Application of lactobacillus plantarum in preparation of product for relieving individual oxidative stress and cognitive impairment caused by methylmalonemia

Abstract

The invention discloses an application of lactobacillus plantarum in preparation of a product for relieving individual oxidative stress and cognitive impairment caused by methylmalonic acidmia, the lactobacillus plantarum is preserved in Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No.61163, and the preservation date is August 21, 2020. The lactobacillus plantarum can be applied to preparation of a product for relieving individual oxidative stress and cognitive impairment caused by methylmalonic acidmia. The relieving effect of lactobacillus plantarum CCFM1149 intervention on oxidative damage and cognitive impairment caused by MMA diseases is comprehensively evaluated through MMA model rats, and the relieving effect is specifically reflected as follows: (a) cognitive and memory impairment is relieved; (b) relieving activation of small glial cells in the hippocampus; (c) reducing malondialdehyde levels in serum and / or tissue; and (d) increasing the level of glutathione peroxidase in serum and / or tissues.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to the application of Lactobacillus plantarum in preparing a product for relieving oxidative stress and cognitive impairment caused by methylmalonic aciduria in individuals. BACKGROUND

[0002] Methylmalonic aciduria (MMA) is also known as methylmalonic acidemia, which is a rare genetic metabolic disease that can be prevented and treated. It usually occurs in the neonatal period or infancy. MMA is caused by the 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. Due to the extremely complex pathogenesis of MMA, there is currently no specific treatment for the disease. At present, the treatment of MMA mainly focuses on maintaining metabolic stability, and the combination of drugs and diet is the main treatment strategy. However, there are relatively few studies on the use of antioxidants as an adjunct therapy in the treatment of methylmalonic acidemia.

[0003] Oxidative stress is closely related to various neurodegenerative diseases and congenital metabolic disorders. As a metabolic disease, MMA is characterized by imbalance of redox homeostasis throughout the disease development process. A large number of patient reports and animal model studies have shown that oxidative stress occurs during the progression of MMA. In addition, there is sufficient evidence to suggest that the antioxidant status of cells has an important impact on the development of MMA. As an endogenous toxic metabolic product, MMA not only damages the energy metabolism of rats, but also triggers oxidative stress in the brain, leading to cognitive impairment in rats. For MMA patients, the accumulation of methylmalonic acid not only causes damage to multiple systems, but also disrupts the body's redox balance by triggering oxidative stress and neuroinflammation, so it has an immeasurable negative impact on the patient's life and quality of life.

[0004] Probiotics are a class of dietary supplements with multiple physiological regulation functions. They are widely accepted as important nutritional supplements due to their multiple functions and benefits to the intestinal tract. At the same time, due to their natural non-toxic properties, probiotics are considered a new source of potential antioxidants. In recent years, many studies have focused on the intestinal flora and found that it plays a crucial role in the development of various metabolic diseases in humans. In addition, intestinal flora also has the effect of relieving oxidative stress and neural damage. The accumulation of methylmalonic acid in the body of MMA patients causes oxidative stress, and may also cause varying degrees of cognitive impairment. Therefore, regulating intestinal microbiota through probiotics may provide a new treatment strategy for relieving oxidative stress and neural development disorders in MMA patients. SUMMARY

[0005] In view of the above problems existing in the prior art, the application provides an application of Lactobacillus plantarum in preparing a product for relieving oxidative stress and cognitive impairment of an individual caused by methylmalonic acidemia.

[0006] The technical scheme of the application is as follows:

[0007] The application aims to provide an application of Lactobacillus plantarum in preparing a product for relieving oxidative stress and cognitive impairment of an individual caused by methylmalonic acidemia, wherein the Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No.61163, and the preservation date is August 21, 2020.

[0008] In an embodiment of the application, the product includes a special medical food, a health product or a medicine.

[0009] In an embodiment of the application, the viable bacterial count of the Lactobacillus plantarum in the product is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0010] In an embodiment of the application, when the product is a medicine, the product further includes a pharmaceutical carrier and / or a pharmaceutical excipient.

[0011] In an embodiment of the application, the pharmaceutical carrier includes microcapsules, microspheres, nanoparticles and / or liposomes.

[0012] In an embodiment of the application, the pharmaceutical excipient includes an excipient and / or an additional agent.

[0013] In an embodiment of the application, the excipient includes a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a coloring agent, an absorbent, a diluent, a flocculating agent, a deflocculating agent, a filter aid and / or a release retarder; and the additional agent includes microcrystalline cellulose, hydroxypropyl methyl cellulose and / or refined lecithin.

[0014] In an embodiment of the application, when the product is a special medical food or a health product, the product is a fermentation product prepared by fermentation of the Lactobacillus plantarum.

[0015] In an embodiment of the application, the symptoms of the methylmalonic acidemia patient include oxidative damage and cognitive impairment caused by accumulation of methylmalonic acid in the body.

[0016] The above Lactobacillus plantarum can relieve oxidative stress and cognitive impairment of an individual caused by methylmalonic acidemia.

[0017] In one embodiment of the present invention, the product functions as follows:

[0018] (a) Reduces cognitive and memory impairments in patients with methylmalonic acidemia;

[0019] (b) Alleviating the activation of hippocampal microglia in patients with methylmalonic acidemia;

[0020] (c) Reduce serum and / or tissue malondialdehyde levels in patients with methylmalonic acidemia;

[0021] (d) Increase the level of glutathione peroxidase in the serum and / or tissues of patients with methylmalonic acidemia.

[0022] The beneficial technical effects of this invention are as follows:

[0023] The *Lactobacillus plantarum* CCFM1149 of this invention can significantly alleviate oxidative damage and cognitive impairment in MMA model rats, improve their behavioral performance in open field, novel object recognition, and Y-maze, alleviate microglia activation in the hippocampus, reduce malondialdehyde levels in serum and / or tissues, and increase glutathione peroxidase levels in serum and / or tissues, thereby contributing to the relief of oxidative damage and cognitive impairment in rats. *Lactobacillus plantarum* CCFM1149 can serve as a functional probiotic for adjuvant treatment of MMA, providing new insights for clinical intervention and alleviating oxidative and cognitive impairment in MMA, and has promising industrial prospects. Attached Figure Description

[0024] Figure 1 Flowchart for animal model creation in experiments;

[0025] Figure 2 The behavioral performance of rats in each group after intervention;

[0026] Figure 3 Open field trajectory diagrams for rats in each group;

[0027] Figure 4 Immunohistochemical staining results of rat brain tissue in each group;

[0028] Figure 5 To investigate the effect of intervention on serum antioxidant capacity in rats of different groups;

[0029] Figure 6 To investigate the effects of intervention on the antioxidant capacity of brain tissue in each group of rats;

[0030] Figure 7 To investigate the effect of intervention on the antioxidant capacity of liver tissue in each group of rats;

[0031] Figure 8 To investigate the effect of intervention on the antioxidant capacity of kidney tissue in rats of different groups. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0033] Preservation of biological materials

[0034] A strain of Lactobacillus plantarum (CCFM1149), taxonomically named Lactobacillus plantarum, was deposited on August 21, 2020, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 61163), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0035] The methylmalonic acid used in the following examples was purchased from Sigma-Aldrich.

[0036] The culture media involved in the following examples are as follows:

[0037] 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.

[0038] MRS solid medium: Add 15-20g of agar to the MRS liquid medium.

[0039] A comprehensive evaluation of the effects of Lactobacillus plantarum CCFM1149 on alleviating oxidative stress and improving neurocognitive impairment in MMA rats was conducted after intervention in MMA model rats (blank group, model group, Lactobacillus plantarum CCFM1149 group, and ascorbic acid (VC) group, 7-8 rats in each group).

[0040] The behavioral testing methods involved in the following embodiments are as follows:

[0041] 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.

[0042] 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%.

[0043] 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%).

[0044] Immunohistochemical detection of rat brain:

[0045] 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.

[0046] Detection of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) in rat serum and liver tissue:

[0047] The tests were performed using ELISA kits, all purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.

[0048] Example 1: Lactobacillus plantarum CCFM1149 alleviates cognitive impairment in MMA model rats

[0049] The specific steps are as follows:

[0050] 1. Preparation of oral bacterial solution

[0051] Frozen *Lactobacillus plantarum* CCFM1149, 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. *Lactobacillus plantarum* CCFM1149 was anaerobically cultured for 24 hours, 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.

[0052] 2. Animal experiments:

[0053] 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.

[0054] After the birth of pregnant rats, they were randomly divided into a control group, a model group, a *Lactobacillus plantarum* CCFM1149 group, and an ascorbic acid (VC) group. Except for the control group, the other three groups were modeled 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. The CCFM1149 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 .

[0055] Table 1 Animal grouping information

[0056] Serial number Group Gavage treatment 1 Blank group Gavage with normal saline 2 Model group Gavage with normal saline 3 CCFM1149 group intraperitoneally with 1 x 10 9 CFU / mL Lactobacillus plantarum CCFM1149 4 VC group Gavage with 500 mg / kg ascorbic acid

[0057] After the intervention, behavioral experiments were conducted in open field, new object recognition, and the Y-maze. The behavioral results are as follows: Figure 2 As shown, compared with the model group, the blank group achieved higher evaluation scores in all three behavioral experiments, indicating that the blank group rats 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 *Lactobacillus plantarum* CCFM1149 group (30.58±15.83 min) significantly increased the dwell time in the central region (P<0.05), indicating that *Lactobacillus plantarum* CCFM1149 can alleviate the anxiety of the model rats and enhance their exploratory abilities, as shown in their movement trajectory diagram. Figure 3 In the novel object recognition experiment, the cognitive index of MMA rats treated with *Lactobacillus plantarum* CCFM1149 was significantly improved (P<0.05). 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 *Lactobacillus plantarum* CCFM1149 group (0.57±0.08) significantly increased the percentage of alternations (P<0.01), thus improving the rats' memory level. In conclusion, *Lactobacillus plantarum* CCFM1149 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.

[0058] Example 2: Lactobacillus plantarum CCFM1149 inhibits microglia activation in the brain of MMA model rats

[0059] 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.

[0060] The activation status of microglia in the hippocampus was detected by immunohistochemical staining, such as... Figure 4As 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 *Lactobacillus plantarum* CCFM1149 reduced the number of activated microglia. These results indicate that CCFM1149 intervention partially inhibited methylmalonic acid-induced microglia activation, but did not completely alleviate it.

[0061] Example 3: Lactobacillus plantarum CCFM1149 enhances antioxidant capacity in MMA model rats

[0062] 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.

[0063] Depend on Figure 5 It can be seen that intervention with Lactobacillus plantarum CCFM1149 significantly reduced the MDA level in the serum of MMA rats (P<0.05), significantly increased the GSH-PX level (P<0.05), and showed a decreasing trend in SOD level.

[0064] Depend on Figure 6 It was found that intervention with Lactobacillus plantarum CCFM1149 significantly reduced the levels of MDA (P<0.05) and SOD (P<0.05) in the brains of MMA rats, and significantly increased the level of GSH-PX (P<0.05).

[0065] Depend on Figure 7 It can be seen that after intervention with Lactobacillus plantarum CCFM1149, the SOD level in the liver of MMA rats was significantly reduced (P<0.01), the GSH-PX level was significantly increased (P<0.01), and the MDA level showed a decreasing trend.

[0066] Depend on Figure 8 It can be seen that after intervention with Lactobacillus plantarum CCFM1149, the levels of MDA and SOD in the kidneys of MMA rats tended to decrease, while the level of GSH-PX tended to increase.

[0067] The above results indicate that *Lactobacillus plantarum* CCFM1149 intervention inhibited lipid oxidation in rat blood and / or tissues induced by methylmalonic acid, alleviated oxidative stress, and helped 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, *Lactobacillus plantarum* CCFM1149 exhibits good antioxidant activity during intervention in MMA model rats.

[0068] 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. Use of Lactobacillus plantarum in the manufacture of a product for alleviating oxidative stress and cognitive impairment induced by methylmalonic acidemia in an individual, characterized in that, The Lactobacillus plantarum CCFM1149 is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No.61163, and the preservation date is August 21, 2020.

2. Use according to claim 1, characterized in that, The product includes a special medical food, a health product or a drug.

3. Use according to claim 1, characterized in that, The viable cell count of Lactobacillus plantarum in the product is not less than 1 x 10 9 CFU / mL or 1 x 10 9 CFU / g.

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

5. Use according to claim 4, characterized in that, The drug carrier includes microcapsules, microspheres, nanoparticles and / or liposomes.

6. Use according to claim 4, characterized in that, The pharmaceutical excipient includes an excipient and / or an additional agent.

7. Use according to claim 6, characterized in that, The excipient includes a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a coloring agent, an absorbent, a diluent, a flocculating agent, a deflocculating agent, a filter aid and / or a release retarder; and the additional agent includes microcrystalline cellulose, hydroxypropyl methyl cellulose and / or refined lecithin.

8. The use according to claim 2, characterized in that, When the product is a special medical food or a health product, it is a fermentation product prepared by fermentation of the Lactobacillus plantarum.

9. The use according to claim 1, characterized in that, The functions of the product include: (a) alleviating cognitive and memory impairment in patients with methylmalonic acidemia; (b) relieving the activation of hippocampal microglia in patients with methylmalonic acidemia; (c) reducing the level of malondialdehyde in the serum and / or tissue of patients with methylmalonic acidemia; (d) increasing the level of glutathione peroxidase in the serum and / or tissue of patients with methylmalonic acidemia.

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