-Gal + probiotic composition of Example 4; Example 5 group: D-Gal + probiotic composition of Example 5; Example 6 group: D-Gal + probiotic composition of Example 6; Example 7 group: D-Gal + probiotic composition of Example 7; Example 8 group: D-Gal + the probiotic composition of Example 8; Example 9 group: D-Gal + the probiotic composition of Example 9; Example 10 group: D-Gal + the probiotic composition of Example 10; Comparative Example 1: D-Gal+ formulation containing only 2'-FL (Comparative Example 1).

[0064] (2) Modeling and drug administration Modeling: Except for the NC group, all other groups were injected subcutaneously with D-galactose (120 mg / kg) daily for 8 consecutive weeks; the NC group was injected with an equal volume of physiological saline.

[0065] Administration: From day 1 of modeling, groups 1-10 and Comparative Example 1 were administered the corresponding preparations by gavage daily for 8 consecutive weeks; the probiotic dosage was uniformly 1 × 10⁻⁶ total live bacteria count. 9 CFU / animal / day; the dosage of the probiotic composition was approximately 90 mg / kg·day; the comparative example group 1 was administered the same amount of 2'-FL (without live bacteria) as Example 4 by gavage; the NC group and the model group were administered the same volume of PBS by gavage; the gavage volume was uniformly 0.2 mL / animal / day.

[0066] (3) Morris water maze test Use a standard circular water maze (120cm in diameter, 30cm in water depth, 22±1℃), with a platform diameter of 10cm, submerged about 1cm below the water surface.

[0067] Orientation and navigation experiment: conducted at the end of week 8 for 5 consecutive days, each mouse was trained 4 times a day, with a maximum swimming time of 90 seconds each time; the escape latency of finding the underwater platform each time was recorded and the daily average was taken; the average escape latency on day 5 was analyzed as an indicator of learning and memory ability.

[0068] Space exploration experiment: On day 6, the platform was removed and the mice were placed in the contralateral quadrant; the percentage of time spent in the original platform quadrant and the number of times the mice crossed the original platform position were recorded within 5 minutes.

[0069] (4) Hippocampal BDNF and IL-6 detection After the behavioral tests, the mice in each group were euthanized, and the hippocampus tissue was quickly harvested, weighed on ice, and then mixed with an appropriate amount of pre-cooled PBS to prepare a 10% tissue homogenate.

[0070] BDNF detection: A commercial mouse BDNF ELISA kit was used, and the procedure was followed according to the instructions. Results are expressed as pg / mg protein.

[0071] IL-6 detection: The mouse IL-6 ELISA kit was used, and the results are expressed as pg / mg protein.

[0072] Table 3. Effects of each example and comparative example on D-galactose-induced cognitive impairment mice. Group Avoiding the incubation period (s, day 5) Time spent in the original platform quadrant (%) Hippocampal BDNF (pg / mg protein) Hippocampal IL-6 (pg / mg protein) NC <![CDATA[25±5 a ]]> <![CDATA[35±4 a ]]> <![CDATA[140±15 a ]]> <![CDATA[20±4 a ]]> Model group 60±8 15±3 70±10 45±6 Example 1 Group <![CDATA[45±7 a ]]> <![CDATA[22±3 a ]]> <![CDATA[95±12 a ]]> <![CDATA[35±5 a ]]> Example 2 group <![CDATA[40±6 abc ]]> <![CDATA[24±3 abc ]]> <![CDATA[105±13 abc ]]> <![CDATA[32±5 abc ]]> Example 3 Group <![CDATA[38±6 abc ]]> <![CDATA[25±3 abc ]]> <![CDATA[110±12 abc ]]> <![CDATA[30±4 abc <!-- 11 -->]]> Example 4 group <![CDATA[30±5 ab ]]> <![CDATA[30±3 ab ]]> <![CDATA[125±14 ab ]]> <![CDATA[25±4 ab ]]> Example 5 group <![CDATA[39±6 abc ]]> <![CDATA[24±3 abc ]]> <![CDATA[108±11 abc ]]> <![CDATA[31±4 abc ]]> Example 6 group <![CDATA[42±7 abc ]]> <![CDATA[23±3 abc ]]> <![CDATA[100±10 abc ]]> <![CDATA[33±5 abc ]]> Example 7 group <![CDATA[41±6 abc ]]> <![CDATA[24±3 abc ]]> <![CDATA[106±12 abc ]]> <![CDATA[32±5 abc ]]> Example 8 group <![CDATA[27±5 abc ]]> <![CDATA[33±4 abc ]]> <![CDATA[135±14 abc ]]> <![CDATA[22±4 abc ]]> Example 9 group <![CDATA[32±5 ab ]]> <![CDATA[29±3 ab ]]> <![CDATA[128±13 ab ]]> <![CDATA[24±4 ab ]]> Example 10 group <![CDATA[31±5 ab ]]> <![CDATA[29±3 ab ]]> <![CDATA[127±12 ab ]]> <![CDATA[24±4 ab ]]> Comparative Example 1 <![CDATA[50±7 ac ]]> <![CDATA[18±3 ac ]]> <![CDATA[85±11 ac ]]> <![CDATA[38±5 ac ]]> Note: a: P<0.05 compared with the model group; b: P<0.05 compared with Example 1 group; c: P<0.05 compared with Example 4 group.

[0073] Compared with the NC group, the model group showed a significantly prolonged escape latency and a significantly shortened time spent in the original platform quadrant; hippocampal BDNF was significantly reduced and IL-6 was significantly increased, indicating that D-galactose successfully induced cognitive impairment and neuroinflammation. The probiotic combination alone in Example 1 significantly improved the above indicators: escape latency was shortened to 45 seconds, and the time spent in the original platform quadrant increased to 22%; BDNF increased to 95 pg / mg, and IL-6 decreased to 35 pg / mg, indicating that the combination of *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35 itself has certain neuroprotective and cognitive-improving effects.

[0074] The probiotic compositions of Examples 2-7 were generally superior to those of Example 1: except for Example 4, the escape latency of each group was further shortened to the range of 38-42s, and the original plateau quadrant dwell time was increased to 23-25%; BDNF was generally increased to 100-110 pg / mg, and IL-6 was decreased to 30-33 pg / mg, indicating that the addition of different oligosaccharides can enhance the gut-brain axis regulation and neuroprotective effects of this probiotic composition.

[0075] Example 4, using 2'-fucosylated lactose, showed a significant improvement effect: the escape latency was only 30s, close to the normal group's 25s, significantly better than Example 1 and other oligosaccharide groups; the original plateau quadrant dwell time reached 30%, significantly close to the normal group's 35%, and significantly higher than other examples; hippocampal BDNF increased to 125pg / mg, close to the normal group level, and significantly higher than Example 1, as well as Example 2, 7, etc.; IL-6 decreased to 25pg / mg, close to the normal group's 20pg / mg, and significantly lower than Example 1 and other oligosaccharide groups.

[0076] Furthermore, Examples 8-10 combined 2'-fucosylated lactose with different types of oligosaccharides to form a dual prebiotic combination. Among them, Example 8 showed the most outstanding performance: the escape latency was shortened to about 27 seconds, and the original platform quadrant dwell time was increased to about 33%, both of which were very close to the normal control group; hippocampal BDNF increased to about 135 pg / mg, and IL-6 decreased to about 22 pg / mg. Statistical analysis showed that the differences were statistically significant compared with the model group, Example 1, and Example 4 (P<0.05).

[0077] Examples 9 and 10 also show further improvements over Example 4: the escape latency is slightly lower than that of Example 4, the original platform quadrant dwell time and BDNF level are slightly higher than those of Example 4, and IL-6 is slightly lower than that of Example 4. However, the overall improvement is not as great as that of Example 8, suggesting that there is a compatibility optimization between 2'-FL and different oligosaccharides. Among them, the combination with resistant dextrin can produce the best cognitive improvement and anti-inflammatory effect in the model of this invention.

[0078] Although Comparative Example 1 (only 2'-fucosylated lactose, no probiotics) showed some improvement in various indicators, its effect was significantly weaker than that of Example 4: the escape latency was 50s, which was only slightly better than the model group and far inferior to Example 4; the original platform quadrant dwell time was 18%, BDNF was 85pg / mg, and IL-6 was 38pg / mg, all of which were significantly worse than those of Example 4.

[0079] It is noted that although 2'-fucosylated lactose, as a human milk oligosaccharide, has certain potential to regulate intestinal microecology and provide mild neuroprotection, it is difficult for it to achieve the technical effects of the combination of the present invention on its own.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A probiotic composition with neuroprotective and cognitive-improving functions, characterized in that: Including at least one of Lactobacillus reuteri HBM11-69 and Lactobacillus rhamnosus HBM11-35.

2. The probiotic composition with neuroprotective and cognitive-improving functions as described in claim 1, characterized in that: It also includes oligosaccharides; the oligosaccharides are at least one of galactooligosaccharides, resistant dextrin, 2'-fucosylated lactose, xylooligosaccharides, isomaltooligosaccharides, and inulin.

3. The method for preparing the probiotic composition with neuroprotective and cognitive-improving functions as described in claim 1, characterized in that, Includes the following steps: S1. Inoculate *Lactobacillus reuteri* HBM11-69 and *Lactobacillus rhamnosus* HBM11-35 into the culture medium, respectively, at a volume of 1-5% of the medium. Incubate at 30-40℃ under anaerobic conditions for 10-24 hours until the viable count in their respective fermentation broths reaches 1×10⁻⁶. 8 -1×10 10 CFU / mL was used to obtain the fermentation broth; Centrifuge the fermentation broth at 4℃-10℃ and 3000-12000×g for 5-20 min, discard the supernatant, and collect the cell precipitate. Resuspend the bacteria in a cryoprotectant solution to achieve a viable count of 1 × 10⁻⁶. 10 -1×10 12 CFU / mL; then the bacterial suspension was pre-frozen at -30~-50℃ for 2-8h, and then freeze-dried at a vacuum degree ≤100Pa for 12-36h to obtain freeze-dried powder of Lactobacillus reuteri HBM11-69 and freeze-dried powder of Lactobacillus rhamnosus HBM11-35. S2. Based on the viable count, mix HBM11-69 lyophilized powder and HBM11-35 lyophilized powder at a viable count ratio of 0.5-2:1 to obtain a mixed lyophilized powder; then mix the mixed lyophilized powder with oligosaccharides at a mass ratio of 1:5-15 until the total viable count of the resulting composite powder is 1×10⁻⁶. 8 -1×10 11 CFU / g yielded a probiotic composition with neuroprotective and cognitive-improving functions.

4. The method for preparing the probiotic composition with neuroprotective and cognitive-improving functions as described in claim 3, characterized in that: The culture medium was MRS liquid medium; at the fermentation endpoint, the viable count of each strain was ≥1×10⁻⁶. 9 CFU / mL.

5. The method for preparing the probiotic composition with neuroprotective and cognitive-improving functions as described in claim 3, characterized in that: The protective agent is composed of disaccharides and dairy solids; the disaccharides are selected from at least one of trehalose and sucrose; the dairy solids are selected from at least one of skim milk powder and whole milk powder; the mass fraction of the disaccharides and dairy solids in the protective agent is 5-20% each.

6. The method for preparing the probiotic composition with neuroprotective and cognitive-improving functions as described in claim 3, characterized in that: In step S2, the ratio of viable bacteria count between HBM11-69 lyophilized powder and HBM11-35 lyophilized powder is 1:

1.

7. The method for preparing the probiotic composition with neuroprotective and cognitive-improving functions as described in claim 3, characterized in that: The mass ratio of the mixed freeze-dried powder to oligosaccharides is 1:

9.

8. The method for preparing the probiotic composition with neuroprotective and cognitive-improving functions as described in claim 3, characterized in that: The oligosaccharide is selected from one or more of galactooligosaccharides, resistant dextrin, 2'-fucosylated lactose, xylooligosaccharides, isomaltooligosaccharides, and inulin.

9. The method for preparing the probiotic composition with neuroprotective and cognitive-improving functions as described in claim 8, characterized in that: The oligosaccharide is a mixture of resistant dextrin and 2'-fucosylated lactose.

10. The method for preparing the probiotic composition with neuroprotective and cognitive-improving functions as described in claim 3, characterized in that: When two oligosaccharides are combined, 2'-fucosylated lactose accounts for 10-40% of the total mass of oligosaccharides.

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