CN114181868A

CN114181868A - A kind of Bifidobacterium adolescentis and its cultivation method and its application in promoting the growth of neurons in the dorsal root ganglion

Info

Publication number: CN114181868A
Application number: CN202111653648.5A
Authority: CN
Inventors: 刘庆军; 沈鹤霄; 李国龙; 张帆; 刘慧敏; 周先锋
Original assignee: Maintain Biomedical Wuhan Co ltd
Current assignee: Maintain Biomedical Wuhan Co ltd
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-03-15

Abstract

The invention discloses bifidobacterium adolescentis, a culture method thereof and application thereof in promoting growth of neuronal cells of dorsal root ganglia, relates to the field of microorganisms, provides a bifidobacterium adolescentis strain MN-84 capable of producing gamma-aminobutyric acid with high yield, is a nerve inhibitor in a nervous system, has potential influence on excitation degree of neurons, and has great regulation effect on improving adverse emotions such as depression of human and the like; MN-84 is also potentially involved in the regulation of the human nervous system by promoting the growth of dorsal root ganglion neuronal cells.

Description

Bifidobacterium adolescentis, culture method thereof and application thereof in promoting growth of neuronal cells of dorsal root ganglia

Technical Field

The invention relates to the field of microorganisms, and particularly relates to bifidobacterium adolescentis, a culture method thereof and application thereof in promoting growth of neuronal cells of dorsal root ganglia.

Background

Dorsal Root Ganglion (DRG) belongs to peripheral sensory ganglion, dorsal root ganglion neuron is primary afferent neuron for pain sensation of trunk and limbs, and has functions of transmitting and regulating body sensation, receiving and transmitting nociception. In the pain generation process, the dorsal root ganglion plays an important role in a pain mechanism as a primary neuron of pain afferent, is mainly expressed in dorsal root ganglion neurons, and an ion channel and a receptor thereof closely related to the pain mechanism are key for realizing targeted analgesia of the dorsal root ganglion.

Dorsal root ganglion neurons express a variety of ion channels or receptors and may have the following functions. (1) Transduction: transient receptor potential channels, sodium ion channels, acid sensitive ion channels, ATP sensitive ion channel receptors, which transduce noxious stimuli into electrical impulses, expressed in the peripheral region of dorsal root ganglion neurons. (2) Conduction: sodium and potassium channels participate in the transport of action potentials. (3) Regulation of synaptic transmission: voltage-gated calcium ion channels (VGCC) and glutamate receptors (GluR) expressed on the primary afferent presynaptic terminal of the spinal cord dorsal horn regulate neurotransmitter release. After nerve injury, both injured and uninjured dorsal root ganglion neurons exhibit increased excitability and ectopic discharge, and this abnormal spontaneous discharge may be associated with altered dorsal root ganglion neuron voltage-gated sodium ion channel (VGSC) distribution, density, and function resulting from nerve injury.

The role of dorsal root ganglion in pain mechanism and pain treatment are always the focus of research in the pain field, so that the development of a substance for the growth of neuronal cells of dorsal root ganglion is of great significance for the research of the role of dorsal root ganglion in pain mechanism and pain, and provides a way for the relevant treatment scheme.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide bifidobacterium adolescentis, a culture method thereof and application thereof in promoting growth of neuronal cells of dorsal root ganglia.

The invention is realized by the following steps:

in a first aspect, the embodiment of the present invention provides a bifidobacterium adolescentis, the preservation number of which is CGMCC No. 23906.

In a second aspect, embodiments of the present invention provide a method for culturing bifidobacterium adolescentis as described in the previous embodiments, which comprises anaerobically culturing the bifidobacterium adolescentis.

In a third aspect, embodiments of the present invention provide the use of bifidobacterium adolescentis as described in the previous embodiments in the manufacture of a product for promoting neuronal cell growth in the dorsal root ganglion.

In a fourth aspect, embodiments of the present invention provide a product for promoting neuronal cell growth in dorsal root ganglia, the active ingredient of which comprises bifidobacterium adolescentis as described in the preceding embodiments.

In a fifth aspect, embodiments of the present invention provide the use of bifidobacterium adolescentis as described in the preceding embodiments for promoting neuronal cell growth in the dorsal root ganglion, which is not directly aimed at the diagnosis or treatment of disease.

In a sixth aspect, embodiments of the present invention provide a use of bifidobacterium adolescentis as described in the previous embodiments in the preparation of a product for high yield of γ -aminobutyric acid.

In a seventh aspect, the present invention provides a high-yield gamma-aminobutyric acid product, wherein the active ingredient of the product comprises bifidobacterium adolescentis as described in the previous embodiments.

In an eighth aspect, embodiments of the present invention provide the use of bifidobacterium adolescentis as described in the previous embodiments for the production of gamma-aminobutyric acid.

The invention has the following beneficial effects:

the invention provides a bifidobacterium adolescentis strain MN-84 which can produce gamma-aminobutyric acid with high yield, is a nerve inhibitor in a nervous system, has potential influence on the excitation degree of neurons, and has great regulation effect on improving the bad mood of human depression and the like; MN-84 is also potentially involved in the regulation of the human nervous system by promoting the growth of dorsal root ganglion neuronal cells.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the results of whether or not the supernatants of the different strains of example 2 have glutamate decarboxylase GAD activity.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

First, the embodiment of the present invention provides Bifidobacterium adolescentis MN-84, Bifidobacterium adolescentis, with a collection number of CGMCC No.23906, a collection address of No. 3, institute of microbiology, institute of china academy of sciences, zip code: 100101; the preservation date is 2021, 11 months and 15 days.

The sequence of the 16s rDNA of Bifidobacterium adolescentis MN-84 is as follows (5 '-3'): GCGGGGGGGTGCTTACCATGCAGTCGAACGGGATCCCAGGAGCTTGCTCCTGGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCAGTTGAACGCATGGCCCTCTGGGAAAGATTTTGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGACGGCCTTCGGGTTGTAAACCGCTTTTGACTGGGAGCAAGCCCTTCGGGGTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGACCATTCCACGGTCTCCGTGTCGGAGCCAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACAGCCCCAGAGATGGGGCCTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTGTGTTGCCAGCACGTCGTGGTGGGAACTCACGGGGGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACACTGTGAGGTGGAGCGGATCCCTTAAAACCGGTCTCAGTTCGGATTGGAGTCTGCAACCCGACTCCATGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACGCCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGTAGCACCCGAAGCCGGTGGCCCAACCTTTTGGGGGGAGCCGTCTAAGGTAGACTCTGAT are provided.

The strain is subjected to whole genome sequencing, and after Blast comparison, no strain with completely the same genome sequence of the strain is found.

The bifidobacterium adolescentis MN-84 is obtained by screening, can produce gamma-aminobutyric acid at high yield, is a nerve inhibitor in a nervous system, has potential influence on the excitation degree of neurons, and has great regulation effect on improving the bad mood of human depression and the like; MN-84 is also potentially involved in the regulation of the human nervous system by promoting the growth of dorsal root ganglion neuronal cells.

Embodiments of the present invention also provide a method for culturing bifidobacterium adolescentis as described in the previous embodiments, which includes performing anaerobic culture on the bifidobacterium adolescentis.

Without limitation, MN-84 may optionally be cultured by methods known for the culture of Bifidobacterium adolescentis.

Preferably, the culture temperature is 32-42 ℃, the culture temperature can be specifically within any one or any two of the ranges of 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃ and 42 ℃, and more preferably 37 ℃, and the culture effect is better.

Preferably, the culture medium adopted by the culture is a culture medium suitable for the bifidobacterium adolescentis;

preferably, the culture medium adopted by the culture is a lactic acid bacteria culture medium MRS.

The invention also provides the application of the bifidobacterium adolescentis as described in the previous embodiment in preparing products for promoting the growth of neuronal cells of dorsal root ganglia.

Preferably, the bifidobacterium adolescentis is at least one of live bacteria, sterilized bacteria, bacteria culture or bacteria fermentation product.

It is understood that the viable bacteria may be a suspension of bifidobacterium adolescentis (viable bacteria state), sterilized, obtained by sterilizing the suspension, cultured by inoculating the bacteria into a seed culture medium, and fermented by inoculating the bacteria or the culture into a fermentation medium.

The dorsal root ganglion DRG is the expansion node of the dorsal root of the spinal cord near the medial surface of each intervertebral foramen and is mainly composed of neuronal cells.

In some embodiments, the dorsal root ganglion may be selected from all vertebrate dorsal root ganglia, preferably mammals, more preferably non-human mammals. Preferably, the dorsal root ganglion is a dorsal root ganglion of a mouse.

Embodiments of the present invention also provide a product for promoting neuronal cell growth in dorsal root ganglia, the active ingredient of which comprises bifidobacterium adolescentis as described in the preceding embodiments.

Preferably, the dorsal root ganglion is a dorsal root ganglion of a mouse.

Optionally, the product is selected from any one of food, pharmaceutical and nutraceutical products.

Preferably, the product may also include another substance for neuronal cell growth having a dorsal root ganglion.

Embodiments of the present invention also provide the use of bifidobacterium adolescentis as described in the preceding embodiments for promoting the growth of neuronal cells of the dorsal root ganglion without the direct purpose of diagnosis or treatment of disease, such as in vitro related research and development and new drug development.

Preferably, the dorsal root ganglion is a dorsal root ganglion of a mouse.

The invention also provides application of the bifidobacterium adolescentis in preparing products for high-yield production of gamma-aminobutyric acid.

The embodiment of the invention also provides a product for high yield of gamma-aminobutyric acid, wherein the active ingredient of the product comprises the bifidobacterium adolescentis as described in the signed embodiment.

The embodiment of the invention also provides application of the bifidobacterium adolescentis in producing gamma-aminobutyric acid.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Bifidobacterium adolescentis MN-84 is prepared from feces of healthy adult people of about 25 years old by mixing with 2.5mg/L lithium mupirocin in lactobacillus culture Medium (MRS) and separating. And (3) standing in a lactic acid bacteria culture Medium (MRS) at the constant temperature of 37 ℃ for overnight culture at the pH of 6.2-6.6.

Example 2

The MN-84 strain promotes the growth detection of rat dorsal root ganglion neuron cells (DRG).

The method comprises the following steps: rat dorsal root ganglion neuronal cells (DRG) were cultured, stimulated with the supernatant of the inactivated strain to be tested, and cell growth was examined by the MTT method.

The MTT method is also called MTT colorimetric method, and is a method for detecting cell survival and growth.

The first principle is as follows: the detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-purple crystalline Formazan (Formazan) and deposit the blue-purple crystalline Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at 490nm wavelength by enzyme linked immunosorbent detector, which can indirectly reflect living cell number. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number. The method is widely used for activity detection of some bioactive factors, large-scale screening of anti-tumor drugs, cytotoxicity test, tumor radiosensitivity determination and the like. It features high sensitivity and economy. The disadvantages are as follows: formazan product produced by reduction of MTT is insoluble in water and needs to be solubilized before detection. This not only increases the workload, but also affects the accuracy of the experimental results, and the organic solvent that dissolves the formazan is harmful to the experimenter. The MTT method can be used to measure relative numbers and relative viability of cells.

Preparation method of MTT solution

MTT full name: 3- < (4,5) -dimethylthiohiazo (-z-y1) -3, 5-di-phenylyttrazolimide; chinese chemical name: 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide salt trade name: thiazole blue, a yellow dye.

The preparation method of the MTT solution comprises the following steps: generally, the concentration of MTT in this method is 5 mg/ml. Therefore, 0.5 g of MTT can be weighed, dissolved in 100ml of Phosphate Buffered Saline (PBS) or phenol red-free medium, filtered through a 0.22 μm filter to remove bacteria in the solution, and stored at 4 ℃ in the dark.

Note that:

1. in the process of preparation and storage, the container is preferably wrapped by aluminum foil paper, and a fluorescent lamp on an ultra-clean bench is generally closed to avoid light during the experiment;

2. dissolving with PBS (pH 7.4) when preparing MTT;

PBS formulation: NaCl8g Ten KCl 0.2g Ten Na₂HPO₄1.44g decaKH₂PO₄Adjusting pH to 7.4 with 0.24g, and fixing volume to 1L;

MTT is prepared in situ, filtered and stored at 4 deg.c in dark for two weeks or prepared into 5mg/ml for long term storage at-20 deg.c to avoid repeated freeze thawing, packed in small amount and packed in dark bag or black paper or tin foil paper to avoid decomposition.

MTT has carcinogenicity, and is careful when in use, the MTT prepared by using transparent film gloves under the condition is best, and needs to be sterile, and is sensitive to bacteria; it does not matter to add time to 96 orifice plates and not to keep out of the sun, and the time of competing is shorter after all, or the light on the operation panel is turned off.

Preparation of MN-84 fermentation liquor

Continuously activating Bifidobacterium adolescentis MN-84 with MRS liquid culture medium for 3 generations, washing with sterile PBS buffer solution, and adjusting density to 3 × 10⁸And (mL). Inoculating 2% volume of MRS liquid culture medium, shaking, sealing, and fermenting in 37 deg.C gas bath constant temperature oscillator; after 1 week, centrifuging the fermentation liquid at 4000r/min for 30min, filtering the supernatant with 0.2 μm microporous membrane filter for sterilization, adjusting pH to 7.2, and subpackaging and freezing for use.

Fourthly, detecting the effect of the MN-84 fermentation liquor on the growth of the rat dorsal root ganglion neuron cells by a medicine MTT method.

1. Collecting logarithmic phase cells, adjusting the concentration of cell suspension, adding 100 mu l of cell suspension into each hole, and paving the plate to adjust the density of the cells to be detected to 1000-10000 holes (the edge holes are filled with sterile PBS);

2.5％CO₂incubating at 37 ℃ until cell monolayer is paved on the bottom of the well (96-well flat bottom plate), sucking 20 mul of cell culture medium, adding 20 mul of bacterial fermentation liquid, and adding the drug after cell adherence, or adding the drug for two hours or half a day, wherein the drug is paved in the afternoon of the previous day and the morning of the next day, generally 5-7 gradients are formed, and each well is 100 mul, and 3-5 multiple wells are arranged;

3.5％CO₂incubation at 37 ℃ for 48 hoursWhen the sample is taken, the sample is observed under an inverted microscope;

4. mu.l of MTT solution (5mg/ml, i.e., 0.5% MTT) was added to each well and incubation was continued for 4 h. If the medicine can react with MTT, centrifuging and removing the culture solution, carefully flushing the medicine for 2-3 times by PBS, and then adding the culture solution containing MTT;

5. terminating the culture, and carefully sucking out the culture solution in the holes;

6. adding 150 μ l dimethyl sulfoxide into each well, and shaking on a shaking bed at low speed for 10min to dissolve the crystals; the absorbance of each well was measured at an enzyme linked immunosorbent assay OD490 nm.

7. At the same time, a zero-setting well (culture medium, MTT, dimethyl sulfoxide) (CK control) and a control well (cells, drug dissolution medium of the same concentration, culture solution, MTT, dimethyl sulfoxide) (blank control) were set.

TABLE 1 results

Remarking: table 1 shows the results of 3 replicates;

the numbers cb7Y2, cb7Y3 and cb39Y4 are 3 groups of the bacterial control group, and the origin method (example 1) and the detection method are the same as those of MN-84.

Example 3

Detecting the gamma-aminobutyric acid (GABA) produced by the strain MN-84.

The method comprises the following steps:

(1) primary screening: after activating and culturing the test strain, the cells were collected and washed 3 times with 0.9% NaC 1. Adding 1% glutamic acid solution into the thallus according to wet weight, adjusting pH to 4.7, reacting for 24h, centrifuging to obtain supernatant, adding mixed acid-base indicator, observing color reaction, and preliminarily judging that the solution turns green and has GAD activity. The solution turned green and had GAD activity, as shown in FIG. 1, and the results indicated that

numbers

1, 2, 4 and 21 (based on the strains shown in Table 2) were test strips showing green color.

(2) And (3) high performance liquid chromatography detection: preparing a gamma-aminobutyric acid standard solution: accurately weighing a gamma-aminobutyric acid standard substance, dissolving the gamma-aminobutyric acid standard substance with ultrapure water to obtain a standard solution, and storing the standard solution in a refrigerator at 4 ℃ for later use. Derivatization of the sample: putting 1mL of standard gamma-aminobutyric acid solution with the concentration of 5mg/mL into a 10mL brown measuring flask, adding 1mL of 0.5mol/L sodium bicarbonate (pH 9.0) solution, then respectively adding 0.4, 0.6, 0.8, 1.0, 1.2 and 1.5mL of 1% (2, 4-dinitrofluorobenzene) FDNB acetonitrile solution, putting the solution into a water bath with the temperature of 60 ℃ for heating for 0.5, 1 and 1.5h in a dark place, taking out, cooling, respectively adding phosphate buffer solution with the pH of 7.0 to 10mL, uniformly mixing, 10000r/min, centrifuging for 15min, filtering through a micropore with the diameter of 0.22 mu m, determining the content of the derivative of the gamma-aminobutyric acid, making a gamma-aminobutyric acid standard curve according to the peak area of a derivative product 2, 4-dinitrofluorobenzene butyric acid, and calculating the content of the gamma-aminobutyric acid according to the standard curve. ③ UPLC chromatographic conditions: mobile phase A: 5mM ammonium formate + 0.1% formic acid water, B: 0.1% formic acid-acetonitrile, column: waters BEH C182.1X 100mm X1.7 μm, flow rate: 0.3mL/min, gradient: 0-1min (5% B); 1-2min (90% B); 2-4.5min (90% B); 4.5-4.6min (5% B); 4.6-7min (stop).

The results are shown in Table 2.

TABLE 2 liquid chromatography test results

Remarking: table 2 shows the results of 3 replicates, and the methods for obtaining Cb7Y3, Cb7Y2, Cb39Y4 were the same as MP-569, specifically, in example 1, and the methods for detection were the same as MP-569.

From the results, the probiotic strain MN-84 can produce gamma-aminobutyric acid in high yield, is a neuroleptic in the nervous system, has potential influence on the excitation degree of neurons, and has great regulation effect on improving the bad mood such as human depression; MN-84 (Bifidobacterium adolescentis) can also potentially participate in the regulation process of the human nervous system by promoting the growth of dorsal root ganglion neuron cells.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Bifidobacterium adolescentis, is characterized in that, its deposit number is CGMCC NO.23906.

2. the culture method of Bifidobacterium adolescentis as claimed in claim 1, is characterized in that, it comprises carrying out anaerobic culture to described Bifidobacterium adolescentis;

Preferably, the temperature of the culture is 32-42°C;

Preferably, the medium used in the culture is a medium suitable for Bifidobacterium adolescentis;

Preferably, the medium used in the culture is MRS medium.

3. The use of Bifidobacterium adolescentis as claimed in claim 1 in the preparation of a product for promoting the growth of neuronal cells of the dorsal root ganglion.

4 . The application according to claim 3 , wherein the Bifidobacterium adolescentis is at least one of living cells, sterilized cells, cell cultures or cell fermentations. 5 .

The application according to claim 3 or 4, wherein the dorsal root ganglion is a mouse dorsal root ganglion.

6. A product for promoting the growth of neuronal cells of dorsal root ganglion, characterized in that its active ingredient comprises Bifidobacterium adolescentis as claimed in claim 1;

Preferably, the Bifidobacterium adolescentis is at least one of living cells, sterilized cells, cell cultures or cell fermentations;

Preferably, the dorsal root ganglion is a mouse dorsal root ganglion.

7. the application of Bifidobacterium adolescentis as claimed in claim 1 in promoting the neuronal cell growth of dorsal root ganglion, and described application is not with the diagnosis or treatment of disease as direct purpose;

Preferably, the dorsal root ganglion is a mouse dorsal root ganglion.

8. the application of Bifidobacterium adolescentis as claimed in claim 1 in preparing the product of high-yielding γ-aminobutyric acid.

9. a product of high-yield γ-aminobutyric acid, characterized in that its active ingredient comprises Bifidobacterium adolescentis as claimed in claim 1;

Preferably, the Bifidobacterium adolescentis is at least one of living cells, sterilized cells, cell cultures or cell fermentations.

10. The application of Bifidobacterium adolescentis as claimed in claim 1 in producing γ-aminobutyric acid.