Application of giant globular Escherichia in promoting intestinal development of piglets

Abstract

The invention relates to the technical field of microorganisms, in particular to giant spheroidin Escherichia and application of the giant spheroidin Escherichia in promoting intestinal development of piglets. The Megasphaera elsdenii provided by the invention is preserved in Guangdong Microbial Culture Collection Center on May 22, 2025, the preservation number is GDMCC No: 66369, and the Megasphaera elsdenii can be used for promoting the development of intestinal tracts and intestinal mucosa of piglets and retarding growth retardation caused by weaning stress. In addition, the in-vitro pure culture supernatant of the giant spheroidin can also improve the viability of the jejunum stem cells and promote the proliferation of the epithelial cells of the pig intestinal tract.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to an application of Megasphaera elsdenii in promoting intestinal development in piglets. Background Art

[0002] During the early weaning stage of piglets, weaning stress is a major issue affecting their growth and development. Therefore, how to alleviate the growth inhibition caused by weaning is an important issue in improving the efficiency of pig farming.

[0003] The colonization of intestinal flora is crucial for the rapid growth and digestive system development of young animals, and can even affect the animals' future growth and development, as well as their market weight. After weaning, the intestinal flora of piglets is susceptible to interference from a variety of internal and external factors, leading to community imbalance and growth retardation. Therefore, early intervention in the intestinal community structure of weaned piglets through microbial therapy can maintain the stability of the piglet's intestinal microecological environment and safeguard intestinal function, which is of great significance in alleviating growth retardation and intestinal function damage caused by weaning stress. However, the current market for pig probiotic products varies greatly, and there is no effective and stable product for promoting intestinal development in weaned piglets.

[0004] It can be seen that it is very necessary to develop pig-derived probiotics that can relieve weaning stress, promote intestinal development and piglet growth. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a Megasphaera elsdenii that promotes the intestinal development of piglets.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a Megasphaera elsdenii, wherein the Megasphaera elsdenii ( Megasphaera elsdenii ) was deposited in Guangdong Provincial Microbiological Culture Collection on May 22, 2025, with the deposit number GDMCC No: 66369.

[0007] The present invention also provides the use of the Megasphaera elsdenii in promoting the intestinal development of piglets.

[0008] The present invention also provides the use of the Megasphaera elsdenii in preparing a drug for promoting intestinal development in piglets.

[0009] The present invention also provides the use of the in vitro pure culture supernatant of Megasphaera elsdenii in promoting the development of intestinal stem cells in piglets.

[0010] The present invention also provides the use of the in vitro pure culture supernatant of Megasphaera elsdenii in promoting the growth of porcine intestinal epithelial cells.

[0011] The present invention has the following beneficial effects: The present invention provides a Megasphaera elsdenii, wherein the Megasphaera elsdenii ( Megasphaera elsdenii ) with a deposit number of GDMCC No. 66369. This Megasphaera elsdenii strain can effectively increase piglet weight and promote intestinal development, particularly in promoting the development of cecal unit weight and unit mucosal weight. It can also significantly enhance the in vitro expansion efficiency of jejunal stem cells. Furthermore, the supernatant of pure in vitro culture of Megasphaera elsdenii can also promote the proliferation of porcine intestinal epithelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 MTT was used to detect the effect of supernatant of pure culture of Megasphaera elsdenii on the viability of porcine intestinal epithelial cells (IPEC-J2).

[0013] Figure 2 To investigate the effects of oral administration of Megasphaera elsdenii on the growth and development of piglets.

[0014] Figure 3 This is a representative image of the in vitro culture of jejunal stem cells in piglets after oral administration of Megasphaera elsdenii.

[0015] Figure 4 Statistical results of in vitro culture of piglet jejunal stem cells. DETAILED DESCRIPTION

[0016] The present invention provides a use of Megasphaera elsdenii 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 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.

[0017] The present invention's Megasphaera elsdenii ( Megasphaera elsdenii ) was deposited on May 22, 2025 in Guangdong Provincial Microbiological Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No: 66369.

[0018] Example 1 Isolation of Megasphaera elsdenii and IPEC-J2 Cell Assay The present invention provides Megasphaera elsdenii, deposited with GDMCC No. 66369, isolated from feces of 234-day-old three-way hybrid pigs at 37°C under anaerobic conditions. The solid culture medium used for isolation was modified GAM broth (supplemented with 15% agar powder) or brain heart infusion (BHI) solid culture medium supplemented with 5% defibrinated sheep blood. Megasphaera elsdenii was cryopreserved in BHI liquid medium containing 20% glycerol or modified GAM liquid medium.

[0019] The steps of the IPEC-J2 cell assay are as follows: Preparation of in vitro pure culture supernatant: Megasphaera elsdenii (GDMCC No: 66369) was inoculated into modified GAM broth and cultured at 37°C under anaerobism for 40 h. The in vitro pure culture solution was centrifuged at 3000 rpm / min for 5 minutes, and the supernatant was collected and filtered through a 0.22 μm filter to prepare the in vitro pure culture supernatant. IPEC-J2 cells were treated with the in vitro pure culture supernatant of Megasphaera elsdenii at a concentration gradient (v / v) of 0.5%, 1%, 2%, 4%, 8%, 16%, and 32%, respectively. IPEC-J2 cells were seeded on 96-well plates at 2000 cells per well and incubated at 37°C under 5% CO2. The cells were cultured in an incubator and the activity of IPEC-J2 cells was detected at 490 nm using the MTT assay at 0 and 24 h. Figure 1 As shown, “**” represents P < 0.01, “***” represents P < 0.001. Figure 1 Results indicate that replacing the supernatant of a pure in vitro culture of M. elsdenii with a concentration greater than 2% significantly increases IPEC-J2 activity, reaching its optimal state at an 8% concentration. Cell-based assays demonstrate that supernatant of a pure in vitro culture of M. elsdenii effectively promotes the proliferation and activity of porcine intestinal epithelial cells, demonstrating the potential of M. elsdenii to promote porcine intestinal development.

[0020] Example 2 Results of the experiment on gavage of piglets with Megasphaera elsdenii 1. Preparation of bacterial suspension: Megasphaera elsdenii, deposited with GDMCC No. 66369, was inoculated into a solid medium consisting of modified GAM and 5% defibrinated sheep blood. After anaerobically incubating at 37°C for 48 h, the bacteria were harvested and resuspended in PBS containing 20% glycerol. This was used as the oral gavage suspension and stored at -20°C until further use. The concentration of the gavage suspension was calculated using a hemocytometer.

[0021] 2. The experimental group used the above bacterial solution for gavage to 21-day-old weaned piglets. The gavage volume for each pig was 5 mL each time, and the bacterial concentration of the gavage solution was 2×10 6 CFU / mL; the control group was gavaged with an equal volume of PBS buffer containing 20% glycerol.

[0022] 3. 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 feed intake, daily weight gain, and diarrhea rate should be recorded. Piglets should be weighed and slaughtered on day 23, and data such as unit intestinal weight and unit mucosal weight of each intestinal segment should be recorded.

[0023] Figure 2 A is the weight change trend of weaned piglets, Figure 2 B is the weight statistics on the 23rd day. Figure 2 A and Figure 2 B It can be seen that compared with the control group, the weight gain of piglets in the experimental group was greater and showed an increasing trend on the 23rd day.

[0024] Figure 2 C is the unit weight of jejunal mucosa, Figure 2 The results show that the experimental group had an increased jejunal mucosal weight compared to the control group. The jejunum is the primary site of nutrient absorption, and its mucosa is composed of circular folds and villi. An increase in the jejunal mucosal weight promotes nutrient digestion and absorption.

[0025] Figure 2 D is the ileum unit weight result, from Figure 2 The results show that the experimental group's ileum weight increased compared to the control group. The ileum, located at the end of the small intestine, primarily absorbs and digests nutrients. This increase in ileum weight also results in a richer and more complete ileal mucosal structure, which promotes nutrient absorption and benefits piglet development.

[0026] Figure 2 E is the unit weight of the cecum, Figure 2 E The results show that the unit weight of the cecum of the piglets in the experimental group was significantly higher than that in the control group.

[0027] Figure 2 F is the unit weight of cecal mucosa, Figure 2 The results showed that the unit weight of cecal mucosa in the experimental group was significantly higher than that in the control group.

[0028] The cecum is located at the beginning of the large intestine, and its main function is microbial fermentation. The microorganisms in the cecum can break down and ferment some nutrients that are not absorbed by the small intestine. Substances such as B vitamins, vitamin K, and short-chain fatty acids produced during the fermentation process are absorbed in the cecum, providing additional nutrition and energy for piglets. Better development of the cecum indicates that piglets have better intestinal function. In weaned piglets, weaning stress may lead to damage of the intestinal mucosal barrier, while piglets gavage with bacterial solution clearly show better intestinal development, which can effectively alleviate the adverse effects of weaning stress.

[0029] Example 3 Results of in vitro organoid culture in piglets after oral administration of Megasphaera elsdenii After piglets were slaughtered on day 23, jejunal segments were harvested and cultured for jejunal organoids. Culture medium was WRN-conditioned medium, maintained at 37°C in a 5% CO2 incubator, with medium exchange every two days. Day 0 was defined as the time of inoculation. Images were taken starting on day 1, and organoid growth was recorded over a five-day period. By comparing data from day 1 with data from day 5, organoid budding, budding efficiency, and budding area were calculated. The number of budding organoids at the initial budding stage (day 3) was counted to calculate organoid expansion efficiency.

[0030] Figure 3 Representative images of jejunal organoid culture in the control and experimental groups on day 1 and day 5.

[0031] Figure 4 A is the statistical result of organoid surface area. There was no significant difference in the surface area of jejunal organoids between the control group and the experimental group on day 1 and day 5.

[0032] Figure 4 B is the statistical result of the expansion efficiency of jejunal organoids. Compared with the control group, the expansion efficiency of jejunal organoids in the experimental group was higher, and there was a significant difference.

[0033] Figure 4 C is the statistical result of the budding efficiency of jejunal organoids. Compared with the control group, the budding efficiency of the jejunal organoids in the experimental group is higher.

[0034] Figure 4 D is the statistical result of the branching coefficient of jejunal organoids. Compared with the control group, the branching coefficient of the jejunal organoids in the experimental group was higher when the budding amount was greater than 0.

[0035] comprehensive Figure 4 The results show that the jejunal stem cells of the piglets in the experimental group have a stronger ability to expand in vitro, indicating that the jejunal stem cells of the piglets treated with the Megasphaera elsdenii gavage of the present invention have better activity and stronger "stemness".

[0036] The present invention has proved that the Megasphaera elsdenii (GDMCC No: 66369) of the present invention has a good bioavailability and good bioavailability. Megasphaera elsdenii ) plays a significant role in promoting intestinal development in piglets, and its in vitro pure culture supernatant can also play a corresponding role.

[0037] 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. A Megasphaera elsdenii, characterized in that The Megasphaera elsdenii ( Megasphaera elsdenii ) was deposited in Guangdong Provincial Microbiological Culture Collection on May 22, 2025, with the deposit number GDMCC No: 66369.

2. Use of Megasphaera elsdenii as claimed in claim 1 in promoting intestinal development in piglets.

3. Use of the Megasphaera elsdenii as claimed in claim 1 in the preparation of a drug for promoting intestinal development in piglets.

4. Use of Megasphaera elsdenii as claimed in claim 1 in promoting the development of intestinal stem cells in piglets.

5. Use of the in vitro pure culture supernatant of Megasphaera elsdenii as claimed in claim 1 in promoting the growth of porcine intestinal epithelial cells.

Images