Pentosaceus shizuokensis R11 and use thereof

Abstract

The present application relates to a kind of pentose pediococcus R11 and its application.The preservation number of the pentose pediococcus R11 is CGMCC No.32715, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is November 20, 2024, and the preservation address is No.3, Beichen West Road, Beijing City, Chaoyang District.The pentose pediococcus R11 and its culture provided by the present application help to prevent and control mycobacterium tuberculosis bovine variant infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to Pediococcus pentosaceus R11 and application thereof. BACKGROUND

[0002] Bovine tuberculosis (BTB) is a chronic infectious disease caused by Mycobacterium bovis (M. bovis), which is mainly transmitted through the respiratory tract and can also be transmitted through excrement such as milk and feces. This disease not only affects the health of cattle herds, leading to death, reduced productivity, etc., but also can be transmitted to humans through animals and their products, and is one of the important sources of human tuberculosis. About 10% of the world's tuberculosis cases are related to bovine tuberculosis bacillus. The transmission routes of bovine tuberculosis include respiratory tract, milk, damaged skin, etc., and the pathogen can infect multiple animals, increasing the risk of cross-species transmission. Controlling bovine tuberculosis is not only necessary to protect the health of cattle herds, but also an important measure to prevent the expansion of public health problems. At present, there is a lack of officially approved safe vaccines for preventing bovine tuberculosis at home and abroad. According to the current regulations in China, once bovine tuberculosis is diagnosed, it needs to be reported to the Ministry of Agriculture and Rural Affairs and disposed of in a harmless manner in a timely manner. Therefore, it is necessary to develop effective means for preventing and controlling bovine tuberculosis in veterinary clinics. SUMMARY

[0003] The technical problem to be solved by the present application is to provide Pediococcus pentosaceus R11, which helps to prevent and control M. bovis infection.

[0004] The technical problem to be solved by the present application is to provide the application of Pediococcus pentosaceus R11.

[0005] To solve the above technical problems, the present application provides Pediococcus pentosaceus R11 with a preservation number of CGMCC No. 32715, a preservation unit of China General Microbiological Culture Collection Center, a preservation time of November 20, 2024, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0006] In some embodiments, the Pediococcus pentosaceus R11 is isolated from sour horse milk in the pastoral area of Inner Mongolia.

[0007] To solve the above technical problems, the present application provides the application of Pediococcus pentosaceus R11 in preparing an antibacterial preparation.

[0008] In some embodiments, the antibacterial agent contains a culture of the Pediococcus pentosaceus R11.

[0009] In some embodiments, the culture is a fermentation broth of the P. pentosus R11.

[0010] In some embodiments, the fermentation broth of the P. pentosus R11 is prepared by the following method:

[0011] The P. pentosus R11 strain is taken out, inoculated into MRS broth medium, cultured for 4-36 hours, centrifuged, and the supernatant is filtered to obtain the fermentation broth of the P. pentosus R11.

[0012] In some embodiments, the bacteriostatic species of the bacteriostatic preparation includes M. tuberculosis bovine variant.

[0013] In addition, the present application also provides a bacteriostatic preparation comprising the P. pentosus R11 or the culture of the P. pentosus R11.

[0014] In addition, the present application also provides a medicine comprising the P. pentosus R11 or the culture of the P. pentosus R11.

[0015] In addition, the present application also provides a feed additive comprising the P. pentosus R11 or the culture of the P. pentosus R11.

[0016] The present application has the following beneficial effects:

[0017] The P. pentosus R11 provided by the present application, especially the fermentation broth containing the P. pentosus R11, has a bacteriostatic effect on M. tuberculosis bovine variant. After the P. pentosus R11 is intervened in mice and then the mice are infected with M. tuberculosis bovine variant, the ability of the mice to resist infection is improved, which is specifically manifested in the following aspects: (1) the body weight loss caused by infection is slowed down; (2) the organ index of the lung and the spleen is significantly reduced; (3) the bacterial load in the lung and the spleen caused by bacterial infection is reduced; (4) the inflammation caused by infection is reduced as shown in pathological sections, and the bacteria in the tissue are reduced as shown in acid-fast staining. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Figure 1 The bacteriostatic effect of the culture of the P. pentosus R11 obtained at different culture times;

[0020] Figure 2To adjust the bacteriostatic effect of the culture of P. pentosaceus R11 on M. tuberculosis var. bovis after adjusting pH;

[0021] Figure 3 To show the weight detection results after the challenge of the culture of P. pentosaceus R11 in Example 4;

[0022] Figure 4 To show the pictures after the challenge of the culture of P. pentosaceus R11 in Example 4;

[0023] Figure 5 To show the calculation results of the spleen organ index of each group in Example 4;

[0024] Figure 6 To show the calculation results of the lung organ index of each group in Example 4;

[0025] Figure 7 To show the calculation results of the bacterial load of the lung after the challenge in Example 4;

[0026] Figure 8 To show the calculation results of the bacterial load of the spleen after the challenge in Example 4;

[0027] Figure 9 To show the enlarged view of the H&E staining section of the lung after the challenge in Example 4;

[0028] Figure 10 To show the enlarged view of the Z&E staining section of the lung after the challenge in Example 4. DETAILED DESCRIPTION

[0029] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in combination with the drawings and examples. The examples of the examples are shown in the drawings, wherein the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The examples described below by referring to the drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application. In addition, it should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0030] Example 1

[0031] This example provides the culture of P. pentosaceus R11, and the specific preparation method is as follows:

[0032] The culture medium involved includes:

[0033] The MRS culture medium is purchased from AOBUX (Boxing);

[0034] 7H9 liquid medium is purchased from Becton, Dickinson and Company, USA;

[0035] 7H10 medium was purchased from Becton, Dickinson and Company;

[0036] The OADC enrichment liquid formula is: Nacl 8.5 g / L, catalase 0.04 g / L, oleic acid 0.5 g / L, D-glucose 20 g / L, BSA-V 50 g / L.

[0037] The taxonomic name of Pediococcus pentosaceus R11 strain is Pediococcus pentosaceus, and the preservation time is November 20, 2024, and the preservation number is CGMCC No. 32715.

[0038] The Mycobacterium tuberculosis bovis involved in the present embodiment and the following embodiments is C68001.

[0039] The method for preparing the culture of Pediococcus pentosaceus R11 (hereinafter referred to as CFS) includes: taking R11 strain from-80℃ refrigerator, activating for two generations on MRS solid medium, and picking single bacteria for overnight culture. Adjust the OD 600 = 1.0 with sterile PBS. Inoculate MRS broth at 2% (v / v), and incubate at 37℃ for 14h-18h to obtain the bacterial solution. Centrifuge at 6000 rpm for 5 min, resuspend the bacterial solution with sterile PBS to OD 600 = 1.0, and obtain the Pediococcus pentosaceus R11 bacterial suspension. Inoculate MRS broth at 2% (v / v), centrifuge at 6000 rpm for 10 min at 4h, 8h, 12h, 24h, and 36h, respectively, take the supernatant, and pass through a 0.22μm filter membrane to obtain the culture (CFS) of Pediococcus pentosaceus R11.

[0040] The culture method of Mycobacterium tuberculosis bovis involved in the following embodiments is as follows: the glycerol bacteria stored in-80℃ refrigerator are taken out, streaked on 7H10 plate, continuously activated for two times, and then single bacteria are picked and inoculated into 7H9 medium (containing 10% OADC enrichment liquid) for culture at 37℃ in a 5% CO2 incubator for 2-3 weeks. When the OD 600 = 0.6-0.8, centrifuge at 6000 rpm for 5 min, and adjust OD 600 = 1.0 with sterile PBS, and obtain the culture.

[0041] The experimental result data is statistically analyzed by using Excel, and single factor variance analysis is performed by using Graph pad prism 10. * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001.

[0042] Example 2

[0043] Bacteriostatic effect of culture of Pediococcus pentosaceus R11 on Mycobacterium tuberculosis var. bovis: The Pediococcus pentosaceus R11 bacterial suspension obtained according to the method provided in Example 1 was inoculated into MRS broth at 2% (v / v), and centrifuged at 6000 rpm for 10 min at 4 h, 8 h, 12 h, 24 h, and 36 h, respectively, to obtain the supernatant, which was filtered through a 0.22 μm filter to obtain 4 h R11_CFS, 8 h R11_CFS, 12 h R11_CFS, 24 h R11_CFS, and 36 h R11_CFS, respectively. The culture of Pediococcus pentosaceus R11 at different time points and 7H9 (containing 10% OADC supplement) were mixed at a ratio of 1:9 to obtain a co-culture medium, and MRS broth was used as a control instead of the culture of Pediococcus pentosaceus R11. Mycobacterium tuberculosis var. bovis with OD 600 = 1.0 was inoculated into the co-culture medium at 2% (v / v), and OD 600 was measured after two weeks. The bacteriostatic effect of the culture of Pediococcus pentosaceus R11 on Mycobacterium tuberculosis var. bovis was observed. The results are shown in Table 1. Figure 1

[0044] As shown in Table 1, the culture of Pediococcus pentosaceus R11 had a significant bacteriostatic effect on Mycobacterium tuberculosis var. bovis, and the bacteriostatic effect increased with the extension of the fermentation time of the culture of Pediococcus pentosaceus R11, and the culture of Pediococcus pentosaceus R11 at 36 h had the best effect. Figure 1 Example 3

[0045] Bacteriostatic effect of culture of Pediococcus pentosaceus R11 on Mycobacterium tuberculosis var. bovis after adjustment of pH

[0046] The 36 h fermentation supernatant was adjusted to the same pH value as MRS broth, and then the culture of Pediococcus pentosaceus R11 at 36 h and 7H9 (containing 10% OADC supplement) were mixed at a ratio of 1:9 to obtain a co-culture medium, and MRS broth was used as a control instead of the culture of Pediococcus pentosaceus R11. Mycobacterium tuberculosis var. bovis with OD 600 = 1.0 was inoculated into the co-culture medium at 2% (v / v), and OD 600 was measured after two weeks. The bacteriostatic effect of the culture of Pediococcus pentosaceus R11 on Mycobacterium tuberculosis var. bovis was observed. The results are shown in Table 2.

[0047] Figure 2 As shown in Table 2, the culture of Pediococcus pentosaceus R11 after adjustment of pH still had a significant bacteriostatic effect on Mycobacterium tuberculosis var. bovis.

[0048] Figure 2 Example 4

[0049] Bacteriostatic effect of culture of Pediococcus pentosaceus R11 on Mycobacterium tuberculosis var. bovis after adjustment of pH

[0050] ​​​Effect of strain and culture of Pediococcus pentosaceus R11 on mice model infected with Mycobacterium bovis var. bovis

[0051] (1) Test animals: healthy female SPF C57BL / 6 mice, 6-8 weeks old (36), the breeding room temperature of these mice is 22-24℃, the humidity is 50-60%, and the light and dark cycle is 12h light and 12h dark. They can eat and drink freely.

[0052] (2) Animal grouping: after 1 week of adaptive feeding, the mice were randomly divided into 6 groups, 6 in each group, and the specific grouping is as follows:

[0053] Control group (PBS gavage + no attack bacteria),

[0054] R11 group (R11 bacterial liquid gavage + no attack bacteria),

[0055] R11C group (R11CFS gavage + no attack bacteria),

[0056] M. bovis group (PBS gavage + attack bacteria),

[0057] R11+M.bovis group (R11 bacterial liquid gavage + attack bacteria),

[0058] R11C+M.bovis group (R11CFS gavage + attack bacteria).

[0059] (3) Intervention method of Pediococcus pentosaceus R11 and CFS

[0060] At the beginning of the experiment, after 1 week of adaptive feeding, the control group and M. bovis group were gavaged with 0.2mL of sterile PBS for 1-2 weeks, once a day; the R11 group and R11+M.bovis group were gavaged with 0.2mL of 1×109 R11 strain for 1-2 weeks, once a day; the R11C group and R11C+M.bovis group were gavaged with 0.2mL of R11CFS for 1-2 weeks, once a day.

[0061] (4) Animal infection method

[0062] Mycobacterium bovis C68001 which was frozen at -80℃ was resuscitated by streaking on 7H10 plate, after two generations of activation, single bacteria were picked and inoculated into 7H9 medium (containing 10% OADC supplement) to culture for 2-3 weeks at 37℃ in a 5% CO2 incubator. When the OD600 of the bacterial solution was 0.6-0.8, the solution was centrifuged at 6000 rpm for 5 min, and then adjusted to OD600 = 1.0 with sterile pbs. Gradient dilution was performed, and then each mouse was intranasally infected with 5000 CFU. After the intervention, the mice were anesthetized with 0.1 mL of Cetep (1:5 dilution), and the M. bovis group, the R11 + M. bovis group, and the R11C + M. bovis group were intranasally infected with 50 ul of bacterial solution (containing 5000 CFU). During the process, the actions were slow, and the 50 ul of bacterial solution was completely inhaled by the mice. After the intranasal instillation, the mice were allowed to wake up.

[0063] Treatment was performed 4 weeks after challenge, and the state of the mice was observed daily, and the weight was measured once a week.

[0064] (5) Sample collection and tissue processing

[0065] After the end of the experiment, the mice were weighed and sacrificed, and the lung, spleen and other tissues were removed aseptically, weighed, and the left lung and lower edge of the spleen were separated and fixed with 10% formalin; the remaining tissues were weighed and placed in a grinding tube.

[0066] (6) Index analysis

[0067] (6.1) Mouse weight detection:

[0068] The results of weight detection after challenge after intervention with culture of Pediococcus pentosaceus R11 are shown in Figure 3 The pictures of weight detection after challenge after intervention with culture of Pediococcus pentosaceus R11 are shown in Figure 4 Compared with the single challenge group, the weight increased significantly after challenge after intervention with Pediococcus pentosaceus R11 and CFS.

[0069] (6.2) Spleen and lung organ index calculation:

[0070] Organ index calculation formula: organ weight / mouse weight

[0071] The results are shown in Figure 5 and Figure 6 Compared with the M. bovis group, the spleen index and lung index of the R11 + M. bovis group and the R11C + M. bovis group decreased, but the results were not significant.

[0072] (6.3) Lung and spleen bacterial load calculation:

[0073] In the grinding tube, 1 mL of sterile PBS was added, homogenized, and gradient diluted, and then plated on 7H10 plates. After 2-3 weeks, the number of colonies was counted, and the organ bacterial load was calculated as the number of colonies / homogenized organ weight*total organ weight.

[0074] As shown in Figure 7 and Figure 8 , after treatment of mice with P. pentosus R11 and CFS, the bacterial load in the spleen and lung was significantly reduced compared with the challenge group alone, and the effect of the P. pentosus R11 treatment group was better than that of the CFS treatment group.

[0075] (6.4) HE staining analysis and acid-fast staining analysis of mouse lung

[0076] The left lung was fixed in 10% formalin, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). The spread of M. bovis in the lung tissue of mice was observed by Ziehl-Neelsen (Z&E) staining. The sections stained by H&E and Z&E were observed under different magnifications. (Magnification: 4X, bar: 500 μm; magnification: 20X, bar: 100 μm; magnification: 50X, bar: 30 μm)

[0077] As shown in Figure 9 and Figure 10 , compared with the control group, the M. bovis group showed extensive inflammatory response in half of the lung lobes, and the bronchoalveolar cavity was filled with inflammatory cells, and epithelioid cell hyperplasia was observed; the R11+M.bovis group showed slight inflammatory foci around the lung vessels, and no other abnormalities were observed; the R11C+M.bovis group showed about one-fourth of the inflammatory area, and a large number of inflammatory cell infiltration and epithelioid cell hyperplasia were observed in the bronchioalveolar. P. pentosus R11 and CFS intervention can alleviate the changes to varying degrees, and the R11+M.bovis group has the best effect.

[0078] In the description of the present specification, the description of the terms "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiments or examples are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0079] The above is the preferred embodiment of the application. It should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which are also considered within the scope of protection of the present application.

Claims

1. A Pediococcus pentosaceus Rl 1 characterized in that, The preservation number of the Pediococcus pentosaceus R11 is CGMCC No. 32715, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is November 20, 2024, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Pediococcus pentosaceus R11 of claim 1 in the preparation of a Mycobacterium bovis (M. bovis) bacteriostatic agent.

3. Use of Pediococcus pentosaceus Rl 1 according to claim 2 for the preparation of a bacteriostatic agent against Mycobacterium bovis, characterized in that, The bacteriostatic agent contains the culture of the Pediococcus pentosaceus R11.

4. Use of Pediococcus pentosaceus Rl 1 according to claim 3 for the preparation of a bacteriostatic agent against Mycobacterium bovis, characterized in that, The culture is a fermentation broth of the Pediococcus pentosaceus R11.

5. Use of Pediococcus pentosaceus Rl 1 according to claim 4 for the preparation of a bacteriostatic agent against Mycobacterium bovis, characterized in that, The fermentation broth of the Pediococcus pentosaceus R11 is prepared by the following method: taking the Pediococcus pentosaceus R11 strain, inoculating into MRS broth medium, centrifuging after 4-36 h of culture, and then filtering the supernatant to obtain the fermentation broth of the Pediococcus pentosaceus R11.

6. A bacteriostatic preparation, characterized by, The bacteriostatic agent contains the culture of the Pediococcus pentosaceus R11.

7. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The bacteriostatic agent contains the culture of the Pediococcus pentosaceus R11.

8. A feed additive, characterized in that, The bacteriostatic agent contains the culture of the Pediococcus pentosaceus R11.

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