Application of bifidobacterium longum subsp. Longum MNH-19749 in preparation of products for preventing and treating nervous system diseases

Abstract

The invention provides application of bifidobacterium longum subsp. Longum MNH-19749 in preparation of products for preventing and treating nervous system diseases, and relates to the technical field of biological medicines. The bifidobacterium longum subsp. Longum MNH-19749 is obtained through long-term strain screening research, and the bifidobacterium longum subsp. Longum MNH-19749 has a better relieving effect on mania, epilepsy, Parkinson's disease and other nervous system diseases. Experiments prove that the inactivated bacteria of the bifidobacterium longum subsp. Longum MNH-19749 can effectively relieve dysfunction of biological models of nervous system diseases such as mania, epilepsy and Parkinson's disease, so that a new basis is provided for immunotherapy of patients with the nervous system diseases, and the bifidobacterium longum subsp. Longum MNH-19749 has good research significance and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of Bifidobacterium longum subspecies longum MNH-19749 in the preparation of products for preventing and treating nervous system diseases. Background Art

[0002] The Gut-Brain Axis refers to the interaction between the intestines and the brain, involving complex interactions between the intestines and the nervous system. The "Gut-Brain Axis" is a two-way signal communication network that is carried out through biochemical signal exchanges between the central nervous system and the gastrointestinal tract. The microorganisms and their metabolites in the Gut-Brain Axis transmit neurotransmitters such as serotonin and dopamine to the brain through channels such as the vagus nerve, and participate in regulating hormone levels. It is an interaction and mutual influence between the brain and the intestine.

[0003] On the one hand, sensory vagus nerve afferent endings distributed in intestinal epithelial cells can sense intestinal microorganisms and transmit signals to the brain; on the other hand, bacterial metabolites, such as short-chain fatty acids, tryptophan, cytokines, neuroactive substances, etc., are released into the blood. Once in the blood, the microbiome and its factors can change peripheral immune cells, promote interactions with the blood-brain barrier (BBB), change the integrity of the BBB, and cross the BBB to affect brain function. Therefore, the intestinal flora has now become a key regulator of immune cells in the gut-brain axis. Deciphering the inflammatory signaling mechanism based on the gut-brain axis is not only crucial for our understanding of neural and immune communication, but also will promote the advancement of immunotherapy for gastrointestinal and neurological diseases.

[0004] Therefore, screening strains that can link gastrointestinal and nervous system diseases through the "gut-brain axis" and rationally preparing products such as probiotic preparations to provide a basis for immunotherapy of neurological diseases has become another breakthrough point in disease treatment today.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an application of Bifidobacterium longum subsp. longum MNH-19749 in the preparation of a product for preventing and treating nervous system diseases. The present invention has been verified by experiments that the inactivated bacteria of Bifidobacterium longum subsp. longum MNH-19749 can effectively alleviate the functional disorders of biological models of nervous system diseases such as mania, epilepsy and Parkinson's disease, thereby providing a new basis for the immunotherapy of patients with the above-mentioned nervous system diseases.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] According to one aspect of the present invention, the present invention provides the use of Bifidobacterium longum subspecies long strain MNH-19749 in the preparation of products for preventing and treating nervous system diseases;

[0009] The deposit number of the Bifidobacterium longum subspecies longum MNH-19749 is GDMCC NO.61113.

[0010] It should be noted that the Bifidobacterium longum subspecies long strain MNH-19749 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 31, 2020, with the deposit number GDMCC NO.61113, the deposit date was July 31, 2020, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0011] The present invention has obtained in the long-term screening research of strains related to the "gut-brain axis" effect that Bifidobacterium longum subspecies longum MNH-19749 has a better alleviating effect on neurological diseases such as mania, epilepsy, and Parkinson's disease. Experimental verification shows that the inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749 can effectively alleviate the dysfunction of biological models of neurological diseases such as mania, epilepsy, and Parkinson's disease through oral administration, thereby providing a new basis for the immunotherapy of patients with the above-mentioned neurological diseases, and has great research significance and application prospects.

[0012] In a preferred embodiment of the present invention, the product for preventing and treating nervous system diseases contains inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749.

[0013] In a preferred embodiment of the present invention, the nervous system diseases include: mania, epilepsy, and Parkinson's disease.

[0014] In a preferred embodiment of the present invention, the product for preventing and treating nervous system diseases is any one of a probiotic, a medicine, a food or a health product.

[0015] As a preferred embodiment, the concentration of inactivated Bifidobacterium longum subspecies longum MNH-19749 in the above-mentioned product for preventing and treating nervous system diseases is 10 5 CFU / mL~10 6 CFU / mL.

[0016] According to one aspect of the present invention, the present invention provides a probiotic agent, wherein the probiotic agent contains inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749;

[0017] Preferably, the concentration of inactivated Bifidobacterium longum subspecies longum MNH-19749 in the probiotic is 10 5CFU / mL~10 6 CFU / mL;

[0018] Preferably, the probiotics further include carriers and excipients acceptable in probiotics.

[0019] As a preferred embodiment, the probiotic is in the form of an inactivated bacterium, and the unit dose of the inactivated bacterium is 10 5 CFU / mL~10 6 CFU / mL.

[0020] According to one aspect of the present invention, the present invention provides a medicine, wherein the medicine contains inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749;

[0021] In a preferred embodiment of the present invention, the concentration of inactivated Bifidobacterium longum subspecies longum MNH-19749 in the drug is 10 5 CFU / mL~10 6 CFU / mL;

[0022] Preferably, the drug further comprises pharmaceutically acceptable carriers and excipients.

[0023] As a preferred embodiment, the dosage form of the drug is an inactivated bactericidal agent, and the unit dose of the inactivated bactericidal agent is 10 5 CFU / mL~10 6 CFU / mL.

[0024] According to one aspect of the present invention, the present invention provides a food, wherein the food contains inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749;

[0025] In a preferred embodiment of the present invention, the concentration of inactivated Bifidobacterium longum subspecies longum MNH-19749 in the food is 10 5 CFU / mL~10 6 CFU / mL;

[0026] Preferably, the food further comprises carriers and auxiliary materials acceptable in food.

[0027] As a preferred embodiment, the dosage form of the food is an inactivated bacterium agent, and the unit dose of the inactivated bacterium agent is 10 5 CFU / mL~10 6 CFU / mL.

[0028] According to one aspect of the present invention, the present invention provides a health product, wherein the health product contains inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749;

[0029] In a preferred embodiment of the present invention, the concentration of inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749 in the health product is 10 5 CFU / mL~10 6 CFU / mL;

[0030] Preferably, the health care product further comprises carriers and excipients acceptable in health care products.

[0031] As a preferred embodiment, the dosage form of the health product is an inactivated bacterium agent, and the unit dose of the inactivated bacterium agent is 10 5 CFU / mL~10 6 CFU / mL.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention has obtained in the long-term screening research of strains related to the "gut-brain axis" effect that Bifidobacterium longum subspecies longum MNH-19749 has a better alleviating effect on neurological diseases such as mania, epilepsy, and Parkinson's disease. Experimental verification shows that the inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749 can effectively alleviate the dysfunction of biological models of neurological diseases such as mania, epilepsy, and Parkinson's disease through oral administration, thereby providing a new basis for the immunotherapy of patients with the above-mentioned neurological diseases, and has great research significance and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 This is a comparison diagram of the effects of the model control group, MNH19749 inactivated group A, and MNH19749 inactivated group B of the zebrafish mania model provided in Example 2 of the present invention;

[0036] Figure 2 This is a comparison diagram of the effects of the model control group, MNH19749 inactivated group A, and MNH19749 inactivated group B of the zebrafish epilepsy model provided in Example 3 of the present invention;

[0037] Figure 3 This is a comparison diagram of the effects of the model control group, MNH19749 inactivated group A, and MNH19749 inactivated group B of the zebrafish Parkinson's disease model provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0038] The present invention provides an application of Bifidobacterium longum subspecies longum MNH-19749 in preparing a product for preventing and treating nervous system diseases;

[0039] The Bifidobacterium longum subspecies longum MNH-19749 of the present invention is a Bifidobacterium longum isolated from the microorganisms of the intestines of healthy people, and its preservation number is GDMCC NO. 61113. It has been fully disclosed in Chinese patent CN116059256A.

[0040] The present invention has no particular limitation on the inactivation method of Bifidobacterium longum subspecies longum MNH-19749, and a conventional inactivation method in the art may be used.

[0041] In the application of the present invention, when the product is a medicine or a probiotic, the medicine preferably also includes a pharmaceutically acceptable excipient. The present invention does not specifically limit the source and type of the excipient, and conventional pharmaceutical excipients in the art can be used. When the product is a food or a health product, the food preferably also includes food-acceptable raw materials and excipients. The present invention does not specifically limit the source and type of the raw materials and excipients, and conventional raw materials and excipients in the food field can be used.

[0042] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0044] Example 1 Preparation of inactivated strain MNH-19749

[0045] (I) Take out 2 mL of MNH-19749 bacterial liquid from a -80°C refrigerator, thaw it, and inoculate it on 80 mL of YM medium at a volume ratio of 5%. Incubate it anaerobically at 37°C for 8 hours to obtain first-class seeds; take 2 mL of first-class seeds and inoculate them on another bottle of 80 mL of YM medium at a volume ratio of 5%. Incubate it anaerobically at 37°C for 16 hours to obtain second-class seeds; take 2 mL of second-class seeds and inoculate them on another bottle of 80 mL of YM medium at a volume ratio of 5%. Incubate it anaerobically at 37°C for 8 hours to obtain third-class seeds.

[0046] (II) Preparation of fermentation medium: Component 1 is composed of the following in terms of concentration (g / L): Rice peptone 15 g / L, Soypeptone 15 g / L, Yeast extract 20 g / L, Glucose·H2O 60 g / L, K2HPO4 2 g / L, Tween 80 1 g / L, Cysteine-HCl·H2O 1 g / L.

[0047] The components of component 2 according to mass (g) are: CaCl2·2H2O 0.25g, MgSO4·7H2O 0.50g, K2HPO4 1.00g, KH2PO4 1.00g, NaHCO3 10.00g, NaCl 2.00g, ddH2O 1000.00g.

[0048] The components of component 3 by volume (mL) are Vitamin K1 0.1 mL, 95% ethanol 20 mL.

[0049] Take 4 L of component 1, 40 mL of component 2, and 0.2 mL of component 3, mix well, adjust the pH to 7.2 with 5 M HCl and 5 M NaOH, and sterilize at 121 °C for 20 min.

[0050] (III) Fermentation centrifugation: The culture solution was transferred to the fermentation medium and cultured in a 5L biochemical reactor for 16 hours at a speed of 100 rpm and a temperature of 37°C. The pH was controlled with 5M NaOH to be not less than 6.5. The speed was adjusted to 300 rpm, the temperature was adjusted to 42°C, the pH was controlled to be not less than 6.0, and the reaction was continued for 8 hours. The culture solution was concentrated by centrifugation and the bacterial cells were collected.

[0051] (IV) Inactivation: Store the bacterial solution at -20°C and dilute to 10% using culture medium. 5 CFU / mL~10 6 CFU / mL and then mixed evenly with glycerol at a mass ratio of 10:1 and stored at -20°C to obtain the inactivated strain MNH-19749.

[0052] The following Examples 2 to 4 use the dosage form of an inactivated bacterium to verify the effect of MNH19749 inactivated bacteria in relieving mania, epilepsy, and Parkinson's disease. The unit dose of the inactivated bacterium is preferably 10 5 CFU / mL~10 6 CFU / mL.

[0053] Example 2 MNH19749 is used to relieve mania

[0054] (I) Select normal developing zebrafish at 48 hpf (hours-post fertilization) and place them in a 6-well cell culture plate, with 15 zebrafish in each well.

[0055] (II) The zebrafish were divided into 5 groups. The model control group, MNH19749 inactivated groups A to C, and MNH19749 live bacteria group were all ingested with the same amount of ISO for modeling (isoproterenol was ingested into the zebrafish by dissolving it in the fish farming water). After taking the test substance for 2 days, the behavior analyzer was used to track and record the movement of the zebrafish and monitor the frequency and angle of the tail swing to confirm that the modeling was successful.

[0056] The above successfully established zebrafish mania model was used for experiments, specifically:

[0057] The model control group was added with 4 mL of zebrafish embryo culture water;

[0058] MNH19749 inactivation group A (referred to as MNH19749-5), add 4mL of 10 5 Zebrafish embryo culture water containing inactivated MNH19749 at 10 CFU / mL.

[0059] MNH19749 inactivation group B (referred to as MNH19749-6), add 4mL of 10 6 Zebrafish embryo culture water containing inactivated MNH19749 at 10 CFU / mL.

[0060] MNH19749 inactivation group C (referred to as MNH19749-3), add 4mL of 10 3 Zebrafish embryo culture water containing inactivated MNH19749 at 10 CFU / mL.

[0061] MNH19749 live bacteria group, add 4mL of 10 6 Zebrafish embryo culture water containing live bacterial solution of MNH19749 at CFU / mL.

[0062] The 6-well cell culture plate of the above test was placed in a biochemical incubator at 28°C for 72 hours, and the newly prepared solution was renewed every 24 hours. After 72 hours, 15 zebrafish larvae were randomly selected from each group and observed and photographed under a fluorescence microscope. The frequency and angle of the zebrafish tail swing were counted. The specific results are shown in Table 1.

[0063] Table 1:

[0064]

[0065] Figure 1This is a comparison chart of the effects of the model control group, MNH19749 inactivated group A, and MNH19749 inactivated group B of the zebrafish mania model provided in this example.

[0066] As can be seen from the above, the model control group zebrafish in this embodiment is significantly different from the MNH19749 inactivated group A (p<0.001), and significantly different from the MNH19749 inactivated group B (p<0.001), while the difference between the MNH19749 inactivated group C and the live bacteria group is not significant. Therefore, the above results show that the MNH19749 inactivated bacteria can significantly reduce the frequency and angle of the tail swing of the ISO-induced zebrafish mania model, and has the effect of alleviating mania, while the inactivated group C and the live bacteria group do not have a significant effect.

[0067] Example 3 MNH19749 is used to relieve epilepsy

[0068] Epilepsy is a chronic brain disease caused by multiple etiologies, characterized by repetitive, paroxysmal, and transient central nervous system dysfunction caused by excessive neuronal discharge. The blood-brain barrier of zebrafish larvae has not yet formed, and the nervous system is highly sensitive to drugs. Combined with the availability of complete behavioral tests and the adaptability of automated behavioral testing equipment hardware and software, zebrafish has become a very important animal model for epilepsy drug screening.

[0069] PTZ (pentylenetetrazole) is a γ-aminobutyric acid transaminase inhibitor that can resist the inhibitory effect of γ-aminobutyric acid on neural activity. It is often used to induce acute attacks of epilepsy-like reactions such as hyperactivity and convulsions in zebrafish larvae and adult fish.

[0070] (I) Select normal developing zebrafish at 48 hpf (hours-post fertilization) and place them in a 6-well cell culture plate, with 15 zebrafish in each well.

[0071] (II) The zebrafish were divided into 5 groups. The model control group, MNH19749 inactivated groups A to C, and MNH19749 live bacteria group were all ingested with the same amount of PTZ for modeling (PTZ was dissolved in the water used for fish farming and ingested into the zebrafish). After taking the test substance for 2 days, the behavior analyzer was used to track and record the movement of the zebrafish and monitor the frequency and angle of the tail swing to confirm that the modeling was successful.

[0072] The above successfully established zebrafish epilepsy model was used for experiments, specifically:

[0073] The model control group was added with 4 mL of zebrafish embryo culture water;

[0074] MNH19749 inactivation group A (referred to as MNH19749-5), add 4mL of 10 5Zebrafish embryo culture water containing inactivated MNH19749 at 10 CFU / mL.

[0075] MNH19749 inactivation group B (referred to as MNH19749-6), add 4mL of 10 6 Zebrafish embryo culture water containing inactivated MNH19749 at 10 CFU / mL.

[0076] MNH19749 inactivation group C (referred to as MNH19749-3), add 4mL of 10 3 Zebrafish embryo culture water containing inactivated MNH19749 at 10 CFU / mL.

[0077] MNH19749 live bacteria group, add 4mL of 10 6 Zebrafish embryo culture water containing live bacterial solution of MNH19749 at CFU / mL.

[0078] The 6-well cell culture plate of the above test was placed in a biochemical incubator at 28°C for 72 hours, and the newly prepared solution was renewed every 24 hours. After 72 hours, 15 zebrafish fry were randomly selected from each group and observed and photographed under a fluorescence microscope. The frequency and angle of the zebrafish tail swing were counted. The specific results are shown in Table 2.

[0079] Table 2:

[0080]

[0081] Figure 2 This is a comparison chart of the effects of the model control group, MNH19749 inactivated group A, and MNH19749 inactivated group B of the zebrafish epilepsy model provided in this example.

[0082] As can be seen from the above, the model control group zebrafish in this embodiment is significantly different from the MNH19749 inactivated group A (p<0.001), and significantly different from the MNH19749 inactivated group B (p<0.001), while the MNH19749 inactivated group C and the live bacteria group are not significantly different. Therefore, the above results show that the MNH19749 inactivated bacteria can significantly reduce the frequency and angle of the tail swing of the PTZ-induced zebrafish epilepsy model, and has the effect of alleviating epilepsy, while the inactivated group C and the live bacteria group do not have a significant effect.

[0083] Example 4 MNH19749 is used to alleviate Parkinson's disease

[0084] Parkinson's disease is a common neurodegenerative disease in middle-aged and elderly people. MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine, MPTP) is now commonly used to replicate the zebrafish model of Parkinson's disease. It will affect the dopamine nervous system of the zebrafish model to varying degrees and cause the main symptoms of Parkinson's disease.

[0085] (I) Select normal developing zebrafish at 48 hpf (hours-post fertilization) and place them in a 6-well cell culture plate, with 15 zebrafish in each well.

[0086] (II) The zebrafish were divided into 5 groups. The model control group, MNH19749 inactivated groups A to C, and MNH19749 live bacteria group were all ingested with the same amount of MPTP for modeling (MPTP was dissolved in the water used for fish farming and ingested into the zebrafish). After taking the test substance for 2 days, the behavior analyzer was used to track and record the movement of the zebrafish and monitor the frequency and angle of the tail swing to confirm that the modeling was successful.

[0087] The above successfully established zebrafish Parkinson's disease model was used for experiments, specifically:

[0088] The model control group was added with 4 mL of zebrafish embryo culture water;

[0089] MNH19749 inactivation group A (referred to as MNH19749-5), add 4mL of 10 5 Zebrafish embryo culture water containing inactivated MNH19749 at 10 CFU / mL.

[0090] MNH19749 inactivation group B (referred to as MNH19749-6), add 4mL of 10 6 Zebrafish embryo culture water containing inactivated MNH19749 at 10 CFU / mL.

[0091] MNH19749 inactivation group C (referred to as MNH19749-3), add 4mL of 10 3 Zebrafish embryo culture water containing inactivated MNH19749 at 10 CFU / mL.

[0092] MNH19749 live bacteria group, add 4mL of 10 6 Zebrafish embryo culture water containing live bacterial solution of MNH19749 at CFU / mL.

[0093] The 6-well cell culture plate of the above test was placed in a biochemical incubator at 28°C for 72 hours, and the newly prepared solution was renewed every 24 hours. After 72 hours, 15 zebrafish fry were randomly selected from each group and observed and photographed under a fluorescence microscope, and the frequency and angle of the zebrafish tail swing were counted. The specific results are shown in Table 3.

[0094] Table 3:

[0095]

[0096] Figure 3 This is a comparison chart of the effects of the model control group, MNH19749 inactivation group A, and MNH19749 inactivation group B of the zebrafish Parkinson's disease model provided in this example.

[0097] As can be seen from the above, the model control group zebrafish in this embodiment has no significant difference compared with the MNH19749 inactivated groups A, C and the live bacteria group (p>0.001), and the difference is significant compared with the MNH19749 inactivated group B (p<0.001). Therefore, the above results show that the MNH19749 inactivated bacteria group B can significantly reduce the frequency and angle of the tail swing of the MPTP-induced zebrafish Parkinson's disease model, and has the effect of alleviating Parkinson's disease, which cannot be achieved by the other unit dose test groups.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Bifidobacterium longum strain MNH-19749 in the preparation of products for the prevention and treatment of nervous system diseases; The deposit number of the Bifidobacterium longum subspecies longum MNH-19749 is GDMCC NO.61113.

2. The use according to claim 1, characterized in that: The product for preventing and treating nervous system diseases contains inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749.

3. The use according to claim 1, characterized in that: The nervous system diseases include: mania, epilepsy, and Parkinson's disease.

4. The use according to claim 1, characterized in that: The product for preventing and treating nervous system diseases is any one of a probiotic, a medicine, a food or a health product.

5. A probiotic, characterized in that: The probiotic contains inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749.

6. The probiotic according to claim 5, characterized in that The concentration of inactivated Bifidobacterium longum subspecies longum MNH-19749 in the probiotic is 10 5 CFU / mL~10 6 CFU / mL; Preferably, the probiotics further include carriers and excipients acceptable in probiotics.

7. A drug, characterized in that The medicine contains inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749; Preferably, the concentration of inactivated Bifidobacterium longum subspecies longum MNH-19749 in the drug is 10 5 CFU / mL~10 6 CFU / mL; Preferably, the drug further comprises pharmaceutically acceptable carriers and excipients.

8. A food, characterized in that The food contains inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749; Preferably, the concentration of inactivated Bifidobacterium longum subspecies longum MNH-19749 in the food is 10 5 CFU / mL~10 6 CFU / mL; Preferably, the food further comprises carriers and auxiliary materials acceptable in food.

9. A health product, characterized in that: The health product contains inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749; Preferably, the concentration of inactivated bacteria of Bifidobacterium longum subspecies longum MNH-19749 in the health product is 105 CFU / mL to 106 CFU / mL; Preferably, the health care product further comprises carriers and excipients acceptable in health care products.

Images