CN113881610B - Bacteroides thetaiotaomicron BP8E and application thereof - Google Patents
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
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- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
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Abstract
The invention belongs to the field of microorganisms and application thereof, and provides bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) BP8E with the preservation number of CGMCC No. 22149. The invention also provides Bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron) Use of BP8E for the manufacture of GABA and the prevention and/or treatment of depression. The Bacteroides thetaiotaomicron BP8E provided by the invention can produce GABA at a high yield under the culture condition without additionally adding glutamic acid, can improve depression-like behaviors of mice, can provide potential strains for probiotic treatment for stress mood disorders such as depression and insomnia, and has great potential market value.
Description
Technical Field
The invention belongs to the field of microorganisms and application thereof, and particularly relates to bacteroides thetaiotaomicron BP8E and application thereof.
Background
Gamma-aminobutyric acid (also known as GABA) is a non-protein natural amino acid, widely found in the nervous system of mammals and also in plants, such as tomatoes, potatoes, grapes, pumpkins, tea leaves, and the like. Fermented foods such as kimchi and fermented brown rice are also rich in GABA.
GABA is an important inhibitory neurotransmitter in the nervous system. Researchers have found that insufficient GABA content can produce emotions such as anxiety, uneasiness, tiredness and anxiety. However, with age, the GABA content in humans tends to decrease. It is known that the GABA content in the human body decreases by about 5% every 10 years. GABA is currently reported to have the following physiological effects: (1) relieving anxiety and inhibiting excitation, (2) promoting metabolism, (3) improving memory, (4) delaying nerve cell aging, (5) caring skin, and (6) regulating blood pressure. The GABA is properly supplemented, so that the mental stress can be well relieved, the mood can be adjusted, and the sleep can be improved. In view of the beneficial physiological effects of GABA and theoretical basis for consolidation, GABA has begun to enter the market of various countries, and as early as 2001, GABA is approved as a new resource food in japan, and then, as early as 2009, GABA is approved as a new resource food in china (publication No. 12 in 2009 in the ministry of health). Until now, the GABA eating application has formally opened the state, GABA is regarded as a new food raw material, is known as 'neural vitamin', and is widely applied to stress-relieving sleep-assisting products for high-pressure people and beauty upgrading products for fashion love.
In addition to direct supplementation of GABA dietary supplements, people may obtain from natural foods such as grains, cruciferous plants, tomatoes, and the like. Microbial fermentation is a hot spot for the current study of GABA synthesis. The intestinal microorganisms inhabit the gastrointestinal system of the host and play a vital role in the metabolism and immune balance of the organism. In addition to lactobacillus and bifidobacterium which contain more GABA-synthesizing strains, the ability of other strains to produce GABA in high yield is still rarely known. Because intestinal microorganisms, particularly intestinal bacteria with probiotic effect, not only have high GABA yield, but also have higher safety, the method for mining and developing the intestinal bacterial strain with high GABA yield has good market application prospect, and simultaneously provides potential bacterial strains for probiotic treatment for stress mood disorders such as depression, insomnia and the like.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention provides a Bacteroides thetaiotaomicron BP8E having a high GABA-producing ability. The Bacteroides thetaiotaomicron can highly produce GABA under the culture condition without additionally adding glutamic acid, the average value of GABA production amount detected by two times of High Performance Liquid Chromatography (HPLC) is 528.6mg/L, and animal experiments show that BP8E can obviously improve depression-like behaviors of mice.
In a first aspect, the present invention provides Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E with preservation number of CGMCC No. 22149。
In a second aspect, the present invention provides the use of a formulation comprising at least one of:
1) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron）BP8E；
2) Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E microbial inoculum;
3) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E viable bacterial suspension;
4) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E dead bacteria suspension;
5) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E metabolite;
6) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E extract;
wherein, the Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E is Bacteroides thetaiotaomicron with preservation number CGMCC No.22149 (described in the first aspect of the invention) ((Bacteroides thetaiotaomicron）BP8E。
In a third aspect, the invention provides the use of a formulation comprising at least one of the following in the manufacture of a product for the prevention and/or treatment of depression:
1) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron）BP8E；
2) Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E microbial inoculum;
3) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E viable bacterial suspension;
4) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E dead bacteria suspension;
5) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E metabolite;
6) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E extract;
wherein, the Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E is CGMCC NoBacteroides thetaiotaomicron of 22149 (c) ((c))Bacteroides thetaiotaomicron）BP8E。
In a fourth aspect, the invention provides a product for use in the prevention and/or treatment of depression, comprising at least one of:
1) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron）BP8E；
2) Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E microbial inoculum;
3) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E viable bacterial suspension;
4) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E dead bacteria suspension;
5) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E metabolite;
6) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E extract;
wherein, the Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E is Bacteroides thetaiotaomicron with accession number CGMCC No.22149 (see the description of the first aspect of the invention) (II)Bacteroides thetaiotaomicron）BP8E。
Preservation description:
the strain name is as follows: BP8E
Latin name:Bacteroides thetaiotaomicron
and (3) classification and naming: bacteroides thetaiotaomicron
The strain number is as follows: 22149
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No. 1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 2021 year, 4 months and 9 days
Registration number of the preservation center: CGMCC number 22149
Bacteroides thetaiotaomicron (I) provided by the present inventionBacteroides thetaiotaomicron) BP8E has good GABA production capacity, and can significantly relieve depression sample of depression model miceThe behavior provides good scientific basis for developing effective anti-depression probiotic products in the future, and has great potential market value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows a mass spectrometric identification of Bacteroides thetaiotaomicron BP8E in the present invention.
FIG. 2 shows the effect of Bacteroides thetaiotaomicron BP8E on the tail suspension experiment in depression model mice in accordance with the present invention. n =8, p <0.05, p < 0.001.
Detailed Description
In order to make the technical solution, objects and advantages of the present invention clearer, the present invention is further described in detail by the following specific embodiments. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict between the prior art and the present disclosure, the present disclosure should control.
Various reagents, materials and the like used in the following examples are commercially available products unless otherwise specified; unless otherwise specified, all the tests and detection methods used in the following examples are conventional in the art and can be obtained from textbooks, tool books or academic journals.
Example 1: isolation and identification of Bacteroides thetaiotaomicron BP8E
Collecting feces samples of healthy adults living in Beijing urban area for a long time, diluting, coating the feces samples on YCFA culture medium, carrying out anaerobic culture at 37 ℃ for 24h, and separating to obtain different single colonies on the plate. And (3) selecting different single colonies by using the sterilized inoculating loops, streaking and purifying the single colonies on a new YCFA solid culture medium plate, and performing anaerobic culture at 37 ℃ for 24 hours to obtain purified single colonies. Coating each purified single colony on a mass spectrum plate, adding lysis solution and a matrix respectively, drying, and performing mass spectrum on a MALDI-TOF MS 1000 mass spectrometer (Autobio, Zheng Zhou AnTu Biotech Co., Ltd.). After the identification, each strain was frozen at-80 ℃ in a strain resource library of this company for later use. Among them, the identification results of Bacteroides thetaiotaomicron BP8E strain according to the present invention are shown in FIG. 1. As can be seen from the results in FIG. 1, the BP8E strain had a very high taxonomic similarity to Bacteroides thetaiotaomicron, and was thus designated as Bacteroides thetaiotaomicron BP8E strain.
Example 2: preparation of Bacteroides thetaiotaomicron BP8E
Coating a BP8E bacterial solution frozen at-80 ℃ on a YCFA solid plate, carrying out inverted culture at 37 ℃ for 24h, inoculating a single bacterial colony in a liquid YCFA culture medium, and carrying out culture at 37 ℃ for 24h to obtain a first generation bacterial solution; inoculating 10% of a first-generation bacterium liquid to a fresh YCFA liquid culture medium, and culturing at 37 ℃ for 24h to obtain a second-generation bacterium liquid; inoculating 10% of the second-generation bacterial liquid into a fresh YCFA liquid culture medium, and culturing at 37 ℃ for 24h to obtain a working bacterial liquid. And (3) centrifuging the working bacterial liquid at 13000 rpm at 4 ℃ for 15 min, collecting the supernatant which is the metabolite of the BP8E strain, and carrying out subsequent experiments, wherein the thallus precipitate is resuspended by using normal saline to obtain the viable bacterial liquid with viable thallus.
Example 3: GABA production amount detection
This example quantitatively detects GABA produced by Bacteroides thetaiotaomicron BP8E strain by paper chromatography and high performance liquid chromatography.
(1) Paper chromatography
The supernatant of Bacteroides thetaiotaomicron BP8E, YCFA medium and GABA standard solutions (0.2, 0.4, 0.8, 1.2, 1.6 and 2.0 g/L) were spotted onto chromatography paper at 4. mu.L each, and then developed in developing solvent (V (n-butanol): V (glacial acetic acid): V (water): 5: 3: 2, ninhydrin dosage 1.2% (w/V)). Immediately after the chromatography is finished, the chromatography paper is put into a 90 ℃ oven for drying and developing for 30 min. Spots corresponding to the standard positions were cut off and eluted with 5 mL of an eluent (V75% ethanol: V0.6% copper sulfate = 38: 2) at 40 ℃ and 50 rpm. And (4) taking 200 mu L of the uniformly mixed eluent, and detecting the light absorption value at 510 nm by using a microplate reader. The concentration of GABA contained in the sample (in mg/L) was calculated from a standard curve drawn with GABA standards.
Through three paper chromatography experiments, the amount of GABA produced by Bacteroides thetaiotaomicron BP8E after 24 hours of culture in YCFA liquid medium (without exogenous L-glutamic acid) is shown in Table 1:
(2) HPLC method
Accurately weighing GABA standard substances, preparing GABA standard solutions with mass concentrations of 0.04, 0.08, 0.12, 0.16 and 0.20 g/L, taking 100 mu L of GABA standard solutions with various concentrations, adding 100 mu L of o-phthalaldehyde into the GABA standard solutions respectively, and incubating at room temperature for at least 90s after fully and uniformly mixing. After the reaction, 20. mu.L of each solution was injected and the absorption peak was measured at 338 nm. And establishing a GABA concentration detection standard curve according to peak area. And then adding o-phthalaldehyde into the diluted BP8E supernatant, incubating, mixing uniformly, injecting a sample, and measuring an absorption peak at 338 nm.
In order to obtain more reliable data on the GABA production amount, two HPLC detections were carried out in total, and the amount of GABA production by Bacteroides thetaiotaomicron BP8E was as shown in Table 2 below.
Table 2 shows that the GABA concentrations detected in two HPLC experiments were 564.6mg/L and 492.7mg/L, respectively, with an average value of 528.6 mg/L. Many strains reported in the literature need exogenous glutamic acid added in a culture system to stimulate the strains to synthesize GABA, while the BP8E strain in the invention can produce GABA (232.8 mg/L, Zhuzontao and the like, screening of bifidobacterium with high aminobutyric acid yield and improving effect on a depression cell model under the condition of no exogenous stimulation, food industry science and technology, 2020), which indicates that the Bacteroides thetaiotaomicron BP8E strain has excellent GABA high-yield capability.
Example 4: influence of Bacteroides thetaiotaomicron BP8E on tail suspension experiment of chronic stress depression model mice
Depression model mice constructed by chronic stress stimulation are the most commonly used animal models in the study of screening depression treatment drugs. Therefore, a depression model mouse constructed by chronic stress stimulation was used as a subject in the following examples.
(1) Grouping and mouse model establishment
32 male Kunming mice were purchased, weighing about 20g, and were randomly divided into 4 groups one week after adaptive feeding: normal group, model group, BP8E live group and fluoxetine hydrochloride group, 8 mice per group. In addition to the normal control group, mice in other groups were given the following stress stimuli in randomized order weekly for 4 weeks according to the chronic unpredictable stress (CUMS) method: fasting for 12h, binding for 1h, wetting padding for 12h, keeping water for 12h, reversing the day and night for 24h, shaking for 1 min, and shocking the soles. BP8E live bacterium group mice each gavage 10 per day9CFU/mL Bacteroides thetaiotaomicron BP8E viable bacterial suspension, lasting 28 days. The mice in the normal group and the model group are given YCFA culture medium corresponding to the gavage, and the mice in the positive group are injected with fluoxetine hydrochloride (10 mg/kg) in the abdominal cavity.
(2) Tail suspension experiment of mice
The mouse tail suspension experiment is a behavior despair model commonly used in pharmacodynamics and is used for evaluating the drug effect of antidepressant drugs, and the change of the depression-like behavior of the mouse is judged by observing the immobility time of the tail suspension of the mouse in the experiment. During the experiment, the adhesive tape is adhered to the position 1cm away from the tail end of the mouse by using the adhesive tape, the mouse is inversely hung, the nose is 20-25cm away from the ground, the despair behavior of the mouse within 6min is recorded by using an animal behavior video analysis system, and the accumulated immobility time(s) within 4min after analysis. In the experiment, mice are separately tested to avoid the mutual visual and sound interference, wherein the judgment standard of the mouse tail suspension motionless time is that the mice stop struggling, are in an upside down suspension state and are still.
The statistical result is shown in fig. 2, in the tail suspension experiment, compared with the normal group, the immobility time of the mouse tail suspension is obviously increased in the model group (p is less than 0.001), compared with the model group, in the BP8E group, the gavage BP8E viable bacteria suspension can obviously reduce the absolute immobility time (p is less than 0.05) of the tested mouse, the average immobility time is reduced from 120s to 94s, and the effect is only slightly weaker than the improvement effect of the drug fluoxetine hydrochloride on the absolute immobility time of the mouse (average immobility time is 85 s), which shows that the bacteroides thetaiotaomicron BP8E strain can obviously relieve the depressive-like behavior of the depression model mouse in the tail suspension experiment.
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 (4)
1. Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E with the preservation number of CGMCC number 22149.
2. Use of a formulation comprising at least one of the following in the manufacture of gamma-aminobutyric acid:
1) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron）BP8E；
2) Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E microbial inoculum;
3) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E viable bacterial suspension;
4) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E dead bacteria suspension;
5) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E metabolite;
6) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E extract;
wherein, the Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron）BP8E is Bacteroides thetaiotaomicron with preservation number CGMCC number 22149 as defined in claim 1 (Bacteroides thetaiotaomicron）BP8E。
3. Use of a formulation comprising at least one of the following in the manufacture of a product for the prevention and/or treatment of depression:
1) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron）BP8E；
2) Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E microbial inoculum;
3) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E viable bacterial suspension;
4) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E dead bacteria suspension;
5) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E metabolite;
wherein, the Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E is Bacteroides thetaiotaomicron with preservation number CGMCC number 22149 as defined in claim 1 (Bacteroides thetaiotaomicron）BP8E。
4. A product for preventing and/or treating depression, comprising at least one of:
1) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron）BP8E；
2) Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E microbial inoculum;
3) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E viable bacterial suspension;
4) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E dead bacteria suspension;
5) bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E metabolite;
wherein, the Bacteroides thetaiotaomicron: (Bacteroides thetaiotaomicron) BP8E is Bacteroides thetaiotaomicron with preservation number CGMCC number 22149 as defined in claim 1 (Bacteroides thetaiotaomicron）BP8E。
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| CN112119167A (en) * | 2018-04-24 | 2020-12-22 | 深圳华大生命科学研究院 | Biomarkers for depression and uses thereof |
| WO2020076043A1 (en) * | 2018-10-08 | 2020-04-16 | 한국생명공학연구원 | Gut microbiota having prophylactic or therapeutic effect on depression and use thereof |
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