Lactobacillus gasseri BDUP, use thereof and product thereof

Abstract

Lactobacillus gasseri BDUP, the use thereof and a product thereof. The Lactobacillus gasseri BDUP can be used for producing GABA and preventing and / or treating depression. The Lactobacillus gasseri BDUP can produce GABA at a high yield under the condition of not additionally adding glutamic acid, can improve depressive behaviors of mice, and can provide potential strains for probiotic treatment for stress mood disorders such as depression and insomnia, and therefore has great potential market value.

Description

Lactobacillus gasseri BDUP, its applications and products Technical Field

[0001] The invention belongs to the field of microorganisms and their applications, and particularly relates to Lactobacillus gasseri BDUP and applications thereof. Background Art

[0002] Gamma-aminobutyric acid (GABA) is a key inhibitory neurotransmitter in the central nervous system. It plays a crucial role in mammalian physiological regulation, including lowering blood pressure, improving sleep, alleviating anxiety, suppressing excitement, enhancing memory, and slowing neurological aging. The human body has a self-regulating system for GABA, and a deficiency can lead to symptoms such as anxiety, restlessness, fatigue, and insomnia. In humans, GABA levels gradually decrease with aging and increased mental stress.

[0003] As research deepens, GABA's physiological functions continue to be elucidated, and it has developed into a novel functional factor, increasingly finding widespread use in industries such as food, medicine, cosmetics, and agriculture. Since 2001, countries around the world have gradually included GABA in food products. Therefore, appropriate GABA supplementation can help alleviate stress, regulate mood, and improve sleep in individuals experiencing high stress.

[0004] Daily GABA intake primarily comes from direct consumption of GABA supplements and daily diet. For example, tomatoes, potatoes, grapes, pumpkin, and some fermented foods, such as kimchi and fermented brown rice, are rich in GABA. Due to its low natural abundance, GABA is difficult to obtain in large quantities from natural sources. Currently, chemical synthesis and microbial fermentation are the main methods used. However, chemically synthesized GABA is prone to residual raw materials, lacks purity, and is unsafe, making it unsuitable for food use. Microbial fermentation is considered a safe and low-cost method for mass production of GABA. Synthesized GABA can be used as a natural functional food ingredient in health foods.

[0005] In recent years, research on the gut microbiome has revealed a strong connection between the brain and the gut, known in medicine as the "gut-brain axis." Gut microbes can influence the brain, such as behavior, appetite regulation, and serotonin metabolism. Alterations in the gut microbiome have been linked to a variety of neurological disorders, including anxiety, multiple sclerosis, autism spectrum disorder, and Parkinson's disease. Furthermore, the homeostasis of the microbiome also influences the expression of neurotransmitters. Some probiotic gut bacteria, such as Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus pentosus, play a crucial role in regulating human health, such as maintaining glucose and lipid metabolism and intestinal homeostasis. Therefore, the discovery and development of gut strains with high GABA production capacity holds great market potential. These strains could not only provide safe GABA to patients with stress-related mood disorders like depression and insomnia, but also enhance the beneficial effects of probiotics in maintaining overall health.

[0006] Summary of the Invention

[0007] In order to solve the above problems existing in the prior art, the present invention provides a Lactobacillus gasseri BDUP with high GABA production capacity.

[0008] According to one aspect of the present invention, a Lactobacillus gasseri BDUP is provided, and its deposit number is CGMCC No.18357.

[0009] According to a second aspect of the present invention, there is provided the use of at least one of the following preparations containing the Lactobacillus gasseri BDUP in the production of GABA:

[0010] 1) Lactobacillus gasseri BDUP;

[0011] 2) Lactobacillus gasseri BDUP agent;

[0012] 3) Lactobacillus gasseri BDUP live bacterial suspension;

[0013] 4) Lactobacillus gasseri BDUP dead cell suspension;

[0014] 5) Lactobacillus gasseri BDUP metabolites;

[0015] 6) Lactobacillus gasseri BDUP extract.

[0016] According to a third aspect of the present invention, there is provided a use of at least one of the following preparations containing Lactobacillus gasseri BDUP3 in the preparation of a product for preventing and / or treating depression:

[0017] 1) Lactobacillus gasseri BDUP;

[0018] 2) Lactobacillus gasseri BDUP agent;

[0019] 3) Lactobacillus gasseri BDUP live bacterial suspension;

[0020] 4) Lactobacillus gasseri BDUP dead bacteria solution;

[0021] 5) Lactobacillus gasseri BDUP metabolites;

[0022] 6) Lactobacillus gasseri BDUP extract.

[0023] According to a fourth aspect of the present invention, there is provided a product for preventing and / or treating depression, comprising at least one of the following of Lactobacillus gasseri BDUP3:

[0024] 1) Lactobacillus gasseri BDUP;

[0025] 2) Lactobacillus gasseri BDUP agent;

[0026] 3) Lactobacillus gasseri BDUP live bacterial suspension;

[0027] 4) Lactobacillus gasseri BDUP dead bacteria solution;

[0028] 5) Lactobacillus gasseri BDUP metabolites;

[0029] 6) Lactobacillus gasseri BDUP extract.

[0030] The Lactobacillus gasseri of the present invention can produce high levels of GABA under culture conditions without the additional addition of glutamate, and animal experiments show that BDUP can significantly improve the depressive-like behavior of mice.

[0031] Description of the preservation of the strain of the present invention:

[0032] Strain name: BDUP

[0033] Latin name: Lactobacillus gasseri

[0034] Classification name: Lactobacillus gasseri

[0035] Strain ID: 18357

[0036] Depository: General Microbiology Center of China Culture Collection Administration

[0037] Abbreviation of depository institution: CGMCC

[0038] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0039] Date of deposit: August 2, 2019

[0040] CGMCC registration number: CGMCC No.18357 BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] FIG1 shows a mass spectrometry identification diagram of Lactobacillus gasseri BDUP in the present invention.

[0043] FIG2 shows a heat map of nucleic acid identity (ANI) analysis of BDUP of the present invention and other Lactobacillus gasseri.

[0044] Figure 3 shows the effects of Lactobacillus gasseri BDUP on depression-like behaviors in depression model mice. (A) Immobility time statistics in the tail suspension test. (B) Immobility time statistics in the forced swim test. n = 8; vs. normal group, ***p < 0.001; vs. model group, ## p<0.01, ### p<0.001. DETAILED DESCRIPTION

[0045] Example 1: Identification of Lactobacillus gasseri BDUP

[0046] Fecal samples were collected from centenarians in Hainan and diluted and spread onto MRS solid plates (10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 20 g glucose, 1 mL Tween-80, 5 g sodium acetate, 2 g dipotassium phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 18 g agar, 1000 mL distilled water). The plates were incubated anaerobically at 37°C for 24 hours to isolate distinct colonies. Using a sterile inoculation loop, distinct colonies were streaked onto new MRS solid medium plates for purification. The plates were then incubated anaerobically at 37°C for 24 hours to obtain purified colonies. Each purified colony was spread onto a mass spectrometry plate, lysate and matrix were added, and the plate was dried before identification by mass spectrometry on a MALDI-TOF MS 1000 mass spectrometer (Autobio, Zhengzhou Antu Biotechnology Co., Ltd.). After identification, each strain will be frozen at -80°C in the applicant's strain resource library for future use.

[0047] The identification results of the Lactobacillus gasseri BDUP strain involved in the present invention are shown in Figure 1. According to the results in Figure 1, the BDUP strain has a very high similarity with Lactobacillus gasseri in terms of classification, and therefore, it is named Lactobacillus gasseri BDUP strain.

[0048] Furthermore, the BDUP bacterial pellet of the present invention was collected by centrifugation, and the bacterial DNA was extracted and sent to a sequencing company. After sequencing, the genome was sorted and the BDUP genome information was compared with the genomes of several Lactobacillus gasseri strains in the NCBI database. It was found that the FastANI (average nucleotide identity) value of the BDUP of the present invention was far greater than 95% (Figure 2). An ANI of 95% is often used as the standard for species identification in analysis. Therefore, it was reconfirmed that the BDUP of the present invention belongs to the species Lactobacillus gasseri.

[0049] Example 2: Preparation of live bacterial liquid and fermentation metabolites of Lactobacillus gasseri BDUP

[0050] The -80°C frozen BDUP bacterial liquid was spread on an MRS solid plate, and after inverted culture at 37°C for 24 hours, a single colony was inoculated into liquid MRS medium (10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium hydrogen citrate, 20 g glucose, 1 mL Tween-80, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 1000 mL distilled water), and cultured at 37°C for 24 hours to obtain the first-generation bacterial liquid; 10% of the first-generation bacterial liquid was inoculated into fresh MRS liquid medium and cultured at 37°C for 24 hours to obtain the second-generation bacterial liquid; 10% of the second-generation bacterial liquid was inoculated into fresh MRS liquid medium and cultured at 37°C for 24 hours to obtain the working bacterial liquid. The working bacterial solution was centrifuged at 13,000 rpm and 4°C for 15 minutes, and the supernatant was collected as the BDUP strain metabolite for subsequent experiments. The bacterial pellet was resuspended in physiological saline to obtain a live bacterial solution containing live bacteria.

[0051] Example 3: GABA detection

[0052] (1) Paper chromatography

[0053] Centrifuge 1 mL of Lactobacillus gasseri BDUP fermentation broth at 4°C, 12,000 rpm for 10 minutes to remove the bacteria. Transfer the supernatant to a fresh centrifuge tube and store for later use. Then, aspirate 4 μL of sample supernatant (two replicates), the corresponding culture medium, and a standard GABA solution of any concentration (0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 g / L) onto filter paper. The spot line was 2.0 cm from the edge of the paper, with 2.0 cm between spots. After the sample spot dried, the paper was developed in a developer (V (n-butanol):V (glacial acetic acid):V (water) = 5:3:2, with 1.2% (w / v) ninhydrin) for 50 minutes (the developer should be approximately 1 cm from the top edge of the paper). After chromatography, the paper was immediately dried in a 90°C oven for 30 minutes to develop color. After color development, cut out the spot corresponding to the standard and elute with 5 mL of eluent (75% ethanol by volume: 0.6% copper sulfate by volume = 38:2) at 40°C and 50 rpm for 40 minutes. After elution, thoroughly mix the eluent and apply 200 μL of the solution to the ELISA plate in triplicate. Measure the absorbance at 510 nm. Substitute the measured absorbance value (y value) into the standard curve equation prepared using the GABA standard solution to calculate the GABA concentration (x, in g / L) in the sample.

[0054] The ability of Lactobacillus gasseri BDUP to metabolize and produce GABA was tested by three times of paper chromatography in MRS liquid medium (without the addition of L-glutamic acid) for 24 hours. The results are shown in Table 1:

[0055] Table 1 GABA production by BDUP strains detected by paper chromatography

[0056] (2) HPLC method

[0057] The pre-column derivatization-HPLC method is currently widely used to detect GABA content. References include but are not limited to "Analysis of nutritional components and γ-aminobutyric acid of germinated brown rice bran and its effect on alleviating physical fatigue", "Determination of γ-aminobutyric acid in soy products and fermented brown rice by OPA derivatization-HPLC", "Determination of γ-aminobutyric acid in sports drinks by pre-column derivatization-high performance liquid chromatography", and "Simultaneous determination of amino acid and monoamine neurotransmitters in serum by pre-column derivatization-high performance liquid chromatography".

[0058] Accurately weigh the GABA standard to prepare GABA standard solutions at concentrations of 0.04, 0.08, 0.12, 0.16, and 0.20 g / L. Add 100 μL of each GABA standard solution to each concentration, mix thoroughly, and incubate at room temperature for 2 minutes. A 20 μL sample of each reaction solution was injected, and the absorbance peak at 338 nm was measured. The peak area was used to construct a GABA concentration standard curve. Next, add OPA to the diluted BDUP supernatant, incubate and mix thoroughly, and then inject the sample.

[0059] The detection conditions were as follows: the detector wavelength was 338 nm; the chromatographic column was a C18 ODS reverse-phase column (250 mm × 4.6 mm, 5 μm), and the column temperature was room temperature; the mobile phase was 52% 0.1 mol / L phosphate buffer (pH 6.0), 46% methanol, and 2% tetrahydrofuran, filtered through a 0.22 μm microporous membrane before use and ultrasonically degassed before use, and the mobile phase flow rate was 1 mL / min.

[0060] The amount of GABA produced by Lactobacillus gasseri BDUP was detected by three high performance liquid chromatography (HPLC) runs and is shown in Table 2 below:

[0061] Table 2. HPLC detection of GABA production by BP8E strain

[0062] Table 2 shows that the average GABA concentration detected by three HPLC experiments was approximately 0.694 g / L. Many strains reported in the literature require the addition of exogenous glutamate to the culture system, while the BDUP strain of the present invention can produce high levels of GABA without exogenous stimulation.

[0063] Example 4: Effects of BDUP on Depressive-like Behaviors in Chronic Stress Depression Model Mice

[0064] The depression model mouse constructed by chronic stress stimulation is the most commonly used animal model in the study of screening depression therapeutic drugs. Therefore, the depression model mouse constructed by chronic stress stimulation was used as the experimental subject in the following examples.

[0065] (1) Grouping and mouse model establishment

[0066] Thirty-two male Kunming mice weighing approximately 20g were purchased and randomly divided into four groups after one week of adaptive feeding: normal group, model group, BDUP live bacteria group, and fluoxetine hydrochloride group, with 8 mice in each group. Except for the normal control group, mice in other groups were subjected to the following stress stimulation in a random order every week for 4 weeks according to the chronic unpredictable stress (CUMS) method: fasting for 12 hours, restraint for 1 hour, wet bedding for 12 hours, water deprivation for 12 hours, day and night reversal for 24 hours, shock for 1 minute, and electric shock to the sole of the foot. Mice in the BDUP live bacteria group were gavaged 10 times a day. 9 CFU / mL of Lactobacillus gasseri BDUP live bacteria for 28 days. The mice in the normal group and the model group were given corresponding MRS culture medium by gavage, and the mice in the positive group were intraperitoneally injected with fluoxetine hydrochloride (10 mg / kg).

[0067] (2) Mouse tail suspension test

[0068] The mouse tail suspension test is a commonly used behavioral despair model in pharmacodynamics, used to evaluate the efficacy of antidepressant drugs. Changes in depressive-like behavior in mice are determined by observing the duration of immobility during tail suspension. During the experiment, mice are taped 1 cm from the end of their tails and hung upside down with their noses 20-25 cm off the ground. An animal behavior video analysis system is used to record the mice's despair behaviors for 6 minutes (min), and the cumulative immobility time (seconds) within 4 minutes is analyzed. During the experiment, mice are tested separately to avoid visual and auditory interference. The criterion for immobility time during tail suspension is that the mice stop struggling, hang upside down, and remain motionless.

[0069] The statistical results are shown in Figure 3A. In the tail suspension test, the immobility time of the mice in the model group was significantly increased compared with the normal group (p<0.001). Compared with the model group, oral administration of live BDUP bacteria in the BDUP group significantly reduced the absolute immobility time of the test mice (p<0.05), with the average immobility time decreasing from ~340s to ~297s. This effect was only slightly weaker than the improvement effect of the drug fluoxetine hydrochloride on the absolute immobility time of mice (average immobility time ~285s).

[0070] (3) Forced swimming test

[0071] The forced swim test is a rodent behavioral test used to evaluate the efficacy of antidepressant drugs / foods. By observing the despair behavior of mice during the experiment, the effects of antidepressant drugs / foods on the mice's depressive-like behavior can be determined. In the experiment, mice were placed in a 20 cm high, 10 cm diameter transparent plexiglass tank filled with (25±1)°C water at a height of 20 cm. One mouse was manipulated at a time, and an animal behavior video analysis system was used to record the mouse's swimming behavior for 6 minutes. The cumulative immobility time (s) of the mouse in forced swimming within 4 minutes was analyzed. The criterion for the immobility time of the mouse in forced swimming was that the mouse floated in the water, stopped struggling, and only made small limb movements to keep its head above the water.

[0072] Statistics of the different times of each group of mice were taken. As shown in Figure 3B, the immobility time of the mice in the model group was significantly increased compared with the normal group (p<0.001). Lactobacillus gasseri BDUP could significantly reduce the absolute immobility time of the test mice. Compared with the model group, the absolute immobility time was reduced from ~308s to ~230s. The average absolute immobility time of the positive drug group was about 210s.

[0073] A major symptom of depression is a decrease in motivated behavior. Swimming and climbing in the forced swim test and tail suspension test are motivated behaviors in animals, while absolute immobility reflects a passive, unmotivated state. Therefore, these results demonstrate that the Lactobacillus gasseri BDUP strain of the present invention can significantly alleviate depressive-like behaviors in depression model mice, providing a strong scientific basis for the future development of effective antidepressant probiotic products.

[0074] In summary, the Lactobacillus gasseri of the present invention can produce high levels of GABA under culture conditions without the addition of additional glutamate. Three times of paper chromatography detection showed that the average GABA production of BDUP was 839.3 mg / L, and animal experiments showed that BDUP can significantly improve the depressive-like behavior of mice.

[0075] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of a preparation containing at least one of the following in the production of γ-aminobutyric acid: 1) Lactobacillus gasseri BDUP; 2) Lactobacillus gasseri BDUP agent; 3) Lactobacillus gasseri BDUP live bacterial suspension; 4) Lactobacillus gasseri BDUP dead bacteria suspension; 5) Lactobacillus gasseri BDUP metabolites; 6) Lactobacillus gasseri BDUP extract; in, The deposit number of the Lactobacillus gasseri BDUP is CGMCC No.18357.

2. Use of a preparation containing at least one of the following in the preparation of a product for preventing and / or alleviating depression: 1) Lactobacillus gasseri BDUP; 2) Lactobacillus gasseri BDUP agent; 3) Lactobacillus gasseri BDUP live bacterial suspension; 4) Lactobacillus gasseri BDUP dead bacteria solution; 5) Lactobacillus gasseri BDUP metabolites; 6) Lactobacillus gasseri BDUP extract; in, The deposit number of the Lactobacillus gasseri BDUP is CGMCC No.18357.

3. A product for preventing and / or alleviating depression, comprising at least one of the following: 1) Lactobacillus gasseri BDUP; 2) Lactobacillus gasseri BDUP agent; 3) Lactobacillus gasseri BDUP live bacterial suspension; 4) Lactobacillus gasseri BDUP dead bacteria solution; 5) Lactobacillus gasseri BDUP metabolites; 6) Lactobacillus gasseri BDUP extract; in, The deposit number of the Lactobacillus gasseri BDUP is CGMCC No.18357.

4. The product according to claim 3, characterized in that The product is a functional food.

5. The product according to claim 3, characterized in that: The products are fermented foods and health foods.

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