Composite probiotic for preventing and treating infectious pneumonia and use thereof

Abstract

The present invention relates to a composite probiotic for preventing and treating infectious pneumonia and use thereof. The composite probiotic for preventing and treating infectious pneumonia consists of the Lactobacillus acidophilus LA85 strain and the Lactobacillus plantarum Lp05 strain. The two strains can cooperate with each other and promote each other, resulting in a synergy in the prevention and treatment of infectious pneumonia. When the same amount of bacteria is used, compared with a strain intervention lacking either strain, the combination of the two strains has a significantly improved effect in the prevention and treatment of infectious pneumonia. Moreover, both strains are probiotics. The product has a high level of safety and a low risk of resistance development.

Description

Composite probiotic for preventing and treating infectious pneumonia and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to a composite probiotic for preventing and treating infectious pneumonia and application thereof. BACKGROUND

[0002] Streptococcus pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus and other bacteria are important pathogens in pneumonia, and sustained inflammatory response can produce excessive reactive oxygen species, which can directly damage nucleic acids, proteins and lipids, and cause irreversible damage to the body. Reactive oxygen species and inflammatory response will start a mutually promoting cycle and spread throughout the body, which can lead to systemic inflammatory syndrome. For the treatment of bacterial pneumonia, inhaled administration can directly deliver antibiotics to the infection area. Although conventional drug therapy can relieve some symptoms, it is often accompanied by significant side effects, which makes it particularly urgent to find safer and more effective alternative treatment methods.

[0003] In recent years, with the in-depth study of the interaction mechanism of the intestinal-lung axis, probiotics, as natural microorganisms that regulate host immune response and improve the balance of intestinal microbial community, have gradually revealed their potential in the prevention and treatment of pneumonia. Intestinal microorganisms are believed to play a key role in regulating local and systemic immune responses of the host. Through the interaction of the intestinal-lung axis, changes in intestinal microorganisms can affect the immune environment of the lungs and thus affect the resistance to respiratory pathogens.

[0004] The application of probiotics in the treatment of infectious pneumonia is still in its early stages, and the prevention and treatment strategies are still few. Therefore, developing more probiotic agents for improving infectious pneumonia can not only provide new ideas and methods for the treatment of infectious pneumonia, but also help to further understand the role of the intestinal-lung axis in diseases and provide scientific basis and technical support for the prevention and treatment of respiratory infectious diseases. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application aims to provide a composite probiotic for preventing and treating infectious pneumonia and application thereof.

[0006] To achieve the object of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a composite probiotic for preventing and treating infectious pneumonia, which is composed of Lactobacillus acidophilus LA85 strain with a preservation number of CGMCC No.1.12735 and Lactobacillus plantarum Lp05 strain with a preservation number of CGMCC No.23547.

[0008] The application creatively develops a brand-new probiotic compound mode and a brand-new strategy for preventing and treating infectious pneumonia, i.e. compound combination of Lactobacillus acidophilus LA85 strain and Lactobacillus plantarum Lp05 strain, and it is found that the two can cooperate with each other, promote each other and synergistically improve the effect in preventing and treating infectious pneumonia, which is specifically shown in the following aspects: (1) significantly inhibiting the growth of infectious microorganisms in the lung and improving the clearance rate of infectious microorganisms; (2) significantly improving the inflammatory response in the lung; (3) significantly improving the survival rate of the infectious pneumonia model mice. In the case of using the same amount of bacteria, compared with the bacterial intervention mode in which any one of the bacteria is missing, the compound of the two bacteria significantly improves the effect in preventing and treating infectious pneumonia. At the same time, both of the bacteria are probiotics, the product is high in safety and is not easy to produce resistance.

[0009] Preferably, the ratio of viable cell count of the LA85 strain to the Lp05 strain is 1:10-10:1, for example 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and other specific point values in the above numerical range can be selected, which will not be repeated here.

[0010] In the second aspect, the application provides a probiotic agent for preventing and treating infectious pneumonia, wherein the strains in the probiotic agent include the compound probiotic bacteria of the first aspect.

[0011] Preferably, the viable cell content of the LA85 strain and the Lp05 strain in the probiotic agent is respectively not less than 1×10 9 CFU / g or 1×10 9 CFU / mL, for example 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×10 10 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), and other specific point values in the numerical range can be selected, which will not be repeated here.

[0012] Preferably, the dosage form of the probiotic agent comprises a solution, a lyophilized powder, a capsule, a tablet or a granule. The dosage form of the probiotic agent involved in the present application is not limited, including the most commonly used solution, lyophilized powder, or further prepared capsule, tablet or granule.

[0013] Preferably, the dosage form of the probiotic agent is a solution, which is prepared by the following method:

[0014] The LA85 strain and the Lp05 strain are inoculated into the culture medium respectively to carry out activation and fermentation culture in sequence to obtain fermentation liquor; the fermentation liquor is centrifuged respectively, resuspended with a solvent to obtain LA85 bacterial suspension and Lp05 bacterial suspension; the LA85 bacterial suspension and the Lp05 bacterial suspension are mixed according to the viable bacterial count ratio to obtain the probiotic agent.

[0015] Preferably, the dosage form of the probiotic agent is a lyophilized powder, which is prepared by the following method:

[0016] The LA85 strain and the Lp05 strain are inoculated into the culture medium respectively to carry out activation and fermentation culture in sequence to obtain fermentation liquor; the fermentation liquor is centrifuged respectively, resuspended with a solvent to obtain LA85 bacterial suspension and Lp05 bacterial suspension; the LA85 bacterial suspension and the Lp05 bacterial suspension are mixed according to the viable bacterial count ratio to obtain the probiotic agent.

[0017] In a third aspect, the present application provides use of the composite probiotic bacteria of the first aspect or the probiotic agent of the second aspect in the preparation of a drug for preventing, improving or treating infectious pneumonia.

[0018] Preferably, the drug further contains an excipient.

[0019] Preferably, the excipient comprises any one or a combination of at least two of a filler, a binder, a wetting agent, a disintegrant, an emulsifying agent, a co-solvent, a solubilizing agent, an osmotic pressure adjusting agent, a coloring agent, a pH adjusting agent, an antioxidant, a bacteriostatic agent or a buffer.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The application creatively develops a brand-new probiotic compound mode and a brand-new strategy for preventing and treating infectious pneumonia, that is, Lactobacillus acidophilus LA85 strain and Lactobacillus plantarum Lp05 strain are compounded and used in combination, and it is found that the two can cooperate with each other, promote each other and synergistically improve the effect in preventing and treating infectious pneumonia, which is specifically shown in the following aspects: (1) significantly inhibiting the growth of infectious microorganisms in the lung and improving the clearance rate of infectious microorganisms; (2) significantly improving the lung inflammatory response; (3) significantly improving the survival rate of the infectious pneumonia model mice. In the case of using the same amount of bacteria, compared with the bacterial species intervention mode in which any one of the bacteria is missing, the compound of the two bacteria significantly improves the effect in preventing and treating infectious pneumonia. At the same time, the two bacteria are probiotics, the product is high in safety and is not easy to produce resistance.

[0022] The LA85 strain involved in the application is classified as Lactobacillus acidophilus, is preserved in the China General Microbiological Culture Collection Center, is preserved on July 20, 2020, has a preservation number of CGMCC No.1.12735 and is located at No.3, Beichen West Road, Chaoyang District, Beijing.

[0023] The Lp05 strain involved in the application is classified as Lactobacillus plantarum, is preserved in the China General Microbiological Culture Collection Center, is preserved on October 9, 2021, has a preservation number of CGMCC No.23547 and is located at No.3, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0024] Fig. 1 is a statistical result graph of the number of white blood cells in the bronchoalveolar lavage fluid of the mice in each group 6 hours after being infected with Pseudomonas aeruginosa;

[0025] Fig. 2 is a statistical result graph of the number of white blood cells in the bronchoalveolar lavage fluid of the mice in each group 24 hours after being infected with Pseudomonas aeruginosa;

[0026] Fig. 3 is a statistical result graph of the number of Pseudomonas aeruginosa in the lung tissue of the mice in each group 24 hours after being infected with Pseudomonas aeruginosa. DETAILED DESCRIPTION

[0027] The technical solutions of the application are further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the application and should not be regarded as specific limitations on the application.

[0028] The medium formula involved in the following examples is as follows:

[0029] MRS medium: 10 g / L proteose peptone, 10 g / L beef extract, 20 g / L glucose, 2 g / L sodium acetate, 5 g / L yeast powder, 2 g / L diammonium hydrogen citrate, 2.6 g / L K2PO4·3H2O, 0.1 g / L MgSO4·7H2O, 0.05 g / L MnSO4, 1 mL / L Tween 80, 0.5 g / L cysteine hydrochloride.

[0030] The LA85 strain involved in the following examples is classified as Lactobacillus acidophilus, and is preserved in the China General Microbiological Culture Collection Center, with a preservation time of July 20, 2020, a preservation number of CGMCC No. 1.12735, and an address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0031] The Lp05 strain involved in the following examples is classified as Lactobacillus plantarum, and is preserved in the China General Microbiological Culture Collection Center, with a preservation time of October 9, 2021, a preservation number of CGMCC No. 23547, and an address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0032] The method for preparing the bacterial suspension involved in the following examples is as follows: inoculate the desired strain in liquid medium, incubate at 37°C for 24 h for activation, and continuously activate for 2 times to obtain an activation liquid; inoculate the activation liquid in liquid medium at an inoculation amount of 5% (v / v), incubate at 37°C for 24 h to obtain a bacterial liquid; centrifuge the bacterial liquid at 4°C at 5000 rpm for 10 min, filter to obtain bacterial bodies, resuspend the bacterial bodies with PBS solution, and obtain the bacterial suspension.

[0033] The test result data is statistically analyzed using the ggplot2 of R language, **** represents p<0.0001, *** represents p<0.001, ** represents p<0.01, * represents p<0.05, and NS. represents no significant difference.

[0034] Example 1

[0035] This example explores the symptom improvement ability of the composite probiotic bacteria on the infectious pneumonia mouse model:

[0036] (1) Test animals: healthy male ICR mice, 6 weeks old (56), these mice are raised in a controlled environment, the room temperature is maintained at 21-23°C, the humidity is 40-60%, and a 12h light / dark cycle is followed. They can eat and drink water at will.

[0037] (2) Animal grouping: After the mice were adaptively fed for 1 week, they were randomly assigned into 7 groups, 8 in each: control group, model group, LA85 group (intervened with LA85 bacterial solution), Lp05 group (intervened with Lp05 bacterial solution), LA85+Lp05 group (intervened with LA85 bacterial solution and Lp05 bacterial solution), ATCC11975+Lp05 group (intervened with commercially available Lactobacillus acidophilus ATCC11975 bacterial solution and Lp05 bacterial solution), LA85+ATCC8014 group (intervened with LA85 bacterial solution and commercially available Lactobacillus plantarum ATCC8014 bacterial solution).

[0038] (3) Animal modeling and intervention method:

[0039] The control group and each bacterial solution intervention group were inoculated with 30 μL (15 μL per nostril) of 0.9% sterile normal saline (for the control group) or each group of bacterial solution (the number of viable bacteria was 1 x 10 9 CFU / mouse) in the mouse nasal cavity 18 h before surgical anesthesia. The model group was not treated. Among them, after the mice in each bacterial solution intervention group and the model group were anesthetized by intraperitoneal injection of 7% chloral hydrate solution (10 mg / kg according to body weight), 20 μL of Pseudomonas aeruginosa (ATCC9027) bacterial suspension (concentration of 1 x 10 8 CFU / mL) (10 μL per nostril) was inoculated in the nasal cavity.

[0040] (4) Index analysis:

[0041] (4.1) Detection of white blood cells in bronchoalveolar lavage fluid:

[0042] After 6 h and 24 h of infection, respectively, 4 mice in each group were sacrificed after anesthesia, and the liquid (referred to as BAL fluid) was collected by tracheal puncture and injection of 500 μL of 0.9% sterile normal saline for bronchoalveolar lavage in a sterile environment. The number of white blood cells in the bronchoalveolar lavage fluid of each group was counted using a cell counter.

[0043] The results are shown in Figures 1 and 2. As can be seen from the figures, compared with the control group, the number of white blood cells in the bronchoalveolar lavage fluid of the model group mice increased significantly, indicating that the mice infected with Pseudomonas aeruginosa had obvious inflammation in the lungs, and the model was successfully constructed. After intervention by each group of probiotics, the number of white blood cells in the bronchoalveolar lavage fluid of the mice decreased to varying degrees, among which the LA85+Lp05 group had the most obvious decreasing trend, which was better than the Lp05 group and the LA85 group, indicating that the LA85 strain and the Lp05 strain had synergistic effect in relieving infectious pneumonia.

[0044] (4.2) Detection of pathogenic bacteria in the lungs:

[0045] The lungs of infected mice for 24 h were excised, homogenized with 0.9% sterile normal saline, and tested with Organotial mouse lung organ culture medium kit (Aibisheng Shanghai Biotechnology Co., Ltd.) on MRS agar plates for total lung homogenate, and the number of P. aeruginosa and probiotics was determined by serial dilution. Incubate at 37°C for 48 h under anaerobic conditions. Mass spectrometry was used to determine the number of P. aeruginosa colonies in the total lung homogenate, and the logarithmic value was measured.

[0046] The results are shown in Figure 3. As can be seen from the figure, compared with the control group, the number of P. aeruginosa in the lung tissue of the model group mice increased significantly, indicating that the model group mice had severe P. aeruginosa infection in the lungs. After intervention with probiotics in each group, the number of P. aeruginosa in the lung tissue of mice decreased to varying degrees, and the number in the LA85+Lp05 group was less than that in the Lp05 group and the LA85 group, indicating that the LA85 strain and the Lp05 strain had synergistic effects on inhibiting the growth of P. aeruginosa and relieving infectious pneumonia.

[0047] Example 2

[0048] This example investigates the effect of composite probiotics on the survival of mice with infectious pneumonia:

[0049] (1) Test animals: healthy male ICR mice, 6 weeks old (35), which were raised in a controlled environment with room temperature maintained at 21-23°C and humidity of 40-60%, following a 12h light / dark cycle. They had free access to food and water.

[0050] (2) Animal grouping: After 1 week of adaptive feeding, the mice were randomly assigned to 7 groups of 5 each: control group, model group, LA85 group (intervened with LA85 bacterial solution), Lp05 group (intervened with Lp05 bacterial solution), LA85+Lp05 group (intervened with LA85 bacterial solution and Lp05 bacterial solution), ATCC11975+Lp05 group (intervened with commercially available Lactobacillus acidophilus ATCC11975 bacterial solution and Lp05 bacterial solution), LA85+ATCC8014 group (intervened with LA85 bacterial solution and commercially available Lactobacillus plantarum ATCC8014 bacterial solution).

[0051] (3) Animal modeling and intervention method:

[0052] The control group and each bacterial solution intervention group were inoculated with 30μL (15μL per nostril) of 0.9% sterile normal saline (control group) or each group of bacterial solution (viable bacterial count was 1×10 9CFU / only), the model group is not treated. Among them, each bacterial liquid intervention group and the model group of mice are anesthetized by intraperitoneal injection of 7% chloral hydrate solution (10 mg / kg according to the body weight) after nasal inoculation of 20 μL of Pseudomonas aeruginosa (ATCC9027) bacterial suspension (concentration is 1×10 8 CFU / mL) (10 μL per nostril).

[0053] (4) Index analysis:

[0054] (4.1) Survival of mice:

[0055] The survival rate of mice in each group is counted within 7 days after the mice are infected with Pseudomonas aeruginosa, and the results are shown in Table 1 as follows:

[0056] Table 1

[0057] From the data results in Table 1, it can be seen that the composite probiotic bacteria involved in the present application can significantly improve the survival of mice after being infected with Pseudomonas aeruginosa, and the LA85+Lp05 group is better than other bacterial liquid intervention groups, which indicates that the LA85 strain and the Lp05 strain can cooperate with each other, promote each other, and synergistically enhance the effect in preventing and treating infectious pneumonia.

[0058] The applicant declares that the technical solutions of the present application are illustrated by the above examples, but the present application is not limited to the above examples, that is, it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0059] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and within the technical concept scope of the present application, various simple modifications of the technical solutions of the present application can be made, and these simple modifications all belong to the protection scope of the present application.

[0060] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined by any suitable means without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A compound probiotic for preventing and treating infectious pneumonia, characterized in that, The compound probiotic for preventing and treating infectious pneumonia consists of Lactobacillus acidophilus LA85 strain with accession number CGMCC No.1.12735 and Lactobacillus plantarum Lp05 strain with accession number CGMCC No.23547.

2. The compound probiotic for preventing and treating infectious pneumonia according to claim 1, characterized in that, The ratio of viable cells of the LA85 strain to the Lp05 strain is 1:10-10:

1.

3. A probiotic agent for preventing and treating infectious pneumonia, characterized in that, The strains in the probiotic agent include the compound probiotics described in claim 1 or 2.

4. The probiotic agent according to claim 3, characterized in that, The viable bacterial content of the LA85 strain and the Lp05 strain in the probiotic agent is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

5. The probiotic agent according to claim 3, characterized in that, The dosage forms of the probiotic agent include solutions, lyophilized powders, capsules, tablets, or granules.

6. The probiotic agent according to claim 5, characterized in that, The probiotic agent is in the form of a solution, which is prepared by the following method: The LA85 and Lp05 strains were inoculated into the culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged and resuspended in a solvent to obtain LA85 bacterial suspension and Lp05 bacterial suspension. The LA85 bacterial suspension and Lp05 bacterial suspension were mixed according to the live bacteria ratio to obtain the probiotic agent.

7. The probiotic agent according to claim 5, characterized in that, The probiotic agent is in the form of a lyophilized powder, which is prepared by the following method: The LA85 and Lp05 strains were inoculated into the culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged, mixed with a protectant, and then freeze-dried to obtain LA85 and Lp05 bacterial powders. The LA85 and Lp05 bacterial powders were mixed according to the live cell count ratio to obtain the probiotic agent.

8. The use of the compound probiotic as described in claim 1 or 2 or the probiotic agent as described in any one of claims 3-7 in the preparation of a medicament for the prevention, improvement or treatment of infectious pneumonia.

9. The application according to claim 8, characterized in that, The drug also contains excipients.

10. The application according to claim 9, characterized in that, The excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

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