A composite probiotic preparation for treating secretory otitis media

Abstract

The invention discloses a composite probiotic preparation for treating secretory otitis media, comprising: kumiss-like lactobacillus, long bifidobacterium and other acceptable drugs or food excipients. The kumiss-like lactobacillus is kumiss-like lactobacillus ZW3, which has been deposited in the strain collection center of the Institute of Microbiology, Chinese Academy of Sciences on December 18, 2008, with a deposit number of CGMCC2809. The long bifidobacterium is preferably CGMCC 15014. The composite probiotic preparation of the invention has precise ingredients and is safe, and is particularly effective in treating secretory otitis media.

Description

Technical Field

[0001] The invention relates to a composite probiotic preparation for treating secretory otitis media, and belongs to the technical field of microorganisms. Background Art

[0002] Secretory otitis media, also known as exudative otitis media, mucous otitis media, catarrhal otitis media, etc., is a non-suppurative disease of the middle ear and one of the common diseases of the ear, nose and throat. Secretory otitis media is characterized by middle ear effusion, hearing loss and intact tympanic membrane. It is more common in children. Because ear pain is not obvious, parents only discover and seek medical treatment when the child's hearing is affected, which often delays diagnosis and treatment. The onset of this disease has two characteristics: acute and chronic.

[0003] For patients with normal eardrums, the Eustachian tube is the only channel for the middle ear to communicate with the external environment. Eustachian tube obstruction is an important cause of secretory otitis media. This disease is a non-suppurative disease of the middle ear. The main treatment principle is to control infection and improve the ventilation function of the Eustachian tube. Mucus promoters and Eustachian tube blowing are often used for treatment. However, the best treatment period is often missed because it is not easy to be discovered. Functional ventilation dysfunction caused by edema of the Eustachian tube mucosa caused by allergic reactions is the cause of secretory otitis media.

[0004] Probiotics are active microorganisms that can colonize and benefit the host. They regulate the balance of the host's flora and various functions of the human body, such as immune status, to produce beneficial effects on health. Lactobacillus is a type of Gram-positive rod that ferments carbohydrates to produce lactic acid. It is anaerobic or facultative anaerobic and does not produce spores. It is known that many lactobacilli can reduce immediate allergic reactions through various pathways and methods. As a probiotic, lactobacillus can regulate the body's immune function and restore the body's immune balance, thereby preventing allergic diseases. The current treatment methods for secretory otitis media are mainly antibiotic treatment, nasal constrictors to improve the ventilation function of the Eustachian tube, and oral mucus promoters. With the deepening of research, people have gradually discovered that common secretory otitis media in children is closely related to allergic reactions, but because the ear pain is not obvious, treatment is often delayed, and probiotic preparations for the prevention and treatment of secretory otitis media have not yet been further developed. Summary of the invention

[0005] The purpose of the present invention is to provide a composite probiotic preparation for treating secretory otitis media, which has a significant effect on regulating immunity, especially for secretory otitis media caused by allergies, and has great application potential.

[0006] Chinese patent 201410437848.0 discloses a strain of kumiss-like Lactobacillus ZW3, which was isolated and screened from kefir grains and deposited in the Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences on December 18, 2008, with the deposit number CGMCC2809. The known function of the strain is that it has a very strong function of degrading nitrite. The kumiss-like Lactobacillus ZW3 used in the present invention was donated by researchers from Tianjin University of Science and Technology.

[0007] The Bifidobacterium longum CGMCC 15014 used in the present invention was purchased from the China General Microbiological Culture Collection Center, with a preservation date of September 3, 2004 and a preservation number of CGMCC 15014. In the process of research and practice, the present invention team found that kumiss-like Lactobacillus ZW3 has a strong anti-allergic function, and the combined oral administration of kumiss-like Lactobacillus ZW3 and Bifidobacterium longum CGMCC 15014 can effectively enhance the anti-allergic reaction, especially has a very good preventive and therapeutic effect on secretory otitis media caused by rapid-acting allergic reactions.

[0008] The technical problem to be solved by the present invention can be achieved through the following technical solutions.

[0009] A composite probiotic preparation for treating secretory otitis media, comprising:

[0010] Kisses-like Lactobacillus, Bifidobacterium longum and other acceptable pharmaceutical excipients.

[0011] The kumiss-like lactobacillus is kumiss-like lactobacillus ZW3, and the strain has been deposited in the Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences on December 18, 2008, with the deposit number: CGMCC2809.

[0012] The Bifidobacterium longum is preferably Bifidobacterium longum CGMCC 15014.

[0013] The composite bacterial agent can be made into oral preparations, capsules and the like.

[0014] Advantages of the present invention:

[0015] The composite probiotic preparation of the present invention has a definite curative effect, can be taken safely, can be directly made into an oral preparation, can also be made into capsules, etc., and is convenient to eat. The present invention has a significant anti-allergic effect, especially for the problem of secretory otitis media, can be taken for a long time, and can regulate the body's immunity. DETAILED DESCRIPTION

[0016] The embodiments of the present invention are described in detail below. The embodiments are only used to explain the present invention and are not to be construed as limiting the present invention.

[0017] Specific embodiments of the present invention are described below.

[0018] Example 1

[0019] Activated bacteria:

[0020] Kumiss Lactobacillus ZW3: Take a ring of Kumiss Lactobacillus ZW3 and inoculate it into MRS solid medium and culture it anaerobically at 37℃ for 24h. Pick a single colony with good growth in the plate and inoculate it into MRS liquid medium for activation. Culture it anaerobically for 10 8 cfu / ml kumiss-like Lactobacillus seed solution.

[0021] Bifidobacterium longum CGMCC 15014: The activation method is the same as that of Lactobacillus kumiss. Finally, it is configured to 10 8 cfu / ml Bifidobacterium longum CGMCC 15014 seed liquid.

[0022] Common Bifidobacterium longum: Bifidobacterium longum CICC6201, purchased from China Industrial Microbiological Culture Collection Management Center. The activation method is the same as that of Lactobacillus kumiss. Finally, it is prepared into 10 8 cfu / ml common Bifidobacterium longum seed solution.

[0023] Example 2

[0024] The operation of activating the strain is the same as in Example 1.

[0025] Modeling of secretory otitis media in mice: 35 female Kunming mice, with a body weight of 22-30g, sensitive auricular reflexes, no history of noise exposure and drug feeding. Randomly divided into 7 groups (5 mice in each group), there was no statistical difference in the weight of mice among the groups. The 6 groups were blank group, model group, positive control group, ZW3 intervention group, CGMCC 15014 intervention group, ZW3+CGMCC 15014 intervention group, and ZW3+ordinary long bifidobacterium intervention group. The experimental group was given 1.2mg ovalbumin and 5.14mg aluminum hydroxide dissolved in 0.2ml PBS for intraperitoneal injection, once on the first day and the eighth day for systemic sensitization. After anesthesia on the 15th day, 0.1mg of OVA was dissolved in 10mlPBS, and 10ul was injected into the tympanic cavity through the front and lower part of the tympanic membrane. After 24h, anesthesia was repeated and the injection was repeated to observe the state of the tympanic membrane and perform ear stimulation. PBS was used instead in the blank group for systemic sensitization and ear stimulation stages.

[0026] After the secretory otitis media mice were successfully modeled, erythema, vascular striae, turbidity of the tympanic membrane and surrounding tissues were observed, and a fluid level appeared in the tympanic cavity. The mice that failed to model and those that failed to meet the standards were eliminated. The specific treatment of the mice in each group was as follows:

[0027] Blank group and model group: Each mouse was intragastrically administered with 0.2 ml of normal saline each time;

[0028] Positive control group: each mouse was given 0.5 mg / kg dexamethasone intraperitoneally;

[0029] ZW3 intervention group: Each mouse was given 0.2 ml 10 8 cfu / ml ZW3 bacterial solution was intragastrically administered;

[0030] CGMCC 15014 intervention group: each mouse was given 0.2 ml 10 8 cfu / ml of CGMCC 15014 bacterial solution was intragastrically administered;

[0031] ZW3+CGMCC 15014 intervention group: each mouse was given 0.1 ml 10 8 cfu / ml ZW3+0.1ml10 8 cfu / ml of CGMCC 15014 bacterial solution was intragastrically administered;

[0032] ZW3+ Bifidobacterium longum intervention group: Each mouse was given 0.1 ml 10 8 cfu / ml of ZW3+0.1ml 10 8 cfu / ml of common Bifidobacterium longum CICC6201 bacterial solution was gavaged intragastrically.

[0033] Oral gavage or intraperitoneal injection of dexamethasone once a day for 10 consecutive days. The blood of mice was collected by tail artery method before the relevant treatment of each group. The blood of mice was collected immediately after the mice in each group were killed on the 11th day, and the total serum IgE concentration before and after the experiment was determined using ELISA kit. The results are shown in Table 1. Statistical analysis: SPSS11.0 was used for statistical analysis, and the results of quantitative data were expressed as mean ± standard deviation, and the inter-group t test was used.

[0034] Table 1 Detection of IgE before and after treatment

[0035]

[0036]

[0037] Note: t test, 1: P < 0.05 (compared with the blank group); 2: P < 0.05 (compared with the model group)

[0038] Before treatment, the IgE levels of the modeling group were higher in all groups, significantly higher than the blank group. After different treatments were performed in each group. There was a statistically significant difference between the ZW3 intervention group and the model group (P<0.05), indicating that the kumiss-like lactobacillus ZW3 had a down-regulating effect on the IgE of the ovalbumin-induced allergic model; while there was no statistical difference between the CGMCC 15014 intervention group and the model group, indicating that the anti-allergic effect was unique to ZW3. The ZW3+CGMCC 15014 intervention group was lower than the positive control group and other intervention groups, and even slightly lower than the positive control group treated with dexamethasone, with a statistically significant difference (P<0.05); while the ZW3+ordinary long bifidobacterium intervention group and the ZW3 intervention group did not change much in terms of the decline, indicating that only the combination of ZW3+CGMCC 15014 would significantly downregulate IgE, and ZW3+CGMCC 15014 had a synergistic effect, which was unexpectedly able to significantly reduce the IgE level of mice with secretory otitis media.

[0039] Example 3

[0040] The changes in the tympanic membrane images of the mice in each group in Example 2 were continuously and dynamically observed on the 3rd, 6th and 9th days after modeling.

[0041] Blank group: The tympanic membrane was translucent, and the handle of the malleus and the light cone were clearly discernible.

[0042] Model group: On the 3rd, 6th and 9th days, the tympanic membrane was congested and edematous, the light cone disappeared, and fluid level was visible in the tympanic cavity.

[0043] Positive control group: On the 3rd day, the tympanic membrane congestion improved, the light cone disappeared, and the fluid level was visible in the tympanic cavity. On the 6th day, the congestion and edema improved compared with the previous day, the light cone disappeared, and bubbles were still visible in the tympanic cavity of 2 mice, which was considered to be partial absorption of the fluid. On the 9th day, the tympanic membranes of all mice were clearly marked, and no tympanic effusion was observed.

[0044] ZW3 intervention group: On the 3rd day, the tympanic membrane congestion improved, the light cone disappeared, and the fluid level was visible in the tympanic cavity. On the 6th day, the congestion and edema improved, the light cone disappeared, and the fluid level was visible in the tympanic cavity. On the 9th day, the congestion and edema improved compared with the previous day, and bubbles were visible in the tympanic cavity.

[0045] CGMCC 15014 intervention group: On days 3, 6, and 9, the tympanic membrane was congested and edematous, the light cone disappeared, and fluid level was visible in the tympanic cavity.

[0046] In the ZW3+CGMCC 15014 intervention group, on the third day, tympanic membrane congestion improved, light cones disappeared, and bubbles were observed in the tympanic cavity of one mouse. On the sixth day, congestion and edema improved, and bubbles were observed in the tympanic cavity of all five mice. On the ninth day, the tympanic membranes of all mice were clearly marked, and no signs of tympanic effusion were observed.

[0047] ZW3+Ordinary Bifidobacterium longum intervention group: On the 3rd day, tympanic membrane congestion improved, light cones disappeared, and fluid level was visible in the tympanic cavity. On the 6th day, congestion and edema improved, light cones disappeared, and fluid level was visible in the tympanic cavity. On the 9th day, congestion and edema improved compared with the previous day, and bubbles were visible in the tympanic cavity, which was considered to be partial absorption of fluid.

[0048] It can be seen that there is no difference between the CGMCC 15014 intervention group and the modeling group. The ZW3 intervention group can alleviate the congestion and edema of the tympanic membrane of otitis media to a certain extent, but improvement can be seen on the 9th day. The ZW3+CGMCC 15014 intervention group can have a significant effect on the 6th day, and the effect is slightly stronger than the dexamethasone group. It is speculated that CGMCC 15014 may specifically accelerate the disappearance of allergic substances in ZW3, promote blood circulation, accelerate the relief of edema, and quickly relieve edema and middle ear exudate caused by allergies.

[0049] Example 4

[0050] The mice in each group in Example 2 were killed on the 11th day, and the anatomical structures of the bilateral auditory bullae and Eustachian tubes of the mice were completely exposed and collected using micro-ophthalmic scissors, and the tissues were dehydrated, embedded, sliced, stained, and sealed. The middle ear tissue was sliced ​​perpendicular to the tympanic membrane, and the thickness of the proximal Eustachian tube mucosa and the tympanic cavity mucosa was measured. The results are shown in Table 2 below:

[0051] Table 2 Detection of mucosal thickness of mice in different groups after treatment

[0052] Thickness of the proximal Eustachian tube mucosa (um) Tympanic mucosal thickness (um) Blank Group 16.34±2.12 5.05±1.01 Model Group <![CDATA[31.32±4.23 1 ]]> 10.24±2.06 Positive control group <![CDATA[20.82±3.93 1，2 ]]> <![CDATA[6.25±1.85 1，2 ]]> ZW3 intervention group <![CDATA[26.63±2.99 1，2 ]]> <![CDATA[8.18±2.24 1，2 ]]> CGMCC 15014 intervention group <![CDATA[30.97±4.02 1 ]]> <![CDATA[9.99±1.88 1，2 ]]> ZW3+CGMCC 15014 intervention group <![CDATA[19.80±5.73 1，2 ]]> <![CDATA[5.88±2.02 1，2 ]]> ZW3+ Bifidobacterium longum intervention group <![CDATA[25.29±3.03 1，2 ]]> <![CDATA[8.35±1.36 1，2 ]]>

[0053] Note: t test, 1: P < 0.05 (compared with the blank group); 2: P < 0.05 (compared with the model group)

[0054] Under the microscope, the Eustachian tube mucosa was a pseudostratified ciliated epithelium, with neatly and evenly arranged cell nuclei, closely arranged ciliated columnar epithelium and goblet cells interspersed therein. The submucosal layer of the modeling group and the CGMCC 15014 intervention group alone was thick, with epithelial cell exfoliation, vascular dilation, and obvious infiltration of eosinophils, lymphocytes, plasma cells, and monocytes. The submucosal layer of the ZW3+Long Bifidobacterium intervention group and the ZW3 intervention group was slightly thicker, and the cell edema was significantly reduced compared with the modeling group. There was a small amount of vascular dilation, a decrease in eosinophils, and lymphocytes and monocytes infiltration. The cell composition of the ZW3+CGMCC 15014 intervention group and the dexamethasone treatment group was close to that of the blank group, with no obvious cell edema and a relatively tight arrangement of cells.

[0055] The results of the measurement of the thickness of the middle ear mucosa showed that the thickness of the eustachian tube mucosa and the tympanic mucosa varied in a similar range. There was a statistically significant difference between the ZW3 intervention group and the model group (P<0.05), indicating that Lactobacillus kumiss ZW3 had a therapeutic effect on the ovalbumin-induced allergic model, which could reduce mucosal edema to a certain extent and promote repair. However, there was no statistical difference between the CGMCC 15014 intervention group and the model group, indicating that this effect was unique to ZW3. The ZW3+CGMCC 15014 intervention group was lower than the positive control group and other intervention groups, and even slightly lower than the positive control group treated with dexamethasone, with a statistically significant difference (P<0.05), indicating that only the combined use of ZW3+CGMCC 15014 would produce a synergistic effect and reduce mucosal edema faster and better.

[0056] In summary, the present invention team found in the process of research and practice that the combined oral administration of kumiss-like Lactobacillus ZW3 and Bifidobacterium longum CGMCC 15014 can effectively enhance the anti-allergic ability of mice, and has a good effect on secretory otitis media. Kumiss-like Lactobacillus ZW3 itself has a very powerful function of decomposing nitrites, and it is speculated that its decomposition ability is strong, and it is speculated that it may be able to quickly decompose toxins and accelerate metabolism in the body. It is not clear how Bifidobacterium longum CGMCC 15014 enhances the oral anti-allergic effect of kumiss-like Lactobacillus ZW3, and it is speculated that it may be related to intestinal metabolism.

[0057] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0058] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A composite probiotic preparation for treating secretory otitis media, comprising: kumiss-like Lactobacillus ZW3, Bifidobacterium longum CGMCC 15014 and other acceptable pharmaceutical excipients.

2. The composite probiotic preparation according to claim 1, characterized in that: The composite probiotic preparation can be made into an oral liquid.

3. The composite probiotic preparation according to claim 1, characterized in that: The composite probiotic preparation can be made into capsules.

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