Min pig source lactobacillus reuteri LRM2293 and application thereof in anti-inflammatory reaction

Abstract

The invention discloses a Min pig source lactobacillus reuteri LRM2293 strain and an application of the Min pig source lactobacillus reuteri LRM2293 strain in anti-inflammatory reaction. The Min pig source lactobacillus reuteri is named as Lactobacillus reuteri LRM2293, and is preserved in the China Center for Type Culture Collection, the address is Wuhan University, Wuhan, China, the preservation number is CCTCC NO: M 20242331, and the preservation time is October 25, 2024. Research finds that the lactobacillus reuteri LRM2293 has the capacity of transforming bile acid in vitro and can be used as a candidate strain of probiotics. In vitro, a culture supernatant of the lactobacillus reuteri LRM2293 can be used for reducing the inflammatory reaction of IPEC-J2 cells; in vivo, the LRM2293 strain can be colonized in each intestinal segment of a piglet, and can repair an intestinal barrier and relieve intestinal injury of the inflammatory piglet, so that the problem of antibacterial drug resistance caused by abuse of antibiotics is expected to be reduced, and a technical means is provided for development of a novel functional product.

Description

Technical Field

[0001] The invention relates to a strain of Lactobacillus reuteri from pigs, and also relates to the use of the Lactobacillus reuteri from pigs in anti-inflammatory reactions. The invention belongs to the technical field of microorganisms. Technical Background

[0002] In livestock and poultry farming, a variety of factors such as the environment, bacterial and viral infections may cause animal enteritis. These diseases not only affect the growth performance of livestock and poultry, but may also cause death in severe cases, and lead to a decline in the quality of livestock products. The resulting economic losses have posed a huge challenge to the development of the breeding industry. Piglets face a variety of stress factors during the weaning period, and changes in nutritional sources are one of the most important factors. These changes can easily lead to a disorder in the composition of piglet intestinal microorganisms, which in turn cause intestinal inflammation, damage to the mucosal barrier, and intestinal dysfunction, thereby affecting their growth and development. For example, pathogenic microorganisms such as Salmonella and Escherichia coli in the environment can invade weaned piglets, leading to increased expression of pro-inflammatory factors such as TNF-α and IL-1β in the intestine, which in turn causes intestinal inflammation in piglets. When the piglet intestine is in an inflammatory state for a long time, it is more conducive to the growth and reproduction of Enterobacteriaceae. Because the gastrointestinal tract is not yet mature, weaned piglets are under a variety of stress after early weaning, which leads to significant changes in their intestinal morphology, structure, and physiological function. Stress in weaned piglets may also cause their immune function to be impaired, thereby causing structural and functional disorders of the digestive system.

[0003] Compared with foreign imported pig breeds, local Chinese pig breeds have better environmental adaptability. Min pigs in Northeast China are one of the important local pig breeds in my country. They are famous for their strong resistance to stress, roughage, cold and disease. Their unique intestinal microbiome structure is closely related to these excellent characteristics. Studies have shown that compared with Landrace piglets, Min piglets have better resistance to diarrhea during weaning.

[0004] In recent decades, as the largest "bacterial reservoir" in animals, the benefits that intestinal flora can bring to the host have attracted more and more attention from researchers. People collectively refer to these beneficial strains as probiotics, which can benefit the host when ingested by the host in sufficient quantities. Probiotics are widely distributed in nature and there are many species, including more than 400 species in the human body. Probiotics are roughly divided into five types: lactobacilli (Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus delbrueckii, etc.), bifidobacteria (Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium infantis, etc.), bacilli (Bacillus licheniformis, Bacillus subtilis, Bacillus natto, etc.), streptococci (Streptococcus faecalis, Streptococcus lactis, Streptococcus thermophilus, etc.) and others (Leuconostoc, Pediococcus acidilactici, etc.). At present, the most commonly used probiotics are mainly some lactic acid bacteria such as yeast, lactobacillus, and bifidobacteria. The intestinal microbiota plays a key role in the host's development, nutrition, metabolism, physiology, and immune function by promoting nutrient absorption and producing molecules that can interact with the host's transporters, enzymes, and receptors. Some bacterial-derived metabolic and immune signaling molecules, including bile acids and indole derivatives, have anti-inflammatory, immunomodulatory, and intestinal epithelial barrier effects.

[0005] In the pig industry, antibiotics are widely used as feed additives to improve the growth performance of pigs and prevent or treat intestinal infections. Although antibiotics play a key role in preventing intestinal infections, their improper use may disrupt the balance of intestinal flora. Lactic acid bacteria are common commensal bacteria in animals, with good colonization ability, and can induce immune regulation, prevent physiological stress, inhibit pathogens, regulate microbiota, and improve the barrier function of the intestinal epithelium. Therefore, they are suitable for development as microecological preparations for practical applications. Summary of the invention

[0006] The purpose of the present invention is to provide a pig-derived Lactobacillus reuteri LRM2293 capable of resisting inflammatory response and a use thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention analyzed the differences in the flora between Min pigs and Sanyuan pigs by 16s high-throughput sequencing, and isolated a strain of Lactobacillus reuteri from the colon contents of Min pigs, named Lactobacillus reuteri LRM2293. The study found that the strain showed excellent stress resistance, and its culture supernatant had an inhibition rate of more than 50% on enterotoxigenic Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Streptococcus suis, and had the ability to convert bile acid, showing potential as a candidate strain of probiotics. In an in vitro inflammation model, the culture supernatant and UDCA of LRM2293 both had anti-inflammatory effects. In piglets, LRM2293 can colonize in various intestinal segments of piglets, repair intestinal villi and tight junction structures, and increase the number of goblet cells, thereby maintaining the stability of the intestinal barrier and reducing intestinal damage caused by inflammation.

[0009] Therefore, based on the above research, the present invention proposes a strain of Lactobacillus reuteri from Min pigs, named Lactobacillus reuteri LRM2293, classified and named Lactobacillus reuteri LRM2293, deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with a deposit number of CCTCCNO: M 20242331, and a deposit date of October 25, 2024.

[0010] Furthermore, the present invention also proposes the use of the pig-derived Lactobacillus reuteri or its culture supernatant in the preparation of anti-inflammatory drugs.

[0011] Furthermore, the present invention also proposes the use of the Lactobacillus reuteri from pigs or its culture supernatant in the preparation of drugs or reagents for inhibiting enterotoxigenic Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Streptococcus suis.

[0012] Furthermore, the present invention also proposes the use of the pig-derived Lactobacillus reuteri or its culture supernatant in the preparation of a drug for treating piglet intestinal inflammation.

[0013] Among them, preferably, the pig-derived Lactobacillus reuteri can colonize in various intestinal segments of piglets, repair intestinal villi and tight junction structures, increase the number of goblet cells, and inhibit the production of pro-inflammatory factors in the body, thereby alleviating the body's inflammatory state, maintaining the stability of the intestinal barrier, and reducing intestinal damage caused by inflammation.

[0014] Among them, preferably, the culture supernatant is obtained by inoculating the Minzhu-derived Lactobacillus reuteri into MRS liquid culture medium at a volume ratio of 1:100, culturing at 37°C for 24 hours, centrifuging, and removing cells and impurities.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention isolates a strain of Lactobacillus reuteri LRM2293 with good probiotic potential from the colon contents of pigs. As a common probiotic, Lactobacillus reuteri can not only be used in feed fermentation, but also its metabolism produces antibacterial active substances such as organic acids and bacteriocins, which can effectively inhibit the production of pathogenic microorganisms, thereby improving the disease resistance of piglets. The present invention uses lipopolysaccharide (LPS) to establish an IPEC-J2 cell inflammation model and a weaned piglet intestinal inflammation model, respectively, and explores the protective effect of Lactobacillus reuteri LRM2293 and its culture supernatant on piglets with intestinal inflammation. The results show that Lactobacillus reuteri LRM2293 has the ability to convert bile acid in vitro and can be used as a candidate strain of probiotics. In vitro, Lactobacillus reuteri LRM2293 and its culture supernatant can reduce the inflammatory response of IPEC-J2 cells; in vivo, the LRM2293 strain can colonize in various intestinal segments of piglets, and can repair the intestinal barrier and reduce intestinal damage in inflammatory piglets. It is not only expected to reduce the problem of antimicrobial resistance caused by the abuse of antibiotics, but also can provide technical means for the development of new functional products. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Distribution diagram of the community structure of Weimin pigs (A) and Sanyuan pigs (B);

[0018] Figure 2 Heat map of the top 10 species ranked by abundance in colon contents of Min pigs and Sanyuan pigs;

[0019] A: relative abundance at the phylum level; B: relative abundance at the class level; C: relative abundance at the order level; D: relative abundance at the family level; E: relative abundance at the genus level; F: relative abundance at the species level;

[0020] Figure 3 The colony morphology and Gram staining results of Lactobacillus reuteri LRM2293 (1000×);

[0021] Figure 4 This is the growth curve and acid production trend diagram of Lactobacillus reuteri LRM2293;

[0022] Figure 5 This is the evaluation result of the antibacterial effect of Lactobacillus reuteri LRM2293;

[0023] Figure 6 It is the scatter plot of OPLS-DA model scores of CON group versus La group;

[0024] The horizontal axis in the figure represents the predicted principal component score of the first principal component, showing the differences between sample groups, and the vertical axis represents the orthogonal principal component score, showing the differences within the sample group;

[0025] Figure 7 This is the Z-score graph of the CON group versus the La group;

[0026] The horizontal axis represents the Z value, and the vertical axis represents the metabolite. The specific formula is: z = (x-μ) / σ: where x is a specific score, μ is the mean, and σ is the standard deviation;

[0027] Figure 8 This is the radar chart of CON group versus La group;

[0028] Fig. 9 The results show the effects of different LPS stimulation time on the IL-6 and TNF-α levels in IPEC-J2 cells;

[0029] Fig.10 The results show the effects of different Lactobacillus reuteri and UDCA concentrations on IPEC-J2 cell activity;

[0030] Fig.11 The results show the effects of Lactobacillus reuteri LRM2293 and UDCA on the inflammatory response of IPEC-J2 cells induced by LPS;

[0031] Fig.12 It is the clinical symptoms and autopsy changes;

[0032] Fig.13 It is the positive rate result of Lactobacillus reuteri LRM2293 marked by CFDA-SE;

[0033] Fig.14 The results of the detection of Lactobacillus reuteri LRM2293 colonization in the intestine of piglets;

[0034] Fig.15 This is a photo of HE-stained sections of piglet intestine;

[0035] Fig.16 The results of the changes in the length of villi, crypt depth and villus / crypt ratio in the small intestine of piglets;

[0036] Fig.17 This is a photo of AB-PAS stained sections of piglet small intestine;

[0037] Fig.18 This is the statistical result of the changes in the number of goblet cells in the small intestine of piglets;

[0038] Fig.19 This is a photo of the morphology of piglet intestinal villi under a scanning electron microscope;

[0039] Fig. 20Ultrastructural photographs observed by transmission electron microscopy;

[0040] Fig.21 This is the effect of Lactobacillus reuteri LRM2293 on the expression of inflammatory cytokines in the colon of inflamed piglets. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention is clearly and completely described. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1 Analysis of the difference in intestinal flora between Min pigs and Sanyuan pigs

[0043] 1.1 Krona analysis of community structure

[0044] In order to compare the differences in colon flora between the Min pig group and the Sanyuan pig group, the colon contents of the weaned Min pig (Min) and Sanyuan pig (DLY) were collected for 16S high-throughput sequencing, and Krona was used to visualize the species annotation results. In the display results, the circles from the inside to the outside represent different classification levels, and the size of the fan represents the relative proportion of different OTU annotation results. The results are shown in Figure 1 As shown, there are differences in the intestinal flora between Min pigs and Sanyuan pigs. Among the intestinal flora of Min pigs, Firmicutes accounts for the largest proportion, and Lactobacillus accounts for the largest proportion under this phylum.

[0045] 1.2 Heatmap of different species

[0046] Statistical analysis was performed at various classification levels such as phylum, class, order, family, genus, and species, and heat maps were drawn based on the relative abundance of different species. Based on the relative abundance of 6 colon content samples from Min pigs and Sanyuan pigs, we displayed a heat map of the top 10 species in terms of abundance. The clustering tree on the left side of the heat map shows the clustering of species, with red representing a higher relative abundance and blue representing a lower relative abundance. The results are shown in Figure 1. Figure 2As shown in the figure, the bacterial community structure of Min pigs and Sanyuan pigs at different taxonomic levels is significantly different. Specifically, at the phylum level, the bacteria in the colon of Min pigs are mainly concentrated in Fibrobacterota, Proteobacteria, and Firmicutes, and the relative abundance is higher than that of Sanyuan pigs. The bacteria in the colon of Sanyuan pigs are mainly concentrated in Bacteroidota and Verrucomicrobiota, and the relative abundance is higher than that of Min pigs. At the class level, the bacteria in the colon of Min pigs are mainly concentrated in Negativicutes and Fusobacteriia, and the relative abundance is higher than that of Sanyuan pigs. The bacteria in the colon of Sanyuan pigs are mainly concentrated in Bacteroidia, and the relative abundance is higher than that of Min pigs. At the order level, the colon bacteria of Min pigs were mainly concentrated in Lactobacillales, Fusobacteriales, Eubacteriales, etc., with a higher relative abundance than Sanyuan pigs; the colon bacteria of Sanyuan pigs were mainly concentrated in Verrucomicrobiales, Coriobacteriales, etc., with a higher relative abundance than Min pigs. At the family level, the colon bacteria of Min pigs were mainly concentrated in Fusobacteriaceae, Enterobacteriaceae, Lactobacillaceae, etc., with a higher relative abundance than Sanyuan pigs; the colon bacteria of Sanyuan pigs were mainly concentrated in Bacteroidaceae, Prevotellaceae, Oscillospiraceae, etc., with a higher relative abundance than Min pigs. At the genus level, the bacteria in the colon of Min pigs were mainly concentrated in Lactobacillus, Fusobacterium, Muribaculaceae, etc., with a higher relative abundance than Sanyuan pigs, and the bacteria in the colon of Sanyuan pigs were mainly concentrated in Prevotella, etc., with a higher relative abundance than Min pigs. At the species level, the bacteria in the colon of Min pigs were mainly concentrated in Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus johnsonii, etc., with a higher relative abundance than Sanyuan pigs, and the bacteria in the colon of Sanyuan pigs were mainly concentrated in some unidentified bacteria, with a higher relative abundance than Min pigs.

[0047] Example 2 Isolation and biological characteristics of Lactobacillus reuteri LRM2293 from pigs

[0048] 2.1 Isolation and purification of LRM2293

[0049] A strain of Lactobacillus reuteri was isolated from the colon contents of pigs. Figure 3 As shown in the figure, after culturing on the MRS plate for 24 hours, a milky white colony with a smooth, moist surface and slight protrusions was formed; its morphology was observed by Gram staining, and the results showed that the strain was blue-purple, which was a Gram-positive bacterium (G + ), with a short rod-shaped morphology, named Lactobacillus reuteri LRM2293 (abbreviated as "LRM2293"), and deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M20242331, and the deposit date is October 25, 2024.

[0050] 2.2 Determination of growth curve and acid production trend of LRM2293

[0051] The isolated Lactobacillus reuteri LRM2293 was inoculated into MRS liquid medium at a ratio of 1:100 and cultured at 37°C for 24 h. The bacterial suspension was taken every 2 h to measure the OD 600 The plate count method was used to calculate the number of viable bacteria and draw a bacterial growth curve. Figure 4 As shown in the figure, 0-2h is the lag phase, 2-12h is the logarithmic growth phase, 12-20h is the plateau phase, and after 20h it enters the decline phase. From the acid production trend curve, it can be seen that the acid production capacity shows a linear trend during the logarithmic growth phase of the bacteria, indicating that the acid production capacity is the strongest, and the initial pH value of the culture solution can be reduced from 5.7 to 4.3 after 24h of culture.

[0052] 2.3 Antibacterial effect of LRM2293 culture supernatant

[0053] The culture fluid of Lactobacillus reuteri LRM2293 obtained in Section 2.2 was centrifuged to remove cells and impurities to obtain the culture supernatant. The results showed that the culture supernatant of LRM2293 had a good inhibitory effect on enterotoxigenic Escherichia coli K88, Staphylococcus aureus, Salmonella typhimurium and Streptococcus suis, with an inhibition rate of more than 50% ( Figure 5 ).

[0054] 2.4 Acid and bile salt resistance test

[0055] After Lactobacillus reuteri LRM2293 was incubated in MRS (pH2, pH3) and MRS (containing 0.1% and 0.3% pig bile salt) liquid culture medium for 3 hours, the live bacteria count method was used to detect the tolerance of LRM2293 to different pH conditions and different pig bile salt concentrations. The results in Table 1 show that the strain LRM2293 has good stress resistance.

[0056] Table 1 Results of acid and bile resistance of LRM2293

[0057]

[0058]

[0059] 2.5 Artificial gastrointestinal fluid tolerance test

[0060] After oral ingestion, lactic acid bacteria need to undergo the combined action of pepsin in the stomach and trypsin in the intestines to play a probiotic role in the animal body. After LRM2293 was cultured in simulated gastric fluid for 2 hours, it was transferred to simulated intestinal fluid for 6 hours, and its survival rate was measured every 2 hours. The specific results are shown in Table 2, indicating that Lactobacillus reuteri LRM2293 can still survive after gastrointestinal digestion.

[0061] Table 2 Lactobacillus reuteri LRM2293 artificial gastrointestinal fluid tolerance results

[0062]

[0063] 2.6 In vitro bile acid conversion capacity of LRM2293

[0064] 2.6.1 Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA)

[0065] Bile acids play an important role in anti-inflammatory research, mainly by binding to nuclear receptors (such as FXR and TGR5) to regulate immune responses, reduce the level of inflammatory mediators, and affect the composition and function of intestinal microbiota. They may reduce chronic inflammation by reducing tissue damage, improving metabolic status, and potential applications in autoimmune and inflammatory diseases.

[0066] In order to quantitatively analyze the conversion of bile acids in the gallbladder by Lactobacillus reuteri LRM2293, UHPLC-MS / MS bile acid high-throughput targeted quantitative detection was used for analysis. The bile acids in the gallbladder before conversion were the CON group (n=4), and the bile acids after conversion by LRM2293 were the La group (n=4). Through OPLS-DA analysis, orthogonal variables that were not related to the categorical variables in the metabolites were filtered out, and non-orthogonal variables and orthogonal variables were analyzed separately to obtain information on the degree of correlation between the inter-group differences in metabolites and the experimental groups. Figure 6 As shown, the differences within the CON group and the La group were similar, but the differences between the groups were large, and all samples were within the 95% confidence interval.

[0067] 2.6.2 Distribution of differential metabolites among different groups

[0068] In order to clarify the distribution of differential metabolites between the CON group and the La group, the metabolites in different samples were normalized by calculating the Z-score value. The distribution of each metabolite between different groups can be seen very intuitively. The results are as follows: Figure 7 As shown in the data, the bile acids with relatively high distribution levels in the La group include ursodeoxycholic acid (UDCA), ursocholic acid, lithocholic acid, isohyodeoxycholic acid, 7-ketodeoxycholic acid, 6-ketolithocholic acid, 3-epideoxycholic acid, 12-ketolithocholic acid, etc.; the bile acids with relatively high distribution levels in the CON group include tauroω-muricholic acid, tauroβ-muricholic acid, tauroω-muricholic acid, glycolithocholic acid, etc. These results suggest that Lactobacillus reuteri LRM2293 has the ability to transform bile acids, which may be related to its ability to inhibit inflammatory responses.

[0069] 2.6.3 Analysis of TOP10 differential metabolites

[0070] In order to analyze the most significant difference between the CON group and the La group, and determine the most important secondary bile acid metabolized by the intestinal flora of the pig, we calculated the corresponding ratio for the quantitative value of each group of metabolites, and took the logarithm transformation with base 2, and took the top 10 differential bile acid metabolites with the highest up- and down-regulation folds for display. The results are shown in Figure 8 As shown in Figure 2, the most significantly upregulated bile acid in the La group was ursodeoxycholic acid (UDCA), with a fold difference of 2. 14.83 times.

[0071] Example 3 Exploring the anti-inflammatory ability of LRM2293 and UDCA at the IPEC-J2 cell level

[0072] 3.1 Establishment of LPS-induced IPEC-J2 cell inflammation model

[0073] IPEC-J2 cells were stimulated with 1 μg / mL LPS. Fig. 9As shown, LPS could induce a significant increase in proinflammatory factors IL-6 and TNF-α at 12h (P<0.001).

[0074] 3.3 Detection of the optimal concentration of LRM2293 and UDCA on IPEC-J2 cells

[0075] In order to eliminate the damage caused by UDCA and LRM2293 supernatant to IPEC-J2 cells, the UDCA concentration was adjusted to four concentration gradients of 0.125 mM-1 mM, and the LRM2293 bacterial concentration was adjusted to 10 3 -10 8 CFU / mL, and the cell activity was determined using the CCK8 method after incubation with IPEC-J2 for 4 hours. Fig.10 As shown, 1 mM UDCA and 10 6 CFU / mL bacterial concentration was used for subsequent experiments.

[0076] 3.4 LRM2293 strain and UDCA reduce the inflammatory response of IPEC-J2 cells

[0077] like Fig.11 As shown in Figure 2, LPS stimulation led to a significant increase in the levels of IL-6 and TNF-α in IPEC-J2 cells (P<0.001). 6 When IPEC-J2 cells were treated with CFU / mL of LRM2293 or 1mM UDCA for 4h and then stimulated with LPS for 12h, the mRNA expression levels of these two inflammatory cytokines were significantly decreased (P<0.001), suggesting that Lactobacillus reuteri LRM2293 and UDCA may play a potential role in inhibiting LPS-induced cellular inflammatory response.

[0078] Example 4 Study on the protection of the intestinal tract of piglets with inflammation by strain LRM2293

[0079] 4.1 Clinical symptoms and autopsy results

[0080] Twelve healthy weaned piglets aged 21 days were randomly divided into three groups: (1) blank control group (CON): basal diet + saline; (2) LPS group (LPS): basal diet + saline; (3) protection group (LRM2293 + LPS): basal diet + Lactobacillus reuteri LRM2293 culture solution (10 9 After 7 days of feeding, the piglets were deprived of food and water for 12 h, and LPS (100 μg / kg) was injected intraperitoneally into the LPS group and the protection group to establish an acute inflammation model. The piglets were killed 6 h later to collect samples.

[0081] like Fig.12As shown in the figure, the intestines of the LPS-treated group showed obvious pathological changes, the liver was obviously congested, the intestinal wall was swollen to varying degrees, and there were watery feces in the cecum and colon. In contrast, the intestines of the protected group showed normal color and shape, with little congestion and bleeding, no intestinal inflation and swelling, and relatively dry feces in the colon.

[0082] 4.2 Detection of LRM2293 colonization in the intestines of piglets after oral administration

[0083] Lactobacillus reuteri LRM2293 was labeled with fluorescent dye CFDA-SE, and the labeling positive rate was detected by flow cytometry. The results are as follows Fig.13 As shown in Table 3 and Fig.14 As shown in the figure, LRM2293 colonization could be detected in the intestinal mucus of the duodenum, jejunum, ileum and colon of piglets fed with LRM2293 bacterial solution for one week. This result showed that it had good colonization ability in the jejunum of piglets.

[0084] Table 3 Determination of the colonization ability of LRM2293 in piglets

[0085]

[0086] Note: Different superscript letters in the same row indicate significant differences among groups (P<0.05).

[0087] 4.3 Analysis of intestinal pathological tissue sections

[0088] In order to study the protective effect of Lactobacillus reuteri LRM2293 on the small intestine of inflammatory piglets, the results of HE staining of intestinal sections were observed and it was found that the intestinal villus structure of the piglets in the control group was relatively complete, and no inflammatory cell infiltration was observed (see Fig.15 The small intestine of the piglets in the LPS group showed a series of pathological changes. The lesions of the small intestine were mainly manifested in the damage of the intestinal villi: the villi became shorter and blunt at the tip, the epithelial cells showed necrosis, dissolution and shedding, there were a lot of cell fragments in the intestinal cavity, and inflammatory cell infiltration was obvious (red arrows indicate inflammatory infiltration, black arrows indicate epithelial cell dissolution and necrosis); the crypt structure of the colon in the control group was intact, but after LPS stimulation, the crypt structure was obviously destroyed and the number decreased.

[0089] The villus height, crypt depth and their ratio of the small intestine were measured and compared. Fig.16As shown in the figure. It was found that after intraperitoneal injection of LPS in piglets, the villus height of the duodenum decreased significantly (P<0.001), and the crypt depth of the jejunum decreased significantly (0.01<P<0.05); compared with the LPS group, the intestinal villus height of piglets in the group fed with Lactobacillus reuteri LRM2293 increased significantly (P<0.001), and the villus-crypt ratio of the jejunum and ileum increased (0.001<P<0.01 or 0.01<P<0.05), indicating that Lactobacillus reuteri LRM2293 can increase the intestinal absorption surface area by increasing the villus length, improve the absorption efficiency of nutrients, and at the same time enhance the intestinal barrier function and reduce the penetration of harmful substances.

[0090] 4.4 Effect of LRM2293 on goblet cells in the intestine of LPS-induced inflammatory piglets

[0091] Goblet cells are distributed among mucosal columnar epithelial cells, and their main function is to synthesize and secrete mucin to form a mucosal barrier to protect epithelial cells. To explore the effect of LPS stimulation on the number of goblet cells in the small intestinal mucosal epithelial cells of piglets, AB-PAS staining was used to observe the goblet cells in the intestine, and the results are as Fig.17 shown. In AB-PAS staining, goblet cells appear purple-red due to the presence of a large number of polysaccharide molecules, but sometimes they may be stained blue due to the different pH of the secreted mucus. Three complete intestinal villi were randomly selected and the number of cells positive for PAS staining was counted. The results are as Fig.18 shown. Analysis found that the LPS group significantly reduced the number of goblet cells in the ileum (0.001<P<0.05), while the protective effect of LRM2293 significantly alleviated the decrease in the number of goblet cells caused by LPS stimulation (0.001<P<0.01 or 0.01<P<0.05).

[0092] 4.5 Protective effect of LRM2293 strain on intestinal villi

[0093] The intestinal villus morphology of piglets in different groups was observed by scanning electron microscopy, and the results are shown in Fig.19 . Compared with the control group, the intestinal villi of piglets in the LPS group were severely damaged, showing collapse and fracture, with incomplete structure, and the top of the villi was severely damaged, and the damage to the duodenum and ileum was more significant. The intestinal villi of piglets in the protection group were arranged more neatly and densely, with complete structure. The morphology of the crypt openings in the control group was regular and the mucosa was relatively flat and smooth, while the crypt openings in the colon of the LPS group became larger and narrower, and were overall more crowded, and the situation in the prevention group improved.

[0094] 4.6 Effect of LRM2293 on the ultrastructure of the intestine of inflammatory piglets

[0095] To further understand the protective effect of LRM2293 on LPS-induced intestinal inflammation in piglets, transmission electron microscopy was used to detect ultrastructural changes in the small intestine of piglets. Red arrows indicate microvilli, yellow arrows indicate mitochondria, and green arrows indicate tight junctions. Fig. 20 As shown, the intestinal microvilli of the piglets in the LPS group were arranged loosely, atrophied and broken; the tight junctions between intestinal epithelial cells were loose, with large gaps, and the tight junctions were blurred under the electron microscope; the mitochondria in the cells were obviously swollen and rounded, and the cristae were blurred. The intestinal microvilli of the control group and the protection group were arranged neatly and tightly, with few breaks; the tight junctions between intestinal epithelial cells were clear and without gaps; the mitochondria had normal morphology, a large number, and clearly visible cristae. The above results show that the intervention of LRM2293 significantly alleviated the damage of LPS stimulation to the tight junctions between intestinal microvilli and epithelial cells in piglets.

[0096] 4.7 Effect of LRM2293 on the expression of inflammatory factors in colon tissue of inflamed piglets

[0097] To prove that Lactobacillus reuteri LRM2293 has a protective effect against LPS-induced acute intestinal inflammation in piglets, several inflammatory factors were measured by RT-qPCR. Fig.21 Compared with the control group, the levels of IL-6 and TNF-α in the colon tissue of piglets in the LPS group were significantly increased (P<0.001), while the protection group reversed this phenomenon, indicating that LRM2293 can inhibit the production of pro-inflammatory factors in piglets caused by LPS, thereby alleviating the inflammatory state of the body.

Claims

1. A strain of Lactobacillus reuteri from Min pigs, named Lactobacillus reuteri LRM2293, deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M 20242331 and a deposit date of October 25, 2024.

2. Use of the pig-derived Lactobacillus reuteri or its culture supernatant as claimed in claim 1 in the preparation of anti-inflammatory drugs.

3. Use of the Lactobacillus reuteri derived from pigs or its culture supernatant as claimed in claim 1 in the preparation of drugs or reagents for inhibiting enterotoxigenic Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Streptococcus suis.

4. Use of the pig-derived Lactobacillus reuteri or its culture supernatant as claimed in claim 1 in the preparation of a medicament for treating intestinal inflammation in piglets.

5. The use according to claim 4, characterized in that The Lactobacillus reuteri derived from Min pigs can colonize in various intestinal segments of piglets, repair intestinal villi and tight junction structures, increase the number of goblet cells, and inhibit the production of pro-inflammatory factors in the body, thereby alleviating the body's inflammatory state, maintaining the stability of the intestinal barrier, and reducing intestinal damage caused by inflammation.

6. The use according to any one of claims 2 to 5, characterized in that: The culture supernatant is obtained by inoculating the pig-derived Lactobacillus reuteri described in claim 1 into an MRS liquid culture medium at a volume ratio of 1:100, culturing at 37° C. for 24 hours, then centrifuging and removing cells and impurities.

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