Bifidobacterium longum subsp. infantis with improved cognitive function and application thereof

Abstract

The present application belongs to the technical field of microorganisms, and particularly relates to a Bifidobacterium longum subsp. infantis with improved cognitive function and application thereof. The Bifidobacterium longum subsp. infantis with improved cognitive function provided by the present application is named Bifidobacterium longum subsp. infantis BD223, is preserved in the Guangdong Microbial Culture Collection Center, and has a preservation date of February 9, 2026 and a preservation number of GDMCC No: 67827. The Bifidobacterium longum subsp. infantis provided by the present application has the ability to produce 5-methyltetrahydrofolate at a high yield. Further, the postbiotic preparation prepared by fermentation of the Bifidobacterium longum subsp. infantis is rich in active folic acid of natural origin, can supplement key methyl donors required for brain development through a natural biological pathway, and thus effectively improves cognitive ability and solves the problems of limited absorption and safety of chemically synthesized folic acid.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bifidobacterium longum infantis with cognitive-improving functions and its applications. Background Technology

[0002] Brain development is the most crucial physiological process in the growth and development of infants and young children. Among these processes, myelination of nerve fibers is an important marker of nervous system maturation. Myelin, like the insulation layer of an electrical wire, ensures the high-speed and precise transmission of nerve signals. Impaired or delayed myelination is closely related to cognitive impairment, motor coordination disorders, and various neurodevelopmental diseases.

[0003] B vitamins, especially folic acid (vitamin B9), are essential micronutrients for neural development. Folic acid, as a carrier of one-carbon units, participates in nucleotide synthesis and the production of S-adenosylmethionine (SAM), thereby regulating DNA and histone methylation. Studies have shown that the maturation of oligodendrocytes (cells that form myelin sheaths) and the expression of myelin genes are strictly regulated by epigenetic modifications, and an adequate supply of folic acid in its appropriate form is crucial for myelin formation.

[0004] Currently, most folic acid supplements on the market are chemically synthesized oxidized folic acid. However, synthetic folic acid requires conversion by hepatic dihydrofolate reductase to be utilized by the body. Infants and young children have lower activity of this enzyme, leading to the accumulation of unmetabolized folic acid in the body, posing a potential safety risk. In contrast, some specialized probiotics can produce reduced, naturally occurring active folic acid (such as 5-methyltetrahydrofolate) through de novo synthesis, which has been proven to have higher bioavailability and safety compared to oxidized folic acid.

[0005] While there are reports on probiotics producing vitamins in the current technology, there is still a lack of research on the screening of specific strains that can produce high levels of active folic acid, as well as the mechanisms by which specific strains promote myelination and improve cognitive function.

[0006] Therefore, developing a specific strain that produces high levels of natural active folic acid and its related formulations is of great scientific significance and application value for solving the problems of limited absorption and safety of chemically synthesized folic acid, and can also provide a technical basis for the development of drug formulations rich in active folic acid. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a strain of *Bifidobacterium longum* subsp. *infantii* with cognitive-improving properties and its applications. The *Bifidobacterium longum* subsp. *infantii* provided by this invention has a high production capacity of 5-methyltetrahydrofolate. Further, a post-biotic preparation prepared from *Bifidobacterium longum* subsp. *infantii* is rich in naturally sourced active folic acid, which can supplement the key methyl donor required for brain development through a natural biological pathway, thereby improving cognitive function and promoting myelin formation. This solves the problems of limited absorption and safety of chemically synthesized folic acid and is suitable for preparing products rich in active folic acid.

[0008] To achieve the above objectives, the first aspect of the present invention adopts the following technical solution:

[0009] A subsp. infantis of Bifidobacterium longum with cognitive-improving properties, named Bifidobacterium longum subsp. infantis BD223, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on February 9, 2026, with accession number GDMCC No: 67827.

[0010] The second aspect of this invention is the following technical solution:

[0011] Application of *Bifidobacterium longum* subsp. *infantii* as described above in the fermentation preparation of 5-methyltetrahydrofolate.

[0012] The third aspect of this invention is the following technical solution:

[0013] A post-biotic preparation rich in 5-methyltetrahydrofolate, said post-biotic preparation being prepared by a method comprising the following steps:

[0014] (1) The activated bacterial solution of Bifidobacterium longum subsp. infantis was inoculated into a fermentation medium for fermentation culture to obtain a fermentation broth; wherein, the name of the Bifidobacterium longum subsp. infantis is Bifidobacterium longum subsp. infantis BD223, the depositary institution is Guangdong Provincial Microbial Culture Collection Center, the deposit date is February 9, 2026, and the deposit number is GDMCC No: 67827;

[0015] (2) The fermentation broth is subjected to heat shock treatment, followed by enzymatic hydrolysis to obtain enzymatically hydrolyzed fermentation broth;

[0016] (3) The enzymatic fermentation broth is subjected to solid-liquid separation treatment, and then the supernatant is collected and dried to obtain the post-biotic preparation.

[0017] As a preferred embodiment, in step (1), the concentration of *Bifidobacterium longum* subsp. infantis in the activated bacterial solution is (1~5)×10⁻⁶. 7 CFU / mL; the inoculation amount of the activated bacterial solution in the fermentation medium is 3%~8%v / v, more preferably 5%v / v.

[0018] As a preferred embodiment, in step (1), the fermentation medium includes MRS basal medium and additives added to the MRS basal medium; the additives, based on their concentration in the MRS basal medium, include: 15-25 g / L lactose, 50-200 mg / L para-aminobenzoic acid, and 20-30 mg / L guanine. In this step, by adding lactose as a carbon source and para-aminobenzoic acid as a folic acid synthesis precursor, the production of 5-methyltetrahydrofolate can be increased.

[0019] As a further preferred embodiment, the additives include: 20 g / L lactose, 100 mg / L para-aminobenzoic acid, and 25 mg / L guanine.

[0020] MRS basal medium is a conventional culture medium in the art, and technicians can prepare it according to standard formulations. This invention does not impose any particular limitations on its composition. As a preferred embodiment, the composition of the MRS basal medium is: peptone 10-15 g / L, yeast extract 5-7 g / L, beef extract 5-7 g / L, glucose 16-20 g / L, sodium acetate 4-6 g / L, ammonium citrate 2-2.3 g / L, Tween 80 0.8-1.2 g / L, magnesium sulfate 0.55-0.65 g / L, manganese sulfate 0.03-0.07 g / L, dipotassium hydrogen phosphate 1.8-2.2 g / L, L-cysteine ​​0.4-0.6 g / L, with water as the substrate.

[0021] As a preferred option, in step (1), the fermentation culture process conditions are: fermentation temperature of 35~38℃, fermentation pH of 6.0~7.0, and fermentation under anaerobic conditions for 24~72h.

[0022] As a further preferred option, the fermentation process conditions are: fermentation temperature of 37°C, fermentation pH of 6.5, and fermentation under anaerobic conditions for 48 hours.

[0023] As a preferred embodiment, in step (2), the heat shock treatment process conditions are as follows: the fermentation broth is heated to 92~98℃ and maintained for 10~40 min; the enzyme used in the enzymatic hydrolysis is lysozyme; the enzymatic hydrolysis process conditions are as follows: enzyme addition amount is 0.1%~0.5% w / v, enzymatic hydrolysis temperature is 45~55℃, and enzymatic hydrolysis time is 1~5 h. In this step, heat shock treatment is used to inactivate the bacterial cells and destroy the cell wall structure, thereby promoting the release of intracellular active folic acid. The combination of heat shock treatment and enzymatic hydrolysis can assist in the release of cell contents and further increase the production of 5-methyltetrahydrofolate.

[0024] As a preferred embodiment, in step (3), the solid-liquid separation process is centrifugation or filtration; the drying process is vacuum freeze-drying or spray drying. In this step, the enzymatic fermentation broth is centrifuged or filtered to remove cell fragments, and the supernatant rich in active folic acid is collected and dried to obtain a powdered metabiotic preparation.

[0025] As a further preferred embodiment, the solid-liquid separation process is centrifugation; the drying process is vacuum freeze-drying. The centrifugation speed is 5000~10000 rpm, and the centrifugation time is 5~30 min. The vacuum freeze-drying process conditions are as follows: pre-freezing at -40℃~-50℃ for 1~4 h; then, the vacuum is turned on to 12~18 Pa and the drying temperature is set to -10~-20℃ for a first drying of 18~25 h; then, the vacuum is adjusted to 3~8 Pa and the drying temperature is set to 25~35℃ for a second desorption drying of 2~6 h. This step, using centrifugation combined with vacuum freeze-drying, can retain the biological activity and structural integrity of 5-methyltetrahydrofolate to the greatest extent through low-temperature freezing and sublimation to remove water. After the above treatment, the resulting fermented biogenic powder has good stability.

[0026] The fourth aspect of this invention is the following technical solution:

[0027] The use of an epigenetic agent as described above in the preparation of a medicine for improving cognitive abilities, wherein the improvement of cognitive abilities is achieved by: increasing the new object recognition index, upregulating the level of brain-derived neurotrophic factor, upregulating the level of nerve growth factor, or one or more of these.

[0028] The technical solution of the present invention has the following advantages and beneficial effects:

[0029] The *Bifidobacterium longum* subspecies *Infant* BD223 provided by this invention is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No: 67827. This *Bifidobacterium longum* subspecies *Infant* BD223 possesses unique strain characteristics, exhibiting a complete de novo folic acid synthesis gene cluster and excellent bioactive folic acid synthesis capabilities. It can efficiently synthesize natural bioactive folic acid (5-methyltetrahydrofolate) in folic acid-free culture media, and its bioactive folic acid yield is significantly superior to commercially available *Bifidobacterium* strains or other comparative strains.

[0030] The post-biotic formulation provided by this invention is rich in bioactive folic acid derived from natural bio-fermentation, with 5-methyltetrahydrofolate (5-MTHF) as the main active ingredient, and contains no chemically synthesized folic acid. By optimizing fermentation conditions, using lactose as a carbon source and adding para-aminobenzoic acid as a precursor, this invention significantly improves the strain's ability to synthesize natural bioactive folic acid de novo, with 5-methyltetrahydrofolate accounting for over 80%. Furthermore, considering that folic acid is primarily stored intracellularly, this invention employs an innovative integrated process of heat shock cell disruption, enzymatic hydrolysis, and separation, effectively releasing the intracellular active ingredient and significantly increasing the yield of bioactive folic acid, making it suitable for applications in products rich in bioactive folic acid.

[0031] Meanwhile, animal experiments have shown that the post-biotic formulation of this invention can significantly improve the novel object recognition index in mice and upregulate the expression levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brain. Therefore, this invention provides a safe, efficient, and highly bioavailable natural nutritional intervention for promoting myelin development and improving cognitive function, and is suitable for development and application as a drug to improve cognitive function.

[0032] Therefore, this invention reveals the mechanism by which *Bifidobacterium longum* subsp. infantis and its post-biotic preparations promote cognitive development by regulating brain-derived active substances (BDNF, NGF) and the novel object recognition index. Furthermore, the strain and post-biotic preparations provided by this invention directly participate in the metabolic cycle by synthesizing naturally reduced folic acid, avoiding the metabolic burden of folic acid synthesis. This makes them more suitable for the delicate metabolic systems of infants and young children, and have broad application potential in the development of folic acid-rich products or drugs that improve cognitive abilities. Attached Figure Description

[0033] Figure 1 The effect of different doses of the postbiotic preparation in this invention on the novel object recognition index in mice;

[0034] Figure 2 The effect of different doses of the post-genetic preparation in this invention on the expression level of brain-derived neurotrophic factor (BDNF) in mice;

[0035] Figure 3This invention illustrates the effect of different doses of the post-biotic preparation on the expression level of nerve growth factor (NGF) in mice. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the protection scope of this invention.

[0037] The biological preservation information involved in this invention is as follows:

[0038] Bifidobacterium longum infantis subspecies BD223;

[0039] The Latin name is *Bifidobacterium longum subsp. infantis* BD223;

[0040] The depository is Guangdong Provincial Center for Microbial Culture Collection;

[0041] The deposit date is February 9, 2026;

[0042] The accession number is GDMCC No: 67827;

[0043] The address for storage is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0044] The lysozyme used in this invention is a commercially available product from Xiasheng Enzyme Biotechnology Co., Ltd., model number SDG-2447. The control strain is Bifidobacterium longum BL21, purchased from Weikang Probiotics Co., Ltd. 10% paraformaldehyde was purchased from Shanghai Yuanye Biotechnology Co., Ltd. BDNF and NGF enzyme-linked immunosorbent assay kits were purchased from Shanghai Enzyme Immunosorbent Assay Biotechnology Co., Ltd.

[0045] The modified MRS medium described below is prepared by adding para-aminobenzoic acid and guanine to the basic MRS medium. The preparation method is as follows: Take 12g of peptone, 6g of yeast extract, 6g of beef extract, 18g of glucose, 5g of sodium acetate, 2.15g of ammonium citrate, 1g of Tween 80, 0.58g of magnesium sulfate, 0.05g of manganese sulfate, 2g of dipotassium hydrogen phosphate, 0.5g of L-cysteine, 100mg of para-aminobenzoic acid, and 25mg of guanine. Add pure water to a final volume of 1L, adjust the pH of the system to 6.5, and autoclave at 121℃ for 15min to obtain the modified MRS medium.

[0046] Example 1

[0047] This embodiment provides a strain of Bifidobacterium longum subsp. infantis BD223, which has the function of improving cognitive function. The Latin name is Bifidobacterium longum subsp. infantis BD223. The depositary institution is Guangdong Provincial Center for Microbial Culture Collection, the deposit date is February 9, 2026, and the deposit number is GDMCC No: 67827.

[0048] The isolation, purification, and identification process of Bifidobacterium longum subsp. infantis BD223 involved in this embodiment is as follows:

[0049] The isolates of *Bifidobacterium longum* subsp. infantis BD223 were obtained from the feces of healthy full-term infants in Harbin. Fresh fecal samples were collected and immediately placed in an anaerobic incubator and rapidly transported to the laboratory under low temperature conditions. 1.0 g of fecal sample was weighed and added to 9.0 mL of sterile physiological saline containing 0.05 wt% L-cysteine ​​hydrochloride. The mixture was thoroughly homogenized and shaken to prepare a suspension. The suspension was then serially diluted 10-fold (10... -1 Up to 10 -6 A suitable dilution of bacterial suspension was plated onto modified MRS medium. The plated plates were then placed in an anaerobic workstation and incubated at 37°C for 48–72 hours. After the incubation period, single colonies exhibiting typical morphological characteristics of Bifidobacterium (e.g., round, raised, milky white, smooth surface) were selected. 16S rRNA gene sequencing and phylogenetic analysis confirmed that the isolated strain was *Bifidobacterium longum* subsp. *infantis*, and it was named *Bifidobacterium longum* subsp. *infantis* BD223.

[0050] The nucleotide sequence of the above-mentioned *Bifidobacterium longum* subsp. *infantii* BD223, identified by 16S rRNA, is shown in SEQ ID NO. 1:

[0051]

[0052] Example 2

[0053] This embodiment provides a post-biotic preparation rich in 5-methyltetrahydrofolate, which is prepared by a method including the following steps:

[0054] (1) Activation and fermentation culture of Bifidobacterium longum subsp. infantis BD223

[0055] The *Bifidobacterium longum* subsp. *infantii* BD223 from Example 1 was removed from a cryopreservation tube at −80°C and rapidly thawed at 37°C. Under aseptic conditions, the strain was inoculated into modified MRS medium and cultured anaerobically at 37°C for 24 h. After culture, the culture was centrifuged at 2000 rpm for 10 min, the supernatant was discarded, and the resulting bacterial cells were washed twice with sterile PBS buffer. Finally, the bacterial cells were resuspended in PBS buffer, and the bacterial concentration was adjusted to 1 × 10⁻⁶. 7 CFU / mL was used to obtain an activated bacterial solution of Bifidobacterium longum subsp. infantis BD223.

[0056] The activated bacterial suspension of *Bifidobacterium longum* subsp. *infantii* BD223 was inoculated into fermentation medium at an inoculation rate of 5% v / v and anaerobic fermentation was carried out at 37℃ and pH 6.5 for 48 h to obtain the fermentation broth. The fermentation medium was prepared as follows: 12g peptone, 6g yeast extract, 6g beef extract, 18g glucose, 5g sodium acetate, 2.15g ammonium citrate, 1g Tween 80, 0.58g magnesium sulfate, 0.05g manganese sulfate, 2g dipotassium hydrogen phosphate, 0.5g L-cysteine, 20g lactose, 100mg para-aminobenzoic acid, 25mg guanine, and water were added to a final volume of 1L. The pH was adjusted to 6.5, and the mixture was autoclaved at 121℃ for 15 min to obtain the fermentation medium.

[0057] (2) Heat shock, enzymatic hydrolysis and post-treatment

[0058] The fermentation broth obtained in step (1) was heated to 95±2℃ and maintained for 25 min for heat shock treatment. Then, 0.3% (w / v) of lysozyme was added to the fermentation broth obtained after heat shock treatment, and enzymatic hydrolysis was performed at 50℃ for 2 h to obtain the enzymatically hydrolyzed fermentation broth. The above-obtained enzymatically hydrolyzed fermentation broth was centrifuged at 8000 rpm for 15 min, and then the fermentation supernatant was collected and vacuum freeze-dried to obtain the post-biotic preparation rich in 5-methyltetrahydrofolate of this embodiment. The vacuum freeze-drying process conditions were as follows: pre-freezing at -45℃ for 2 h, then opening the vacuum to 15 Pa and adjusting the temperature to -15℃, drying for 20 h, then adjusting the vacuum to 5 Pa and the temperature to 30℃, and drying for 4 h.

[0059] In other embodiments of the present invention, the fermentation parameters can be adjusted according to the actual production scale. Preferred fermentation conditions are: fermentation temperature 35-38°C, fermentation pH 6-7, and fermentation time 24-72 h. Fermentation under these conditions yields a fermentation broth rich in 5-methyltetrahydrofolate.

[0060] In the above embodiments, centrifugation is used to achieve solid-liquid separation, causing cell debris to settle and removing large cell debris and insoluble impurities, resulting in a clear fermentation supernatant rich in extracellular and intracellular folic acid. In other embodiments, ceramic membrane microfiltration can be used to treat the fermentation broth. Microfiltration, by setting a filter medium with a specific pore size, effectively retains cell debris while allowing small molecule nutrients such as 5-methyltetrahydrofolate to pass through smoothly, thereby obtaining a clear supernatant that achieves the same preparation effect as the present invention.

[0061] In other embodiments, after obtaining a supernatant rich in active folic acid through solid-liquid separation, a stabilization treatment step may be further included. The stabilization treatment includes adding a protective agent and an antioxidant to the supernatant. In one embodiment, 10%–15% of a protective agent, selected from maltodextrin or skim milk powder, is added; simultaneously, 1%–3% of an antioxidant (vitamin C) is added to prevent oxidative degradation of the highly active 5-methyltetrahydrofolate during subsequent processing and storage.

[0062] In the above embodiments, a stable post-biotic preparation is obtained through drying, which is performed using vacuum freeze-drying. In other embodiments, low-temperature spray drying can also be used to obtain a stable post-biotic preparation rich in naturally sourced active folic acid. Therefore, this invention does not impose excessive limitations on the drying process.

[0063] The technical effects of this invention are illustrated below with experimental examples. All experimental data are expressed as mean ± standard deviation and were repeated at least three times. One-way ANOVA was performed on the data between different groups using SPSS 18.0 software, and graphs were plotted using GraphPad Prism 5.0 and Origin 9.0 software. Different lowercase letters indicate statistically significant differences (p < 0.05). Furthermore, in the analysis of different groups, ns indicates no significant difference between groups; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001, all indicating significant differences between groups.

[0064] Experiment 1: Screening of bacterial strains

[0065] 1. Identification of candidate strains

[0066] The candidate strains for this experiment were *Bifidobacterium longum* subsp. infantis BD223 from Example 1 of this invention, and three control strains (*Bifidobacterium longum* subsp. infantis BD221, *Bifidobacterium longum* subsp. infantis BD222, and *Bifidobacterium longum* BL21). The control strains *Bifidobacterium longum* subsp. infantis BD221 and BD222 were isolated from the feces of healthy full-term infants in Harbin City and were screened using the isolation and identification method for *Bifidobacterium longum* subsp. infantis BD223 in Example 1. The control strain *Bifidobacterium longum* BL21 was a commercially available strain. The folic acid production capacity of each candidate strain was tested to screen for the strain with the strongest folic acid production capacity.

[0067] 2. Activation of candidate strains

[0068] Candidate bacterial strains (Bifidobacterium longum subsp. infantis BD223, Bifidobacterium longum subsp. infantis BD221, Bifidobacterium longum subsp. infantis BD222, and Bifidobacterium longum BL21) were taken from cryovials stored at -80℃ and rapidly thawed at 37℃. Under aseptic conditions, the strains were inoculated into modified MRS medium and cultured anaerobically at 37℃ for 24 h. After culture, the cultures were centrifuged at 2000 rpm for 10 min, the supernatant was discarded, and the resulting bacterial cells were washed twice with sterile PBS buffer. Finally, the bacterial cells were resuspended in PBS buffer, and the bacterial concentration was adjusted to 1×10⁻⁶. 7 CFU / mL was used to obtain activated bacterial solutions for each candidate strain.

[0069] 3. Determination of folic acid production

[0070] The activated bacterial cultures of *Bifidobacterium longum* subsp. infantis BD223, BD221, BD222, and BL21 obtained above were inoculated into folic acid-free modified MRS medium at an inoculation rate of 5% v / v and cultured under anaerobic conditions at 37°C and pH 6.5 for 48 h. After culture, the fermentation broth was collected, centrifuged at 8000 rpm for 10 min at 4°C to remove bacterial cells, and the supernatant of each candidate strain was collected for folic acid content detection.

[0071] The folic acid content testing procedure was as follows: equal volumes of phosphate buffer containing 0.1 wt% ascorbic acid and 0.1 wt% dithiothreitol (DTT) were added to the supernatant of each candidate strain to extract folic acid. The extracts were then filtered through a 0.22 μm filter and analyzed by LC-MS / MS. Chromatographic separation was performed using a C18 reversed-phase column (2.1 mm × 100 mm, 1.7 μm). The mobile phase consisted of an aqueous solution (A) containing 0.1 wt% formic acid and an acetonitrile solution (B) containing 0.1 wt% formic acid, with gradient elution at a flow rate of 0.3 mL / min. Mass spectrometry was performed using ESI positive ion mode and MRM scanning. An external standard method was used to establish a standard curve for quantitative analysis of the total folic acid and 5-methyltetrahydrofolate content in the supernatant, and the mass percentage of 5-methyltetrahydrofolate in the total folic acid was obtained. The results of the determination of the total folic acid content and the percentage of 5-methyltetrahydrofolate in the supernatant obtained from the fermentation of different strains are shown in Table 1.

[0072] Table 1. Differences in total folic acid and 5-methyltetrahydrofolate content in the supernatant obtained from fermentation of different strains

[0073] Table 1 shows that different strains exhibited significant differences in growth rate and final biomass level. The results indicated that, under the same culture conditions, the percentage of 5-methyltetrahydrofolate in the fermentation supernatant of *Bifidobacterium longum* subsp. *infantii* BD223 was significantly higher than that of the other control strains tested (p<0.05). This suggests that *Bifidobacterium longum* subsp. *infantii* BD223 possesses outstanding folic acid synthesis capabilities, especially in the efficient production of bioactive folic acid. Therefore, subsequent research will focus on designing a comprehensive fermentation process using *Bifidobacterium longum* subsp. *infantii* BD223 as the target strain.

[0074] Experimental Example 2: Optimization of Fermentation Conditions and Preparation of Metabiotics

[0075] To maximize the production of natural bioactive folic acid (5-methyltetrahydrofolic acid) by Bifidobacterium longum subsp. infantis BD223, this invention further optimizes the composition of the fermentation medium and the culture conditions, and establishes a standardized postbiotic preparation process.

[0076] 1. Optimization of fermentation culture medium

[0077] This experimental example demonstrates the optimization of the basal culture medium to obtain the fermentation medium. The formula of the basal culture medium (g / L) is as follows: peptone 12g, yeast extract 6g, beef extract 6g, glucose 18g, sodium acetate 5g, ammonium citrate 2.15g, Tween 80 1g, magnesium sulfate 0.58g, manganese sulfate 0.05g, dipotassium hydrogen phosphate 2g, L-cysteine ​​0.5g, guanine 25mg, diluted to 1L with pure water, pH adjusted to 6.5, and autoclaved at 121℃ for 15min to obtain the basal culture medium. Note that the peptone, yeast extract, and beef extract were pre-treated with activated charcoal to remove vitamins.

[0078] (i) Optimization of carbon source types

[0079] 20 g / L of glucose, lactose, galactooligosaccharides (GOS), and 2'-fucosylated lactose (2'-FL) were added to the basal culture medium. After fermentation of *Bifidobacterium longum* subsp. *infantii* BD223 using the carbon-added medium, the total folic acid and 5-methyltetrahydrofolate contents were measured (fermentation parameters and folic acid detection methods were the same as in Example 1). The effects of different carbon sources on the production of total folic acid and 5-methyltetrahydrofolate by *Bifidobacterium longum* subsp. *infantii* BD223 are shown in Table 2.

[0080] Table 2. Effects of different carbon sources on total folic acid and 5-methyltetrahydrofolate production from BD223

[0081] Table 2 shows that when lactose is used as the main carbon source, the synthesis of total folic acid and active folic acid by *Bifidobacterium longum* subsp. infantis BD223 is the highest. Considering the matching degree between industrial cost and simulated infant intestinal environment, lactose is preferred as the main carbon source.

[0082] (ii) Concentration optimization of p-aminobenzoic acid

[0083] Para-aminobenzoic acid (p-aminobenzoic acid) is a key precursor in the folic acid biosynthesis pathway. In this invention, 20 g / L lactose was added as a carbon source to the aforementioned basal culture medium, followed by the addition of 10, 20, 50, 100, 150, and 200 mg / L p-aminobenzoic acid. The culture medium containing p-aminobenzoic acid was then used to ferment *Bifidobacterium longum* subsp. *infantii* BD223, and the total folic acid and 5-methyltetrahydrofolate contents were measured (fermentation process parameters and folic acid detection methods were the same as in Example 1). The effects of different concentrations of p-aminobenzoic acid on the production of total folic acid and 5-methyltetrahydrofolate by *Bifidobacterium longum* subsp. *infantii* BD223 are shown in Table 3.

[0084] Table 3. Effects of different concentrations of p-aminobenzoic acid on total folic acid and 5-methyltetrahydrofolate production from BD223

[0085] Table 3 shows that folic acid production peaked when the para-aminobenzoic acid (PABA) concentration was 100 mg / L, approximately 1.8 times higher than the lower concentration group. Further increases in PABA concentration did not significantly improve folic acid production. Therefore, the optimal PABA concentration was determined to be 100 mg / L.

[0086] 2. Optimization of fermentation culture conditions

[0087] Activated bacterial solution of Bifidobacterium longum subsp. infantis BD223 (1×10⁻⁶) 7 CFU / mL was inoculated at 5% (v / v) into the optimized fermentation medium (composition same as in Example 2), and fermented at 37°C and pH 6.5 for 12, 24, 48, 72, and 96 h, respectively. The total folic acid and 5-methyltetrahydrofolate contents were then measured (fermentation process parameters and folic acid detection methods were the same as in Example 1). The effects of different fermentation times on the production of total folic acid and 5-methyltetrahydrofolate by Bifidobacterium longum subsp. infantis BD223 are shown in Table 4.

[0088] Table 4. Effects of different fermentation times on the production of total folic acid and 5-methyltetrahydrofolate by BD223

[0089] Table 4 shows that, based on the combined analysis of total folic acid accumulation and 5-methyltetrahydrofolate conversion rate, 48 hours is the optimal fermentation time. In the first 48 hours of fermentation, total folic acid synthesis is in the logarithmic growth phase, reaching a relatively high yield (2.75 mg / L) by 48 hours, with the proportion of the active ingredient 5-methyltetrahydrofolate reaching its peak (89.52%). Although the total folic acid accumulation is higher at 96 hours, there is no significant difference between 48 and 72 hours. Furthermore, prolonged fermentation leads to a significant increase in time and energy costs, and the proportion of active products is no longer optimized. Therefore, based on the principles of high efficiency, high yield, and high quality, 48 hours is determined to be the optimal fermentation endpoint.

[0090] 3. Optimization of cell cell disruption, release, and inactivation processes

[0091] Since the natural folic acid synthesized by *Bifidobacterium longum* subsp. infantis is mainly stored intracellularly, traditional solid-liquid separation can lead to the loss of active ingredients. Therefore, this invention employs an integrated process of heat shock combined with enzymatic hydrolysis after fermentation. Specifically, the fermentation broth obtained from step (1) of Example 2 is rapidly heated to 95°C and maintained for 25 minutes for heat shock treatment. By controlling the above heat shock treatment conditions, on the one hand, the microorganisms in the fermentation broth are irreversibly inactivated, ensuring the safety of the postbiotic product; on the other hand, the heat effect is used to destroy the cell wall and cell membrane structure, promoting the full release of highly active intracellular 5-methyltetrahydrofolate (5-MTHF) into the fermentation broth system.

[0092] Following the heat shock treatment, this invention further incorporates an enzymatic hydrolysis-assisted process. Specifically, after the heat shock treatment, the fermentation broth is cooled to 50°C, and 0.3% (w / v) of lysozyme is added for 2 hours of enzymatic hydrolysis. After the above heat shock and enzymatic hydrolysis treatments, an enzymatically hydrolyzed fermentation broth is obtained. The obtained enzymatically hydrolyzed fermentation broth is centrifuged at 8000 rpm for 15 minutes, and then the fermentation supernatant is collected for the determination of total folic acid and 5-methyltetrahydrofolate content (the detection method is the same as in Experimental Example 1). The uncentrifuged fermentation broth is further subjected to microbial testing.

[0093] Meanwhile, control groups were set up with only heat shock treatment, only enzymatic hydrolysis treatment, and a control group without heat shock treatment and enzymatic hydrolysis treatment. Other process conditions remained unchanged. The contents of total folic acid and 5-methyltetrahydrofolate in the obtained fermentation supernatant were detected to investigate the effects of heat shock treatment and enzymatic hydrolysis on the production of total folic acid and 5-methyltetrahydrofolate by BD223. The results are shown in Table 5.

[0094] Table 5. Effects of heat shock treatment and enzymatic hydrolysis on the production of total folic acid and 5-methyltetrahydrofolate by BD223.

[0095] Table 5 shows that no culturable live bacteria were detected in the fermentation broth after heat shock treatment combined with enzymatic hydrolysis, and the content of extracellular free 5-methyltetrahydrofolate was significantly increased compared with other control groups (p<0.05). Therefore, it can provide a raw material basis rich in target metabolites for subsequent separation steps.

[0096] Experiment Example 3: Animal Experiments to Promote Cognitive Abilities

[0097] The experimental samples were the post-biotic preparations rich in 5-methyltetrahydrofolate obtained in Example 2 of this invention, which were dissolved in sterile saline to prepare drug solutions of different concentrations as needed for the experiment. The experimental animals were healthy newborn SPF-grade C57BL / 6J mice, purchased from the Second Affiliated Hospital of Harbin Medical University, and experiments began at 7 days of age. All experimental animals were housed in a standard animal facility environment with a temperature controlled at 22±2℃ and a relative humidity of 50%–60%, using 12-hour light / dark alternating lighting, and with free access to food and water.

[0098] The experimental animals were randomly divided into four groups: a blank control group, a low-dose group, a medium-dose group, and a high-dose group, with six animals in each group. All animals were administered the treatment via gavage. The intervention methods for each experimental group were as follows: the low-dose group received the post-biotic preparation from Example 2 via gavage at a dose of 50 mg / kg; the medium-dose group received the post-biotic preparation from Example 2 via gavage at a dose of 150 mg / kg; and the high-dose group received the post-biotic preparation from Example 2 via gavage at a dose of 500 mg / kg. The post-biotic preparation for each group was pre-dissolved in physiological saline. The blank control group received an equal volume of sterile physiological saline via gavage.

[0099] After four weeks of continuous intervention and completion of behavioral tests, all mice were euthanized after a 24-hour fast. Whole brain tissue was rapidly extracted, and the hippocampus and cerebral cortex were separated for further use. A portion of brain tissue was also collected and fixed in 10% paraformaldehyde for histological analysis. A suitable amount of hippocampus was ground in liquid nitrogen and used. Following the manufacturer's instructions, the levels of rat brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured using an enzyme-linked immunosorbent assay (ELISA) kit, thereby performing molecular signaling analysis of the mouse brain.

[0100] 1. Measurement of cognitive abilities in mice

[0101] Cognitive ability is a crucial manifestation of higher functions of the central nervous system, primarily encompassing learning ability, memory formation, and information retrieval. To evaluate the cognitive function and learning / memory abilities of mice in different treatment groups, a novel object recognition test was used to conduct behavioral tests and systematically assess changes in cognitive ability. The novel object recognition test mainly reflects the mouse's ability to recognize and retain novel stimuli, and its performance depends on the functional state of the hippocampus and related cortical regions. Generally, an improvement in the novel object recognition index indicates enhanced cognitive function and learning / memory abilities in mice; conversely, a decrease in related indicators may suggest cognitive impairment. Therefore, the novel object recognition test can comprehensively evaluate the cognitive ability and neurological development of mice. The results of the novel object recognition index measured after the novel object recognition test for each group of mice are shown below. Figure 1 As shown.

[0102] Figure 1 The results showed that the novel object recognition index of mice in all treatment groups was higher than that in the blank control group, with the high-dose group showing the most significant increase. Specifically, the novel object recognition index of the low-dose group was slightly higher than that of the blank control group, but the difference was not significant (ns); the novel object recognition index of the medium-dose group and the high-dose group was significantly higher than that of the blank control group (*p<0.05), and the novel object recognition index of the high-dose group was higher than that of the medium-dose group (***p<0.001), showing a dose-dependent trend.

[0103] The above results indicate that all intervention groups significantly improved the novel object recognition index of mice, thereby improving their cognitive function and learning and memory abilities, with the high-dose group showing the most significant effect.

[0104] 2. Changes in signaling molecules in the mouse brain

[0105] Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are important neurotrophic factors in the central nervous system of rats, participating in neuronal growth, differentiation, synaptic plasticity, and the maintenance of cognitive function. Changes in their levels can reflect potential changes in the neurological function and cognitive abilities of mice. To evaluate the changes in neurotrophic factor levels in the brains of mice in different treatment groups, the expression levels of BDNF and NGF were detected using ELISA, thereby systematically assessing changes in central nervous system function in mice. The results of the BDNF and NGF expression level measurements are shown below. Figure 2 and Figure 3 As shown.

[0106] Figure 2 The results showed that the BDNF content in the brains of mice in all treatment groups was higher than that in the blank control group, with the high-dose group showing the most significant increase. Specifically, the BDNF level in the low-dose group was not significantly different from that in the blank control group (ns); the BDNF levels in the medium-dose group and the high-dose group were significantly higher than those in the blank control group (***p<0.001, ****p<0.0001), and the high-dose group was higher than that in the medium-dose group (****p<0.0001), showing a clear dose-dependent trend.

[0107] Figure 3 The results showed that there was no significant difference in NGF levels between the low-dose group and the control group (ns), and the difference between the medium-dose group and the control group was also not significant (ns). However, the NGF level in the high-dose group was significantly higher than that in the control group (*p<0.05). Furthermore, the NGF level in the high-dose group was significantly higher than that in the medium-dose group (*p<0.05), and the medium-dose group showed a certain upward trend compared with the low-dose group (*p<0.05).

[0108] The above results indicate that all intervention groups significantly increased the levels of BDNF and NGF in the mouse brain, which is beneficial for further improving nerve metabolism and protecting the myelin sheath structure, thereby improving the neurological function and cognitive ability of mice. The high-dose group showed the most significant effect.

[0109] In summary, the *Bifidobacterium longum* subsp. infantis provided by this invention has the ability to produce high levels of 5-methyltetrahydrofolate. Furthermore, the postbiotic preparation made from *Bifidobacterium longum* subsp. infantis through fermentation is rich in naturally sourced active folic acid, which can supplement the key methyl donor required for brain development through a natural biological pathway, promote cognitive function and learning and memory abilities, and improve neurological function. This addresses the limitations in absorption and safety issues associated with chemically synthesized folic acid, making it suitable for preparing products rich in active folic acid.

[0110] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A *Bifidobacterium longum* subspecies *Infantile* with cognitive-improving properties, characterized in that... The name of the Bifidobacterium longum infant subspecies is Bifidobacterium longum infant subspecies ( Bifidobacterium longum subsp. infantis BD223, deposited at Guangdong Provincial Center for Microbial Culture Collection, deposited on February 9, 2026, with accession number GDMCCNo: 67827.

2. The application of *Bifidobacterium longum* subsp. *infantitidis* as described in claim 1 in the fermentation preparation of 5-methyltetrahydrofolate.

3. A post-biotic preparation rich in 5-methyltetrahydrofolate, characterized in that, The post-biotic preparation is prepared using a method comprising the following steps: (1) The activated bacterial solution of *Bifidobacterium longum* subsp. infantis is inoculated into a fermentation medium for fermentation culture to obtain a fermentation broth; wherein, the name of the *Bifidobacterium longum* subsp. infantis is *Bifidobacterium longum* subsp. infantis (…). Bifidobacterium longum subsp. infantis BD223, deposited at Guangdong Provincial Center for Microbial Culture Collection, deposited on February 9, 2026, with accession number GDMCC No: 67827; (2) The fermentation broth is subjected to heat shock treatment, followed by enzymatic hydrolysis to obtain enzymatically hydrolyzed fermentation broth; (3) The enzymatic fermentation broth is subjected to solid-liquid separation treatment, and then the supernatant is collected and dried to obtain the post-biotic preparation.

4. The post-biotic preparation rich in 5-methyltetrahydrofolate according to claim 3, characterized in that, In step (1), the concentration of Bifidobacterium longum subsp. infantis in the activated bacteria solution is (1-5) x 10 7 CFU / mL; and the inoculation amount of the activated bacteria solution in the fermentation medium is 3%-8% v / v.

5. The post-biotic preparation rich in 5-methyltetrahydrofolate according to claim 3, characterized in that, In step (1), the fermentation medium includes MRS basal medium and additives added to the MRS basal medium; The additives, based on their concentration in the MRS basal medium, include: 15-25 g / L lactose, 50-200 mg / L para-aminobenzoic acid, and 20-30 mg / L guanine.

6. The post-biotic preparation rich in 5-methyltetrahydrofolate according to claim 3, characterized in that, In step (1), the fermentation culture process conditions are: fermentation temperature of 35~38℃, fermentation pH of 6.0~7.0, and fermentation under anaerobic conditions for 24~72h.

7. The post-biotic preparation rich in 5-methyltetrahydrofolate according to any one of claims 3 to 6, characterized in that, In step (2), the heat shock treatment process conditions are: heating the fermentation broth to 92~98℃ and maintaining it for 10~40min; the enzyme used in the enzymatic hydrolysis treatment is lysozyme; the enzymatic hydrolysis treatment process conditions are: enzyme addition amount is 0.1%~0.5%w / v, enzymatic hydrolysis temperature is 45~55℃, and enzymatic hydrolysis time is 1~5h.

8. The post-biotic preparation rich in 5-methyltetrahydrofolate according to any one of claims 3 to 6, characterized in that, In step (3), the solid-liquid separation process is centrifugation or filtration; the drying process is vacuum freeze drying or spray drying.

9. The use of a post-natal agent as described in any one of claims 3 to 8 in the preparation of a medicament for improving cognitive abilities during brain development, characterized in that, The improvement in cognitive abilities during brain development is achieved through one or more of the following: increasing the new object recognition index, upregulating brain-derived neurotrophic factor levels, and upregulating nerve growth factor levels.

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