Paracteroides faecium capable of relieving senescence and application of paracteroides faecium

Abstract

The invention discloses parabacteroides faecium capable of relieving senescence and application of the parabacteroides faecium, and belongs to the technical field of microorganisms and the technical field of medicines. Animal experiments prove that Parabacteroides vervae CCFM1428 has the effects of improving the weakness index of a mouse, increasing peroxide dismutase in the liver of the mouse, reducing malonaldehyde, increasing the level of glutathione peroxidase (GSH-px), increasing the level of catalase, reducing an inflammatory factor IL-6, reducing an inflammatory factor TNF-alpha and increasing the level of acetylcholine in the brain of the mouse, and has the effects of improving the weakness index of the mouse, increasing the liver peroxide dismutase of the mouse, reducing malonaldehyde, increasing the level of glutathione peroxidase (GSH-px) and the level of catalase. The gripping force of the mouse is improved. The strain disclosed by the invention has a huge application prospect in preparation of products for relieving senescence.

Description

Technical Field

[0001] The invention relates to a strain of Parabacteroides faecium capable of alleviating aging and application thereof, and belongs to the technical field of microorganisms and the technical field of medicine. Background Art

[0002] Human aging is a complex process characterized by the decline of human functions over time and a decrease in the quality of life. Aging is the main risk factor for most chronic diseases in humans, including cardiovascular disease, sarcopenia, osteoporosis, cancer, and neurodegenerative diseases. At present, against the background of accelerated aging, there is still a certain difference between healthy life expectancy and life expectancy, and healthy aging has become an issue that needs to be addressed urgently.

[0003] In recent years, the study of microbiome has attracted widespread attention in the field of aging. Among the 12 most recently released markers of aging, the disorder and imbalance of intestinal microbiome is one of the important markers of aging. In addition, aging also brings a series of negative effects on the body, such as increased levels of oxidative stress and inflammation, muscle loss, and weakened muscle strength. Current methods of alleviating aging mainly focus on drug intervention and lifestyle, and lack innovative methods for the microbiome, so there are some limitations, such as adverse drug reactions and drug compliance.

[0004] So far, one of the main challenges facing existing technologies is to find a safe, non-side-effect, and non-dependent treatment method that can relieve aging-related symptoms by regulating intestinal flora, regulating the body's oxidative stress and inflammatory response levels, and relieving common aging-related symptoms such as muscle loss and reduced strength, thereby effectively improving the body's health level and alleviating aging. Intervention using microorganisms is a recognized treatment method with high safety and low dependence, but no microbial species have been found that can effectively relieve the body's aging symptoms. Summary of the invention

[0005] The present invention provides a strain of Parabacteroides merdae CCFM1428, and conducts animal test efficacy verification on the effect of alleviating aging of the strain.

[0006] The first object of the present invention is to provide a strain of Parabacteroides merdae CCFM1428, wherein the Parabacteroides merdae CCFM1428 is deposited in Guangdong Province Microbiological Culture Collection Center with a deposit number of GDMCC No: 65154.

[0007] In one embodiment of the present invention, the Parabacteroides merdae CCFM1428 is isolated from human feces.

[0008] In one embodiment of the present invention, the growth characteristics of the Parabacteroides merdae CCFM1428 are as follows: the strain is an anaerobic bacterium, and after being inoculated into the culture medium, it is cultured at a culture temperature of 37° C. for 24 hours.

[0009] In one embodiment of the present invention, the culture medium is BHI medium.

[0010] In one embodiment of the present invention, the anti-aging effect of CCFM1428 was evaluated based on various biochemical indices of 20 experimental group mice (blank group, model group, spermidine group, and Parabacteroides faecium CCFM1428 group, 5 mice in each group).

[0011] The second object of the present invention is to provide a microbial preparation containing the Parabacteroides faecium CCFM1428.

[0012] In one embodiment of the present invention, the microbial preparation contains one or more of living cells, fermentation broth, lysate or extract of the Parabacteroides faecium CCFM1428.

[0013] The third object of the present invention is to provide a medicine containing the Parabacteroides faecium CCFM1428 or the microbial preparation.

[0014] In one embodiment of the present invention, the amount of Parabacteroides faecium CCFM1428 added to the drug is not less than 2×10 8 CFU / mL or 2×10 8 CFU / g.

[0015] The fourth object of the present invention is to provide a method for preparing the microbial preparation, which is obtained by mixing the cultured Parabacteroides faecium CCFM1428 cells with a protective agent.

[0016] In one embodiment of the present invention, the protective agent includes one or more of skimmed milk powder, glycerol, maltodextrin, trehalose, and sodium L-glutamate.

[0017] The fifth object of the present invention is to provide the use of the Parabacteroides faecium CCFM1428 or the microbial preparation in the preparation of a drug for alleviating aging symptoms.

[0018] In one embodiment of the present invention, the delaying of aging includes reducing the level of oxidative stress, reducing the inflammatory response, reducing the frailty index, and increasing the level of acetylcholine in the brain.

[0019] In one embodiment of the present invention, the amount of Parabacteroides merdae CCFM1428 added to the drug is not less than 2×10 8 CFU / mL or 2×10 8 CFU / g.

[0020] In one embodiment of the present invention, the medicine contains the Parabacteroidesmerdae CCFM1428, a drug carrier and / or a pharmaceutical excipient.

[0021] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0022] In one embodiment of the present invention, the pharmaceutical excipients include one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0023] In one embodiment of the present invention, the dosage form of the drug is granules, capsules, tablets, pills or liquid preparations.

[0024] In one embodiment of the present invention, the drug is used to regulate blood sugar, regulate intestinal flora, regulate oxidative stress response, reduce inflammatory response, reduce frailty index, bring health benefits and thus delay aging.

[0025] In one embodiment of the present invention, the drug is used in at least one of (a) to (i):

[0026] (a) reducing the frailty score index of aging individuals;

[0027] (b) reduce the level of MDA in liver tissue of aging individuals;

[0028] (c) increasing the level of SOD enzyme in liver tissue of aging individuals;

[0029] (d) increasing the level of CAT enzyme in liver tissue of aging individuals;

[0030] (e) increasing the level of GSH-px enzyme in liver tissue of aging individuals;

[0031] (f) increasing the level of acetylcholine in the brain tissue of aging individuals;

[0032] (g) reducing the expression level of pro-inflammatory factors in colon tissue of aging individuals, including IL-6 and TNF-alpha;

[0033] (h) Improving grip strength in aging individuals.

[0034] (i) Increase the abundance of beneficial bacteria in the intestines of aging individuals and improve the structure of the intestinal flora.

[0035] Beneficial Effects

[0036] The present invention relates to a strain of Parabacteroides merdae CCFM1428 that can bring health benefits and thus alleviate the aging of mice. After the Parabacteroides merdae CCFM1428 provided by the present invention is used to act on aging mice, the aging symptoms of the mice can be significantly alleviated, which is specifically embodied in:

[0037] (1) Significantly reduced the frailty index of aging mice by 32.05%.

[0038] (2) Significantly increased the level of superoxide dismutase (SOD) in the liver of aged mice by 13.26%.

[0039] (3) Significantly reduced the level of malondialdehyde (MDA) in the liver of aged mice by 23.15%.

[0040] (4) Significantly increased the level of glutathione peroxidase (GSH-px) in the liver of aged mice by 149.78%

[0041] (5) Significantly increased the level of catalase (CAT) in the liver of aged mice by 58.93%.

[0042] (6) Reduced the IL-6 level in the colon homogenate of aged mice by 28.29%.

[0043] (7) Reduced TNF-alpha levels in colon homogenates of aged mice by 23.83%.

[0044] (8) Increased acetylcholine levels in the brains of aged mice by 183.92%.

[0045] (9) Improved the grip strength of aged mice by 13.46%.

[0046] (10) It increased the abundance of beneficial bacteria in the intestinal flora of aged mice and improved the composition of the intestinal flora.

[0047] Therefore, Parabacteroides merdae CCFM1428 has great application prospects in the preparation of products that alleviate aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Frailty index of experimental mice in different groups;

[0049] Figure 2 : Superoxide dismutase (SOD) levels in the liver of experimental mice in different groups;

[0050] Figure 3 : Malondialdehyde (MDA) levels in the liver of different groups of experimental mice;

[0051] Figure 4 : Levels of glutathione peroxidase (GSH-px) in the liver of experimental mice in different groups;

[0052] Figure 5 :The levels of catalase (CAT) in the liver of different groups of experimental mice;

[0053] Figure 6 :IL-6 levels in colon homogenates of experimental mice in different groups;

[0054] Figure 7 :TNF-alpha levels in the colon of different groups of experimental mice;

[0055] Figure 8 :Acetylcholine levels in the brains of experimental mice in different groups;

[0056] Fig. 9 : The grip strength levels of experimental mice in different groups;

[0057] Fig.10 :Animal experiment flow chart;

[0058] Fig.11 : Stacked diagram of intestinal flora species in different groups of mice.

[0059] Deposit of biological materials:

[0060] A strain of Parabacteroides merdae CCFM1428, taxonomically named Parabacteroides merdae, was deposited in the Guangdong Microbial Culture Collection Center on September 20, 2024, with the deposit number GDMCCNo: 65154. The deposit address is the 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. DETAILED DESCRIPTION

[0061] The C57BL / 6J male mice involved in the following examples were purchased from Beijing Weitonglihua Animal Experiment Technology Co., Ltd., and the D-galactose and spermidine involved in the following test examples were purchased from Aladdin. The BHI culture medium involved in the following examples was purchased from Sinopharm. The Parabacteroides faecium CCFM1428 involved in the following examples was deposited in the Strain Collection Center of the Biotechnology Center of the School of Food Science and Engineering of Jiangnan University.

[0062] The method for detecting the number of viable bacteria involved in the following embodiments is based on the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection".

[0063] The preparation method of the D-galactose solution involved in the following embodiments is as follows:

[0064] D-galactose solution: Weigh a certain amount of D-galactose, dissolve it in sterile saline to prepare D-galactose, and inject subcutaneously at 700 mg / kg body weight and 1000 mg / kg body weight.

[0065] Example 1: Screening and identification of Parabacteroides merdae CCFM1428

[0066] 1. Isolation and identification of Parabacteroides merdae CCFM1428

[0067] The strain was isolated from the feces of healthy adults, gradedly diluted with physiological saline, spread on BHI solid culture medium, and inverted in an anaerobic incubator at 37°C for 48 hours. According to the colony morphology, a single colony was picked and streaked on the BHI culture medium for culture, and inverted in an anaerobic incubator at 37°C for culture for 48 hours. A single colony was picked and inoculated into 5 mL of liquid BHI culture medium, and incubated in an anaerobic incubator at 37°C for 48 hours. 1.5 mL of the bacteria was centrifuged at 6000 r / min for 3 minutes to remove the supernatant, and 1 mL of 30% sterile glycerol was added for storage. At the same time, a 1.5 mL portion of the bacteria was centrifuged, the supernatant was removed, and then resuspended in sterile water for strain identification. The strain was sequenced and analyzed, and the sequence obtained by sequencing was compared with the nucleic acid sequence in Genbank. The result showed that the nucleic acid sequence similarity with that of Parabacteroides merdae was greater than 99%. The strain was named Parabacteroides merdae CCFM1428, and the Parabacteroides merdae CCFM1428 has been preserved in Guangdong Provincial Microbial Culture Collection Center with a preservation number of GDMCC No: 65154.

[0068] 2. Morphology of Parabacteroides merdae CCFM1428

[0069] Parabacteroides merdae CCFM1428 was inoculated into BHI solid medium with L-cysteine ​​hydrochloride, and cultured anaerobically at 37°C for 48 hours. The colonies were observed and the bacteria were observed under a microscope. It was found that the colonies were milky white, smooth in surface, convex in the middle, and neat at the edges.

[0070] 3. Preservation of Parabacteroides merdae CCFM1428

[0071] The strain was cultured in BHI medium, and Parabacteroides faecium was cultured in an anaerobic incubator at 37°C for 48 hours for one generation. Single colonies on the solid medium of the second generation were selected and liquid cultured for the third generation under the same conditions as above. The tube containing the bacterial solution was centrifuged at 4°C and 8000g for 2 minutes, the supernatant was discarded, the precipitate was retained, and 1 mL of sterile 30% glycerol solution was added, and it was stored in a -80°C refrigerator after shaking.

[0072] Parabacteroides merdae CCFM1428 was inoculated into BHI liquid culture medium and cultured at 37°C for 48 h. The bacterial solution was then inoculated into fresh BHI liquid culture medium at an inoculum size of 4% (v / v), and cultured under the same conditions for 36 h. The bacteria were centrifuged at 8000 r / min for 15 min, washed with 0.9% saline and centrifuged again at 8000 r / min for 10 min, the bacteria were collected, resuspended with 30% (v / v) glycerol solution, and resuspensions were prepared respectively, which were frozen at -80°C for use.

[0073] 4. Preparation of Parabacteroides merdae CCFM1428 bacterial suspension

[0074] Preparation of bacterial suspension for intragastric administration: When Parabacteroides merdae CCFM1428 was used for intragastric administration to mice, the prepared resuspension was taken out from -80°C, centrifuged at 4°C, 8000r / min for 10min, the supernatant was discarded, and the bacterial suspension was washed with 0.9% saline and resuspended to obtain a bacterial suspension for intragastric administration. The bacterial concentration was: 2×10 8 CFU / mL.

[0075] Example 2: Effect of Parabacteroides merdae CCFM1428 on the Alleviation of Frailty Index in Aging Mice

[0076] Eight-week-old SPF-grade C57BL / 6J male mice were housed in the Experimental Animal Center of Jiangnan University, fed with ordinary feed, maintained at a constant temperature of 20-26°C, humidity of 40%-70%, noise level less than or equal to 60 dB, animal illumination of 15-20 LX, 12 h light, and 12 h dark (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0077] The experimental design is 20 weeks in total. The specific process is as follows: Fig.10After entering the breeding facility, the mice were first adapted for one week. Galactose (CAS No. 59-23-4, Aladdin) was used to establish an aging model from 2 to 13 weeks. The control group mice were subcutaneously injected with sterile saline, and the other treatment groups were subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile saline. Because mice will develop a certain resistance to D-galactose, 1000 mg / kg BW / d of subcutaneous injection was continued from 14 to 20 weeks. At the same time, each group of mice was treated with the corresponding experiments. The control group and the model group were gavaged with 200 μL of sterile saline every day. The volume of injection liquid was the same for all groups.

[0078] The details are as follows:

[0079] Blank group: After entering the breeding facility, the mice were first adapted for one week. From 2 to 13 weeks, the mice in the blank group were subcutaneously injected with sterile saline; from 14 to 20 weeks, the mice were subcutaneously injected with sterile saline and gavaged with 200 μL of sterile saline daily.

[0080] Model group: After entering the breeding facility, mice were first adapted for one week. From 2 to 13 weeks, the model group was subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile saline; from 14 to 20 weeks, 1000 mg / kg BW / d of D-galactose dissolved in sterile saline was subcutaneously injected, and 200 μL of sterile saline was gavaged daily; food and water were freely available.

[0081] Experimental group: After entering the breeding facility, mice were first adapted for one week. From 2 to 13 weeks, the model group was subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile saline; from 14 to 20 weeks, the mice were subcutaneously injected with 1000 mg / kg BW / d of D-galactose dissolved in sterile saline, and gavage with 200 μL of Parabacteroides merdae CCFM1428 bacterial suspension (bacterial concentration was 2×10 8 CFU / mL); free access to food and drinking water.

[0082] Drug group: After entering the breeding facility, mice were first adapted for one week. From 2 to 13 weeks, the model group was subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile saline; from 14 to 20 weeks, they were subcutaneously injected with 1000 mg / kg BW / d of D-galactose dissolved in sterile saline, and were gavaged with spermidine 0.02 mg / kg body weight every day; they were allowed to eat and drink water freely.

[0083] Before killing the mice, the frailty of each aging mouse was scored, including the assessment of 13 variables (Table 1). The frailty index (FI) was calculated based on the scores of individual variables, and the specific scoring criteria were as follows: for each variable, 0 points represented no defect, 0.5 points represented mild defects, and 1 point represented severe defects. The total score was calculated for evaluation, and the total score ranged from 0 to 13.

[0084] Table 1: Mouse weakness index table

[0085]

[0086] Depend on Figure 1 It can be seen that the average frailty index of the blank group was 2.06, the average frailty index of the model group was 4.68, the average frailty index of the CCFM1428 group was 3.18, and the average frailty index of the spermidine group was 2. Compared with the model group, CCFM1428 reduced the frailty index of mice by 32.05%.

[0087] The above experimental results show that Parabacteroides merdae CCFM1428 can effectively reduce the frailty index of mice and improve the frail state of mice.

[0088] Example 3: Effect of Parabacteroides merdae CCFM1428 on the level of superoxide dismutase (SOD) in the liver of aged mice

[0089] The specific steps refer to Example 2. After the experiment, the mice were killed, the mouse livers were removed and placed in liquid nitrogen, stored in a -80°C refrigerator, and the tissues were rinsed with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood and cut into pieces. Weigh about 1.2g of fresh tissue, add PBS at a weight-to-volume ratio of 1:9, add 1mL of universal protease inhibitor (Biyuntian) to every 100mL of this PBS, add an equal amount of grinding beads to each tissue, use a pre-cooled tissue grinder mold, and grind thoroughly on ice until no solids are completely eliminated. Further freeze-thaw repeatedly to fully lyse. Centrifuge the homogenate at 5000×g for 5-10 minutes, and take the supernatant and divide it into portions for later use. BCA method is used to measure protein concentration. Take 1.2 ml of protein standard solution (0.5 g bovine serum albumin, dissolved in distilled water and fixed to 100 ml, to make a 5 mg / ml solution, diluted ten times when used) and add it to a tube of protein standard (30 mg BSA). After fully dissolving, prepare a 25 mg / ml protein standard solution. Take an appropriate amount of 25 mg / ml protein standard and dilute it to a final concentration of 0.5 mg / ml. According to the number of samples, weigh 10 g BCA (1%), 20 g Na 2 CO 3·H 2 O(2%), 1.6gNa 2 C 4 H 4 O 6 ·2H 2 O (0.16%), 4 g NaOH (0.4%), 9.5 g NaHCO3 (0.95%), add water to 1 L, use NaOH or solid NaHCO 3 Adjust pH to 11.25) and add 1 volume of BCA reagent B (take 2g CuSO 4 ·5H 2 O (4%), add distilled water to 50ml) (50:1) to prepare an appropriate amount of BCA working solution and mix thoroughly. Select an appropriate concentration within the standard concentration of 0-1.5mg / mL to make a standard curve, and obtain the appropriate dilution multiple from the sample pre-experiment. In the formal experiment, add 20μL of sample or standards of different concentrations to each well, add 200μL BCA working solution to each well, and incubate at 37℃ in the dark for 30 minutes. Use an enzyme reader to measure the absorbance at 562nm. Calculate the sample protein concentration.

[0090] The superoxide dismutase (SOD) content in mouse liver homogenate was detected according to the instructions of the liver superoxide dismutase (SOD) ELISA kit. The results are as follows Figure 2 shown.

[0091] The results show:

[0092] Depend on Figure 2 It can be seen that the average value of liver superoxide dismutase (SOD) in the blank group was 27.90ng / mg protein, the average value of liver superoxide dismutase (SOD) in the model group was 16.254ng / mg protein, the average value of liver superoxide dismutase (SOD) in the CCFM1428 group was 24.03ng / mg protein, and the average value of liver superoxide dismutase (SOD) in the spermidine group was 29.51ng / mg protein. Compared with the model group, the liver SOD level of mice in the CCFM1428 group increased by 47.85%.

[0093] The above experimental results show that Parabacteroides merdae CCFM1428 can effectively increase the level of superoxide dismutase (SOD) in mouse liver and improve the weak state of mice.

[0094] Example 4: Effect of Parabacteroides merdae CCFM1428 on reducing the level of malondialdehyde (MDA) in the liver of aged mice

[0095] Specific implementation method Referring to Example 3, after obtaining mouse liver homogenate, the malondialdehyde (MDA) content in the mouse liver homogenate was detected according to the instructions of the liver malondialdehyde (MDA) level ELISA kit. The results are as follows: Figure 3 shown.

[0096] The results show:

[0097] Depend on Figure 3 It can be seen that the average liver malondialdehyde (MDA) value of the blank group was 21.95nmol / mg protein, the average liver malondialdehyde (MDA) value of the model group was 34.71nmol / mg protein, the average liver malondialdehyde (MDA) value of the CCFM1428 group was 26.68nmol / mg protein, and the average liver malondialdehyde (MDA) value of the spermidine drug group was 44.38nmol / mg protein. Compared with the model group, the liver malondialdehyde (MDA) value of mice in the CCFM1428 group decreased by 23.15%, and the improvement effect of Parabacteroides faecium CCFM1428 on MDA was better than that of drugs.

[0098] The above experimental results show that Parabacteroides merdae CCFM1428 can effectively reduce the level of malondialdehyde (MDA) in mouse liver and improve the weak state of mice.

[0099] Example 5: Effect of Parabacteroides merdae CCFM1428 on the level of glutathione peroxidase (GSH-px) in the liver of aged mice

[0100] Specific implementation method Referring to Example 3, after obtaining mouse liver homogenate, the content of glutathione peroxidase (GSH-px) in the mouse liver homogenate was detected according to the instructions of the liver glutathione peroxidase (GSH-px) level ELISA kit. The results are as follows: Figure 4 shown.

[0101] The results show:

[0102] Depend on Figure 4 It can be seen that the average liver glutathione peroxidase (GSH-px) value of the blank group was 180.25U / mg protein, the average liver glutathione peroxidase (GSH-px) value of the model group was 77.07U / mg protein, the average liver glutathione peroxidase (GSH-px) value of the CCFM1428 group was 192.50U / mg protein, and the average liver glutathione peroxidase (GSH-px) value of the spermidine group was 161.38U / mg protein. Compared with the model group, the liver malondialdehyde (MDA) value of mice in the CCFM1428 group increased by 149.78%, reaching a level equivalent to that of the blank group.

[0103] The above experimental results show that Parabacteroides merdae CCFM1428 can effectively increase the level of glutathione peroxidase (GSH-px) in mouse liver and improve the weak state of mice.

[0104] Example 6: Effect of Parabacteroides merdae CCFM1428 on the level of catalase (CAT) in the liver of aged mice

[0105] Specific implementation method Referring to Example 3, after obtaining mouse liver homogenate, the catalase (CAT) content in the mouse liver homogenate was detected according to the instructions of the catalase (CAT) level ELISA kit. The results are as follows: Figure 5 shown.

[0106] The results show:

[0107] Depend on Figure 5 It can be seen that the average liver catalase (CAT) value of the blank group was 539.19pg / mg protein, the average liver catalase (CAT) value of the model group was 258.53pg / mg protein, the average liver catalase (CAT) value of the CCFM1428 group was 410.89pg / mg protein, and the average liver catalase (CAT) value of the spermidine drug group was 575.71pg / mg protein. Compared with the model group, the liver malondialdehyde (MDA) value of mice in the CCFM1428 group increased by 58.93%.

[0108] The above experimental results show that Parabacteroides merdae CCFM1428 can effectively increase the level of catalase (CAT) in mouse liver and improve the weak state of mice.

[0109] Example 7: Effect of Parabacteroides merdae CCFM1428 on the reduction of IL-6 levels in colon homogenates of aged mice

[0110] Specific implementation method Referring to Example 2, after killing the mouse, the mouse colon was removed and placed in liquid nitrogen, stored in a -80°C refrigerator, and the tissue was rinsed with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood and cut into pieces. Weigh about 1.2g of fresh weight tissue, add PBS at a weight-to-volume ratio of 1:9, add 1mL of universal protease inhibitor (Biyuntian) to every 100mL of this PBS, add an equal amount of grinding beads to each tissue, use a pre-cooled tissue grinder mold, and grind thoroughly on ice until no solids are completely gone. Further freeze and thaw repeatedly to fully lyse. Centrifuge the homogenate at 5000×g for 5-10 minutes, and take the supernatant and divide it into portions for later use. BCA method is used to measure protein concentration. Take 1.2 ml of protein standard solution (0.5 g bovine serum albumin, dissolved in distilled water and fixed to 100 ml, to make a 5 mg / ml solution, dilute ten times when used) and add it to a tube of protein standard (30 mg BSA). After fully dissolving, prepare a 25 mg / ml protein standard solution. Take an appropriate amount of 25 mg / ml protein standard and dilute it to a final concentration of 0.5 mg / ml. According to the number of samples, weigh 10 g BCA (1%), 20 g Na 2 CO 3 ·H 2 O(2%), 1.6gNa 2 C 4 H 4 O 6 ·2H 2 O (0.16%), 4 g NaOH (0.4%), 9.5 g NaHCO3 (0.95%), add water to 1 L, use NaOH or solid NaHCO 3 Adjust pH to 11.25) and add 1 volume of BCA reagent B (take 2g CuSO 4 ·5H 2 O (4%), add distilled water to 50ml) (50:1) to prepare an appropriate amount of BCA working solution and mix thoroughly. Select an appropriate concentration within the standard concentration of 0-1.5mg / mL to make a standard curve, and obtain the appropriate dilution multiple from the sample pre-experiment. In the formal experiment, add 20μL of sample or standards of different concentrations to each well, and add 200μL BCA working solution every well, incubate at 37℃ in the dark for 30 minutes. Use an enzyme reader to measure the absorbance at 562nm. Calculate the sample protein concentration.

[0111] The IL-6 content in the mouse colon homogenate was detected according to the instructions of the IL-6 ELISA kit. Figure 6 shown.

[0112] The results show:

[0113] Depend on Figure 6It can be seen that the average IL-6 in the colon homogenate of the blank group was 306.95pg / mg protein, the average IL-6 in the colon homogenate of the model group was 366.24pg / mg protein, the average IL-6 in the colon homogenate of the CCFM1428 group was 262.63pg / mg protein, and the average IL-6 in the colon homogenate of the spermidine drug group was 312.41pg / mg protein. Compared with the model group, the IL-6 value in the colon homogenate of the mice in the CCFM1428 group decreased by 28.19%.

[0114] The above experimental results show that Parabacteroides merdae CCFM1428 can effectively reduce IL-6 in the colon homogenate of mice and improve the weak state of mice.

[0115] Example 8: Effect of Parabacteroides merdae CCFM1428 on reducing TNF-alpha levels in colon homogenates of aged mice

[0116] Specific implementation method Referring to Example 7, after obtaining the mouse colon homogenate, the TNF-alpha content in the mouse colon homogenate was detected according to the instructions of the TNF-alpha ELISA kit. The results are as follows: Figure 7 shown.

[0117] The results show:

[0118] Depend on Figure 7 It can be seen that the average TNF-alpha in the colon homogenate of the blank group was 281.00pg / mg protein, the average TNF-alpha in the colon homogenate of the model group was 324.56pg / mg protein, the average TNF-alpha in the colon homogenate of the CCFM1428 group was 247.22pg / mg protein, and the average TNF-alpha in the colon homogenate of the spermidine group was 267.28pg / mg protein. Compared with the model group, the TNF-alpha value in the colon homogenate of the mice in the CCFM1428 group decreased by 23.83%.

[0119] The above experimental results show that Parabacteroides merdae CCFM1428 can effectively reduce TNF-alpha in mouse colon homogenate and improve the weak state of mice.

[0120] Example 9: Effect of Parabacteroides merdae CCFM1428 on the elevation of acetylcholine levels in brain homogenates of aged mice

[0121] Specific implementation method Referring to Example 2, after killing the mouse, remove the mouse brain and place it in liquid nitrogen, store it in a -80°C refrigerator, rinse the tissue with pre-cooled PBS (0.01M, pH=7.4), remove residual blood, and cut it into pieces. Weigh about 1.2g of fresh weight tissue, add PBS at a weight-to-volume ratio of 1:9, add 1mL of universal protease inhibitor (Biyuntian) to every 100mL of this PBS, add an equal amount of grinding beads to each tissue, use a pre-cooled tissue grinder mold, and grind it thoroughly on ice until no solid matter is completely gone. Further freeze and thaw repeatedly to fully lyse. Centrifuge the homogenate at 5000×g for 5-10 minutes, and take the supernatant and divide it into portions for later use. BCA method is used to measure protein concentration. Take 1.2 ml of protein standard solution (0.5 g bovine serum albumin, dissolved in distilled water and fixed to 100 ml, to make a 5 mg / ml solution, dilute ten times when used) and add it to a tube of protein standard (30 mg BSA). After fully dissolving, prepare a 25 mg / ml protein standard solution. Take an appropriate amount of 25 mg / ml protein standard and dilute it to a final concentration of 0.5 mg / ml. According to the number of samples, weigh 10 g BCA (1%), 20 g Na 2 CO 3 ·H 2 O(2%), 1.6gNa 2 C 4 H 4 O 6 ·2H 2 O (0.16%), 4g NaOH (0.4%), 9.5gNaHCO 3 (0.95%), add water to 1L, and use NaOH or solid NaHCO 3 Adjust pH to 11.25) and add 1 volume of BCA reagent B (take 2g CuSO 4 ·5H 2 O (4%), add distilled water to 50ml) (50:1) to prepare an appropriate amount of BCA working solution and mix thoroughly. Select an appropriate concentration within the standard concentration of 0-1.5mg / mL to make a standard curve, and obtain the appropriate dilution multiple from the sample pre-experiment. In the formal experiment, add 20μL of sample or standards of different concentrations to each well, add 200μL BCA working solution to each well, and incubate at 37℃ in the dark for 30 minutes. Use an enzyme reader to measure the absorbance at 562nm. Calculate the sample protein concentration.

[0122] According to the instructions of the acetylcholine kit, the acetylcholine content in the mouse brain homogenate was detected. Figure 8 shown.

[0123] The results show:

[0124] Depend on Figure 8It can be seen that the average acetylcholine in the brain homogenate of the blank group was 842.99μg / mg protein, the average acetylcholine in the brain homogenate of the model group was 440.88μg / mg protein, the average acetylcholine in the brain homogenate of the CCFM1428 group was 1251.77μg / mg protein, and the average acetylcholine in the brain homogenate of the spermidine group was 401.33μg / mg protein. Compared with the model group, the acetylcholine value of the brain homogenate of the mice in the CCFM1428 group increased by 183.92%.

[0125] The above experimental results show that Parabacteroides merdae CCFM1428 can effectively increase the level of acetylcholine in mouse brain homogenate and improve the weak state of mice.

[0126] Example 10: Effect of Parabacteroides merdae CCFM1428 on the grip strength of aged mice

[0127] Specific implementation method Referring to Example 2, before killing the mouse, the mouse was placed flat on the T-frame of the grip strength meter (Jinan Yiyan Technology Co., Ltd.), the mouse tail was grasped and slowly pulled backwards, and the data was read. The results were as follows: Fig. 9 shown.

[0128] The results show:

[0129] Depend on Fig. 9 It can be seen that the average grasping force of mice in the blank group was 101.26g, the average grasping force of mice in the model group was 80.54g, the average grasping force of mice in the Parabacteroides faecium CCFM1428 group was 91.38g, and the average grasping force of mice in the spermidine drug group was 68.27g. Therefore, Parabacteroides faecium CCFM1428 has a better effect on improving the grasping force of mice than drugs, and can restore the grasping force to a level comparable to that of the control group.

[0130] The above experimental results show that Parabacteroides merdae CCFM1428 can effectively increase the grip level of mice and improve the weak state of mice.

[0131] Example 11: Parabacteroides merdae CCFM1428 increases the abundance of beneficial bacteria in the intestinal tract of mice and improves the composition of the intestinal flora of mice

[0132] Specific implementation method Referring to Example 2, before killing the mice, the mouse feces were collected, and the total DNA from the feces was extracted using the QIAamp DNAStoolMini Kit (Qiagen, Hilden, Germany), and the purity and integrity of the DNA was detected by 1% agarose gel electrophoresis; metagenomic sequencing was performed on the DNBSEQ-T7 platform of Beijing Novogene Co., Ltd. The original sequencing reads were used to remove sequencing adapters and low-quality sequences using Trimmomatic (version 0.39). The filtered sequences were aligned with the human reference genome using BWA (version 0.7.17), Samtools (version 1.9), and BEDTools (version 2.30.0) to remove host sequences. MetaPhlAn (version 4.0.6) was used to annotate the intestinal flora species of the sequences that had undergone quality control and host removal operations to obtain information on the abundance of mouse intestinal species. The stacked graph was drawn using the mean of each group. The results are as follows Fig.11 shown.

[0133] The results show:

[0134] Depend on Fig.11 It can be seen that compared with the model group and the blank group mice, Parabacteroides merdae CCFM1428 significantly increased the abundance of Bifidobacterium pseudolongum in the mouse intestine, which is a beneficial bacteria that can improve immunity.

[0135] The above experimental results show that Parabacteroides merdae CCFM1428 can effectively increase the abundance of beneficial bacteria in the intestinal flora of mice and improve the composition of the intestinal flora of mice.

[0136] Example 12: Preparation of Parabacteroides merdae CCFM1428 microbial preparation

[0137] Preparation of liquid microbial preparation: Parabacteroides merdae CCFM1428 was cultured under anaerobic conditions for 24 h, the culture medium was removed by centrifugation, and the Parabacteroides merdae CCFM1428 cells were resuspended in sterile saline to ensure that the content of Parabacteroides merdae CCFM1428 was not less than 2×10 8 CFU / mL, prepare bacterial suspension. If necessary, one or more of skimmed milk powder, glycerol, maltodextrin, trehalose, and sodium L-glutamate can be added to the bacterial suspension as a protective agent.

[0138] Preparation of solid microbial preparation: The cultured Parabacteroides faecium CCFM1428 cells are mixed with a protective agent and then freeze-dried and sprayed to obtain a solid microbial preparation. The protective agent includes one or more of skimmed milk powder, glycerol, maltodextrin, trehalose, and sodium L-glutamate. The number of Parabacteroides faecium CCFM1428 in the obtained microbial preparation is not less than 2×10 8 CFU / g.

[0139] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Parabacteroides merdae CCFM1428, characterized in that: The faecal bacteria CCFM1428 was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 20, 2024, with the collection number GDMCCNo: 65154.

2. A microbial preparation containing the Parabacteroides faecium CCFM1428 according to claim 1, characterized in that: The microbial preparation contains one or more of the living cells, fermentation broth, lysate or extract of the Parabacteroides faecium CCFM1428.

3. A medicine containing the Parabacteroides faecium CCFM1428 according to claim 1, or a medicine containing the microbial preparation according to claim 2.

4. The drug according to claim 3, characterized in that In the drug, the amount of Parabacteroides faecium CCFM1428 added is not less than 2×10 8 CFU / mL or 2×10 8 CFU / g.

5. A method for preparing the microbial preparation according to claim 2, characterized in that: The cultured Parabacteroides faecium CCFM1428 cells are mixed with a protective agent to obtain the product.

6. The method according to claim 5, characterized in that The protective agent includes one or more of skimmed milk powder, glycerol, maltodextrin, trehalose, and sodium L-glutamate.

7. Use of the Parabacteroides faecium CCFM1428 according to claim 1 or the microbial preparation according to claim 2 in the preparation of a drug for alleviating aging symptoms.

8. The use according to claim 7, characterized in that: The medicine contains the Parabacteroides merdae CCFM1428, a drug carrier and / or a pharmaceutical excipient.

9. The use according to claim 8, characterized in that: The drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; the pharmaceutical excipients comprise one or more of fillers, binders, wetting agents, disintegrants, lubricants and flavoring agents.

10. The use according to any one of claims 7 to 9, characterized in that: The drug is used for at least one of (a) to (i): (a) reduce the frailty score index of aging individuals; (b) reduce the level of MDA in liver tissue of aging individuals; (c) increasing the level of SOD enzyme in liver tissue of aging individuals; (d) increasing the level of CAT enzyme in liver tissue of aging individuals; (e) increasing the level of GSH-px enzyme in liver tissue of aging individuals; (f) increasing the level of acetylcholine in the brain tissue of aging individuals; (g) reducing the expression level of pro-inflammatory factors in colon tissue of aging individuals; the pro-inflammatory factors include IL-6 and TNF-alpha; (h) improving grip strength in aging individuals; (i) Increase the abundance of beneficial bacteria in the intestines of aging individuals and improve the structure of the intestinal flora.

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