CN113215034B - Bifidobacterium breve capable of relieving Alzheimer's disease and increasing content of fecal acetic acid and application thereof - Google Patents

Abstract

The invention discloses a strain of Bifidobacterium breve capable of relieving Alzheimer's disease and increasing fecal acetic acid content and its application, belonging to the field of microorganisms. The Bifidobacterium breve CCFM1179 provided by the present invention can significantly improve the cognitive and memory impairments of Alzheimer's mice, reduce the content of Aβ1-42 in the brains of Alzheimer's mice, and increase the levels of Alzheimer's mice The levels of brain-derived neurotrophic factor and postsynaptic density protein in the brain increase the content of serotonin in the serum of Alzheimer's mice; and can regulate the intestinal flora and its metabolites. The Bifidobacterium breve CCFM1179 provided by the invention can be used to prepare a pharmaceutical composition for relieving dementia, Alzheimer's disease and improving cognitive impairment, and can also be used as a microecological preparation in medicine, fermented food or health care product, and has a very wide range of application prospects.

Description

A strain of Bifidobacterium breve that can alleviate Alzheimer's disease and increase fecal acetate content and its application

technical field

The invention relates to a Bifidobacterium breve capable of relieving Alzheimer's disease and increasing fecal acetic acid content and its application, belonging to the field of microorganisms.

Background technique

Alzheimer's disease, also known as senile dementia, is a neurodegenerative disease clinically manifested as a decline in learning and cognitive abilities and a gradual loss of self-care ability. The clinical features of Alzheimer's disease are insidious onset, progressive aggravation, and death due to complications within 10-20 years of onset. Alzheimer's disease accounts for 60-80% of all dementia patients and is the most common type of dementia.

Regarding the pathogenesis of Alzheimer's disease, there are currently more than a dozen hypotheses in modern medicine. The most studied are the theory of cholinergic loss, the theory of Tau protein hyperphosphorylation, the theory of oxidative stress, the theory of beta amyloid toxicity, Inflammatory factor theory, etc. The two most characteristic pathological changes in Alzheimer's disease are senile plaques formed by Aβ deposition and neurofibrillary tangles caused by accumulation of abnormally phosphorylated Tau protein in cells. Since 1992, the amyloid cascade hypothesis has played an important role in explaining the pathogenesis of Alzheimer's disease. Aβ is considered to be a key factor in the pathogenesis of Alzheimer's disease, and Aβ1-42 easily forms fibrillar amyloid aggregates, which are known as the main component of senile plaques.

Currently, about 50 million people worldwide suffer from Alzheimer's disease, and the number of this group is expected to continue to grow. However, the development of Alzheimer's drugs has been slow. Current treatments for Alzheimer's disease include drug therapy, immunotherapy, and dietary supplementation with natural extracts. Acetylcholinesterase inhibitor (AChEI) is by far the most widely used Alzheimer's drug in clinical practice. Among the five drugs approved by the FDA for the treatment of Alzheimer's disease, in addition to memantine, which is an NMDA receptor antagonist, the other four are Tacrine, Donepezil, Galanthamine, and Kabbah. Rivastigmine is an acetylcholinesterase inhibitor. Clinically, the five currently FDA-approved drugs for Alzheimer's disease are all symptomatic treatment drugs, and the drugs that affect the disease process are still in the preliminary research stage. In addition, the effect of a single drug is not significant, and the side effects of neurological drugs are relatively large. Therefore, it is particularly important to explore methods of intervening in the early stage of Alzheimer's disease to delay the disease process, which has a very broad market potential.

The "brain-gut axis" is a new concept proposed in recent years. As a two-way communication system between intestinal bacteria and the brain, it mainly regulates the function and behavior of the brain through neural pathways, endocrine pathways and immune pathways. With the deepening of the research on the "brain-gut axis", it has been found that probiotics can regulate the composition of intestinal flora through the vagus nerve, neuroimmune system, neurotransmitters, and microbial metabolites, thereby alleviating neurological diseases. Clinical trials have confirmed that probiotics, prebiotics and fecal transplantation can modulate the composition of intestinal flora and effectively improve neurological diseases such as depression and autism in rodents. At present, the types of "psychotic probiotics" known to be used for the prevention and treatment of neurological diseases are too scarce; secondly, due to the complexity of intestinal flora and the difficulty of research methods, there are few clinical studies on intestinal flora, and its participation in The mechanism of body metabolism and interaction is not yet clear; therefore, it is very important to screen out a probiotic that can mediate gut flora to alleviate Alzheimer's disease, and to explore its functions in depth to develop probiotic products with higher health value. important. In addition, the development of psychoactive probiotics will also open up new avenues and solutions for alleviating Alzheimer's disease through dietary interventions.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a strain of Bifidobacterium breve CCFM1179 (Bifidobacterium breve), which was preserved in the Guangdong Provincial Microbial Culture Collection Center on March 31, 2021, and the preservation address is No. 59, No. 100, Xianlie Middle Road, Guangzhou City. Building 5, Guangdong Institute of Microbiology, the deposit number is GDMCC No.61589.

The bacterial strain of the present invention has the following effects:

(1) Improve mammalian learning and cognitive impairment;

(2) Delay the progression of Alzheimer's disease in mammals;

(3) Mediating the role of brain-gut axis in regulating intestinal flora and its metabolites.

The second object of the present invention is to provide a composition containing the Bifidobacterium breve CCFM1179.

In one embodiment, the number of cells of Bifidobacterium longum subsp. breve in the composition is > 1×10 ⁸ CFU/g or 1×10 ⁸ CFU/mL.

In one embodiment, the composition is a functional food or drug.

In one embodiment, the functional food is prepared by the Bifidobacterium breve participating in fermentation.

In one embodiment, the composition is a fermented food, including solid food, liquid food or semi-solid food.

In one embodiment, the types of fermented foods include dairy products, soy products or fruit and vegetable products.

In one embodiment, the dairy products include fermented milk, flavored fermented milk, fermented milk beverages, etc., cream, cheese, milk-containing beverages or milk powder; the soy products include soy milk or soy milk powder; the fruit and vegetable products include Fruit and vegetable products prepared from at least one of cabbage, white radish, cucumber, sugar beet, yellow peach or bayberry products as raw materials.

In one embodiment, the medicament further contains a pharmaceutically acceptable carrier.

In one embodiment, the carrier includes one or more of fillers, binders, wetting agents, disintegrating agents, lubricants, and flavoring agents commonly used in medicine.

In one embodiment, the dosage form of the drug is granule, capsule, tablet, pill or oral liquid.

The invention also provides the application of the Bifidobacterium breve in the preparation of fermented foods and health care products for relieving cognitive impairment, delaying the progression of Alzheimer's disease, and regulating intestinal flora.

In one embodiment, the application is to use the Bifidobacterium breve as a main ingredient to prepare food or medicine that can be ingested into the human body.

In one embodiment, the delaying the progression of Alzheimer's disease comprises at least one of (a) to (d):

(a) reducing β-amyloid-42 (Aβ1-42) deposition in mammalian brain;

(b) increasing the level of neurotransmitters in the mammalian brain;

(c) increasing synaptic protein levels in mammalian brain;

(d) increasing the content of serotonin (5-HT) in mammalian serum;

In one embodiment, the gut flora includes, but is not limited to, Akkermansia muciniphila.

In one embodiment, the mammal includes, but is not limited to, a human.

The present invention also provides a bacterial agent containing the Bifidobacterium breve CCFM1179, which is a powder obtained by drying the bacterial liquid containing the Bifidobacterium breve CCFM1179.

In one embodiment, the drying refers to vacuum freeze drying or other bacterial liquid drying processes.

In one embodiment, the number of Bifidobacterium breve cells in the bacterial preparation is ≥1×10 ⁸ CFU/g.

The present invention also provides a method for preparing Bifidobacterium breve CCFM1179 inoculum. The method inoculates Bifidobacterium breve CCFM1179 in the medium according to the inoculum amount of 2-4% based on the mass of the medium, and then inoculates it in the medium. Incubate for 24-48h under anaerobic conditions at a temperature of 35-39°C, wash 2-4 times with phosphate buffer with a pH of 6.8-7.2, and resuspend with a cell protectant to make the bacterial concentration reach 10 ¹⁰ CFU/mL; then, let The suspension is pre-cultured under anaerobic conditions at a temperature of 37° C. for 50-70 minutes, then pre-frozen at -15-20° C. for 8-14 hours, and finally vacuum freeze-dried to obtain the fermentation inoculum.

In one embodiment, the medium is MRS medium containing 0.05% L-cysteine hydrochloride, pH 6.8±0.2.

In one embodiment, the medium is sterilized at 119-123°C for 15-25 min.

In one embodiment, the protective agent is a lyophilized solution prepared from a solution containing 100g/L-150g/L skimmed milk powder, 100g/L-150g/L maltodextrin, 140g/L-160g/L trehalose Protective agent.

Beneficial effects of the present invention

The Bifidobacterium breve CCFM1179 of the invention has good growth characteristics, can significantly improve the cognitive and memory impairment of Alzheimer's mice, reduce the content of Aβ1-42 in the brain of Alzheimer's mice, and improve Alzheimer's disease. The levels of brain-derived neurotrophic factor and postsynaptic density protein in the brains of mice with Alzheimer's disease significantly increased the content of serotonin in the serum of mice with Alzheimer's disease, and increased the flora in the feces of mice with Alzheimer's disease The level of the metabolite acetate can effectively regulate the structure of the intestinal flora in Alzheimer's disease mice, improve the species diversity of the intestinal flora, and increase the relative relative value of the beneficial intestinal bacterium Akkermansia muciniphila. abundance. Therefore, the Bifidobacterium breve CCFM1179 of the present invention can be used to prepare a pharmaceutical composition for relieving dementia, Alzheimer's disease, and improving cognitive impairment, and can also be used as a microecological preparation in medicines, fermented foods or health products. Very broad application prospects.

biological material preservation

Bifidobacterium breve CCFM1179 (Bifidobacterium breve), classified as Bifidobacterium breve, was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 31, 2021, and the preservation address is 5th Floor, Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Provincial Institute of Microbiology, the deposit number is GDMCC No.61589.

Description of drawings

Figure 1 shows the behavioral changes of mice after the intervention of Bifidobacterium breve CCFM1179. (A) The percentage of free alternation in the Y maze test; (B) The escape latency of the mice in each group in the water maze test; (C) The ratio of the time that the mice in each group stayed in the quadrant of the original platform in the water maze test; (D) Latency of mice in the memory retention test of the dark avoidance experiment; among them, compared with the model group, *P<0.05, **P<0.01.

Figure 2 shows the content of amyloid beta (Aβ1-42) in the hippocampus of mice after the intervention of Bifidobacterium breve CCFM1179. Among them, compared with the model group, *P<0.05.

Figure 3 shows the changes of synaptic protein content in mouse hippocampus after Bifidobacterium breve CCFM1179 intervention. (A) content of brain-derived neurotrophic factor (BDNF); (B) content of postsynaptic density protein 95 (PSD95); among them, compared with model group, *P<0.05, **P<0.01, ** *P<0.001.

Figure 4 shows the changes in the content of serotonin (5-HT) in the serum of mice after the intervention of Bifidobacterium breve CCFM1179. Among them, compared with the model group, *P<0.05.

Figure 5 shows the changes in the concentration of acetic acid in mouse feces after the intervention of Bifidobacterium breve CCFM1179. Among them, compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

Figure 6 shows the changes in the alpha diversity of microbiota in mouse feces after the intervention of Bifidobacterium breve CCFM1179. Among them, compared with the model group, **P<0.01, ***P<0.001.

Figure 7 shows the changes in the β-diversity of microflora in mouse feces after the intervention of Bifidobacterium breve CCFM1179. Principal coordinate analysis based on Bray-Curtis distance, nonparametric multivariate analysis of variance (PERMANOVA) P<0.004. Among them, the dots of the same color represent samples from the same group; the farther the distance between samples in the two-dimensional coordinate axis, the greater the difference in species abundance.

Figure 8 shows the changes in the relative abundance of Akkermansia muciniphila in the intestinal tract of mice after the intervention of Bifidobacterium breve CCFM1179. Among them, compared with the model group, *P<0.05, **P<0.01.

Detailed ways

Example 1 Screening and identification of Bifidobacterium breve CCFM1179

(1) Isolation and screening of strains:

(1) Use a disposable sterile urinal to collect the feces of a 10-month-old male infant who was exclusively breastfed in Wuxi City, Jiangsu Province. In the culture medium of cysteine, enriched in an anaerobic incubator (N ₂ :CO ₂ :H ₂ =80:10:10) for 12h;

(2) The fecal sample was diluted with sterile physiological saline and then applied to MRS+ mass percentage (0.05%-0.1%) L- On the solid plate of cysteine hydrochloride, cultivate for 24-48h;

(3) Select a single colony that conforms to the basic form of bifidobacteria to carry out streak purification on a plate, and screen and isolate the selected strain;

(4) The above single colony was cultured in liquid MRS+mass percentage (0.05%-0.1%) cysteine medium for 24 hours, followed by Gram staining, and Gram-positive bacteria were selected for subsequent tests.

(2) Preliminary identification of bifidobacteria: fructose-6-phosphate phosphatidase assay

(1) culturing the lactic acid bacteria screened in step (1) in liquid MRS+mass percentage (0.05%-0.1%) cysteine culture solution for 24h, then taking 1mL of culture and centrifuging at 8000rpm for 2min;

(2) Wash twice with 0.05M KH ₂ PO ₄ solution containing 0.05% (mass percent) cysteine at pH 6.5;

(3) Resuspend in 200 μL of the above-mentioned phosphate buffer added with 0.25% (mass percentage) Triton X-100;

(4) Add 50 μL of a mixture of 6 mg/mL sodium fluoride and 10 mg/mL sodium iodoacetate and 50 μL of 80 mg/mL fructose-6-phosphate, and incubate at 37°C for 1 h;

(5) Add 300 μL of hydroxyamine hydrochloride with a concentration of 0.139 g/mL and pH 6.5, and place at room temperature for 10 min;

(6) Add 200 μL of 15% (mass percent) trichloroacetic acid and 4M HCl respectively;

(7) Add 200 μL of 0.1M HCl containing 5% (mass percent) ferric chloride, if the system quickly turns red, it is F6PPK positive, and it can be preliminarily concluded that it is Bifidobacterium.

(3) Molecular biological identification of bifidobacteria

(1) Take 1 mL of the bacterial cells screened in step (2) and activated for 3 generations (culturing for 12-48 h) for bacterial species identification, centrifuge at 6000 r/min for 3 min, and discard the supernatant to obtain bacterial cells.

(2) After adding 1 mL of sterile water to blow and wash the bacterial cells, centrifuge at 10,000 r/min for 1 min, discard the supernatant to obtain bacterial cells, add 500 μL of sterile water to resuspend, and use it as a bacterial liquid template.

(3) 16S rDNA PCR system:

A. Bacterial 16S rDNA, 20 μL PCR reaction system:

27F, 0.5 μL; 1492R, 0.5 μL; Taq enzyme, 1 μL; template, 1 μL; ddH20, 8 μL.

B. PCR conditions:

94℃ for 5min; 94℃ for 30s; 55℃ for 30s; 72℃ for 2min; 72℃ for 10min; step2-4 30×; 12℃ for 2min.

(3) Prepare 1% agarose gel, then mix the PCR product with 10000×Loading buffer, load 2μL, run at 120V for 30min, and then perform gel imaging;

(4)将16S rDNA的PCR产物进行测序分析，其测序结果为ATCCTAGGGGAGCGGCGTTCTGCAGAGCGGACGGGTCACCTTGACCGGGTCAGTCACACCGGCGGCCAGCAGGTCCTCGTAGGTGTCGGTGGCGGCGTTGAAGCCTTCGCCATCGGGCAGAGAACGAACCTTGTTGATCACCACGTCGCCGGACACGCCAGCATTCTCGGCAATCTGCTTGATCGGGGCTTCGATGGCGCGGAACACGATGGCGGCACCGGTAGCCTCTTCGCCGGTCAGGGAGGTGACGGCCTCAGCCTTCTCGGCCTTGGCAGCAGCCTGAACGAGGGCCACGCCACCGCCGGGCAGCAGGCCTTCCTCAATGGCGGCCTTGGCGTTACGCACGGCATCTTCGATGCGGTGCTTGCGCTCCTTGGCCTCGACCTCGGTGGCAGCGCCGACCTTGATGACAGCCACGCCGCCGGCCAGCTTGGCCAGACGCTCCTGCAGCTTCTCACGATCGTTAATCGGAA(如SEQ ID NO.1所示)，并将得到的序列结果使用BLAST在GenBank中进行搜索和相似性比对，选取测序As a result, it was identified as a strain of Bifidobacterium breve, which was stored at -80°C for future use.

(4) Preparation of Bifidobacterium breve suspension

The bacterial solution after activation for 3 generations was inoculated into 1 L of liquid MRS medium with 2% inoculum, and then incubated in an anaerobic incubator at 37° C. for 24 h after shaking and mixing. Centrifuge at 8000g/min and 4°C for 15min. After removing the supernatant, wash twice with sterile normal saline containing 0.05%-0.1% L-cysteine hydrochloride. Centrifuge under the same conditions. After removing the supernatant, it was stored in 30% glycerol and frozen at -80°C for one week. Before animal experiments, the bacterial solution was centrifuged at 6000 r/min for 5 min, washed twice with sterile saline, resuspended in 10% skim milk, shaken evenly, and determined by plate pouring method for initial and one-week cryopreservation. the number of viable bacteria. The formula of MRS medium is: beef extract 10g; tryptone 10g; yeast powder 5g; glucose 20g; anhydrous sodium acetate 5g; MgSO ₄ ·7H ₂ O 0.1g; MnSO ₄ ·H ₂ O 0.05g; Ammonium 2 g; K ₂ HPO ₄ ·3H ₂ O 2.6 g; Tween 80 1 mL; L-cysteine hydrochloride 0.8 g; adjust pH to 6.8±0.2; Autoclave at 115°C for 20min.

Experimental results: The initial viable count was 9.5×10 ⁹ CFU/mL, and after 1 week, the viable count was 7.6×10 ⁹ CFU/mL, and the order of magnitude did not change, indicating that freezing the bacterial solution would not affect the experiment. , which can be used in animal experiments.

Example 2 Bifidobacterium breve CCFM1179 improves behavioral indicators in mice with Alzheimer's disease

Thirty-two 7-week-old male C57BL/6J mice were taken. After one week of acclimatization, an animal model of Alzheimer's disease was established by one-time microinjection of Aβ1-42 protein into the hippocampus of the brain. On the 3rd day after modeling, the animals were divided into groups. There were four groups: control group (injected with equal volume of normal saline), model group, drug intervention group, and CCFM1179 intervention group, each containing 8 mice. Animal grouping and treatment methods are shown in Table 1.

Table 1 Animal experiment grouping and treatment methods

AD mouse model induced by microinjection of Aβ1-42 protein into the brain: After the mice were adapted for one week, the mice in each group were anesthetized with isoflurane (induction concentration of 3%, maintenance concentration of 1%), and the top of the mice was shaved. After sterilizing with 75% alcohol, it was fixed on the brain stereotaxic apparatus. The scalpel made an incision along the midsagittal direction to expose the skull. The skull was wiped with a medical cotton swab and hydrogen peroxide to expose the criss-cross area of the front funnel. Find the location of the front funnel and make a mark. Referring to the stereotaxic map of the mouse brain, a hole was punched at 2.0mm posterior to the anterior chimney and 1.8mm beside the midline. The 5 μL micro-injector was inserted into the needle 2.0 mm vertically downward from the surface of the skull, the injected sample volume was 1 μL, and the injection speed was 0.2 μL/min. The model group, drug intervention group and CCFM1179 intervention group were injected with oligomeric peptide Aβ1-42 at a concentration of 2 μg/μL, and the control group was injected with the same amount of sterile normal saline. After injection, the needle was retained for 5 min and then slowly withdrawn. After the wound is sutured, intramuscular injection of penicillin sodium is given at 40,000 units/only for about 2-3 days. The entire process is sterile.

Probiotics gavage: take the activated 2-generation Bifidobacterium CCFM1179 and culture it at 37 °C for 24 h, then centrifuge at 4 °C and 8000 r/min for 3 min to collect the bacteria, discard the supernatant and resuspend it with 10% sterilized skim emulsion Bacteria, so that the concentration of lactic acid bacteria reached 5 × 10 ⁹ CFU/mL. The gavage volume was 0.2 mL/only/day.

Beginning in the fifth week, behavioral testing was performed on all mice. Including Y maze experiment, water maze experiment, dark avoidance experiment. The specific implementation methods and results are as follows:

(1) Y-maze experiment:

The Y-maze test can simultaneously observe the escape conditioning ability and spatial discrimination ability of animals, and can be used to evaluate the discriminative learning, working memory and reference memory of animals. The Y-maze experiment consists of two stages. The first stage is the training period: the mice are put into the Y-shaped maze, and they move freely in the starting arm and other arms for 10 minutes. After the training, the mice are returned to the rearing cage. 24 The next phase of testing takes place after hours. The second phase was the detection phase: the mice were placed in the maze from the center point and freely moved in the three arms for 8 minutes. The video analysis system records the total number of arm advances and the number of consecutively entering three different arms, and calculates the alternation percentage according to the formula: alternation percentage=the number of consecutively entering three different arms/(total arm advancing times-2)×100%.

The experimental results are shown in Figure 1A. The percentage of free alternation in the Y-maze test of the mice in the model group was reduced to 48.41%, showing the behavioral characteristics of discriminating memory impairment. The gavage of CCFM1179 could significantly improve the above behavioral impairments, and the effect was similar to that of donepezil hydrochloride. The intervention groups were comparable.

(2) Water maze experiment:

The water maze experiment forces the experimental mice to swim, so as to learn to find the platform hidden in the water, and then test the learning and memory ability of the experimental animals on the sense of spatial position and direction (spatial orientation). It is the first choice for behavioral research, especially learning and memory research. Classic experiment.

The water maze circular pool is divided into four quadrants, and the water temperature is maintained at 23-25°C. Water maze adaptive training was performed on day 0, and positioning cruise experiment was performed on days 1-5, 4 times a day. During the positioning cruise experiment, the mice were placed into the pool from four random different entry points facing the pool wall, and the time required for the mice to enter the water to find a hidden underwater platform and stand on it (escape latency) and The total distance moved during this period. If the mice failed to find the hidden platform 60s after entering the water, they were gently guided to the platform with a long stick and allowed to stand on the platform for 30s, and the escape latency was recorded as 60s. Then, on the sixth day, the platform was removed, and a space exploration experiment was performed to record the number of times the mice crossed the original platform, the time spent in the quadrant where the original platform was located, and the movement distance within 60 s.

The experimental results are shown in Figures 1B and 1C. The escape latency of the mice in the model group was significantly increased to 51.06s in the water maze test, indicating that the spatial memory and learning and memory of the mice in the model group were impaired; gavage with CCFM1179 could significantly improve the above behavioral disorders. , the incubation period was reduced to 37.6s. In addition, the percentage of the total time spent in the quadrant of the CCFM1179 group was significantly increased to 68.21% (Fig. 1C), and the effect was significantly better than that of the donepezil hydrochloride drug intervention group (50.68%). .

(3) Darkness avoidance experiment:

After the water maze test, the mice rested for 1 day, and then carried out the dark avoidance test, which lasted for 2 days. Day 1 was a single-shock training period. The mice were placed in a bright box. After 10 s, the door between the light and dark boxes was opened. Most strains of mice have a strong exploratory behavior, like the dark and the dark. Therefore, mice will quickly enter the camera obscura. Once the mouse has completely entered the dark box, close the access door and start the electric shock (0.3mA, 2s); let the mouse stay in the dark box for 10s (to allow the animal to form an association between the box and the electric shock), put the mouse back into the cage Inside. Day 2 was the memory retention test period. The mice were placed in a bright box with the middle door open, but no electric shock was given. Record the time when the mouse penetrated into the dark box, which is recorded as the incubation period.

The experimental results are shown in Figure 1D. The latency of the mice in the model group was significantly shortened to 10.6s, indicating that the memory retention ability of the mice was impaired, and CCFM1179 intervention could significantly extend the latency of the mice to 33.04s. Relieve its memory impairment, the effect is better than the drug group's 14.67s.

Example 3 CCFM1179 reduces Aβ protein deposition in the brain of Alzheimer's mice

Mice grouping, modeling and treatment methods were the same as those in Example 2. After the behavioral experiment of mice, the mice were intraperitoneally injected with 1% sodium pentobarbital solution on the next day to make them anesthetized, the mice were sacrificed, and the mouse brain tissues were collected and separated on ice. hippocampus. Take a certain quality of fresh hippocampal tissue, add 9 times the volume of sterile PBS buffer (equivalent to 1 g of tissue plus 9 ml of homogenate), use a tissue homogenizer to homogenize, and centrifuge the tissue fluid at 3000 g for 15 min. The content of Aβ1-42 in hippocampus was detected by ELISA kit.

The experimental results are shown in Figure 2. The results show that Bifidobacterium breve CCFM1179 intervention can significantly reduce the deposition of Aβ1-42 protein in the hippocampus of model mice, and the remission effect is comparable to that of donepezil hydrochloride drug intervention group.

Example 4 Bifidobacterium breve CCFM1179 increases the level of neurotransmitters in the brain of mice with Alzheimer's disease

Take the brain tissue obtained in Example 3 with a certain quality, add 9 times the volume of sterile PBS buffer (equivalent to 1 g of tissue plus 9 ml of homogenate), use a tissue homogenizer to homogenize, and the tissue fluid is centrifuged at 3000 g and 15 min. In the supernatant, the content of brain-derived neurotrophic factor BDNF in brain tissue was detected by ELISA kit.

The experimental results are shown in Figure 3A. The results showed that Bifidobacterium breve CCFM1179 intervention significantly reversed the reduction of BDNF levels in the brains of Alzheimer's disease model mice, and the elevation of BDNF could promote neurogenesis and reduce inflammation in diseased brains .

Example 5 Bifidobacterium breve CCFM1179 increases the level of synaptic proteins in the brain of Alzheimer's mice

Take the brain tissue obtained in Example 3 with a certain quality, add 9 times the volume of sterile PBS buffer (equivalent to 1 g of tissue plus 9 ml of homogenate), use a tissue homogenizer to homogenize, and the tissue fluid is centrifuged at 3000 g and 15 min. In the supernatant, the content of postsynaptic density protein PSD-95 in brain tissue was detected by ELISA kit.

The experimental results are shown in Figure 3B. The results showed that the intervention of Bifidobacterium breve CCFM1179 significantly reversed the decrease in the level of PSD-95 in the brain of Alzheimer's disease model mice, which was increased to 1.507pg compared to 1.325pg/mL in the model group. /mL, and the improvement effect of CCFM1179 on PSD95 in the brain was significantly better than the 1.402pg/mL of donepezil hydrochloride, which was comparable to the normal control group.

Example 6 Bifidobacterium breve CCFM1179 can increase the content of 5-HT in the serum of Alzheimer's mice

Mice grouping, modeling and treatment methods were the same as those in Example 2. After the mice were sacrificed after the behavioral experiment, the collected blood of the mice was left for 2 hours, centrifuged at 3000 × g for 15 minutes to obtain serum, and the content of serotonin (5-HT) in the serum was detected by ELISA kit. 5-HT in peripheral tissues is important for maintaining normal intestinal motility. 5-HT in peripheral tissues can stimulate 5-HT2 receptors in gastrointestinal smooth muscle, or act on 5-HT4 receptors in ganglion cells in the intestinal wall, causing gastrointestinal smooth muscle contraction, increasing gastrointestinal tension, and accelerating intestinal peristalsis. ; The reduction of 5-HT in peripheral tissue is directly related to constipation, gastrointestinal discomfort and other diseases.

The experimental results are shown in Figure 4. In the serum of Alzheimer's disease model mice, the level of 5-HT decreased significantly to 36.95 pg/mL, indicating that the intestinal peristalsis function was impaired; after the intervention of Bifidobacterium breve CCFM1179, serum The level of 5-HT in the middle was significantly increased to 47.70pg/mL, which not only showed better cognitive and memory protection functions, but also restored the normal intestinal peristalsis function of mice, while the drug donepezil hydrochloride had no significant intervention effect and did not recover. to normal levels.

Example 7 Bifidobacterium breve CCFM1179 can increase the content of acetic acid in the feces of Alzheimer's mice

Mice grouping, modeling and treatment methods were the same as those in Example 2. After the mouse behavioral experiment, the mouse feces were collected and frozen at -80°C, and the content of acetic acid in the mouse feces was detected by GC-MS. The specific method is as follows: take 50 mg of mouse feces sample, add 500 μL of saturated NaCl solution to soak and mix well; add 20 μL of 10% sulfuric acid solution for acidification, then add 800 μL of ether to shake, centrifuge (13000rpm, 15min, 4°C); take the upper layer of ether phase, add 0.25g of anhydrous sodium sulfate to dry; fully shake and let stand for 30min, centrifuge again (13000rpm, 15min, 4°C); suck the supernatant and add it to the gas phase vial for gas analysis.

The GC-MS conditions are as follows: use a Rtx-Wax column (column length 30m, inner diameter 25μm); carrier gas is He, flow rate is 2mL/min; injection volume is 1μL, and the temperature is increased to 140°C at 7.5°C/min, and then at 60°C. The temperature was raised to 200 °C for 3 min/min, and the ionization temperature was 20 °C; the analysis was performed in full scan mode, and the standard curve was measured by the external standard method, and then the concentration of acetic acid was calculated.

The experimental results are shown in Figure 5. The acetic acid content in the feces of the mice in the model group was significantly lower than that in the control group, with statistical significance. The intervention of Bifidobacterium breve CCFM1179 significantly increased the acetic acid content in the feces of the mice to 18.187 μmol/g, which was significantly higher than that of the control group. The model group was 6.787 μmol/g higher, and the acetic acid content was 2.031 μmol/g higher than the control group.

Example 8 The regulatory effect of Bifidobacterium breve CCFM1179 on the intestinal flora of Alzheimer's mice

Mice grouping, modeling and treatment methods were the same as those in Example 2. After the mouse behavioral experiment, the mouse feces were collected. Extract the bacterial genome from mouse feces according to the operating steps of the MP kit; take the mouse feces genome as the template, the upstream primer 520F (5′-AYTGGGYDTAAAGNG-3′) and the downstream primer 802R (5′-TACNVGGGTATCTAATCC-3′) as Primers were used to amplify the V3-V4 region fragment of 16S rDNA; the PCR product was purified by agarose gel electrophoresis, and the target band gel was recovered according to the instructions of the QIAquick GelExtraction Kit; the sample DNA was detected according to the Qubit DNA3.0 kit Then, the library was constructed according to the TurSeq DNA LT Sample Preparation Kit and its instructions; finally, the Illumina Miseq sequencer was sequenced according to the instructions of the MiSeq Regent Kit. After sequencing, species classification annotation was performed on the QIIME platform, and the data was analyzed on the MicrobiomeAnalyst platform, and the alpha diversity and beta diversity of the samples were calculated.

The α-diversity within the flora was characterized by Chao1 index and species richness (Observed richness). The results are shown in Figure 6. Bifidobacterium breve CCFM1179 can significantly up-regulate the α-diversity of intestinal flora and improve the species abundance of intestinal flora. The β-diversity of the microbiota was evaluated by principal coordinates analysis (PCoA) (Fig. 7), and the results showed that the intestinal microbiota of Alzheimer's disease mice was significantly different from that of normal mice, and that after Bifidobacterium breve CCFM1179 intervention The intestinal microbiota was separated from the microbiota of the model mice to a certain extent, and there was a tendency to transform to the normal mouse microbiota.

In addition, the gut microbial abundance of Akkermansia muciniphila in the feces of mice in the Alzheimer's disease model group was significantly reduced, and the intake of Bifidobacterium breve CCFM1179 could significantly increase the relative abundance by 3 times around, while the drug donepezil hydrochloride had no significant change in its abundance. The main metabolite of Akkermansia muciniphila is propionic acid, and its colonization in the gut is closely related to the health of the host. It can improve obesity, inflammation in diabetic patients, and insulin resistance and glucose tolerance. Adverse symptoms can also regulate the body's immune response and maintain metabolic balance in the body.

Example 9 Utilize Bifidobacterium breve CCFM1179 of the present invention to manufacture fermented food containing the bacteria

The fresh vegetables or fruits are washed and squeezed, including one or a mixture of the above-mentioned products from cabbage, white radish, cucumber, beet, yellow peach and bayberry as raw materials. The raw material after being squeezed is sterilized at high temperature for an instant, and after sterilization at a temperature of 140° C. for 2 seconds, the temperature is immediately lowered to 37° C., and then the Bifidobacterium breve CCFM1179 of the present invention or a bacterial agent starter containing the strain is connected. , so that the concentration of Bifidobacterium breve CCFM1179 reaches more than 10 ⁶ CFU/mL, and refrigerated and stored at a temperature of 4° C., thus obtaining a fruit and vegetable beverage containing the viable Bifidobacterium breve CCFM1179 of the present invention.

The Bifidobacterium breve CCFM1179 of the present invention is used as a fermenting microorganism to produce fermented food, and the fermented food includes solid food, liquid food and semi-solid food. The fermented food includes dairy products, soy products, fruit and vegetable products, and the dairy products include fermented dairy products (fermented milk, flavored fermented milk, fermented milk beverages, etc.), cream, cheese, milk-containing beverages, and milk powder; the soybean Products include soybean milk and soybean milk powder; the fruit and vegetable products include cabbage, white radish, cucumber, beet, yellow peach, and bayberry products.

According to the methods described in Examples 2-8, the effect of fermented food on improving Alzheimer's symptoms was verified. The results showed that the fermented food could improve the cognitive and memory impairment of Alzheimer's mice, reduce Alzheimer's The content of Aβ1-42 protein in the brain of mice with Alzheimer's disease, increased the levels of brain-derived neurotrophic factor and postsynaptic density protein in the brain of mice with Alzheimer's disease, and increased the serum 5- The content of serotonin has an auxiliary effect; the fermented food can increase the level of acetic acid metabolite in the feces of Alzheimer's mice, improve the function of gastrointestinal motility, and can regulate the intestinal flora of Alzheimer's mice. structure, improve the species diversity of the intestinal flora, and increase the relative abundance of the intestinal beneficial bacterium Akkermansia muciniphila.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> A Bifidobacterium breve that can alleviate Alzheimer's disease and increase fecal acetate content and its application

<130> BAA210490A

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 473

<212> DNA

<213> Bifidobacterium breve

<400> 1

atcctagggg agcggcgttc tgcagagcgg acgggtcacc ttgaccgggt cagtcacacc 60

ggcggccagc aggtcctcgt aggtgtcggt ggcggcgttg aagccttcgc catcgggcag 120

agaacgaacc ttgttgatca ccacgtcgcc ggacacgcca gcattctcgg caatctgctt 180

gatcggggct tcgatggcgc ggaacacgat ggcggcaccg gtagcctctt cgccggtcag 240

ggaggtgacg gcctcagcct tctcggcctt ggcagcagcc tgaacgaggg ccacgccacc 300

gccgggcagc aggccttcct caatggcggc cttggcgtta cgcacggcat cttcgatgcg 360

gtgcttgcgc tccttggcct cgacctcggt ggcagcgccg accttgatga cagccacgcc 420

gccggccagc ttggccagac gctcctgcag cttctcacga tcgttaatcg gaa 473

Claims (10)

1. Bifidobacterium breve: (Bifidobacterium breve) CCFM1179, deposited at 31.3.2021 in Guangdong province of microbial cultures Collection, with the deposit number GDMCC NO: 61589.

2. Composition containing Bifidobacterium breve CCFM1179 of claim 1, wherein the cell count of Bifidobacterium breve CCFM1179 in the composition is not less than 1 x 10 ⁸ CFU/g or 1X 10 ⁸ CFU/mL。

3. The composition of claim 2, wherein the composition is a functional food or a pharmaceutical.

4. The composition of claim 2, wherein the composition is a fermented food product; the fermented food is solid food, liquid food or semisolid food.

5. The composition of claim 4, wherein the fermented food product is a dairy product, a soy product, or a fruit and vegetable product.

6. The microbial agent of claim 1, wherein the microbial agent is a powder obtained by drying a bacterial liquid containing bifidobacterium breve CCFM 1179.

7. Use of the bifidobacterium breve CCFM1179 of claim 1, or the composition of any one of claims 2 to 5, or the microbial preparation of claim 6 for the preparation of a medicament for delaying alzheimer's disease, wherein the medicament comprises the bifidobacterium breve CCFM1179 of claim 1 and a pharmaceutically acceptable carrier.

8. The use according to claim 7, wherein the carrier is one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents, which are generally used in medicine.

9. The use according to claim 7, wherein the delayed Alzheimer's disease is selected from at least one of (a) - (d):

(a) reducing beta-amyloid polypeptide-42 deposition in the brain of a mammal;

(b) increasing brain-derived neurotrophic factor BDNF level in the brain of a mammal;

(c) increasing post-synaptic density protein levels in the brain of the mammal;

(d) increasing the content of 5-hydroxytryptamine in the serum of mammals.

10. Use of a bifidobacterium breve CCFM1179 as claimed in claim 1, or a composition as claimed in any one of claims 2 to 5, or a bacterial preparation as claimed in claim 6 in the preparation of a product for modulating intestinal flora.

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