CN110643542B

CN110643542B - Lactobacillus reuteri capable of relieving Th2 reaction of allergic asthma and application thereof

Info

Publication number: CN110643542B
Application number: CN201911023858.9A
Authority: CN
Inventors: 陈卫; 陆文伟; 李凌智; 翟齐啸; 张秋香; 赵建新; 杭锋; 张灏
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2021-01-29
Anticipated expiration: 2039-10-25
Also published as: CN110643542A

Abstract

The invention discloses lactobacillus reuteri capable of relieving Th2 reaction of allergic asthma and application thereof, and belongs to the technical field of microorganisms and medicines. The Lactobacillus reuteri (Lactobacillus reuteri) has the function of relieving allergic asthma, and is specifically represented as follows: (1) the lung inflammatory reaction of allergic asthma mice is obviously reduced; (2) remarkably inhibiting the production of total immunoglobulin IgE in the serum of an allergic asthma mouse; (3) the content of IL-5 in the lung of an allergic asthma mouse is obviously reduced; (4) the content of IL-13 in the lung of an allergic asthma mouse is obviously reduced; (5) obviously increases the content of propionic acid in the intestinal tract of an allergic asthma mouse, so that the Lactobacillus reuteri has great application prospect in preparing products for preventing and/or treating the allergic asthma.

Description

Lactobacillus reuteri capable of relieving Th2 reaction of allergic asthma and application thereof

Technical Field

The invention relates to lactobacillus reuteri capable of relieving Th2 reaction of allergic asthma and application thereof, belonging to the technical field of microorganisms and medicines.

Background

Allergic asthma is a common clinical disease and a difficult disease to treat, and the disease course is delayed and healed, and is mainly characterized in that various inflammatory cells (eosinophils, mast cells, T lymphocytes and the like) infiltrate in the airway, and some inflammatory mediators secreted by the cells amplify inflammatory cascade reaction, increase Airway Hyperreactivity (AHR) and stimulate mucus secretion, so that airway obstruction and airway remodeling are caused. In susceptible patients, the inflammation may cause recurrent wheezing, shortness of breath, chest tightness and/or cough, which may lead to respiratory arrest and respiratory failure, which are complications and endanger the life of the patient.

Asthma prevalence increases year by year in developing countries. Its onset is influenced by many factors, such as exposure route, genetic susceptibility, allergen dose, etc. Studies have shown that 85% of asthmatic patients are often allergic to House Dust Mite (HDM). Allergen enters into body for sensitization, the ratio of Th1/Th2 cells in body is unbalanced, Th0 cells tend to differentiate into Th2 cells, and secretion of Th2 factors such as IL-5 and IL-13 is increased, so that body is in sensitization state. When the organism contacts the allergen again, B cells induce the secreted IgE to be combined with the surfaces of basophils and mast cells, and a series of substances such as histamine, leukotriene and the like are released, so that allergic symptoms are generated. The Th2 type cytokines IL-5 and IL-13 play an important role in initiating and maintaining the pathophysiological response process of asthma.

With the intensive study of intestinal flora, a plurality of studies prove that allergic asthma is related to the disturbance of the intestinal flora, the discovery of intestinal flora of healthy people and patients with allergic asthma is compared, beneficial bacteria in intestinal tracts are reduced, harmful bacteria are increased, metabolites in intestinal tracts are changed, such as the reduction of short-chain fatty acids, and the like, the study detects the concentration of the short-chain fatty acids in feces of patients with allergic asthma, and the discovery shows that the concentrations of propionic acid, butyric acid and isobutyric acid in the intestinal tracts of patients with allergic asthma are obviously reduced compared with the concentrations of the propionic acid, the butyric acid and the isobutyric acid in the intestinal tracts of the patients with allergic asthma, and meanwhile, cytokines interleukin 5 and interleukin 13 at the Th2 type allergic reaction level are in negative. It is marked that propionic acid produced by the metabolism of the intestinal flora is beneficial for reducing Th2 response in allergic asthma.

Currently, the means of treating allergic asthma is usually controlled by drugs including bronchodilators, anti-inflammatory drugs and probiotic formulations. Among them, most of the bronchodilatory drugs and anti-inflammatory drugs are administered by intravenous injection or inhalation, so that the patient's compliance with these drugs is poor, which results in repeated attacks of the patient's disease and severe complications such as emphysema and pulmonary heart disease in the patient over time.

Although the probiotic preparation is relatively safe and healthy, most of the probiotic preparations on the market have the defects of slow effect taking and undefined action relationship because the probiotics such as lactobacillus gasseri, lactobacillus rhamnosus, bifidobacterium animalis and the like which are discovered at present and have the symptom of allergic asthma have poor effect or undefined action effect.

Therefore, a probiotic with strong Th2 response reducing and allergic asthma relieving capabilities is urgently needed to solve the problems of slow effect taking and undefined action relation of the existing probiotic preparation for allergic asthma

Disclosure of Invention

In order to solve the problems, the first object of the invention is to provide a Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 strain, wherein the Lactobacillus reuteri CCFM1072 strain is preserved in Guangdong province microbial culture collection center in 8.8.2019, and the preservation number is GDMCC No.60735, and the preservation address is No. 59 building 5 of Michelia Torrens No. 100 Hospital, Guangzhou city.

The Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 is obtained by separating human excrement from the Lai area of Shandong, the strain is subjected to sequencing analysis, the 16S rRNA sequence of the strain is shown as SEQ ID NO.1, the sequence is subjected to nucleic acid sequence comparison in NCBI, and the result shows that the strain is the Lactobacillus reuteri and is named as the Lactobacillus reuteri CCFM 1072.

The microorganism morphological characteristics of the Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 are as follows: the cells were slightly irregular, rounded-ended Campylobacter, non-motile, non-sporulating.

The second purpose of the invention is to provide the application of the Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 in preparing products for preventing, relieving, improving and/or treating allergic asthma.

In one embodiment, the viable count of Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 in the product is not less than 1 × 10⁶CFU/mL。

In one embodiment, the pharmaceutical product comprises Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072, a pharmaceutical carrier and/or a pharmaceutical excipient.

In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and an additive.

In one embodiment of the present invention, the pharmaceutical excipients comprise an anti-adhesive agent, a penetration enhancer, a buffering agent, a plasticizer, a surfactant, an antifoaming agent, a thickener, an encapsulating agent, an absorbent, a humectant, a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a pH adjuster, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an integrating agent, an osmotic pressure adjuster, a stabilizer, a glidant, a flavoring agent, a preservative, a foaming agent, a suspending agent, a coating material, a flavoring agent, a diluent, a flocculating agent and deflocculating agent, a filter aid, and a release retardant.

In one embodiment of the present invention, the additive comprises microcrystalline cellulose, hydroxypropylmethylcellulose, and refined lecithin.

In one embodiment of the present invention, the dosage form of the pharmaceutical product comprises granules, capsules, tablets, pills or oral liquid.

In one embodiment, the product comprises a food, pharmaceutical or nutraceutical.

In one embodiment, the food product comprises a dairy, soy or fruit and vegetable product produced using a starter culture comprising Lactobacillus reuteri (CCFM 1072).

In one embodiment, the food product is a solid beverage comprising Lactobacillus reuteri (CCFM 1072).

In one embodiment of the present invention, the preparation method of the leavening agent is: inoculating Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 into a culture medium according to the inoculation amount accounting for 2-4% of the total mass of the culture medium, and culturing at 37 ℃ for 18h to obtain a culture solution; centrifuging the culture solution to obtain thalli; washing the thallus with phosphate buffer solution with pH of 7.2 for 3 times, and then resuspending with lyophilized protectant (the mass ratio of lyophilized protectant to thallus is 2:1) to obtain resuspension; and (3) freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain the fermentation agent of the Lactobacillus reuteri (Lactobacillus reuteri) CCFM 1072.

In one embodiment of the invention, the medium comprises 87.7% water, 10% skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract solubles by total mass of the medium.

In one embodiment of the invention, the pH of the medium is 6.8.

In one embodiment of the present invention, the ratio of the skimmed milk powder to maltodextrin to sodium L-glutamate is 8-10: 1.

Has the advantages that:

the invention screens out a Lactobacillus reuteri (CCFM 1072), and the Lactobacillus reuteri (CCFM 1072) has the function of relieving allergic asthma and is specifically embodied as follows:

(1) the lung inflammation score of the allergic asthma mouse is reduced from 3.25 to 1.5;

(2) the production of total immunoglobulin IgE in the serum of an allergic asthma mouse is reduced to 354.38 mu g/mL from 403.19;

(3) the content of IL-5 in the lung of the allergic asthma mouse is reduced from 213.19 to 61.40 pg/mL;

(4) the content of IL-13 in the lung of the allergic asthma mouse is reduced from 1044.90 to 442.50 pg/mL;

(5) the content of propionic acid in the intestinal tract of allergic asthma mice is increased from 5.01 to 7.77 mu mol/g,

therefore, the Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 has great application prospect in preparing products (such as food, medicine or health food and the like) for preventing and/or treating allergic asthma.

Biological material preservation

A strain of Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 is classified and named as Lactobacillus reuteri, is preserved in Guangdong province microorganism strain preservation center in 8.8.2019, has the preservation number of GDMCC No.60735, and has the preservation address of No. 59 floor 5 of Michelia Torresiae No. 100 in Guangzhou city.

Drawings

FIG. 1 shows the pathological lung tissue scores of different groups of experimental mice.

FIG. 2 shows the content of immunoglobulin IgE in the serum of different groups of experimental mice.

FIG. 3 shows the IL-5 content in alveolar lavage fluid of different groups of experimental mice.

FIG. 4 shows the IL-13 content in the vacuolar lavage fluid of different groups of experimental mice.

FIG. 5 shows the content of propionic acid in intestinal tract of different groups of experimental mice.

Detailed Description

MRS solid medium (g/L): 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate₂PO₄·3H₂O 2.6g/L、MgSO₄·7H₂O 0.1g/L、MnSO₄0.05g/L, Tween 801 mL/L, agar 20g/L and cysteine hydrochloride 0.5 g/L.

MRS liquid medium (g/L): 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate₂PO₄·3H₂O 2.6g/L、MgSO₄·7H₂O 0.1g/L、MnSO₄ 0.05gL, Tween 801 mL/L and cysteine hydrochloride 0.5 g/L.

The detection methods referred to in the following examples are as follows:

the detection method of viable count comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus is adopted.

And (3) an acidity detection method: the national standard GB 431334-.

The strains in the examples are described in patent application publication No. CN 109628359A; lactobacillus reuteri l.reuteri1 and l.reuteri2 are strains isolated by the same method.

Example 1: screening and identification of lactobacillus reuteri

The method comprises the following specific steps:

1. screening:

taking human excrement from Laizhou Shandong as a sample, pretreating the sample, storing the pretreated sample in 30% glycerol at-80 ℃, taking out and unfreezing the sample, uniformly mixing the sample, sucking 0.5mL of the sample, adding the sample into 4.5mL of the sample, performing gradient dilution by using 0.9% physiological saline, selecting a proper gradient dilution liquid, coating the gradient dilution liquid on an MRS solid culture medium, culturing the gradient dilution liquid at 37 ℃ for 48h, selecting a typical bacterial colony, streaking and purifying the typical bacterial colony on the MRS solid culture medium, selecting a single bacterial colony, transferring the single bacterial colony to the MRS liquid culture medium for enrichment, and preserving the single bacterial colony by using 30% glycerol to obtain a strain CCFM 1072.

2. And (3) identification:

the 16S rDNA of the CCFM1072 is amplified and sequenced, the sequence is compared with the nucleotide sequence in NCBI, and the result shows that the strain is Lactobacillus reuteri and is named as Lactobacillus reuteri CCFM 1072.

Example 2: culture of Lactobacillus reuteri

The method comprises the following specific steps:

lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 is inoculated into an MRS solid culture medium and cultured for 48 hours at 37 ℃, the bacterial colony is observed and the thallus is observed under a microscope, and the bacterial colony is milk white, irregular, round and convex and smooth, the shape of the thallus is a slightly irregular and round bent bacillus with a tail end, and the single, paired and small clusters exist usually.

Inoculating Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 into an MRS liquid culture medium, culturing at 37 ℃ for 18h, transferring into a fresh MRS liquid culture medium, culturing under the same conditions for 18h, centrifuging the thallus for 15min at 6000g, washing the thallus with 0.9% physiological saline, centrifuging again for 10min at 6000g to obtain the thallus, resuspending the thallus with 30% sucrose solution, and freezing at-80 ℃ for later use.

Example 3: effect of Lactobacillus reuteri on pulmonary inflammation in allergic asthma mice

The method comprises the following specific steps:

the SPF-grade BALB/c female mice of 6 weeks old are divided into 5 groups, namely a normal group, a model group and an experimental group, wherein the experimental group comprises a CCFM1072 prevention group of Lactobacillus gasseri CCFM1072, an L.reuteri1 prevention group of Lactobacillus gasseri 1 and an L.reuteri2 prevention group of

Lactobacillus gasseri

2, 6 mice in each group are bred in the center of experimental animals of south Jiangtian university and fed with common feed, the constant temperature is 21-26 ℃, the humidity is 40-70%, the noise is less than or equal to 60dB, and the animal illumination is 15-20LX (all animal experimental procedures are examined and approved by the university of animal welfare and ethics administration of south Jiangtian).

The experiment was performed for 6 weeks (5 days per week), and the model was made at 2-6 weeks, and the model group and the experimental group mice were anesthetized with 25. mu.g/10. mu.L of HDM (house dust mite extract) per day, and the normal group was treated with 10. mu.L of sterile physiological saline alone as a control.

In 1-6 weeks, the experimental group is gavaged with 0.25mL per day (viable count is 1X 10)⁹CFU), normal and model groups gavage only 0.25mL sterile saline as control, all groups being free to drink and ingest.

After the experiment is finished, the mouse is killed by anesthesia, the thoracic cavity is dissected, the agarose solution is injected into the left lung of the mouse, the left lung is taken out and placed in a 4% paraformaldehyde solution for fixation, paraffin embedding and HE staining are carried out, the left lung is sent to a pathology department for lung inflammation degree evaluation, and the inflammation degree evaluation result is shown in figure 1.

As shown in fig. 1, after mice were fed with the strain CCFM1072 of the present invention, inflammatory cell infiltration was significantly reduced and lung histopathological score was reduced by 54%, which is significantly lower than that of the model group (p < 0.05) and 46% lower than that of the l.reuteri1 and l.reuteri2 groups, which is closer to the blank group, indicating that the l.reuteri CCFM1072 reduces the inflammatory response of the lungs of asthmatic mice.

The experimental results show that compared with L.reuteri1 and L.reuteri2, the Lactobacillus reuteri CCFM1072 can obviously improve the pulmonary inflammation of asthmatic mice and relieve asthma symptoms.

Example 4: effect of Lactobacillus reuteri on Total IgE in serum of allergic asthma mice

The method comprises the following specific steps:

the SPF-grade BALB/c female mice of 6 weeks old were divided into 6 groups, which were a normal group, a model group, and an experimental group, respectively, wherein the experimental group included CCFM1072 prevention group of lactobacillus gasseri CCFM1072, l.reuteri CCFM1040 prevention group of lactobacillus gasseri CCFM1040, l.reuteri1 prevention group of lactobacillus reuteri1, and l.reuteri2 prevention group of

lactobacillus gasseri

2, and 6 animals per group were housed in the center of experimental animals of south of the river university, fed with a common feed, kept at a constant temperature of 21-26 ℃, humidity of 40-70%, noise of 60dB or less, and animal illuminance of 15-20LX (all animal experimental procedures were reviewed and approved by the animal welfare and ethics administration committee of south of the river university).

After the experiment, anesthetizing the mouse, collecting blood by eyeballs, standing for 2h, centrifuging at 3000rpm of a centrifuge for 10min, collecting serum in a clean centrifuge tube, and freezing and storing at-20 ℃.

The Total IgE content in the serum is detected by a Total IgE ELISA kit, and the detection result is shown in figure 2.

As shown in figure 2, after the strain CCFM1072 of the invention is fed, the content of the total IgE in the blood serum of the mice is reduced to 354 mug/ml which is obviously lower than that of the model group to 403 mug/ml (p is less than 0.05), the content of the total IgE in the blood serum of the mice is equivalent to that of the blank group to 344 mug/ml, the content of the total IgE in the blood serum is superior to that of the L.reuteri1 to 365 mug/ml, and the content of the L.reuteri2 to 394 mug/ml and the content of the L.reuteri CCFM1040 to 410 mug/ml which are not obviously different from that of the model group.

The experimental results show that the lactobacillus reuteri CCFM1072 can obviously reduce the total IgE in the serum of an asthmatic mouse and improve anaphylactic reaction, but the lactobacillus reuteri L.reuteri2 and L.reuteri CCFM1040 have no effect.

Example 5: effect of Lactobacillus reuteri on IL-5 in alveolar lavage fluid of mice with allergic asthma

The method comprises the following specific steps:

the SPF-grade BALB/c female mice of 6 weeks old were divided into 6 groups, which were a normal group, a model group, and an experimental group, respectively, wherein the experimental group included l.reuteri CCFM 1040-prevention group of lactobacillus gasseri CCFM1040, CCFM 1072-prevention group of lactobacillus reuteri CCFM1072, l.reuteri 1-prevention group of lactobacillus gasseri 1, and l.reuteri 2-prevention group of

lactobacillus gasseri

2, and 6 animals per group were housed in the center of experimental animals of south of the river, fed with a common feed, kept at a constant temperature of 21-26 ℃, humidity of 40-70%, noise of 60dB or less, and animal illuminance of 15-20LX (all animal experimental procedures were reviewed and approved by the animal welfare and ethics administration committee of south of the river).

After the experiment, the mouse is killed by anesthesia, placed on an anatomical plate, the head and four are fixed, the chest and the neck are dissected, the trachea and the two lungs of the mouse are exposed, the right lung is ligated, the trachea cannula is performed by an indwelling needle, 0.3mL precooled PBS is absorbed by a 1mL syringe, the left lung of the mouse is lavaged for 3 times, the recovery amount is more than or equal to 80%, and the lavaging liquid is collected in a clean Eppendorf tube. Collecting supernatant in a centrifuge at 4 deg.C and 1000rpm for 5min, subpackaging in Eppendorf tube, freezing at-20 deg.C, detecting IL-5 content in alveolar lavage fluid by ELISA kit, and the detection result of IL-5 content in alveolar lavage fluid is shown in FIG. 3,

as shown in figure 3, after mice are fed with the strain CCFM1072 of the invention, the content of IL-5 in alveolar lavage fluid is significantly reduced by 71 percent (p is less than 0.05) compared with a model group, and has no significant difference with a normal group, and is respectively reduced by 60 percent and 40 percent better than CCFM1040 and L.reuteri2, which indicates that the strain of the invention can inhibit Th2 reaction; the IL-5 content in the pulmonary alveolar lavage fluid of the L.reuteri1 group is not obviously different from that of the model group, and is only reduced by 13 percent.

Combining the above experimental results, lactobacillus reuteri CCFM1072 can significantly down-regulate typical Th 2-related cytokines in alveolar lavage fluid of asthmatic mice to normal level, and is significantly better than the other three strains of lactobacillus reuteri CCFM1040, l.reuteri1 and l.reuteri 2.

Example 6: effect of Lactobacillus reuteri on IL-13 in alveolar lavage fluid of mice with allergic asthma

The method comprises the following specific steps:

Lactobacillus gasseri

After the experiment, the mouse is killed by anesthesia, placed on an anatomical plate, the head and four are fixed, the chest and the neck are dissected, the trachea and the two lungs of the mouse are exposed, the right lung is ligated, the trachea cannula is performed by an indwelling needle, 0.3mL precooled PBS is absorbed by a 1mL syringe, the left lung of the mouse is lavaged for 3 times, the recovery amount is more than or equal to 80%, and the lavaging liquid is collected in a clean Eppendorf tube. Collecting supernatant in a centrifuge at 4 deg.C and 1000rpm for 5min, subpackaging in Eppendorf tubes, freezing at-20 deg.C, and detecting IL-13 content in alveolar lavage fluid by ELISA kit, wherein the detection result of IL-13 content in alveolar lavage fluid is shown in FIG. 4.

As shown in FIG. 4, after mice were fed with the strain CCFM1072 of the present invention, the IL-13 content in alveolar lavage fluid was reduced by 58%, which was significantly reduced (p < 0.05) compared to the model group, and was not significantly different from the normal group, indicating that the strain of the present invention was able to suppress the Th2 response; and the content of IL-13 in the alveolar lavage fluid of the L.reuteri1 and L.reuteri2 groups is reduced by only 15 percent and 37 percent, and has no significant difference with the model group.

In combination with the above experimental results, lactobacillus reuteri CCFM1072 can significantly reduce the typical Th 2-related cytokine up-regulated in alveolar lavage fluid of asthmatic mice to normal level, especially reduce the IL-13 level which can stimulate airway epithelial cells to produce a large amount of mucus, and is significantly better than the other two strains of lactobacillus reuteri l.reuteri1 and l.reuteri 2.

Example 7: effect of Lactobacillus reuteri on propionic acid in intestinal tract of allergic asthma mice

The method comprises the following specific steps:

the SPF-grade BALB/c female mice of 6 weeks old were divided into 6 groups, namely a normal group, a model group and an experimental group, wherein the experimental group comprises a CCFM1072 prevention group of Lactobacillus gasseri CCFM1072, a L.reuteri CCFM1040 prevention group of Lactobacillus gasseri CCFM1040, a L.reuteri1 prevention group of Lactobacillus gasseri 1 and a L.reuteri2 prevention group of

Lactobacillus gasseri

2, and 6 animals in each group were bred in the center of the Experimental animals of south Jiangnan university, fed with a common feed, kept at a constant temperature of 21-26 ℃, a humidity of 40-70%, a noise of 60dB or less, and an animal illuminance of 15-20LX (all animal experimental procedures were examined and approved by the animal welfare and ethics administration Committee of south Jiangnan university).

After the experiment was completed, the mice were anesthetized, the abdominal cavity was dissected, the cecum contents were taken out in a clean centrifuge tube and frozen at-20 deg.C

The content of propionic acid in the caecum content was measured by GC-MS, and the results are shown in FIG. 5, and the average content (μ M/g) of each propionic acid group: blank 4.05, model 5.01, CCFM 10727.77, CCFM 10403.87, l.reuteri 15.35, l.reuteri 23.12.

As shown in fig. 5, after mice were fed with the strain CCFM1072 of the present invention, the content of propionic acid in cecum content was significantly increased by 55% (p < 0.05) compared to the model group, which is significantly higher than that of blank and model groups, while lactobacillus reuteri l.reuteri2 group did not show significant increase, and CCFM1040 and l.reuteri1 groups were decreased by 50% and 42%, respectively. The strain CCFM1072 of the invention can promote the production of propionic acid in intestinal tract.

In combination with the above experimental results, lactobacillus reuteri CCFM1072 significantly up-regulated the propionic acid content in short chain fatty acids. Lactobacillus reuteri CCFM1072 is significantly superior to l.reuteri1 and l.reuteri2 in promoting the intestinal production of propionic acid.

Example 8: preparation of solid beverage containing lactobacillus reuteri CCFM1072

Inoculating Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 into a culture medium according to the inoculation amount accounting for 3 percent of the total mass of the culture medium, and culturing at 37 ℃ for 18 hours to obtain a culture solution; centrifuging the culture solution to obtain thalli; cleaning the thallus with phosphate buffer solution with pH of 7.2 for 3 times, and then re-suspending with trehalose lyophilized protectant with trehalose concentration of 100g/L (mass ratio of lyophilized protectant to thallus is 2:1) to obtain re-suspension; and (3) freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 powder.

Will contain 10⁹Mixing the powder of the CFU Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 bacteria with maltodextrin, wherein the total mass of the powder of the CCFM1072 bacteria and the maltodextrin is 1 g, and obtaining the solid beverage rich in the Lactobacillus reuteri (Lactobacillus reuteri) CCFM 1072.

Taking 10g (equivalent to a total irrigation 10)¹⁰CFU) the solid beverage containing the Lactobacillus reuteri CCFM1072 is dissolved again by normal saline, the volume is constant to 20 ml, each mouse is filled with 200 microliters of the normal saline every day for two weeks, the content of total IgE in the serum of the mouse with allergic asthma, inflammatory cell infiltration in lung tissues and the production of inflammatory cytokines can be effectively reduced, the content of propionic acid in intestinal tracts is improved, and the solid beverage has excellent effect of relieving the allergic asthma.

Example 9: preparation of cow milk containing Lactobacillus reuteri CCFM1072

The method comprises the following specific steps:

inoculating Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 into a culture medium according to the inoculation amount accounting for 3 percent of the total mass of the culture medium, and culturing at 37 ℃ for 18 hours to obtain a culture solution; centrifuging the culture solution to obtain thalli; washing the bacteria with phosphate buffer solution with pH of 7.2 for 3 times, and suspending with trehalose lyophilized protectant with trehalose concentration of 100g/L until the bacteria concentration reaches 1 × 10¹⁰CFU/mL to obtain a bacterial suspension; placing the suspension at 37 deg.C for 60min for pre-culturing, and lyophilizing by lyophilization method to obtain Lactobacillus reuteri CCFM1072 leaven; wherein, the cultureThe culture medium comprises 87.7 percent of water, 10 percent of enzyme hydrolysis skim milk, 0.5 percent of glucose, 1.5 percent of tryptone and 0.3 percent of yeast extract solution by mass; the pH of the medium was 6.8;

sterilizing skim milk at 95 deg.C for 20min, cooling to 4 deg.C, adding Lactobacillus reuteri CCFM1072 starter to make viable bacteria concentration of Lactobacillus reuteri CCFM1072 in skim milk reach 1 × 10⁹CFU/mL, and refrigerating at 4 deg.C to obtain cow milk containing viable Lactobacillus reuteri CCFM 1072.

200 microliter of cow milk containing live lactobacillus reuteri CCFM1072 bacteria is taken to perform intragastric administration on the mouse for 3 weeks continuously, so that the content of total IgE in serum of the mouse with allergic asthma, inflammatory cell infiltration in lung tissues and inflammatory cytokine production can be effectively reduced, the content of propionic acid in intestinal tracts is increased, and the cow milk has a good effect on relieving the allergic asthma.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> Lactobacillus reuteri capable of relieving Th2 reaction of allergic asthma and application thereof

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tcgagcctca gcgtcagttg cagaccagac agccgccttc gccactggtg ttcttccata 780

tatctacgca ttccaccgct acacatggag ttccactgtc ctcttctgca ctcaagtcgc 840

ccggtttccg atgcacttct tcggttaagc cgaaagcttt cacatcagac ctaagcaacc 900

gcctgcgctc gctttacgcc caataaatcc ggataacgct tgccacctac gtattaccgc 960

ggctgctggc acgtagtagc cgtgactttc tggttggata ccgtcactgc gtgaacagtt 1020

actctcacgc acgtcttctc aacaacagag ctttacgagc cgaaa 1065

Claims

1. The Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 is characterized in that the Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 is preserved in Guangdong province microbial strain preservation center in 8.8.2019, and the preservation number is GDMCC No.60735, and the preservation address is No. 59 building 5 of Michelia Torrens No. 100, Guangzhou city.

2. Use of Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 according to claim 1 for the preparation of a product for the prevention, alleviation, amelioration and/or treatment of allergic asthma; the product is a food and/or a pharmaceutical product.

3. The use according to claim 2, wherein the viable count of Lactobacillus reuteri (CCFM 1072) in the product is not less than 1 x 10⁶CFU/mL。

4. A composition comprising the Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 of claim 1.

5. The composition of claim 4, wherein the composition is a pharmaceutical composition comprising a pharmaceutical carrier and/or a pharmaceutical excipient.

6. Food product comprising a Lactobacillus reuteri (Lactobacillus reuteri) CCFM1072 according to claim 1.

7. A starter is characterized in that the preparation method comprises the following steps: inoculating the lactobacillus reuteri of claim 1 into a culture medium according to an inoculation amount accounting for 2-4% of the total mass of the culture medium, culturing at 35-37 ℃ for 12-24 h, collecting thalli, re-suspending with a freeze-drying protective agent, and freeze-drying by a vacuum freezing method to obtain the lactobacillus reuteri starter.