CN117965398A

CN117965398A - Composite probiotics with mood stabilization effect and application thereof

Info

Publication number: CN117965398A
Application number: CN202410370908.5A
Authority: CN
Inventors: 方曙光; 董瑶; 齐咏梅; 司文瑾
Original assignee: WeCare Probiotics Co Ltd
Current assignee: WeCare Probiotics Co Ltd
Priority date: 2024-03-29
Filing date: 2024-03-29
Publication date: 2024-05-03
Anticipated expiration: 2044-03-29
Also published as: CN117965398B

Abstract

The invention relates to a compound probiotic with a mood-stabilizing effect and application thereof, wherein the compound probiotic with the mood-stabilizing effect consists of a bifidobacterium animalis subspecies Bifidobacterium animalis subsp. The two strains can cooperate and promote each other, and the two strains are synergistic in the aspect of mood stabilization efficacy, and are specifically expressed in: effectively regulate and restore the equilibrium state of neurotransmitters, providing a biochemical basis for mood stabilization; significantly reducing stress response in the body, and alleviating the physiological mechanism of mood disorders by reducing the level of stress hormones such as corticosterone; brings about significant positive changes in behavioral aspects, including reduction of anxiety and depression behavior, exhibiting a combined emotional state improving effect.

Description

Composite probiotics with mood stabilization effect and application thereof

Technical Field

The invention belongs to the technical field of probiotics, and relates to a compound probiotic with an emotion stabilizing effect and application thereof.

Background

Mood disorders such as anxiety and depression affect people's daily life and mental health, and traditional treatment methods, including drug therapies such as selective 5-hydroxytryptamine reuptake inhibitors (SSRIs) and psychological therapies such as Cognitive Behavioral Therapies (CBT), are effective to some extent, but have drawbacks such as slow onset of treatment, significant side effects, limited therapeutic effects, etc., often result in poor patient treatment compliance, thus limiting the wide application of these treatments.

With the deep research of human microbiome, the concept of "intestinal brain axis" is gradually widely accepted by the scientific community. This concept reveals the existence of a complex two-way communication system between the gut microbiota and the brain, the health status of the gut directly affecting mood and cognitive function. Against this background, probiotics have shown great potential in improving mood disorders as an effective means of regulating intestinal microbial balance.

Probiotic strains can positively affect mood stabilization through a variety of mechanisms including, but not limited to, improving intestinal barrier function, modulating the immune system to reduce inflammatory responses, and modulating the production of neurotransmission substances, all through signaling on the intestinal brain axis. Despite significant advances in the study of probiotics in mood regulation, there is still a lack of probiotic formulations on the market that are specifically directed to mood stabilization. Most existing probiotic products are mainly focused on improving intestinal health or immunomodulation, and the application potential of probiotics in the field of mental health cannot be fully explored. Therefore, the development of a probiotic agent with mood stabilization effect is particularly urgent and necessary.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide composite probiotics with the function of stabilizing emotion and application thereof, in particular to composite probiotics with the function of stabilizing emotion, a probiotic agent with the function of stabilizing emotion and application thereof in preparing products for preventing, improving or treating depression and/or anxiety.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

In a first aspect, the invention provides a compound probiotic with mood-stabilizing effect, which consists of a bifidobacterium animalis subspecies Bifidobacterium animalis subsp lactis BLa36 strain with the preservation number of CGMCC No. 24029 and a bifidobacterium breve Bifidobacterium breve BBr strain with the preservation number of CGMCC No. 12915.

The invention creatively develops a brand-new probiotic compound mode and a brand-new anxiolytic and antidepressant strategy, namely, the bifidobacterium animalis subspecies Bifidobacterium animalis subsp. Lactis BLa36 strain and bifidobacterium breve Bifidobacterium breve BBr strain are compounded and combined, and the two strains are found to be matched with each other, mutually promoted and synergistically enhanced in the aspect of mood stabilization, and the invention is specifically characterized in that: (1) Effectively regulate and restore the equilibrium state of neurotransmitters, providing a biochemical basis for mood stabilization; (2) Significantly reducing stress response in the body, and alleviating the physiological mechanism of mood disorders by reducing the level of stress hormones such as corticosterone; (3) Brings about significant positive changes in behavioral aspects, including reduction of anxiety and depression behavior, exhibiting a combined emotional state improving effect.

Under the condition of consistent using bacterial load, compared with the intervention mode of single BLa36 strain or single BBr60 strain, the effect of the combination of the two bacteria on mood stabilization is obviously improved. Therefore, the compound probiotics have good prospect in preparing products for preventing, relieving or treating anxiety and/or depression. Meanwhile, both bacteria are probiotics, the product safety is high, and the resistance is not easy to generate.

The preparation method of the composite probiotics adopts a technical method conventional in the field, and can be exemplified by: activating BLa36 strain or BBr60 strain, and then respectively inoculating the activated BLa36 strain or BBr60 strain into a culture medium for culture to obtain a culture solution; centrifuging the culture solution, and re-suspending the bacteria to obtain bacterial suspension; mixing the two bacterial suspensions according to the ratio of the viable bacteria number. Or further adding a protective agent for freeze drying to obtain a freeze-dried bacterial powder product.

Preferably, the ratio of the viable count of the strain Bla36 to that of the strain BBr60 is 1:20-20:1, such as 1:20、1:18、1:15、1:12、1:10、1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1、12:1、15:1、18:1、20:1, and other specific values within the numerical range can be selected, and will not be described in detail herein.

In a second aspect, the present invention provides a probiotic with mood stabilising effect, the strain of which comprises the complex probiotic of the first aspect.

Preferably, in the probiotic agent, the total number of viable bacteria is not less than 1×10 ¹⁰ CFU/mL or 1×10 ¹⁰ CFU/g, for example 1×10¹⁰CFU/g（CFU/mL）、2×10¹⁰CFU/g（CFU/mL）、5×10¹⁰CFU/g（CFU/mL）、8×10¹⁰CFU/g（CFU/mL）、1×10¹¹CFU/g（CFU/mL）、5×10¹¹CFU/g（CFU/mL）、1×10¹²CFU/g（CFU/mL）、1×10¹³CFU/g（CFU/mL）、1×10¹⁴CFU/g（CFU/mL）, and other specific values within the numerical range can be selected, which will not be described in detail herein.

Preferably, the formulation of the probiotic agent comprises freeze-dried powder, capsules, tablets or granules.

The formulation of the probiotics related to the invention is not limited, and comprises the most commonly used freeze-dried powder, or further prepared capsules, tablets or granules. The lyophilized powder can be prepared by the following method:

activating BLa36 strain and BBr60 strain, and then respectively inoculating the activated BLa36 strain and BBr60 strain into a culture medium for culture to obtain a culture solution; centrifuging the culture solution to obtain thalli; re-suspending the thalli by using a freeze-drying protective agent to obtain re-suspension; freeze-drying the heavy suspension, and mixing according to a proportion to obtain the finished product.

Preferably, the medium includes an MRS medium.

Preferably, the MRS medium includes, in concentration: 8-12 g/L of peptone, 8-12 g/L of beef extract, 15-25 g/L of glucose, 10-20 g/L of lactose, 3-7 g/L of yeast powder, 1-3 g/L、K₂PO₄·3H₂O 2-3 g/L、MgSO₄·7H₂O 0.05-0.2 g/L、MnSO₄ 0.01-0.1 g/L、 Tween 80 0.5-2 mL/L of diammonium hydrogen citrate and 0.1-1 g/L of cysteine hydrochloride.

Preferably, the lyophilization is by vacuum freezing.

Preferably, the probiotic agent further comprises a lyoprotectant and/or a prebiotic.

Preferably, the lyoprotectant comprises any one or a combination of at least two of skim milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

The prebiotic comprises any one or a combination of at least two of fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, isomalto-oligosaccharide, soy oligosaccharide, inulin, spirulina, arthrospira, coriolus versicolor polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.

In a third aspect, the present invention provides the use of a complex probiotic according to the first aspect or a probiotic according to the second aspect in the manufacture of a product for the prevention, amelioration or treatment of depression and/or anxiety.

Preferably, the product further comprises auxiliary materials.

Preferably, the auxiliary material is selected from any one or a combination of at least two of a filler, a binder, a wetting agent, a disintegrating agent, an emulsifying agent, a cosolvent, a solubilizer, an osmotic pressure regulator, a colorant, a pH regulator, an antioxidant, a bacteriostatic agent or a buffering agent.

Compared with the prior art, the invention has the following beneficial effects:

The invention creatively develops a brand-new probiotic compound mode and a brand-new anxiolytic and antidepressant strategy, namely, the bifidobacterium animalis subsp Bifidobacterium animalis subsp. Lactis BLa36 strain and bifidobacterium breve Bifidobacterium breve BBr strain are combined, and the two strains are found to be matched with each other, mutually promoted and synergistically enhanced in the aspect of stabilizing emotion efficacy, and under the condition of consistent using bacterial load, compared with the single BLa36 strain or single BBr60 strain intervention mode, the effect of the two bacteria in the aspect of emotion stabilization is obviously improved. Therefore, the compound probiotics have good prospect in preparing products for preventing, relieving or treating anxiety and/or depression. Meanwhile, both bacteria are probiotics, the product safety is high, and the resistance is not easy to generate.

Drawings

FIG. 1 is a graph showing the statistical results of open arm time ratio (%) for each group of mice in the elevated plus maze experiment;

FIG. 2 is a graph showing statistics of the number of times each group of mice enters the open arm in the overhead plus maze experiment;

FIG. 3 is a graph of the statistical results of the immobility time(s) of each group of mice in the tail suspension experiment;

FIG. 4 is a graph showing the statistical results of the immobility time(s) of each group of mice in the forced swimming experiment;

FIG. 5 is a graph showing the statistical results of serum corticosterone content in each group of mice;

FIG. 6 is a graph showing the statistical result of the gamma-aminobutyric acid content of hippocampus of each group of mice;

FIG. 7 is a graph showing the statistical results of the hippocampal 5-hydroxytryptamine content of each group of mice;

FIG. 8 is a graph showing the statistical results of the hippocampal 5-hydroxyindoleacetic acid content of each group of mice.

The BLa36 strain related by the invention is classified and named as bifidobacterium animalis subsp Bifidobacterium animalis subsp. The korean district North Star, beijing city, part No. 1, no. 3.

The BBr60 strain related by the invention is classified and named as bifidobacterium breve Bifidobacterium breve, the preservation unit is China general microbiological culture Collection center, the preservation time is 2016, 08 and 29 days, the preservation number is CGMCC No. 12915, and the address is: the korean district North Star, beijing city, part No. 1, no. 3.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

The BLa36 strain according to the following examples is classified and named as bifidobacterium animalis subsp Bifidobacterium animalis subsp. The korean district North Star, beijing city, part No. 1, no. 3.

The BBr60 strain related to the following examples is classified and named as bifidobacterium breve Bifidobacterium breve, the preservation unit is China general microbiological culture Collection center (China Committee for culture Collection of microorganisms), the preservation time is 2016/08/29, the preservation number is CGMCC No. 12915, and the address is: the korean district North Star, beijing city, part No. 1, no. 3.

Peptone, beef extract, glucose, lactose, yeast powder, diammonium hydrogen citrate, K ₂PO₄·3H2O、MgSO₄·7H₂O、MnSO₄, tween 80 and cysteine hydrochloride referred to in the examples below were purchased from the national pharmaceutical group chemical company.

The following examples relate to the following media:

MRS Medium (g/L): 10g/L of peptone, 10g/L of beef extract, 15g/L of glucose, 15g/L of lactose, 5g/L of yeast powder, 1mL/L of ammonium citrate 2g/L、K₂PO₄·3H₂O 2.6g/L、MgSO₄·7H₂O 0.1g/L、MnSO₄ 0.05g/L、 Tween 80 and 0.5g/L of cysteine amino acid salt.

The bacterial suspensions referred to in the following examples: inoculating the required strain into MRS liquid culture medium, culturing at 37deg.C for 24 hr for activation, and continuously activating for 2 times to obtain activating solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing at 37 ℃ for 24 hours to obtain bacterial solution; centrifuging the bacterial liquid at 6000g for 10min, and re-suspending the bacterial body by using PBS.

Statistical analysis of test result data using ggplot of R language, # represents p <0.001 compared to control group; compared to the model group, p <0.001, p <0.01, p <0.05, ns.

Examples

The present example explores the mood stabilization ability of complex probiotics on sleep disturbance model mice:

Sleep disorders, such as poor sleep quality and insufficient sleep, have a direct link to mood disorders (including anxiety and depression) that can significantly affect mood reactivity and restorability, and thus mood stabilization. By simulating a sleep disturbance model of human sleep disturbance states, causal relationships between sleep and emotion can be deeply explored, and how sleep disturbance affects mood stabilization through neurotransmitter changes, hormonal level fluctuations, and functional changes in brain mood adjustment areas.

(1) Test animals: SPF-grade male ICR mice (70, purchased from Shanghai laboratory animal center) were 18-22g in body weight. These mice were kept under a controlled environment, maintained at room temperature of 22±2 ℃ and humidity of 55% ±5%, following a 12h light/dark cycle. They can eat and drink water at will. All experimental procedures involving mice were in compliance with the ethical guidelines for animal care and use prescribed by the Shanghai laboratory animal Care and animal Experimental center. In the experiment, a roller sleep disturbance instrument is used for carrying out sleep disturbance on a mouse, and a food box and a drinking nozzle are arranged in the roller so as to ensure the basic life requirement of animals.

(2) Grouping animals: after 1 week of adaptive feeding in the above mice, the mice were randomly divided into 7 groups (10 per group): control group (CTL group), model group (MC group), BLa36 group (S1 group), BBr60 group (S2 group), commercial bifidobacterium animalis subspecies lactis strain ATCC27536 group (S3 group), complex bacteria group 1 (BLa 36+ BBr60 group, viable count ratio 1:1, S4 group), complex bacteria group 2 (ATCC 27536+ BBr60 group, viable count ratio 1:1, S5 group).

(3) Animal modeling and intervention method:

The CTL group does not receive sleep disturbance stress and only is used for lavage to administer distilled water; the MC group receives sleep disturbance stress and irrigates the stomach with distilled water; the probiotic group received sleep disturbance and was fed with purified water containing 1X 10 ⁹ CFU/mL of bacterial liquid (1X 10 ⁹ CFU/day). After all mice were acclimatized for 7 days, other mice except for the control group were placed in the roller for continuous acclimatization for 3 days, 3 hours per day (roller rotation speed 1 r/min). After the end of the adaptation period, mice were placed in a sleep disruptor for 28 days of sleep disruption modeling and probiotic dosing was started on the first day until the end of the experiment.

(4) Behavioral evaluation:

after modeling and administration, behavioural tests including overhead plus maze tests, tail suspension tests and forced swimming tests were performed to evaluate the effects of sleep disturbances and drug interventions on the behaviour of mice.

(4.1) Elevated plus maze experiment: the elevated plus maze test can be used to evaluate anxiety behavior in animals, specifically by: mice were placed in the central region of the maze facing the open arms, animals were allowed to move freely for 6 minutes, and the time ratio (%) of each group of mice at the open arms and the number of times of entering the open arms were counted.

As shown in fig. 1 and 2, it can be seen that the model mice exhibited significant anxiety behavior in the experiment, particularly with a significant reduction in both residence time and number of entries in the open arm, as compared to the control group, and these behavioral changes indicated that the model mice were characterized by significant mood disorders. After probiotic intervention, especially in the S4 group, the above behavioural indexes all show significant reversion. In particular, the residence time and the number of entries in the experiment of the mice of the probiotic intervention group are significantly increased, which indicates that the probiotic intervention effectively improves the mood disorder condition of the mice and reduces the anxiety behavior.

(4.2) Tail suspension experiment:

The tail tip (2 cm a from the tail tip) of the mouse was attached to the suspension rod (30 a cm a from the ground) with adhesive tape. The mouse is hung by touching, and the mouse can be struggled to try to escape from the dilemma, but can not get trapped in the hopeless state and still stand still, so as to show depression-like behaviors, and the longer the standing time is, the more depression is caused. Athletic activity was monitored by a video tracking system for 6 min a and the immobility time (immobility defined as movement without voluntary or evasive) of each animal during the test was calculated.

As shown in fig. 3, it can be seen from the graph that the mice in the model group showed significant depression symptoms compared with the control group, and the mice in the model group showed significant increase in the time ratio of immobility in the experiment, and the behavioral indexes showed significant reversal after the intervention of probiotics, especially in the S4 group. This suggests that probiotic intervention effectively improves the mood disorder status of the mice, alleviating depression-like behaviour.

(4.3) Forced swimming experiment: a30 cm glass jar was used, containing 23-25℃water. All mice were forced to swim 15 min for training on the first day. The next day, mice were placed in the same environment 10 min. The motor activity was monitored using a video tracking system and the immobility time of each animal during the test was calculated (immobility was defined as the immobility condition except for the floating action required to keep the head above the water surface).

As shown in fig. 4, it can be seen from the graph that the mice in the model group showed significant depression symptoms compared with the control group, and the mice in the model group showed significant increase in the time ratio of immobility in the experiment, and the behavioral indexes showed significant reversal after the intervention of probiotics, especially in the S4 group. This suggests that probiotic intervention effectively improves the mood disorder status of the mice, alleviating depression-like behaviour.

(5) Sample collection:

Mice were bled 30 minutes after dosing on the day after the behavioral tests, and serum was left to stand at 4 ℃ for 4 hours, centrifuged, and stored at-80 ℃ for later use. Subsequently, brain tissue collection was performed, and the hippocampal tissue of interest was taken out and immediately after that, the sample was transferred to a-80 ℃ refrigerator for preservation. Sample pretreatment involves homogenization of brain tissue using an ultrasonic disruptor, protein precipitation by addition of an internal standard solution and trifluoroacetic acid, and subsequent supernatant is used for neurotransmitter content determination.

(6) And (3) index detection:

(6.1) Using an ELISA kit (Wohan purity Biotechnology Co., ltd.), quantitative determination of serum corticosterone concentration was performed according to the manufacturer's instructions.

The results are shown in fig. 5, in which serum corticosterone levels were significantly elevated in the model group compared to the control group, and this change indicates an enhanced physiological response in the stress state of the model mice, reflecting a highly stressed response in the body in the mood disorder state. Serum corticosterone levels were significantly reduced following probiotic intervention, especially in group S4. This result shows that probiotic intervention can effectively alleviate stress response of mice with model of mood disorder, reduce excessive corticosterone level, thereby stabilizing mood at physiological level and alleviating negative effects caused by stress.

(6.2) Neurotransmitter (gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA)) levels in the hippocampus of mice were quantitatively determined using high performance liquid chromatography.

The results, as shown in fig. 6, 7 and 8, indicate that the levels of the hippocampal neurotransmitters gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were significantly reduced in the model group compared to the control group, and this result indicates that the balance of the hippocampal neurotransmitters was disrupted in the state of mood disorder, particularly the level of transmitters closely related to mood regulation was reduced, reflecting the neurochemical basis of mood disorder. The level of the hippocampal neurotransmitter gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) of the mice after the intervention of probiotics is obviously increased, and the effect of the S4 group is better. This suggests that probiotic intervention can effectively regulate the neurochemical environment of model mice of mood disorders, restore the equilibrium state of neurotransmitters, thereby molecularly stabilizing the mood against mood disorders.

The applicant states that the technical solution of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be implemented by the above embodiments. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

1. The compound probiotics with the mood-stabilizing effect is characterized by comprising a bifidobacterium animalis subspecies Bifidobacterium animalis subsp. Lacti BLa36 strain with the preservation number of CGMCC No. 24029 and a bifidobacterium breve Bifidobacterium breve BBr strain with the preservation number of CGMCC No. 12915.

2. The compound probiotic with mood-stabilizing efficacy according to claim 1, characterized in that the ratio of viable count of the BLa36 strain to BBr60 strain is 1:20-20:1.

3. A probiotic with mood stabilising effect, characterised in that the strain in the probiotic comprises a complex probiotic according to claim 1 or 2.

4. A probiotic with mood stabilising efficacy according to claim 3, wherein the total number of viable bacteria in the probiotic is not less than 1 x 10 ¹⁰ CFU/mL or 1 x 10 ¹⁰ CFU/g.

5. A probiotic with mood stabilising effect according to claim 3, wherein the probiotic is in a dosage form comprising a lyophilized powder, capsule, tablet or granule.

6. A probiotic with mood stabilising effect according to claim 3, wherein the probiotic further comprises a lyoprotectant and/or a prebiotic.

7. The mood-stabilizing probiotic according to claim 6, wherein the lyoprotectant comprises any one or a combination of at least two of skim milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol;

8. Use of a complex probiotic according to claim 1 or 2 or a probiotic according to any one of claims 3 to 7 for the preparation of a product for the prevention, amelioration or treatment of depression and/or anxiety.

9. The use according to claim 8, wherein the product further comprises adjuvants.

10. The use according to claim 9, wherein the adjuvant is selected from any one or a combination of at least two of fillers, binders, wetting agents, disintegrants, emulsifiers, co-solvents, solubilisers, tonicity adjusting agents, colorants, pH adjusting agents, antioxidants, bacteriostats or buffers.