Bifidobacterium animalis with depression improving effect and application of bifidobacterium animalis

Abstract

The invention relates to the technical field of microorganisms, in particular to bifidobacterium animalis with a depression improving effect and application of the bifidobacterium animalis. Experimental results show that the bifidobacterium animalis CP-9 for preventing and relieving depression, provided by the invention, can reduce the content of proinflammatory cytokines in serum of depression mice. Therefore, the bifidobacterium animalis CP-9 has a good development prospect in the aspect of preparing products for preventing and / or treating depression or in the aspect of preparing products for reducing the content of pro-inflammatory cytokines in serum of depressive mice.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to an animal bifidobacterium with depression-improving efficacy and application thereof. Background Art

[0002] Currently, monoamine neurotransmitter reuptake inhibitors (MARIs), such as fluoxetine and paroxetine, are commonly used clinically as antidepressants. However, while these drugs are effective, they are also associated with side effects such as nausea, vomiting, insomnia, and anxiety. Therefore, the development of an effective product that can alleviate depression with minimal side effects is urgent.

[0003] Probiotics are a general term for active microorganisms that colonize the host and exert beneficial effects. They regulate the balance of intestinal flora, promote the conversion and absorption of nutrients, or synthesize related metabolites, thereby producing individual microorganisms or a defined mixture of microorganisms that are beneficial to the host's vital activities. Intestinal bacteria can influence the nervous system through their metabolites. The intestine produces neurotransmitters that the brain uses for psychological processes such as emotion and memory. Studies have found that probiotics can regulate mood and, to a certain extent, alleviate depression. However, there are currently few reports of probiotics with significant therapeutic effects on depression, and related products still require further development. Summary of the Invention

[0004] In response to the technical problem that existing probiotic products are not significantly effective in treating depression, the present invention provides a new application of Bifidobacterium animalis CP-9 for preventing and alleviating depression.

[0005] The present invention discloses an animal bifidobacterium (Bifidobacterium animalis subsp. Lactis) CP-9 and an application of a bacterial agent containing the animal bifidobacterium (Bifidobacterium animalis subsp. Lactis) CP-9 in preparing a product for reducing the content of pro-inflammatory cytokines in the serum of depressed mice. The animal bifidobacterium (Bifidobacterium animalis subsp. Lactis) CP-9 has a collection number of CCTCC M2014588. The Chinese invention patent CN105985918A - an anti-obesity lactic acid bacteria strain and an application thereof in a food composition and a pharmaceutical composition - has been disclosed.

[0006] The present invention discloses application of Bifidobacterium animalis subsp. Lactis CP-9 and a bacterial agent containing Bifidobacterium animalis subsp. Lactis CP-9 in preparing a product for preventing and / or treating depression. The accession number of Bifidobacterium animalis subsp. Lactis CP-9 is CCTCC M2014588.

[0007] Preferably, the bacterial agent also includes maltooligosaccharide.

[0008] Preferably, the preparation method of the bacterial agent is as follows:

[0009] (1) activating Bifidobacterium animalis subsp. Lactis CP-9 on a modified MRS plate medium, inoculating the activated Bifidobacterium animalis subsp. Lactis CP-9 into a modified MRS liquid medium, and cultivating a bacterial liquid of Bifidobacterium animalis subsp. Lactis CP-9;

[0010] (2) After centrifuging the bacterial solution, the bacterial cells were collected, washed with sterile physiological saline, and resuspended in reconstituted skim milk to obtain a Bifidobacterium animalis subsp. Lactis CP-9 suspension; adjusting the concentration of the suspension, and freeze-drying to obtain Bifidobacterium animalis subsp. Lactis CP-9 bacterial powder;

[0011] (3) Compounding the Bifidobacterium animalis subsp. Lactis CP-9 bacterial powder with isomaltooligosaccharide to prepare the Bifidobacterium animalis subsp. Lactis CP-9 bacterial agent.

[0012] Preferably, in step (1), the inoculation amount of Bifidobacterium animalis subsp. Lactis CP-9 is 1%.

[0013] Preferably, in step (1), the culture temperature is 37° C. and the culture time is 18 h.

[0014] Preferably, in step (2), the concentration of the suspension is adjusted to 1.0×10 9 ~2.0×10 9 cfu / mL.

[0015] Preferably, in step (3), the ratio of Bifidobacterium animalis subsp. Lactis CP-9 bacterial powder to isomaltooligosaccharide is 3:1.

[0016] Beneficial effects of the present invention

[0017] The present invention provides a strain of Bifidobacterium animalis CP-9 for preventing and alleviating depression. Experimental results show that it can reduce the levels of pro-inflammatory cytokines in the serum of depressed mice. Therefore, Bifidobacterium animalis CP-9 has promising development prospects for the preparation of products for preventing and / or treating depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the result of ELISA detection of neurotransmitter 5-HT;

[0019] Figure 2 Diagram for preparation and analysis of hippocampal pathological sections;

[0020] Figure 3 mRNA expression in mouse hippocampus. DETAILED DESCRIPTION

[0021] Example 1 Preparation of bacterial agent

[0022] (1) Preparation of modified MRS liquid medium and modified MRS plate medium: 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of potassium dihydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 0.5 g of L-cysteine and 1000 mL of distilled water were mixed, the pH was adjusted to natural pH, the bacterial solution was stirred, and then sterilized at 121°C and 0.1 MPa for 20 min to prepare the modified MRS liquid medium; the modified MRS plate medium was prepared by adding 15 g / L agar to the modified MRS liquid medium, pouring it into a plate and letting it cool.

[0023] (2) Activating Bifidobacterium animalis CP-9 on a modified MRS plate medium, inoculating the activated Bifidobacterium animalis CP-9 into a modified MRS liquid medium at a 1% inoculum rate, and culturing in an anaerobic incubator at 37° C. for 18 h to obtain a bacterial solution;

[0024] (3) After centrifugation of the bacterial solution, the cells were collected, washed with sterile saline, and resuspended in 15% (w / w) reconstituted skim milk to obtain a suspension; the concentration of the suspension was adjusted to 1.0×10 9 ~2.0×10 9 cfu / mL, freeze-dried to obtain Bifidobacterium animalis CP-9 bacterial powder;

[0025] (4) The above-mentioned animal Bifidobacterium CP-9 bacterial powder is compounded with oligosaccharide to prepare the animal Bifidobacterium CP-9 bacterial agent.

[0026] According to different dosage requirements, by adjusting the compounding ratio of animal Bifidobacterium CP-9 powder and isomaltooligosaccharide, products with different animal Bifidobacterium CP-9 contents can be obtained.

[0027] Example 2 Preventive Effect of Animal Bifidobacterium CP-9 on Depression in Mice

[0028] 1. Establishment of a Depression Mouse Model

[0029] Forty 6- to 8-week-old SPF-grade BALB / cA healthy male mice were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. and subjected to one-week adaptive feeding. The breeding conditions were: sufficient food and water, temperature of 25°C, 12 hours of light and 12 hours of darkness.

[0030] Experimental mice were intervened by gavage of normal saline in the blank control group and the model group, and drugs in the positive control group. The other three groups were gavage of 1×10 7～ 1×10 9 CFU / d probiotics were administered orally for 21 days at 8:00-9:00 AM.

[0031] Modeling method: The experimental group was fed for 21 days before modeling. Five experimental groups were intraperitoneally injected with 0.2 mL of LPS solution 1 hour after oral gavage intervention; the control group was intraperitoneally injected with an equal volume of sterile saline 1 hour after oral gavage intervention. LPS injection intervention was performed once.

[0032] Model establishment success test: 4 hours after intraperitoneal injection of LPS, mice were subjected to behavioral tests, using the sugar water preference test, forced swim test, and behavioral tests as evaluation criteria.

[0033] 2. Behavioral assessment

[0034] (1) Sugar water preference test

[0035] Preparation before the experiment: 1% sucrose solution and pure water were respectively divided into 50 mL centrifuge tubes, and 6 groups of mice were placed from ordinary cages into experimental cages, and each mouse was housed individually.

[0036] Acclimation Phase: Before the formal experiment, mice were first given two solutions: a 2% (w / w) sucrose solution and pure water. The acclimation phase lasted 72 hours. To prevent the mice from habituating to a side-biased orientation, the position of the sucrose solution and pure water was switched every 12 hours. After 72 hours, the mice were deprived of food and water for 6 hours.

[0037] Formal experiment: Each cage of mice was given 2% sucrose solution and pure water. After 12 hours, the total mass of liquid consumed by each water bottle was measured and the sucrose preference index was calculated using the following formula:

[0038] Sucrose preference index = (sucrose solution consumption / total liquid consumption) × 100%

[0039] The experimental data obtained were expressed as "mean ± standard deviation" and analyzed using statistical software (SPSS26.0). When the comparisons among multiple groups met the normal distribution and homogeneity of variance, one-way ANOVA was used for analysis of variance.

[0040] The results are shown in Table 1. Compared with the blank group, the sucrose preference index of other groups was significantly reduced (P < 0.01), indicating that the depressed mouse model was successfully established. The sucrose preference index of the treatment group increased compared with the model group, and there was a significant difference, indicating that taking animal Bifidobacterium CP-9 has a certain preventive effect on depression in mice.

[0041] (2) Forced swimming test

[0042] Place mice in cylindrical glass bottles (25 cm high, 10 cm in diameter). Maintain the water level at 16 ± 2 cm and the temperature at 26 ± 2°C. Use black cardboard to separate adjacent cylinders to eliminate interference. After 2 minutes, observe the cumulative inactivity time of the mice over a 4-minute period.

[0043] The results are shown in Table 1. Compared with the blank group, except for group C3, the rest groups had longer immobility time, indicating that the depression mouse model was effective and that groups C1 and C3 had a certain preventive effect on depression in mice.

[0044] Table 1 Preventive effect of animal Bifidobacterium CP-9 on depression in mice

[0045] Group Sugar water preference index Forced swim test immobility time / total time Blank control group 0.78±0.03** 0.28±0.05** Model Group 0.44±0.05 0.41±0.04 Positive control group 0.54±0.02** 0.28±0.07** Group C1 0.53±0.03* 0.37±0.11 Group C2 0.6±0.03** 0.34±0.03* Group C3 0.48±0.03 0.27±0.04**

[0046] Note: Compared with the M model group, * has significant difference (P < 0.05), ** has significant difference (P < 0.01).

[0047] Example 3 Effect of Animal Bifidobacterium CP-9 on the Concentrations of Proinflammatory Cytokines IL-1, IL-6, and TNF-α in Serum of Depressed Mice

[0048] The impact of degree

[0049] After the behavioral experiments, mice were anesthetized with an intraperitoneal injection of 10% chloral hydrate. Eyeballs were removed and blood was collected. The blood was allowed to stand for 2 hours and then refrigerated at 4°C. After the blood was clearly separated, it was centrifuged at 3500 rpm for 15 minutes at 4°C. The supernatant was collected and stored at -80°C until further use. Serum levels of cytokines IL-1, IL-6, and TNF-α were measured according to the ELISA kit instructions. Data were expressed as mean ± standard deviation and analyzed using SPSS 26.0. One-way ANOVA was used for comparisons between groups when normal distribution and homogeneity of variance were met. Results were considered statistically significant with P < 0.05.

[0050] Table 2 Proinflammatory cytokine levels in mice in each group

[0051]

[0052] Note: Compared with the M model group, * has significant difference (P < 0.05), ** has significant difference (P < 0.01); compared with the K blank group, # has significant difference (P < 0.05), ## has significant difference (P < 0.01).

[0053] According to the data in Table 2, compared with the blank group, the serum levels of IL-1, IL-6, and TNF-α in the model group mice were significantly increased, indicating that the inflammatory indicators in the depressed mice were elevated (P < 0.01). Compared with the model group, the serum levels of IL-1, IL-6, and TNF-α in the mice in the animal Bifidobacterium CP-9 group were significantly reduced (P < 0.01). This suggests that animal Bifidobacterium CP-9 can reduce the levels of proinflammatory cytokines IL-1, IL-6, and TNF-α in depressed mice and has a certain alleviating effect on depression in mice.

[0054] ELISA detection of neurotransmitter 5-HT

[0055] The results are as follows Figure 1 As shown in the results, compared with the blank group, the 5-HT level in the serum of the mice in the depression model group was significantly decreased (P<0.01); compared with the depression model group, the 5-HT levels in the serum of the mice in the fluoxetine group (positive group) and the CP-9 groups at different doses were increased (P<0.01), and the 5-HT level was the highest in the CP-9 medium-dose group. In addition, the efficacy of the CP-9 groups at different doses was comparable to that of the fluoxetine group.

[0056] Group A (neuroinflammation) hippocampal pathological section preparation and analysis

[0057] Mouse hippocampus tissue sections were observed. Figure 2As can be seen, the hippocampal cells in the blank group were structurally intact, while the cells in the model group showed condensed nuclei and disorganized arrangement. The positive group had significantly fewer degenerated cells compared to the model group. The results of the various CP-9 concentration groups need to be analyzed in conjunction with other data.

[0058] mRNA expression in mouse hippocampus

[0059] Methods: Mice were anesthetized with chloral hydrate for 24 hours after LPS modeling, and then decapitated. The hippocampal tissue was removed and stored at -80°C for later use. The mRNA expression of four targets, HTRIA, NF-κB, MAPK14, and Nr3c1, in the hippocampal tissue of mice was detected. The results are as follows: Figure 3 shown.

Claims

1. Use of Bifidobacterium animalis subsp. Lactis CP-9 and a bacterial agent containing Bifidobacterium animalis subsp. Lactis CP-9 in the preparation of a product for reducing the content of pro-inflammatory cytokines in the serum of depressed mice, characterized in that: The accession number of the Bifidobacterium animalis subsp. Lactis CP-9 is CCTCC M2014588.

2. Use of Bifidobacterium animalis subsp. Lactis CP-9 and a bacterial agent containing Bifidobacterium animalis subsp. Lactis CP-9 in the preparation of a product for preventing and / or treating depression, characterized in that: The accession number of the Bifidobacterium animalissubsp.Lactis CP-9 is CCTCC M2014588.

3. The use according to claim 1 or 2, characterized in that The bacterial agent also includes maltooligosaccharide.

4. The use according to claim 1 or 2, characterized in that The preparation method of the bacterial agent is as follows: (1) activating Bifidobacterium animalis subsp. Lactis CP-9 on a modified MRS plate medium, inoculating the activated Bifidobacterium animalis subsp. Lactis CP-9 into a modified MRS liquid medium, and cultivating a bacterial liquid of Bifidobacterium animalis subsp. Lactis CP-9; (2) After centrifuging the Bifidobacterium animalis subsp. Lactis CP-9 bacterial solution, collecting the bacterial cells, washing them with sterile physiological saline, and resuspending them in reconstituted skim milk to obtain a Bifidobacterium animalis subsp. Lactis CP-9 suspension; adjusting the concentration of the suspension, and freeze-drying the suspension to obtain Bifidobacterium animalis subsp. Lactis CP-9 bacterial powder; (3) Compounding the Bifidobacterium animalis subsp. Lactis CP-9 bacterial powder with isomaltooligosaccharide to prepare the Bifidobacterium animalis subsp. Lactis CP-9 bacterial agent.

5. The use according to claim 4, characterized in that In step (1), the inoculation amount of Bifidobacterium animalis subsp. Lactis CP-9 was 1%.

6. The use according to claim 4, characterized in that In step (1), the culture temperature is 37°C and the culture time is 18 hours.

7. The use according to claim 4, characterized in that In step (2), the concentration of the suspension was adjusted to 1.0×10 9 ~2.0×10 9 cfu / mL.

8. The use according to claim 4, characterized in that In step (3), the ratio of Bifidobacterium animalis subsp. Lactis CP-9 bacterial powder to isomaltooligosaccharide is 3:1.

Images