Bacillus subtilis, bacterial agent and composition and their application in preparing products for preventing and treating endometritis

Abstract

The present invention is applicable to the field of microbial technology, and provides a Bacillus subtilis, a bacterial agent and a composition and their application in preparing a product for preventing and treating endometritis. Bacillus subtilis M2022, taxonomically named Bacillus subtilis Bacillus subtilis ; The deposit number is CGMCC No.28555; the deposit date is September 25, 2023. Bacillus subtilis M2022 can alleviate the inflammatory response of uterine tissue caused by subacute rumen acidosis, reduce the levels of neutrophils, TNF‑α and IL‑1β in uterine lavage fluid, thereby alleviating the occurrence of endometritis in dairy goats. It has potential application prospects in the prevention and treatment of endometritis in dairy goats.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Bacillus subtilis, a bacterial agent and a composition, and applications thereof in preparing a product for preventing and treating endometritis. Background Art

[0002] Endometritis can cause significant morbidity and economic losses worldwide. It may lead to problems such as dystocia and infertility. The etiology of endometritis is not fully understood and needs further study. In order to meet the nutritional needs of dairy goats and improve their production performance, the excessive use of high-concentrate feed in modern intensive management is an important factor leading to subacute ruminal acidosis. Subacute ruminal acidosis is mainly characterized by a decrease in ruminal pH, which is associated with an increase in the concentration of lipopolysaccharide (LPS) in the rumen. LPS can destroy the epithelial barrier of the rumen and diffuse into the peripheral circulation, leading to a systemic inflammatory response. Previous studies have found that subacute ruminal acidosis can lead to increased levels of LPS and TNF-α in the rumen, serum, and endometrial tissues, and cause endometrial damage by reducing the expression of tight junction proteins. In addition, subacute ruminal acidosis can increase the expression of nuclear transcription factors and proinflammatory cytokine mRNA in uterine tissues, confirming the presence of uterine inflammation. It can be seen that subacute ruminal acidosis is an important factor inducing endometritis, and alleviating its symptoms may be an effective way to treat endometritis.

[0003] Bacillus is a type of Gram-positive bacteria that can produce spores. As a probiotic, the lipopeptides secreted by Bacillus help to resist bacteria, fight inflammation and regulate immunity. As a type of Bacillus, Bacillus subtilis has a strong flora regulation effect, which can promote the abundance of probiotics, inhibit the colonization of harmful bacteria, and thus ensure the health of the body. Bacillus subtilis is often used as a feed additive for livestock and poultry, and has the effects of killing or inhibiting pathogenic bacteria, antiviral, regulating immune response, improving intestinal flora, enhancing intestinal barrier function, and promoting nutrient utilization. In addition, adding Bacillus subtilis to livestock and poultry feed can improve their immune level, promote the development of immune organs, and activate innate immunity and adaptive immunity. Bacillus subtilis can inhibit the expression of downstream proteins of the inflammatory signaling pathway by secreting surfactant, thereby preventing the inflammatory response process. However, there is currently no relevant research on the relief of endometritis caused by subacute rumen acidosis by Bacillus subtilis. In order to overcome the defects in the prior art for preventing and treating endometritis, the present invention provides a Bacillus subtilis, a bacterial agent and a composition and their application in preparing a product for preventing and treating endometritis. Summary of the invention

[0004] The object of the present invention is to provide a Bacillus subtilis, a bacterial agent and a composition and their use in the preparation of a product for preventing and treating endometritis, aiming to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A Bacillus subtilis, wherein the Bacillus subtilis is Bacillus subtilis M2022, taxonomically named Bacillus subtilis Bacillus subtilis ; The deposit number is CGMCC No.28555; it was deposited in the General Microbiology Center of China Culture Collection Administration on September 25, 2023, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] A bacterial agent, comprising the above-mentioned Bacillus subtilis, Bacillus subtilis fermentation liquid or sterile fermentation liquid after centrifugation.

[0008] A composition comprising the above-mentioned Bacillus subtilis or the above-mentioned bacterial agent.

[0009] An application of the above-mentioned Bacillus subtilis, the above-mentioned bacterial agent or the above-mentioned composition.

[0010] Furthermore, the application is one or more of the following:

[0011] a. Application in the preparation of products for improving rumen pH;

[0012] b. Application in the preparation of products for alleviating subacute rumen acidosis;

[0013] c. Application in the preparation of products for reducing uterine neutrophil content;

[0014] d. Application in the preparation of products for reducing inflammatory cell infiltration in uterine tissue;

[0015] e. Application in the preparation of products that reduce inflammatory cytokines in uterine tissue;

[0016] f. Application in the preparation of products for preventing and treating endometritis in dairy goats.

[0017] Furthermore, the product is feed, feed additive or veterinary drug.

[0018] Furthermore, the number of viable Bacillus subtilis in the product is not less than 1×10 9 CFU / mL.

[0019] Furthermore, the product is applied to ruminants, which are cattle, sheep or deer.

[0020] Furthermore, the cattle are dairy cows, yellow cattle, buffaloes or yaks; the sheep are sheep or goats; and the deer are red deer, elk, reindeer or sika deer.

[0021] Furthermore, the goats are dairy goats.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The Bacillus subtilis discovered in the present invention can alleviate subacute rumen acidosis in ruminants, reduce the content of LPS, and alleviate the inflammatory response of the uterus, and has potential application prospects in the field of prevention and treatment of endometritis in dairy goats. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the rumen pH value of the control group, model group, intervention treatment group and intervention control group.

[0025] Figure 2 is the content of neutrophils in the uterine washing fluid of the control group, model group, intervention treatment group and intervention control group.

[0026] Figure 3 These are pathological sections of uterine tissues in the control group, model group, intervention treatment group and intervention control group.

[0027] Figure 4 The pathological sections of uterine tissues in the control group, model group, intervention treatment group and intervention control group were scored.

[0028] Figure 5 is the LPS content in uterine washing fluid of the control group, model group, intervention treatment group and intervention control group.

[0029] Figure 6 is the TNF-α content in uterine washing fluid of the control group, model group, intervention treatment group and intervention control group.

[0030] Figure 7 is the content of IL-1β in uterine washing fluid of the control group, model group, intervention treatment group and intervention control group. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0033] Feeding method: Before the experiment, 40 lactating goats with similar body weight (34.65±5.46 kg) were placed in the experimental field for 2 weeks of adaptation. Feed was fed at fixed time and place at two time periods at 07:00 and 18:00 every day, and drinking water was free. During this period, corn and straw were fed in a ratio of 4:6 to adapt to the environment.

[0034] At the beginning of the experiment, the dairy goats were randomly divided into 4 groups (control group, model group, intervention treatment group and intervention control group), with 10 goats in each group.

[0035] In the first phase of the experiment (15 days), all groups of dairy goats were fed corn and straw in a weight ratio of 4:6. Mixed feed was fed to adapt to this diet for 15 consecutive days, with feed fed at 7:00 and 18:00 every day, and free drinking water. During this period, the ratio of corn meal to corn straw was 4:6 to adapt to the environment.

[0036] In the second phase of the experiment (60 days), the control group and the intervention control group were fed with corn and straw in a weight ratio of 4:6; the model group and the intervention treatment group were fed with corn and straw in a weight ratio of 7:3. The goats were fed continuously for 60 days, with feeds fed at 7:00 and 18:00 every day, and free drinking water. Uterine lavage fluid samples were collected, and the neutrophil content and inflammatory cytokine concentration in the lavage fluid were detected to establish a high-concentrate feed-induced endometritis model in dairy goats.

[0037] In the third stage of the experiment (15 days), the goats in each group were gavaged with the following prescription, with a gavage dose of 100 mL, once every afternoon for 15 consecutive days.

[0038] (1) Control group: PBS, 100 mL.

[0039] (2) Model group: PBS, 100 mL.

[0040] (3) Intervention treatment group: 1 mL PBS contained 1×10 8 CFU Bacillus subtilis M2022, 1×10 8 CFU Bacillus subtilis M2022 / 1mL PBS, 100 mL.

[0041] (4) Intervention control group: 1 mL PBS contained 1×10 8 CFU Bacillus subtilis M2022, 1×10 8 CFU Bacillus subtilis M2022 / 1mL PBS, 100 mL.

[0042] At the end of the experiment, rumen fluid was collected from dairy goats using rumen fluid collection tubes to evaluate the pH of the rumen fluid. Uterine wash fluid was collected, and part of the wash fluid sample was used to detect the number of neutrophils, while the other part was stored in liquid nitrogen. Uterine tissue was collected and divided into small pieces, and the uterine samples were stored in 4% paraformaldehyde.

[0043] Embodiment 1, Bacillus subtilis Effects of M2022 on rumen fluid pH in dairy goats;

[0044] Experimental method: 100 mL of rumen fluid from dairy goats was collected through a rumen fluid collection tube, and the rumen pH was tested using a pH meter.

[0045] Experimental results: Figure 1 As shown in the figure, the pH value of the rumen fluid of dairy goats in the model group was lower than 5.6, proving that the subacute rumen acidosis model was successfully established. At the same time, compared with the control group, the intervention control group did not significantly affect the rumen fluid pH. Bacillus subtilis M2022 did not affect the pH of rumen fluid. In addition, compared with the model group, the pH of rumen fluid in the intervention treatment group increased significantly. The above results show that Bacillus subtilis M2022 can restore the phenomenon of decreased rumen pH caused by high concentrate feed, thereby alleviating subacute rumen acidosis in dairy goats.

[0046] Embodiment 2, Bacillus subtilis Effect of M2022 on the neutrophil content in uterine washing fluid of dairy goats;

[0047] Experimental method: Place fresh uterine lavage fluid into a clean 10 mL centrifuge tube. Use a pipette to evenly apply a small amount of liquid on a glass slide and quickly dry it with a flame to avoid cell deformation. Then stain the smear. Fix the smear in Diff Quick Fixive for 20 s. Stain with Diff Quick I for 5-10 s, remove immediately, and stain with DiffQuickII for 10-20 s. Then dehydrate in 95% ethanol for 5 s, 100% ethanol I for 5 s, 100% ethanol II for 30 s, xylene for 1 min, and then seal. Finally, observe the smear under an optical microscope. The statistical method is to determine the proportion of neutrophils per 200 cells, and select 5 areas for each smear for detection.

[0048] Experimental results: The neutrophil content in uterine washing fluid is a key indicator to measure whether endometritis occurs. Figure 2 As shown in the figure, compared with the control group, high-concentrate feed can significantly increase the content of neutrophils. It can be seen that subacute rumen acidosis can induce endometritis in dairy goats. In addition, compared with the model group, gavage Bacillus subtilis M2022 can significantly reduce the content of neutrophils. Bacillus subtilis M2022 treatment alone did not affect the content of healthy uterine neutrophils.

[0049] Embodiment 3, Bacillus subtilis Effects of M2022 on pathological damage of uterine tissue in dairy goats;

[0050] Experimental methods: Uterine tissue samples were fixed with 4% paraformaldehyde for 48 h. Then, 0.5-1 cm of uterine tissue was placed in an embedding cassette and rinsed with tap water. The embedding cassette was immersed in a 50% ethanol solution, and then immersed in 75%, 85%, 90%, 95%, 100% ethanol and xylene for 1 h. Then, the tissue was embedded in paraffin and cut into slices of approximately 4 μm in series. The slices were placed in xylene for 10 min for dewaxing, and placed in 95%, 85%, 70%, and 50% alcohol for 2 min, respectively. Then, they were immersed in hematoxylin for 10 min, washed, and immersed in eosin staining solution for 5 min. The slices were dehydrated with 70%, 85%, 95%, and 100% alcohol, and placed in xylene to make them transparent. Finally, they were sealed with neutral resin and observed under an optical microscope. Uterine tissue was processed according to the histopathological scoring criteria for endometritis (inflammatory cell infiltration, endometrial cell damage and proliferation, endometrial edema, and endometrial congestion). Each histological feature was scored according to the epithelial histopathological scoring criteria, with a score range of 0-5.

[0051] Experimental results: In order to further clarify the damaging effect of high-concentration diet on uterine tissue, H&E staining was used to detect the pathological changes of uterine tissue. Figure 3 and Figure 4 As shown in the figure, compared with the control group, the uterine tissue of the goats in the model group showed inflammatory cell infiltration, severe endometrial damage, uterine myometrium edema, mucosal damage, and a significant increase in the inflammation score. Bacillus subtilis M2022 treatment reduced inflammatory cell infiltration and myometrial edema in the uterine tissue of dairy goats, alleviated uterine mucosal damage, and significantly reduced the inflammatory score. Bacillus subtilis M2022 can alleviate endometrial tissue damage in dairy goats induced by subacute rumen acidosis.

[0052] Embodiment 4, Bacillus subtilis Effects of M2022 on lipopolysaccharide (LPS) in uterine washing fluid of dairy goats;

[0053] Experimental method: First, add 50 μL of sample to the assay well. Then add 100 μL of Avidin-HRP to the well and incubate at 37 °C for 1 h. After washing the wells 5 times with washing solution, add 50 μL of solution A, solution B, and stop solution to each well, and measure the results at 450 nm.

[0054] Experimental results: Subacute rumen acidosis usually leads to "chronic low-grade" inflammation, which in turn causes endometritis. To determine whether subacute rumen acidosis is related to endometritis, we detected the LPS content in uterine washing fluid. Preliminary analysis results showed that compared with the control group and the intervention control group, the model group significantly increased the level of LPS in the uterine washing fluid ( Figure 5 ).and Bacillus subtilis M2022 can alleviate the phenomenon of LPS increase caused by subacute rumen acidosis. The above data show that subacute rumen acidosis can induce the occurrence of endometritis. Bacillus subtilis M2022 can effectively prevent the occurrence of endometritis.

[0055] Embodiment 5, Bacillus subtilis Effect of M2022 on TNF-α in uterine washing fluid of dairy goats;

[0056] Experimental methods: To detect the inflammatory response in uterine lavage fluid, the uterine lavage fluid was centrifuged at 4 °C, 3000 rpm for 15 min. The content of TNF-α in the uterine lavage fluid was determined by ELISA. First, the capture antibody was added to each test well and incubated overnight at 4 °C. 300 μL of wash buffer was added and washed 4 times. 200 μL of assay diluent A was added, incubated for 1 h, and washed 4 times. 100 μL of sample was added to each well and incubated at 37 °C for 2 h. After washing 4 times, 100 μL of detection antibody was added and incubated at 37 °C for 1 h. 100 μL of Avidin-HRP was added and incubated for 30 min. Wash 5 times. After that, solution A and solution B were added and protected from light for 15 min. Finally, 100 μL of stop solution was added to each well and the results were measured at 450 nm within 15 min.

[0057] Experimental results: The concentration of TNF-α in the flushing fluid can reflect the inflammation level of dairy goats and can therefore be used as an indicator of endometritis. Figure 6 As shown in the figure, compared with the control group, the model group significantly increased the content of TNF-α in the rinsing fluid. Bacillus subtilis Treatment with M2022 significantly reduced the level of TNF-α. Bacillus subtle M2022 treatment alone does not affect the content of TNF-α in the uterine washing fluid of healthy women. Bacillus subtle M2022 can effectively alleviate the phenomenon of increased TNF-α content caused by subacute rumen acidosis.

[0058] Embodiment 6, Bacillus subtilis Effect of M2022 on IL-1β in uterine washing fluid of dairy goats;

[0059] Experimental method: The uterine lavage fluid was centrifuged at 4 °C, 3000 rpm for 15 min. The IL-1β content in the uterine lavage fluid was determined by ELISA. First, the capture antibody was added to the test well and incubated overnight at 4 °C. Then, diluent A, 100 μL sample, detection antibody and Avidin-HRP were added respectively and incubated at 37 °C for 1 h. After washing 5 times, solution A, solution B and solution D were added respectively. The results were measured at 450 nm within 15 min.

[0060] Experimental results: In order to further determine the occurrence of endometritis, we detected the content of IL-1β in uterine washing fluid. The preliminary analysis results showed that compared with the control group and the intervention control group, the model group significantly increased the content of IL-1β in uterine washing fluid ( Figure 7 ).and Bacillus subtilis M2022 could reverse the increase of IL-1β content induced by subacute rumen acidosis. Furthermore, compared with the control group, Bacillus subtilis M2022 does not affect the content of IL-1β in the uterine washing fluid. Bacillus subtilis M2022 can effectively prevent the occurrence of endometritis.

[0061] From Examples 1-6, it can be concluded that Bacillus subtilis M2022 can alleviate the inflammatory response of uterine tissue caused by subacute rumen acidosis, reduce the content of neutrophils, TNF-α and IL-1β in uterine washing fluid, and thus alleviate the occurrence of endometritis in dairy goats.

[0062] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. An application of Bacillus subtilis or a bacterial agent or a composition, characterized in that: The Bacillus subtilis is Bacillus subtilis M2022, taxonomically named Bacillus subtilis Bacillus subtilis ; The deposit number is CGMCC No.28555; The deposit date is September 25, 2023; The bacterial agent includes the Bacillus subtilis, the fermentation broth of Bacillus subtilis or the sterile fermentation broth after centrifugation; The composition includes the Bacillus subtilis or the bacterial agent; The application is one or more of the following: a. Application in the preparation of products for increasing the pH value of the rumen of dairy goats; b. Application in the preparation of products for alleviating subacute rumen acidosis in dairy goats; c. Application in the preparation of a product for reducing the neutrophil content in the uterus of dairy goats; d. Application in the preparation of a product for reducing inflammatory cell infiltration in uterine tissue of dairy goats; e. Application in the preparation of products that reduce inflammatory cytokines in uterine tissue of dairy goats; f. Application in the preparation of products for the prevention and treatment of endometritis in dairy goats; The number of viable Bacillus subtilis in the product is not less than 1×10 8 CFU / mL.

2. The use according to claim 1, characterized in that: The product is feed, feed additive or veterinary drug.

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