Ruminococcus buchneri and application of in-vitro pure culture metabolic supernatant thereof in promoting intestinal development of piglets

Abstract

The invention relates to the technical field of microorganisms, in particular to ruminococcus buchneri and application of in-vitro pure culture metabolic supernate of the ruminococcus buchneri in promoting intestinal development of piglets. The invention relates to ruminococcus buchneri, and the collection number of the ruminococcus buchneri is ATCC (American Type Culture Collection) 51896. The ruminococcus buchneri and the in-vitro pure culture metabolic supernate thereof can obviously improve the unit intestinal weight of the cecum of the weaned piglets, and have the trend of increasing the unit intestinal weight of the jejunum and the ileum of the piglets, increasing the average daily feed intake and reducing the feed-to-gain ratio. The in-vitro organoid culture result also shows that the amplification efficiency of the jejunum organoid of the piglet is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to application of Ruminococcus brucei and in vitro pure culture metabolic supernatant thereof in promoting intestinal development in piglets. Background Art

[0002] Under natural conditions, weaning of piglets is a gradual process, usually taking 14-17 days to complete. To adapt to the needs of intensive farming, modern animal husbandry generally adopts early weaning technology to improve the reproductive efficiency of sows by shortening the lactation period. However, growth retardation caused by weaning stress remains a difficult technical bottleneck to overcome. This stress response is mainly manifested in: damage to the intestinal epithelial structure and barrier function, reduced proliferation and differentiation activity of intestinal stem cells; accompanied by intestinal flora imbalance and impaired repair capacity, ultimately inducing intestinal development blockage and reduced digestion and absorption efficiency. This series of pathological changes not only directly weakens the growth performance and disease resistance of piglets, but also poses a continuous threat to the economic benefits of the farming industry and animal welfare standards. Therefore, how to mitigate the adverse effects that occur during the weaning stage of piglets is an important issue in improving production efficiency. Summary of the Invention

[0003] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a method for using Ruminococcus brucei and its in vitro pure culture metabolic supernatant in promoting intestinal development in piglets, aiming to alleviate the pathological conditions of stress and growth retardation in piglets after weaning in the prior art.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides an application example of Ruminococcus brucei in promoting intestinal development in piglets, and its deposit number is ATCC 51896.

[0005] The second aspect of the present invention provides the use of the above-mentioned in vitro pure culture metabolic supernatant of Ruminococcus brucei in promoting the intestinal development of piglets.

[0006] A third aspect of the present invention provides the use of the above-mentioned Ruminococcus brucei in the preparation of a bacterial agent or medicine for promoting intestinal development in piglets.

[0007] A fourth aspect of the present invention provides the use of the metabolic supernatant of the above-mentioned in vitro pure culture of Ruminococcus brucei in the preparation of a bacterial agent or medicine for promoting intestinal development in piglets.

[0008] The fifth aspect of the present invention provides the use of the metabolic supernatant of the above-mentioned pure in vitro culture of Ruminococcus brucei in improving the activity of porcine intestinal epithelial cells.

[0009] The present invention has the following beneficial effects: The present invention provides Ruminococcus brucei ( Ruminococcus bromii) and its in vitro pure culture metabolic supernatant for promoting intestinal development in piglets, which has the accession number ATCC 51896. The Ruminococcus brucei and its in vitro pure culture metabolic supernatant of the present invention can increase the unit intestinal weight of the jejunum and ileum of piglets, the average daily feed intake, reduce the feed-to-weight ratio, and significantly increase the unit intestinal weight of the cecum of piglets. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 MTT was used to detect the effect of supernatant of Ruminococcus brucei pure culture in vitro on the viability of piglet jejunal epithelial cells (IPEC-J2).

[0011] Figure 2 Immunofluorescence (IF) staining was used to detect PCNA in IPEC-J2 cells. + Results of cell number.

[0012] Figure 3 The results of IF detection of E-Cadherin protein expression in IPEC-J2 cells.

[0013] Figure 4 To investigate the effects of oral administration of Ruminococcus brucei and its metabolic supernatant from pure in vitro culture on the growth performance of piglets.

[0014] Figure 5 The study aimed to investigate the effects of oral administration of Ruminococcus brucei and its metabolic supernatant from pure culture in vitro on the unit intestinal weight of piglets.

[0015] Figure 6 Representative images of piglet jejunal organoids gavaged with Ruminococcus brucei and its in vitro pure culture metabolic supernatant on day 1 and day 5 of culture.

[0016] Figure 7 The statistical results of proliferation, differentiation and other indicators of jejunal organoids in piglets were obtained by gavage with Ruminococcus brucei and its in vitro pure culture metabolic supernatant. DETAILED DESCRIPTION

[0017] The present invention provides the use of Ruminococcus brucei and its in vitro pure culture metabolic supernatant for promoting intestinal development in piglets. To further clarify the objectives, technical solutions, and effects of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0018] The Ruminococcus brucei used in the present invention ( Ruminococcus bromii ) was purchased from Guangzhou Kangruisi Biotechnology Co., Ltd., with the accession number being ATCC 51896. After purchase, the cells were frozen in minced meat carbohydrate (CMC) liquid medium containing 20% glycerol.

[0019] Example 1: MTT assay of the effect of the supernatant of Ruminococcus brucei pure culture in vitro on IPEC-J2 cell viability 1. IPEC-J2 Cell Culture and Treatment: IPEC-J2 cells were cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. When cell coverage reached 80% of the well area, cells were digested with 0.25% trypsin solution and passaged at a 1:3 ratio. Subsequent experiments used cells in the logarithmic growth phase at passages 3-5.

[0020] 2. Preparation of in vitro pure culture metabolic supernatant: Ruminococcus brucei with the deposit number ATCC 51896 ( Ruminococcus bromii ) were inoculated into CMC medium and cultured anaerobically at 37°C for 36 hours. The in vitro pure culture solution was centrifuged at 4000 rpm for 20 minutes, and the supernatant was filtered through a 0.22 μm filter plug to obtain the in vitro pure culture metabolic supernatant.

[0021] 3. MTT assay for cell viability: IPEC-J2 cells were cultured at 2×10 3 Cells were seeded at a density of 100 cells / well in 96-well cell culture plates. MTT assays were performed at 0 h (immediate baseline) and 12 h after the experimental intervention. Separate culture plates were used for each assay time point to avoid interference from repeated manipulations. Porcine intestinal epithelial cells (IPEC-J2) were treated with metabolic supernatants from pure in vitro cultures of Ruminococcus brucei at concentrations (v / v) of 0.25%, 0.5%, and 1%, respectively. CMC culture medium controls were also used at corresponding concentrations. Cell viability was determined using the MTT assay at a characteristic wavelength of 490 nm.

[0022] The results are as follows Figure 1 As shown, CON represents the control group, which was treated with standard complete culture medium; the CMC group was treated with complete culture medium containing CMC bacterial culture medium; and the sup group was treated with complete culture medium containing the metabolic supernatant of a pure in vitro culture of Ruminococcus brucei during the stationary phase. When the supernatant of a pure in vitro culture of Ruminococcus brucei was added at a concentration of 0.25% to 1%, the viability of IPEC-J2 cells was significantly improved, and IPEC-J2 activity reached its optimal state at a replacement concentration of 0.5%. Cellular assay results demonstrated that the metabolic supernatant of a pure in vitro culture of Ruminococcus brucei effectively promoted the proliferation and viability of porcine jejunal epithelial cells, revealing the potential of Ruminococcus brucei to promote porcine intestinal development.

[0023] 4. Immunofluorescence staining to detect PCNA in IPEC-J2 cells + Cell number IPEC-J2 cells were treated with complete medium containing 0.5% metabolic supernatant of pure in vitro culture of Ruminococcus brucei and 0.5% CMC medium for 12 h, washed three times with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde solution for 1 h, permeabilized with 0.5% Triton for 10 min, blocked with 10% bovine serum albumin (BSA) solution for 1 h, incubated with PCNA primary antibody (purchased from Cell Signaling Technology) at 4°C for 16 h, incubated with anti-mouse IgG (Zhengneng Biotechnology) at 4°C for 2 h in the dark, and stained with DAPI for 10 min.

[0024] 5. Detection of E-Cadherin protein expression IPEC-J2 cells blocked as described above were taken, and E-Cadherin primary antibody (Zhengneng Biotechnology Co., Ltd.) was added to the cell samples. The cells were incubated at 4°C for 12 h, and then incubated with anti-rabbit IgG secondary antibody (Zhengneng Biotechnology Co., Ltd.) in the dark for 2 h, and stained with DAPI for 10 min.

[0025] The results are as follows Figure 2 and Figure 3 As shown, “*” represents P <0.05. Figure 2 PCNA in IPEC-J2 cells + Cell test results, Figure 3 The results of E-Cadherin protein expression were obtained. Figure 2 and Figure 3 The results show that treatment with 0.5% metabolic supernatant of pure culture of Ruminococcus brucei in vitro can significantly promote the proliferation activity of IPEC-J2 cells and increase the tight junction degree between intestinal cells, thereby promoting intestinal development in piglets.

[0026] Example 2: Animal Experiment 1. Preparation of oral bacterial solution: Inoculate Ruminococcus brucei into CMC liquid medium and culture at 37°C for 36 hours. After centrifugation, collect the bacterial pellet and resuspend it in diluent. Determine the bacterial concentration and adjust the bacterial solution concentration to 1×10 8 CFU / mL; 2. Obtaining the in vitro pure culture metabolic supernatant: Centrifuge the Ruminococcus brucei culture solution after 36 hours of culture at 4000 rpm for 20 minutes, and filter through a 0.22 μm filter plug to obtain the supernatant to obtain the in vitro pure culture metabolic supernatant; 3. Control group (CON group): 21-day-old weaned piglets were gavaged with sterile diluent, with a gavage volume of 5 mL per pig each time; R.brommiGroup: 21-day-old weaned piglets were gavaged with the above-mentioned bacterial solution, with a gavage volume of 5 mL per pig each time; R.brommi Sup group: 21-day-old weaned piglets were gavaged with the above in vitro pure culture metabolic supernatant, with a gavage volume of 5 mL per pig each time.

[0027] 4. Schedule gavage administration: piglets should be gavaged on days 1, 2, 10, and 12, and weighed on days 1, 7, 12, 19, and 23. Throughout the breeding process, data such as free feed intake, daily weight gain, and diarrhea rate should be recorded. After weighing on day 23, piglets should be slaughtered and data such as organ indices, unit intestinal weight of each intestinal segment, and unit mucosal weight should be recorded.

[0028] The results are as follows Figure 4-5 As shown in the figure, "*" represents P <0.05.

[0029] Figure 4 The study was to investigate the effects of Ruminococcus brucei and its in vitro pure culture metabolic supernatant on the growth performance of piglets. Figure 4 A is the weight change trend of weaned piglets, Figure 4 B is the statistical result of the weight of weaned piglets on the 23rd day. Compared with the control group, the experimental group had a greater weight gain, and on the 23rd day, the group treated with the supernatant of pure culture of Ruminococcus brucei in vitro showed a trend of increasing weight. Figure 4 C is the statistical result of the average daily weight gain of piglets, Figure 4 D is the statistical result of the average daily feed intake of piglets. Figure 4 E is the statistical result of the feed-to-weight ratio of piglets. Compared with the control group, the treatment with the metabolic supernatant of pure culture of Ruminococcus brucei in vitro has a tendency to increase the weight of piglets, increase the average daily weight gain of piglets, and reduce the feed-to-weight ratio of piglets. The treatment with Ruminococcus brucei bacterial liquid has a tendency to increase the average daily feed intake of piglets and reduce the feed-to-weight ratio.

[0030] Figure 5 is the unit intestinal weight of each intestinal segment of the piglet. The results show that the treatment with Ruminococcus brucei has a trend of increasing the unit intestinal weight of the piglet jejunum, and the treatment with the metabolic supernatant of Ruminococcus brucei in vitro pure culture has a trend of increasing the unit intestinal weight of the piglet jejunum and ileum. Both treatments can significantly increase the unit intestinal weight of the piglet cecum.

[0031] As the initial segment of the large intestine, the cecum's core physiological function is to provide a fermentation site for intestinal microorganisms. The microbial communities colonizing the cecum are able to perform anaerobically fermentation on nutrients that are not fully digested in the small intestine. The various bioactive substances metabolized through this fermentation process are primarily absorbed and utilized in the cecum and adjacent colon, providing important nutritional supplements and energy sources for piglets. Cecal development is a key indicator for assessing the integrity of piglet intestinal function. During the weaning transition period, piglets often experience significant stress responses, which can lead to impaired structural integrity and functional impairment of the intestinal mucosal barrier. Piglets treated with Ruminococcus brucei and its in vitro pure culture metabolic supernatant by gavage showed significantly better cecal and overall intestinal development than those in the control group. This intervention can effectively enhance intestinal mucosal barrier function, significantly alleviate the adverse changes in intestinal morphology and physiology induced by weaning stress, and promote healthy growth in piglets.

[0032] Example 3: Results of in vitro culture of piglet jejunal organoids by oral administration of Ruminococcus brucei and its in vitro pure culture metabolic supernatant 1. Tissue Processing and Crypt Isolation: After piglets were slaughtered on day 23, a 20 cm segment of jejunum was aseptically removed. The intestinal lumen was longitudinally incised and the contents were repeatedly rinsed with pre-chilled Dulbecco phosphate-buffered saline (DPBS). The mucosal surface mucus and villi were gently scraped off using a sterile glass slide. The intestinal segment was then cut into 2 cm segments and transferred to a 50 mL centrifuge tube containing 25 mL of 4°C pre-chilled DPBS. The segments were washed five times with manual shaking until the wash solution was transparent. Microscopic examination confirmed that only a small amount of debris remained.

[0033] 2. Chelation and Crypt Purification: Replace the buffer with chelation buffer and incubate at 4°C on a horizontal shaker for 40 min. Discard the supernatant and add DPBS containing 1% penicillin-streptomycin. Wash manually with shaking (5 min / wash). Adjust the mechanical force parameters through dynamic microscopic monitoring until the crypt release efficiency is >80% and the integrity is good (crypt morphological integrity ≥90%).

[0034] 3. Crypt Enrichment and Culture: The suspension was collected and allowed to stand on ice for 10 minutes. The pellet was aspirated and resuspended in DMEM medium containing 10% fetal bovine serum and 1% double-antibody-containing culture medium. The pellet was centrifuged at 1000 g for 3 minutes at 4°C. After washing twice, the crypt density was determined using a Neubauer counting plate. 40 crypts were plated onto Matrigel-coated 48-well plates, with 6 wells per well. The plates were incubated at 37°C for 20 minutes. After gelation, 250 μL of complete intestinal organoid culture medium was added and the plates were cultured in a 5% CO2 incubator for 5 days.

[0035] The results are as follows Figure 6 and Figure 7 As shown, Figure 7 In the text, “*” represents P <0.05, “**” represents P <0.01.

[0036] Figure 6 Representative images of piglet jejunal organoids cultured on the 1st and 5th days after oral administration of Ruminococcus brucei and its in vitro pure culture metabolic supernatant.

[0037] Figure 7 A is the statistical result of in vitro organoid formation efficiency. Compared with the CON group, R.brommi Group and R.brommi The in vitro organoid formation efficiency of the sup group was significantly improved. Figure 7 B is the statistical result of in vitro organoid sprouting efficiency. Figure 7 C is the statistical result of the surface area of in vitro organoids. Figure 7 D is the statistical result of the branching coefficient of in vitro organoids. Compared with the CON group, R.brommi The germination efficiency of the sup group showed an increasing trend.

[0038] comprehensive Figure 6 and Figure 7 The results show that treatment with Ruminococcus brucei and its in vitro pure culture metabolic supernatant can promote the ability of piglet jejunal stem cells to expand in vitro, and have a beneficial effect on piglet intestinal development.

[0039] The present invention has proved that the Ruminococcus brucei with the deposit number of ATCC 51896 ( Ruminococcus bromii ) plays a significant role in promoting the intestinal development of piglets, and its in vitro pure culture metabolic supernatant can also play a corresponding role.

[0040] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and inventive concept of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An application of Ruminococcus brucelli in promoting intestinal development in piglets, wherein the Ruminococcus brucelli ( Ruminococcus bromii ) The deposit number is ATCC 51896.

2. Use of the in vitro pure culture metabolic supernatant of Ruminococcus brucei as claimed in claim 1 in promoting intestinal development in piglets.

3. Use of Ruminococcus brucei as claimed in claim 1 in the preparation of a bacterial agent or medicine for promoting intestinal development in piglets.

4. Use of the in vitro pure culture metabolic supernatant of Ruminococcus brucei as claimed in claim 1 in the preparation of a bacterial agent or medicine for promoting intestinal development in piglets.

5. Use of the in vitro pure culture metabolic supernatant of Ruminococcus brucei according to claim 1 in improving the activity of porcine intestinal epithelial cells.

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