Application of bifidobacterium longum in preparation of product for relieving cognitive impairment caused by methylmalonemia

Abstract

The invention discloses application of bifidobacterium longum in preparation of a product for relieving cognitive impairment caused by methylmalonemia, and belongs to the technical field of microorganisms. The Bifidobacterium longum CCFM752 is preserved in the Guangdong Province Microbial Culture Collection Center, the preservation number is GDMCC (China General Microbiological Culture Collection Center) No.61157, and the preservation date is August 21, 2020. The Bifidobacterium longum CCFM752 disclosed by the invention has the advantages that the Bifidobacterium longum CCFM752 can be used for preparing the biological feed additive; the effect of the bifidobacterium longum CCFM752 for relieving the cognitive impairment of the MMA disease is comprehensively evaluated through an MMA model rat, and the effect is specifically reflected as follows: (a) the cognitive and memory impairment is relieved; (b) relieving activation of small glial cells in the hippocampus; (c) reducing the malondialdehyde level in serum and / or tissue to a certain extent; and (d) improving the level of glutathione peroxidase in serum and / or tissues to a certain extent.

Description

Technical Field

[0001] This invention relates to microbial technology, and in particular to the use of a type of Bifidobacterium longum in the preparation of products that alleviate cognitive impairment caused by methylmalonic acidemia. Background Technology

[0002] Methylmalonic aciduria (MMA), also known as methylmalonic acidemia, is a rare, treatable, and preventable inherited metabolic disorder. It is also the most common organic acid metabolism disorder in my country, typically manifesting in the neonatal or infancy period. MMA is listed as number 71 in my country's first batch of rare diseases. A nationwide screening study of 7.82 million newborns showed an MMA detection rate of 1 / 15,213. MMA was first reported in my country in 2000, and diagnostic, treatment, and prevention techniques have gradually been established. It has been determined that simple MMA caused by methylmalonyl-CoA mutase deficiency accounts for one-third of MMA patients in my country, requiring special dietary management, cobalamin, and L-carnitine treatment. MMA is caused by a deficiency of methylmalonyl-CoA mutase or its coenzyme cobalamin, leading to the accumulation of harmful substances such as methylmalonic acid in the body, causing damage to the nervous system and affecting brain development and growth.

[0003] Many neurodegenerative diseases and congenital metabolic disorders are closely related to the gut microbiota. MMA, as a metabolic disease, involves nervous system damage throughout its development. The accumulation of toxic metabolites in the body promotes the excessive production of free radicals, leading to cell damage and dysfunction. For MMA patients, the accumulation of methylmalonic acid not only damages multiple systems but also triggers neuroinflammation and disrupts the body's redox balance, thus having an immeasurable negative impact on their quality of life and well-being. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, this invention provides the application of *Bifidobacterium longum* in the preparation of products that alleviate cognitive impairment induced by methylmalonic acidemia. This invention can alleviate cognitive impairment in MMA model rats.

[0005] The technical solution of the present invention is as follows:

[0006] The purpose of this invention is to provide an application of Bifidobacterium longum in the preparation of products that alleviate cognitive impairment caused by methylmalonic acidemia. The Bifidobacterium longum CCFM752 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 61157 and deposit date of August 21, 2020.

[0007] In one embodiment of the present invention, the product includes special medical foods, health products, or pharmaceuticals.

[0008] In one embodiment of the present invention, the viable count of Bifidobacterium longum in the product is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0009] In one embodiment of the present invention, when the product is a pharmaceutical product, it further includes a drug carrier and / or pharmaceutical excipients.

[0010] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0011] In one embodiment of the present invention, the pharmaceutical excipient comprises excipients and / or additives.

[0012] In one embodiment of the present invention, the excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, absorbents, diluents, flocculants, anti-flocculators, filter aids, and / or release inhibitors; the additives include microcrystalline cellulose, hydroxypropyl methylcellulose, and / or refined lecithin.

[0013] In one embodiment of the present invention, when the product is a special medical food or health product, it is a fermented product obtained by fermentation with the participation of Bifidobacterium longum.

[0014] In one embodiment of the present invention, the product functions as follows:

[0015] (a) Reduces cognitive and memory impairments in patients with methylmalonic acidemia;

[0016] (b) Alleviating the activation of hippocampal microglia in patients with methylmalonic acidemia;

[0017] (c) Reduce serum and / or tissue malondialdehyde levels in patients with methylmalonic acidemia;

[0018] (d) Increase the level of glutathione peroxidase in the serum and / or tissues of patients with methylmalonic acidemia.

[0019] The beneficial technical effects of this invention are as follows:

[0020] The *Bifidobacterium longum* CCFM752 provided by this invention can significantly alleviate cognitive impairment in MMA model rats, improve their behavioral performance in open field, novel object recognition, and Y-maze, and alleviate the activation of microglia in the hippocampus; it also reduces malondialdehyde levels in serum and / or tissues to a certain extent; and increases glutathione peroxidase levels in serum and / or tissues to a certain extent, thereby alleviating cognitive impairment in rats. *Bifidobacterium longum* CCFM752 can serve as a functional probiotic for adjuvant treatment of MMA, providing new insights for clinical intervention and alleviating cognitive impairment in MMA, and has promising industrial prospects. Attached Figure Description

[0021] Figure 1 Flowchart for animal model creation in experiments;

[0022] Figure 2 The behavioral performance of rats in each group after intervention;

[0023] Figure 3 Open field trajectory diagrams for rats in each group;

[0024] Figure 4 Immunohistochemical staining results of rat brain tissue in each group;

[0025] Figure 5 To investigate the effect of intervention on serum antioxidant capacity in rats of different groups;

[0026] Figure 6 To investigate the effects of intervention on the antioxidant capacity of brain tissue in each group of rats;

[0027] Figure 7 To investigate the effect of intervention on the antioxidant capacity of liver tissue in each group of rats;

[0028] Figure 8 To investigate the effect of intervention on the antioxidant capacity of kidney tissue in rats of different groups. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0030] Preservation of biological materials

[0031] A strain of Bifidobacterium longum, CCFM752, taxonomically named Bifidobacterium longum, was deposited on August 21, 2020, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo.61157, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0032] The methylmalonic acid used in the following examples was purchased from Sigma-Aldrich.

[0033] The culture media involved in the following examples are as follows:

[0034] MRS liquid medium (1L): peptone 10g, beef extract 10g, glucose 20g, sodium acetate 5g, yeast extract 5g, diammonium citrate 2g, K2HPO4 2g, MgSO4·7H2O 0.1g, MnSO4·H2O 0.05g, Tween 80 1mL.

[0035] MRS solid medium: Add 15-20g of agar to the MRS liquid medium.

[0036] A comprehensive evaluation of the effect of Bifidobacterium longum CCFM752 on improving neurocognitive impairment in MMA rats was conducted after intervention in MMA model rats (blank group, model group, Bifidobacterium longum CCFM752 group, ascorbic acid (VC) group, 7-8 rats in each group).

[0037] The behavioral testing methods involved in the following embodiments are as follows:

[0038] Open field experiment: Rats were removed from their cages and placed facing away from the experimenter in the central area of ​​an open field consisting of a square gray box with no upper cover, each side measuring 100 cm. During the experiment, the animals' activity in the open field was recorded for 5 minutes, and the time the rats spent in the central area was subsequently analyzed.

[0039] Novel Object Experiment: After being acclimatized for one day in an empty box (the same box used in the open field experiment), rats were placed in a box containing two identical cube-shaped wooden blocks, with their heads facing the opposite direction of the blocks. They were allowed to move freely within the box, and the rats' interactions with the two objects were recorded, including the time spent exploring within 2 cm of the initial block (nose or mouth). The entire process was recorded for 5 minutes. After the experiment, the rats were removed and returned to their original cages. The next day, one of the cube-shaped blocks was replaced with a cylindrical block of the same height and similar size, fixed in the same position. The location of the new object (left or right) was randomly assigned to different rats and groups. Rats were placed in the box again using the same method, and their exploration activities were recorded for 5 minutes. The recorded rat activity videos were analyzed, and the number of times each rat explored the new object and the time spent exploring the old object were analyzed separately to obtain the rat's Recognition Index (RI). RI (%) = Number of times new object was explored / (Number of times new object was explored + Number of times old object was explored) × 100%.

[0040] Y-maze: Rats were placed in the central area of ​​a Y-shaped maze as the starting point and allowed to explore freely for 3 minutes, with the entire movement process recorded. The data was then analyzed to calculate the percentage of rats alternating in the Y-maze (percentage of alternation = number of times the rat entered three different arms consecutively / (total number of arm entries - 2) × 100%).

[0041] Immunohistochemical detection of rat brain:

[0042] Rat brains were uniformly collected and fixed in 4% paraformaldehyde. After 24 hours of fixation, the fixative was removed by rinsing with tap water. Paraffin-embedded sections of the brain were prepared and immunohistochemically analyzed. Finally, the stained sections were mounted with neutral resin. The prepared sections were scanned using a Pannoramic MIDI digital slide scanner.

[0043] Detection of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) in rat serum and liver tissue:

[0044] The tests were performed using ELISA kits, all purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0045] Example 1: Bifidobacterium longum CCFM752 alleviates cognitive dysfunction in MMA model rats

[0046] The specific steps are as follows:

[0047] 1. Preparation of gastric bacterial culture

[0048] Frozen Bifidobacterium longum CCFM752 (stored at -80℃) was inoculated into MRS medium at a 2% inoculation rate in a clean bench. After three generations of activation, the culture was expanded and anaerobically cultured for 24 hours. The culture was then centrifuged at 8000 rpm for 15 minutes to collect the bacterial sludge. The sludge was washed three times with sterilized 0.9% physiological saline, and then centrifuged again under the same conditions. The obtained sludge was resuspended in 30% glycerol solution, counted, and stored at -80℃ for later use. Before gavage, it was diluted with sterilized 0.9% physiological saline to a viable count of 1 × 10⁻⁶. 9 The bacterial solution was obtained at CFU / mL and quickly transported to the animal room for gavage.

[0049] 2. Animal experiments:

[0050] Thirty 5-day-old Wistar rats were selected as experimental animals. Pregnant rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The entire experiment strictly followed the requirements of the national standard for laboratory animal welfare ethics. The animal experimental protocol was approved by the Laboratory Animal Ethics Committee of Jiangnan University (JN.No20241015W0121204

[530] ). The rats were housed in a barrier at the Laboratory Animal Center of Jiangnan University. The temperature inside the barrier was maintained at 20-26℃ and the humidity was controlled at 40%-70%. The light intensity was 15-20 LX, and a 12-hour light-dark cycle was adopted. The environmental noise did not exceed 60dB. All rats in the experimental groups had free access to food and water. The rats were randomly divided into 5 groups, with 5 rats per cage after weaning at 21 days of age.

[0051] After the birthing period, pregnant rats were randomly divided into a control group, a model group, a Bifidobacterium longum CCFM752 group, and an ascorbic acid (VC) group. Except for the control group, the other three groups underwent modeling according to the following protocol: Methylmalonic acid was dissolved in physiological saline, and the pH was adjusted to 7.4 with 6 mmol / L NaOH. From day 5 to day 28 after birth, rats were subcutaneously injected twice daily (8-hour interval) with the methylmalonic acid solution. The injection dose was 10 μl / g, with the following concentrations: 0.72 μmol / g from day 5 to day 12 after birth; 0.89 μmol / g from day 13 to day 19 after birth; and 1.67 μmol / g from day 20 to day 28 after birth. The control group received the same volume of physiological saline. The intervention phase began in the third week (15 days) after the pups' birth and lasted until the end of the fourth week (28 days). The control group and model group received daily gavage with saline solution, while the CCFM752 group and VC group received probiotic gavage intervention according to Table 1, once daily, with a gavage volume of 1 mL / 100g body weight. Fecal samples were collected on days 14, 21, and 28. Specific grouping intervention procedures and experimental protocols are shown in Table 1 and... Figure 1 .

[0052] Table 1 Animal grouping information

[0053]

[0054]

[0055] After the intervention, behavioral experiments were conducted in open field, new object recognition, and Y-maze. The behavioral results are as follows: Figure 2As shown, compared with the model group, the blank group achieved higher evaluation scores in all three behavioral experiments, indicating that the rats in the blank group had better learning and cognitive abilities, confirming the validity of the model. Furthermore, the intervention groups containing antioxidant VC significantly improved the learning and cognitive abilities of the rats. The dwell time in the central region of the open field test indicates the rats' exploratory behavior and anxiety level. Compared with the model group (9.99±7.90 min), the Bifidobacterium longum CCFM752 group (27.59±12.46 min) significantly increased the dwell time in the central region (P<0.05), indicating that Bifidobacterium longum CCFM752 can alleviate the anxiety of the model rats and enhance their exploratory abilities, as shown in their movement trajectory diagram. Figure 3 The percentage of alternations in the Y-maze test is an important indicator of working memory ability in rats. Compared with the model group (0.40±0.09), the Bifidobacterium longum CCFM752 group (0.56±0.09) significantly increased the percentage of alternations in rats (P<0.05), thus improving the rats' memory level. In conclusion, Bifidobacterium longum CCFM752 also improved the learning and cognitive abilities of rats to varying degrees, suggesting that it may have a positive impact on brain development in rats after methylmalonic acid intervention.

[0056] Example 2: Bifidobacterium longum CCFM752 inhibits microglia activation in the brain of MMA model rats

[0057] Following behavioral tests, rat brains were uniformly collected and fixed in 4% paraformaldehyde. After 24 hours of fixation, the fixative was removed by rinsing with tap water. Paraffin-embedded sections of the brain were prepared and immunohistochemically analyzed. Finally, the stained sections were mounted with neutral resin. The prepared sections were scanned using a Pannoramic MIDI digital slide scanner.

[0058] The activation status of microglia in the hippocampus was detected by immunohistochemical staining, such as... Figure 4 As shown, in the control group, the cell bodies of microglia were relatively small and their numbers were small; while in the model group, the cell bodies became larger and rounder, and the microglia were activated and more numerous; intervention with Bifidobacterium longum CCFM752 reduced the number of activated microglia. These results indicate that probiotic intervention partially inhibited methylmalonic acid-induced microglia activation, but did not completely alleviate it.

[0059] Example 3: Bifidobacterium longum CCFM752 enhances antioxidant capacity in MMA model rats

[0060] At the end of the experiment, blood was collected via the abdominal aorta. After standing for 2 hours, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C, and the supernatant was collected. Rat brain, liver, and kidney tissues were collected, ground, and the supernatant was collected to determine their protein content. Competitive ELISA was used to detect MDA concentration, and double-antibody sandwich ELISA was used to determine SOD and GSH-PX activity levels. All procedures were strictly performed according to the kit instructions, and absorbance was read at 450 nm using a microplate reader.

[0061] Depend on Figure 5 It can be seen that intervention with Bifidobacterium longum CCFM752 reduced the levels of MDA and SOD in the serum of MMA rats and increased the level of GSH-PX.

[0062] Depend on Figure 6 It can be seen that intervention with Bifidobacterium longum CCFM752 reduced the levels of MDA and SOD in the brains of MMA rats and increased the level of GSH-PX.

[0063] Depend on Figure 7 It can be seen that intervention with Bifidobacterium longum CCFM752 reduced the levels of MDA and SOD in the liver of MMA rats and increased the level of GSH-PX.

[0064] Depend on Figure 8 It can be seen that intervention with Bifidobacterium longum CCFM752 reduced the levels of MDA and SOD in the kidneys of MMA rats and increased the level of GSH-PX.

[0065] The above results indicate that intervention with Bifidobacterium longum CCFM752 inhibited lipid oxidation in rat blood and / or tissues induced by methylmalonic acid to a certain extent, alleviating oxidative stress and helping to protect brain neurons from inflammatory responses; it also scavenged superoxide and free radicals in rat blood and / or tissues, helping to alleviate oxidative damage and improve the rats' learning and cognitive abilities. In conclusion, Bifidobacterium longum CCFM752 has a certain degree of antioxidant effect during intervention in MMA model rats.

[0066] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. The application of *Bifidobacterium longum* in the preparation of products for alleviating cognitive impairment caused by methylmalonic acidemia, characterized in that, The *Bifidobacterium longum* strain is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 61157 and deposit date of August 21, 2020.

2. The application according to claim 1, characterized in that, Products include special medical foods, health products, or medicines.

3. The application according to claim 1, characterized in that, The viable count of Bifidobacterium longum in the product is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

4. The application according to claim 2, characterized in that, When the product is a pharmaceutical product, it also includes a drug carrier and / or pharmaceutical excipients.

5. The application according to claim 4, characterized in that, Drug carriers include microcapsules, microspheres, nanoparticles, and / or liposomes.

6. The application according to claim 4, characterized in that, Pharmaceutical excipients include excipients and / or additives.

7. The application according to claim 6, characterized in that, The excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, absorbents, diluents, flocculants, anti-flocculators, filter aids, and / or release inhibitors; the additives include microcrystalline cellulose, hydroxypropyl methylcellulose, and / or refined lecithin.

8. The application according to claim 2, characterized in that, When the product is a special medical food or health product, it is a fermented product obtained by fermentation with the participation of Bifidobacterium longum.

9. The application according to claim 1, characterized in that, The product's functions include: (a) Reduces cognitive and memory impairments in patients with methylmalonic acidemia; (b) Alleviating the activation of hippocampal microglia in patients with methylmalonic acidemia; (c) Reduce serum and / or tissue malondialdehyde levels in patients with methylmalonic acidemia; (d) Increase the level of glutathione peroxidase in the serum and / or tissues of patients with methylmalonic acidemia.

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