Lactobacillus plantarum c067 for enhancing memory capacity, fermented food thereof, and use thereof

Abstract

The application discloses Lactobacillus plantarum C067 capable of improving memory, a fermented food thereof and application of the fermented food. The Lactobacillus plantarum C067 can significantly improve the fear memory and spatial memory of an individual, increase the content of antioxidant substances in blood and reduce the level of inflammatory factors, affect the expression of nerve receptors in the brain, promote nerve signal transmission, regulate intestinal flora to increase the abundance of beneficial bacteria and improve the content of intestinal acetic acid and butyric acid. The fermented food also has the effect of significantly improving the memory of an individual. The Lactobacillus plantarum C067 can be used to prepare functional food, health products and medicines with the functions of enhancing, improving memory or improving memory impairment, and has a very wide application prospect.

Description

Technical Field

[0001] This invention relates to Lactobacillus plantarum C067, which enhances memory, its fermented foods, and their applications, belonging to the field of microbial technology. Background Technology

[0002] Learning and memory are the foundation of human cognition and essential functions for individuals to adapt to their social environment and survive better. Cognitive function is crucial for daily life, encompassing not only the abilities to remember, learn, think, and solve problems, but also influencing emotions and behavior. Research shows that cognitive function is closely related to overall health; good cognitive function can improve quality of life, slow the aging process, and reduce the risk of neurodegenerative diseases. Therefore, exploring methods to enhance and protect cognitive function has significant scientific and clinical implications.

[0003] In recent years, numerous studies have indicated that the gut microbiome influences cognitive function through the gut-brain axis. The gut-brain axis is a complex bidirectional communication network connecting the gastrointestinal tract and the central nervous system via endocrine, immune, and neural pathways. Aseptically raised laboratory animals and antibiotic-induced gut microbiota dysbiosis models have demonstrated a causal relationship between gut microbiota disruption and memory impairment. Research suggests that gut microbiota may alter cognitive function by affecting intestinal permeability, systemic inflammation levels, synaptic plasticity, and by modifying neurotransmitter signaling, influencing neural receptors, and related metabolites.

[0004] It is evident that regulating gut microbiota through probiotics to improve brain function and cognitive abilities has become a novel and potential approach. Currently, the known functions of probiotics primarily include regulating gut function and enhancing host nutrient metabolism. Very few probiotics have been reported to improve cognitive abilities, and most focus on disease states of cognitive decline, lacking strains that can enhance the cognitive abilities of healthy individuals. Strains with potential brain function regulation, such as Bifidobacterium, require stringent culture conditions, necessitating a strictly anaerobic environment, which also limits their application. Furthermore, the functions of probiotics exhibit strong strain specificity; which probiotics can improve the cognitive abilities of healthy individuals, and how they enhance memory and cognitive abilities, remains unknown. Therefore, screening for probiotics that improve individual memory and possess good environmental tolerance, and developing functional products that can assist in improving individual memory, holds immense application potential. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides Lactobacillus plantarum C067, which can rapidly proliferate in an aerobic culture environment and has memory-enhancing capabilities, its fermented foods, and their applications. The aim is to solve the technical problems that there are very few known types of probiotics that can improve cognitive impairment, and which probiotics can improve the cognitive abilities of healthy individuals, and how they improve the memory and cognitive abilities of healthy individuals.

[0006] The first technical solution provided by this invention is a Lactiplantibacillus plantarum C067, which was deposited on November 15, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo:65484, and the deposit address is the Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The second technical solution provided by the present invention is a microbial preparation containing Lactobacillus plantarum C067 as described in the first technical solution.

[0008] In some embodiments, the concentration of *Lactobacillus plantarum* C067 in the microbial preparation is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0009] Preferably, the concentration of *Lactobacillus plantarum* C067 in the microbial preparation is not less than 1 × 10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0010] The present invention also provides a third technical solution, which is a fermentation agent containing Lactobacillus plantarum C067 as described in the first technical solution or a microbial preparation as described in the second technical solution.

[0011] In some embodiments, the amount of *Lactobacillus plantarum* C067 added to the fermenting agent is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0012] Preferably, the amount of *Lactobacillus plantarum* C067 added to the fermenting agent is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0013] The fourth technical solution provided by the present invention is a product containing Lactobacillus plantarum C067 as described in the first technical solution or a microbial preparation as described in the second technical solution, or the product is obtained by fermentation using the fermenting agent as described in the third technical solution.

[0014] In some embodiments, the product is a feed additive, food, medicine, or health product.

[0015] In some embodiments, the food is a fermented food containing *Lactobacillus plantarum* C067 as described in the first technical solution or a microbial preparation as described in the second technical solution, or the food is a fermented food obtained by fermentation using a fermenting agent as described in the third technical solution.

[0016] In some embodiments, the fermented food includes dairy products, soy products, fruit and vegetable products, or other fermented foods containing Lactobacillus plantarum C067.

[0017] In some embodiments, the dairy products include milk, sour cream, and cheese.

[0018] In some embodiments, the fruit and vegetable products include one or more products from cucumbers, carrots, beets, celery, cabbage, and other edible fruits and vegetables.

[0019] In some embodiments, the fermented food also contains additives selected from one or more combinations of flavorings, fruit and vegetable juices, herbal tea juices, colorings, acidity regulators, preservatives, antioxidants, thickeners, and sweeteners.

[0020] In some embodiments, the fermented food is processed into solid food, liquid food, or semi-solid food.

[0021] In some embodiments, the feed additive contains *Lactobacillus plantarum* C067 as described in the first technical solution or a microbial preparation as described in the second technical solution.

[0022] In some embodiments, the amount of *Lactobacillus plantarum* C067 added to the feed additive is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0023] Preferably, the amount of *Lactobacillus plantarum* C067 added to the feed additive is not less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0024] The fifth technical solution provided by this invention is the application of Lactobacillus plantarum C067 as described in the first technical solution or the microbial preparation as described in the second technical solution in the preparation of products that improve individual memory ability or improve memory impairment.

[0025] In some implementations, the application includes at least one of the following functions:

[0026] (1) Enhance an individual's ability to remember fear;

[0027] (2) Enhance individual spatial memory ability;

[0028] (3) Increase the levels of superoxide dismutase (SOD) and reduced glutathione (GSH) in the serum of individuals;

[0029] (4) Upregulates the expression of brain-derived trophoblast, glutamate receptor and / or neuropeptide Y receptor in the hippocampus of the individual brain.

[0030] (5) Regulates individual brain immunity and reduces the content of inflammatory factors in individual hippocampal tissue;

[0031] (6) Regulate individual gut microbiota and gut metabolites.

[0032] In some embodiments, the product includes functional foods, pharmaceuticals, or health products.

[0033] In some embodiments, the pharmaceutical product further comprises a drug carrier and / or pharmaceutical excipients.

[0034] In some embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles, and / or liposomes.

[0035] In some embodiments, the pharmaceutical excipient comprises excipients and / or additives.

[0036] In some embodiments, the excipient includes a binder, filler, disintegrant, and / or lubricant.

[0037] In some embodiments, the additives include solubilizers, cosolvents, latent solvents, and / or preservatives.

[0038] In some embodiments, the dosage form of the medicine is powder, granules, capsules, tablets, pills, or oral liquid.

[0039] The sixth technical solution provided by the present invention is the application of Lactobacillus plantarum C067 as described in the first technical solution or the microbial preparation as described in the second technical solution in the preparation of a drug for regulating individual brain immunity.

[0040] In some embodiments, the modulation of an individual's brain immunity includes reducing the levels of TNF-α and IL-1β in the individual's hippocampus.

[0041] In some embodiments, the pharmaceutical product further comprises a drug carrier and / or pharmaceutical excipients.

[0042] In some embodiments, the drug carrier comprises microcapsules, microspheres, nanoparticles, and / or liposomes.

[0043] In some embodiments, the pharmaceutical excipient comprises excipients and / or additives.

[0044] In some embodiments, the excipient includes a binder, filler, disintegrant, and / or lubricant.

[0045] In some embodiments, the additives include solubilizers, cosolvents, latent solvents, and / or preservatives.

[0046] In some embodiments, the dosage form of the medicine is powder, granules, capsules, tablets, pills, or oral liquid.

[0047] The seventh technical solution provided by the present invention is the application of Lactobacillus plantarum C067 as described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of promoters of hippocampal brain-derived nutritional factors, glutamate receptors and / or neuropeptide Y receptors.

[0048] The technical effects of this invention are as follows:

[0049] In animal experiments on mice, taking Lactobacillus plantarum C067 of the present invention can significantly improve an individual's fear memory and spatial memory, increase the content of antioxidants in the blood and reduce the level of inflammatory factors, affect the expression of nerve receptors in the brain, promote nerve signal transmission, regulate the intestinal flora to increase the abundance of beneficial bacteria, and increase the content of acetic acid and butyric acid in the intestine; the fermented food also has the effect of significantly improving an individual's memory.

[0050] The Lactobacillus plantarum C067 described in this invention can be used to prepare functional foods, health products, and pharmaceuticals that enhance or improve memory or improve memory impairment, and has a very broad application prospect.

[0051] Preservation of biological materials

[0052] A strain of Lactiplantibacillus plantarum C067, taxonomically named Lactiplantibacillus plantarum, was deposited on November 15, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNO:65484. The deposit address is Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0053] Figure 1 The effect of Lactobacillus plantarum C067 on the memory ability of mice. (A) Restlessness time in mice during the conditioned fear experiment; (B) Average distance to the platform in the water maze experiment; (C) Number of times the platform was crossed in the water maze experiment; *P<0.05, **P<0.01.

[0054] Figure 2 The effect of *Lactobacillus plantarum* C067 on redox levels in mice. (A) Serum reduced glutathione (GSH) levels; (B) Serum superoxide dismutase (SOD) levels; *P<0.05, **P<0.01

[0055] Figure 3 The effect of *Lactobacillus plantarum* C067 on the levels of inflammatory factors in mouse serum. (A) Serum TNA-α level; (B) Serum IL-1β level; *P<0.05, **P<0.01

[0056] Figure 4 The effects of Lactobacillus plantarum C067 on the expression of neuroreceptors in the mouse brain. (A) Relative expression levels of brain-derived trophic factor (BDNF) mRNA; (B) Relative expression levels of neuropeptide Y receptor 5 (NPY5R) mRNA; (C) Relative expression levels of metabotropic glutamate receptor 1 (mGluR1) mRNA; (D) Relative expression levels of metabotropic glutamate receptor 1 (mGluR3) mRNA; *P<0.05, **P<0.01.

[0057] Figure 5 The effects of Lactobacillus plantarum C067 on the gut microbiota of mice. (A) Relative abundance of community (phylum level); (B) Relative abundance of community (genus level); (C) β-diversity analysis of gut microbiota;

[0058] Figure 6 Effects of Lactobacillus plantarum C067 on intestinal metabolites in mice. (A) Acetic acid concentration; (B) Propionic acid concentration; (C) Butyric acid concentration; *P<0.05, **P<0.01. Detailed Implementation

[0059] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0060] The following embodiments involve test methods:

[0061] 1. Morris water maze experiment

[0062] The experiment lasted for 6 days, with 4 training sessions held at fixed times each day. On the first day, mice were trained to stand on the platform. From the second to the fifth day, the platform was placed in the first quadrant, and mice were placed into the pool facing the wall from any of the four starting points on the pool wall. A free-flowing video recording system recorded the time it took for the mice to find the platform and their swimming path. During the four training sessions, mice were placed into the water from four different starting points (different quadrants). If a mouse could not find the platform within 60 seconds, it was guided to the platform, rested for 10 seconds, and then the next training session was conducted after a 1-minute interval. On the sixth day, the platform was removed, and mice were placed into the water from the same entry point in a specific quadrant. The swimming path of the mice within 60 seconds, the number of times the mice crossed the target quadrant platform, and their swimming speed were recorded.

[0063] 2. Conditioned fear experiment:

[0064] On day 1 of the experiment, mice were placed in an experimental box (the bottom of the box had copper bars and could be electrified). After acclimatization for 2 minutes, they were given a single-frequency sound stimulus (3.0 kHz, 65 dB, 30 s), followed simultaneously by an inescapable foot shock (0.4 mA, 2 s). The sound and shock ended simultaneously. After the experiment, the mice remained in the box for another 2 minutes before being returned to their cages. The bottom of the box was wiped with 75% alcohol after each experiment. 24 hours later, mice that had developed conditioned fear were placed back in the original box without any stimulus, and their environment-induced rigidity behavior was recorded for 3 minutes. 2 hours later, the box environment was changed (a whiteboard was placed at the bottom of the box, and the color of the box walls was changed), and the mice were placed back in the box. After acclimatization, they were given the same intensity of sound stimulus for 3 minutes, and their sound-induced rigidity behavior was recorded for 3 minutes. Rigidity behavior was defined as the absence of any motor behavior other than breathing.

[0065] 3. Analysis of mouse gut microbiota

[0066] Total DNA was extracted from fresh mouse feces using the Stool DNA Kit (OMEGA). Using the mouse fecal genome as a template, the V3-V4 region of 16S rDNA was amplified using upstream primer 338F and downstream primer 806R. Each sample was tested in triplicate. PCR products from the same sample were mixed and analyzed by 2% agarose gel electrophoresis. PCR products were recovered from the gel using the AxyPrep DNA Gel Extraction Kit (AXYGEN) and analyzed by QuantiFluor. TMThe -ST blue fluorescence quantitative quantification system (Promega) was used for detection and quantification. Based on the fluorescence quantitative results, samples were mixed in appropriate proportions according to the sequencing volume requirements of each sample. Subsequently, the sequences were sequenced using an Illumina MiSeq sequencer. Sequences were assembled based on overlap relationships, and sequence quality was controlled and filtered. Operational Taxonomic Unit (OTU) clustering analysis and species annotation were performed on all valid data with 97% consistency. Based on the OTU clustering analysis results, various diversity indices can be analyzed for OTUs, and sequencing depth can be determined. Based on taxonomic information, statistical analysis of community structure can be performed at various taxonomic levels.

[0067] 4. Analysis of short-chain fatty acids in mouse feces

[0068] Accurately weigh 0.1 g of mouse feces, add 1.5 mL of deionized water, vortex to mix, centrifuge at 10,000 rpm for 10 min at 4 °C, and filter the supernatant through a 0.45 mm filter membrane. Then, take 0.9 mL of the sample solution and transfer it to a centrifuge tube containing 0.1 mL of 50% sulfuric acid and 2 mL of internal standard working solution (2-ethyl-butyric acid concentration of 2 mmol / L). After mixing for 5 minutes, centrifuge at 10,000 rpm for 10 min and hold at -20 °C for 20 min. Use the supernatant for the determination of short-chain fatty acids. Analyze the short-chain fatty acids present in the supernatant using a gas chromatography system. Measurement conditions: detector: flame ionization detector (FID); temperature program: 120 °C for 5 min, increase to 250 °C at 15 °C / min, hold for 1 min; FID temperature: 255 °C; carrier gas: N2; flow rate: 1.5 mL / min; sample injection volume: 2 μL; split ratio: 10:1. Quantification was accomplished by measuring the peak areas of acetate, propionate, butyrate, and valerate relative to 2-ethyl-butyric acid, in mg / g feces.

[0069] The culture media involved in the following examples are as follows:

[0070] MRS solid medium: tryptone 10.00 g / L, yeast extract 5.00 g / L, beef extract 10.00 g / L, glucose 20.00 g / L, triammonium citrate 2.00 g / L, anhydrous sodium acetate 5.00 g / L, magnesium sulfate 0.10 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2.00 g / L, Tween 80 1.00 mL / L, agar powder 15.00 g / L, pH adjusted to 6.5.

[0071] MRS liquid culture medium: tryptone 10.00 g / L, yeast extract 5.00 g / L, beef extract 10.00 g / L, glucose 20.00 g / L, triammonium citrate 2.00 g / L, anhydrous sodium acetate 5.00 g / L, magnesium sulfate 0.10 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2.00 g / L, Tween 80 1.00 mL / L, pH adjusted to 6.5.

[0072] The animals involved in the following examples:

[0073] 1. The C57BL / 6J male mice were obtained from Spiford (Suzhou) Biotechnology Co., Ltd.

[0074] Example 1: Strain Screening and Identification

[0075] (I) Isolation and screening of Lactobacillus plantarum in fresh camel milk:

[0076] (1) Select fresh camel milk samples, serially dilute them with sterile PBS buffer, spread them on MRS solid medium with 1% CaCO3, and incubate them in an incubator at 37°C for 48 hours.

[0077] (2) Pick single colonies with obvious transparent zones that are white or milky white, perform Gram staining and microscopic examination, select rod-shaped strains that are Gram-positive, and streak the colonies for purification.

[0078] (3) Pick a single colony, incubate it in MRS liquid medium at 37°C for 48 hours, and then collect the bacterial cells for strain identification.

[0079] (II) Molecular biological identification of lactobacilli:

[0080] (1) Genomic DNA was extracted using a bacterial genome extraction kit;

[0081] (2) PCR amplification of 16S rDNA using universal primers for bacterial strain identification;

[0082] (3) The obtained PCR product was sent to a professional sequencing company for sequencing. The sequencing results were searched and compared in the GeneBank database using BLAST. Combined with its morphological characteristics, it was identified as Lactobacillus plantarum.

[0083] (4) Whole genome sequencing: The whole genome extracted in (1) was sent to a professional sequencing company. The whole genome of the bacteria was sequenced using a second-generation sequencer. The obtained sequence results were searched and compared for similarity in the GeneBank database using BLAST. The sequencing results identified the strain as belonging to Lactobacillus plantarum and named it Lactobacillus plantarum C067. The strain was stored at -80℃ for later use.

[0084] Example 2: Effects of Lactobacillus plantarum C067 on the memory ability of mice

[0085] Forty 6-week-old male C57BL / 6J mice were randomly divided into four groups after one week of acclimatization: a normal control group, a *Lactobacillus plantarum* C067 intervention group, an antibiotic clearance control group, and a post-clearance *Lactobacillus plantarum* C067 intervention group, with 10 mice in each group. The specific animal groupings are as follows:

[0086] Group 1 (control group): Free access to food and water, and gavage administration of control solvent;

[0087] Group 2 (Lactobacillus plantarum C067 intervention group): Free access to food and water, and gavage administration of Lactobacillus plantarum C067;

[0088] Group 3 (antibiotic control group): After 2 weeks of antibiotic treatment, the patients had free access to food and water, and were given the control solution by gavage.

[0089] Group 4 (antibiotic + Lactobacillus plantarum C067 intervention group): After 2 weeks of antibiotic treatment, Lactobacillus plantarum C067 was administered by gavage.

[0090] Antibiotic treatment: Mice in all groups were fed a normal diet. Mice in the antibiotic treatment groups (groups 3 and 4) were administered 0.1 mL / 10 g body weight of antibiotic solution (108.0 mg bacitracin, 108.0 mg neomycin, 43.2 mg penicillin, 21.6 mg meropenem, and 6.48 mg vancomycin dissolved in 4.5 mL distilled water) by gavage. Mice in the other groups were given the corresponding volume of solution. Administration was twice daily (once in the morning and once in the evening) for two weeks.

[0091] Lactic acid bacteria intervention: Activated *Lactobacillus plantarum* C067 was cultured in MRS liquid medium at 37°C for 48 hours. Fresh bacterial culture was collected by centrifugation, the supernatant was discarded, and the bacterial cells were resuspended in PBS to achieve a bacterial concentration of 10⁻⁶. 9 CFU / mL. Administer by gavage once daily in the morning for 4 weeks, with a gavage volume of 0.1 mL / 10g animal body weight.

[0092] After the lactic acid bacteria intervention, behavioral tests were performed on the mice, including the Morris water maze and conditioned fear test. The specific results are as follows:

[0093] (1) Results of the conditioned fear experiment: Figure 1As shown, compared with the control group, feeding with *Lactobacillus plantarum* C067 significantly increased fear memory in mice, with the duration of rigidity increasing from 41.42 seconds to 58.49 seconds, a 1.41-fold increase. In the antibiotic-treated group, the duration of rigidity was reduced, but feeding with *Lactobacillus plantarum* C067 increased it by 1.39 times, from 26.45 seconds to 36.64 seconds. These results indicate that *Lactobacillus plantarum* C067 can significantly improve fear memory in normal animals and also has a beneficial effect on memory in mice with antibiotic-induced gut microbiota dysbiosis.

[0094] (2) The results of the Morris water maze experiment are as follows Figure 1 As shown, the average distances from *Lactobacillus plantarum* C067 intervention to the platform were 35.97 cm and 37.71 cm for mice in the normal group and the antibiotic-treated group, respectively, representing reductions of 14% and 12% compared to the control group. The average number of times mice crossed the platform was 6.40 and 5.33, respectively, which were 1.42 times and 1.57 times that of the control group. These results indicate that *Lactobacillus plantarum* C067 can significantly improve the spatial memory ability of animals.

[0095] Example 3: Effects of Lactobacillus plantarum C067 on redox balance in mice

[0096] The animal model was constructed and grouped according to Example 2. Mice in each group were euthanized, and their serum was collected. The levels of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), and nitric oxide (NO) in the serum were detected using a kit. The results showed ( Figure 2 Feeding mice with *Lactobacillus plantarum* C067 significantly altered the levels of SOD and GSH in their serum. After intervention with *Lactobacillus plantarum* C067, the serum SOD levels in the normal group and the antibiotic-free group were 162.20 U / mL and 161.81 U / mL, respectively, representing increases of 1.31 times and 1.26 times compared to the control. The GSH levels were 24.96 μmol / L and 17.95 μmol / L, respectively, representing increases of 1.46 times and 2.07 times compared to the control. This indicates that *Lactobacillus plantarum* C067 can promote the production of antioxidants in animals, regulate the body's redox balance, and thus enhance cognitive function.

[0097] Example 4: Effects of Lactobacillus plantarum C067 on the expression of neural receptors in the brain

[0098] Animal model construction and grouping were performed according to Example 2. Brain tissue from mice in each group was dissected, and hippocampal tissue was isolated on ice. Total RNA was extracted from the tissues using the RNAiso Plus kit (Takara) and PrimeScript was used. TM RTMaster Mix (Takara) reverse transcribed cDNA using iTaq.TM Universal Quantitative real-time PCR was performed using the Green Supermix kit (Bio-Rad Laboratories) to detect neural receptors in the posterior hippocampus. The housekeeping gene GAPDH was used as an internal reference gene, and the primer sequences used are shown in Table 1.

[0099] Table 1 Primer sequences for real-time PCR

[0100]

[0101] The results are as follows Figure 3 As shown, intervention with *Lactobacillus plantarum* C067 altered the expression of brain-derived nutritional factor (BDNF), neuropeptide Y receptor (NPY5R), and metabolite glutamate receptors (mGluR1, mGluR3) in the hippocampus of mice. In the normal group, the expression levels of NPY5R and mGluR3 increased by 1.67 and 1.56 times, respectively, while in the antibiotic-treated group, the expression levels of BDNF, mGluR1, and mGluR3 increased by 2.06, 1.46, and 1.99 times, respectively. This indicates that *Lactobacillus plantarum* C067 affects signal transduction by influencing the expression of neural receptors, thereby affecting memory.

[0102] Example 5: Immunomodulatory effects of Lactobacillus plantarum C067 on mouse brain

[0103] The animal model was constructed and grouped according to Example 2. Mice were euthanized, and mouse brain tissue was collected. Hippocampal tissue was isolated on ice, and sterile PBS buffer was added. Tissue homogenate was prepared using a tissue homogenizer, and the levels of IL-1β, IL-6, IL-10, and TNF-α were detected using an ELISA kit. The results showed ( Figure 4 Lactobacillus plantarum C067 significantly reduced the levels of TNF-α and IL-1β in the hippocampus of mice. In the normal control group and the antibiotic group, TNF-α levels decreased to 494.89 ng / ug and 317.84 ng / ug, respectively, representing a 2.75-fold and 3.1-fold reduction. In the antibiotic group, IL-1β levels decreased from 11.43 ng / ug to 2.36 ng / ug, a 4.84-fold reduction. This demonstrates that Lactobacillus plantarum C067 can reduce the levels of inflammatory factors in the mouse brain and exhibits a significant anti-inflammatory effect.

[0104] Example 6: Effects of Lactobacillus plantarum C067 on intestinal flora and metabolites in mice

[0105] The animal model was constructed and grouped according to Example 2. Fresh feces from mice were collected for analysis of mouse gut microbiota and metabolites.

[0106] (1) The results of the mouse gut microbiota analysis showed that ( Figure 5Principal coordinates analysis (PCoA) was used to assess the β-diversity of the gut microbiota. The results showed that *Lactobacillus plantarum* C067 intervention altered the gut microbiota structure, normalizing it in antibiotic-treated mice. At the phylum level, feeding *Lactobacillus plantarum* C067 increased the *Verrucous* phylum in both healthy and antibiotic-treated mice; the *Actinomyces* phylum increased in antibiotic-treated mice, which was normalized by *Lactobacillus plantarum* C067 intervention, restoring the gut microbiota structure to normal. At the genus level, *Lactobacillus plantarum* C067 intervention increased the abundance of *Akkermansia* genus. *Akkermansia muciniphila* and other bacteria within this genus have effects such as strengthening the intestinal barrier, improving host metabolic health, regulating the immune system, and anti-inflammation. These effects synergistically enhance the beneficial effects of *Lactobacillus plantarum* C067, thereby improving the host's cognitive abilities.

[0107] (2) Analysis of short-chain fatty acids in mouse feces showed that ( Figure 6 Antibiotic treatment reduced the content of short-chain fatty acids in mouse feces. Intervention with *Lactobacillus plantarum* C067 increased the acetic acid content in the healthy group and the antibiotic group to 14.23 mg / g and 8.86 mg / g, respectively, representing increases of 1.37 times and 1.35 times compared to the control. The butyrate content in the feces of mice in the antibiotic group increased from 0.522 mg / g to 0.87 mg / g, an increase of 1.67 times compared to the control. These results further demonstrate that *Lactobacillus plantarum* C067 can also regulate intestinal health, enhance the immune system, reduce inflammation, inhibit the growth of harmful bacteria, and promote the growth of beneficial bacteria by regulating intestinal acetic acid and butyrate levels, thereby maintaining intestinal flora balance.

[0108] Example 7: Application of Lactobacillus plantarum C067

[0109] Fresh milk and granulated sugar were mixed and homogenized (10:1), sterilized at 140°C for 2 seconds, cooled to 35°C, and then inoculated with the *Lactobacillus plantarum* C067 inoculum prepared in this invention. Fermentation was carried out in a sealed container at 35°C for 4 hours, followed by post-fermentation at 4°C for 12 hours to obtain the final fermented product (bacterial concentration 10). 8 (CFU / mL or higher).

[0110] This invention enables the production and preparation of other fermented foods using Lactobacillus plantarum C067, including solid foods, liquid foods, and semi-solid foods. The fermented foods include dairy products, soy products, and fruit and vegetable products; the dairy products include milk, sour cream, and cheese; the fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products.

[0111] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A type of Lactobacillus plantarum ( Lactiplantibacillus plantarum C067, characterized in that, It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 15, 2024, with accession number GDMCC No:65484.

2. A microbial preparation containing the Lactobacillus plantarum C067 of claim 1.

3. The microbial preparation according to claim 2, characterized in that, In the microbial preparation, the concentration of *Lactobacillus plantarum* C0672 is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

4. A fermenting agent, characterized in that, The fermentation agent contains Lactobacillus plantarum C067 as described in claim 1 or the microbial preparation as described in claim 2 or 3.

5. A product characterized in that, The product is a product containing *Lactobacillus plantarum* C067 as described in claim 1 or a microbial preparation as described in claim 2 or 3, or the product is a product obtained by fermentation using the fermenting agent as described in claim 4; the product is a pharmaceutical product.

6. The use of Lactobacillus plantarum C067 as described in claim 1 or the microbial preparation as described in claim 2 or 3 in the preparation of a medicine for improving individual memory or improving memory impairment.

7. The application according to claim 6, characterized in that, The application includes at least one of the following functions: (1) Enhance an individual's ability to remember fear; (2) Enhance individual spatial memory ability; (3) Increase the levels of superoxide dismutase (SOD) and reduced glutathione (GSH) in the serum of individuals; (4) Upregulates the expression of brain-derived trophoblast, glutamate receptor and / or neuropeptide Y receptor in the hippocampus of the individual brain; (5) Regulates individual brain immunity and reduces the content of inflammatory factors in individual hippocampal tissue; (6) Regulate individual gut microbiota and gut metabolites.

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