Lactococcus lactis with alleviating intestinal injury of mice caused by salmonella and composition and application thereof

Abstract

The application discloses a lactococcus lactis capable of relieving intestinal injury of mice caused by salmonella, a composition and application thereof, and belongs to the technical field of microorganisms. 8 -1.0×10 10 cfu / g of lactococcus lactis subsp. Lactococcus lactis subsp. lactis ) KLDS 4.0325 and hematin; the mass ratio of hematin to KLDS 4.0325 is 1: (50-150). The KLDS 4.0325 can restore intestinal flora disorder caused by salmonella typhimurium infection through aerobic respiration, reduce the load of salmonella typhimurium in the intestinal tract of a host and limit the displacement of the salmonella typhimurium, reduce the expression level of virulence genes of the salmonella typhimurium, promote the autophagy of intestinal cells of mice and inhibit the NF-κB pathway, so that the inflammatory reaction is relieved.

Description

Technical Field

[0001] This invention relates to a lactococcus lactis strain for the prevention and relief of colitis, its composition and application, and particularly to a lactococcus lactis strain for the relief of Salmonella-induced intestinal damage in mice, its composition and application, belonging to the field of microbial technology. Background Technology

[0002] In existing technologies, it is uncertain whether the aerobic respiration metabolism of *Lactococcus lactis* reduces the oxygen content in the host gut. It is also uncertain whether the amount of oxygen consumed by *Lactococcus lactis* is sufficient to inhibit Salmonella growth. Furthermore, the amount of heme added during aerobic respiration metabolism by *Lactococcus lactis* in the host is uncertain. In other words, it is unclear whether *Lactococcus lactis* can prevent and alleviate colitis by restoring the anaerobic environment and maintaining gut microbiota homeostasis in the host gut through aerobic respiration metabolism. This study utilizes *Lactococcus lactis* subsp. *lactis* (L. lactis) KLDS 4.0325, a previously obtained aerobic respiration-capable bacterium, and employs a Salmonella-induced enteritis mouse model. Based on molecular biology, histology, immunology, gut environment, and gut microbiota, it investigates the effects of lactic acid bacteria in preventing and alleviating colitis and explores its mechanism of action. This provides a theoretical basis for developing potential antibiotic alternatives and is of great significance for protecting human health. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a lactococcus lactis strain that can alleviate intestinal damage in mice caused by Salmonella, as well as its composition and application.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0005] A lactococcal composition for alleviating intestinal damage in mice caused by Salmonella includes the following components: a viable count of 1.0 × 10⁻⁶ bacteria. 8 -1.0×10 10 CFU / g of Lactococcus lactis subsp. lactis KLDS 4.0325 and heme; the mass ratio of heme to KLDS 4.0325 was 1:(50-150).

[0006] The use of the composition of the present invention in the preparation of a drug for the prevention of colitis.

[0007] The use of the composition of the present invention in the preparation of a drug for relieving colitis.

[0008] The use of the composition of the present invention in the preparation of a drug for relieving intestinal damage.

[0009] The composition reduces the host's Salmonella load.

[0010] The composition improves the pathological changes in the host gut.

[0011] A type of lactococcus lactis that can alleviate intestinal damage caused by Salmonella in mice, comprising 1.0 × 10⁻⁶ live bacteria. 8 -1.0×10 10 cfu / g of Lactococcus lactis subsp. lactis KLDS4.0325.

[0012] The application of the lactococcus lactis of the present invention in the preparation of drugs for the prevention of colitis.

[0013] The application of the lactococcus lactis of the present invention in the preparation of drugs for relieving colitis.

[0014] The application of the lactococcus lactis of the present invention in the preparation of drugs to alleviate intestinal damage.

[0015] The present invention has the following beneficial effects:

[0016] Compared with the model group, L. lactis KLDS 4.0325 combined with heme can improve the survival rate and body weight of mice, significantly reduce the disease activity index, significantly reduce the Salmonella load in the cecum and colon, and inhibit the expression of virulence genes sipB, sipC and sopE2; significantly increase colon length, reduce organ index and alleviate histopathological reactions; significantly upregulate colonic ZO-1 expression, but downregulate Occludin and Muc2 expression; and significantly reduce serum lipopolysaccharide and C-reactive protein concentrations.

[0017] In summary, *L. lactis* KLDS 4.0325 can restore the intestinal flora imbalance caused by *Salmonella typhimurium* infection through aerobic respiration, reduce the *Salmonella typhimurium* load in the host gut and limit its translocation, decrease the expression level of *Salmonella typhimurium* virulence genes, promote autophagy in mouse intestinal cells and inhibit the NF-κB pathway, thereby alleviating the inflammatory response, significantly reducing serum lipopolysaccharide and C-reactive protein concentrations, and improving host intestinal infection symptoms. Therefore, *L. lactis* KLDS 4.0325 can prevent *Salmonella typhimurium* infection in the host through aerobic respiration. Attached Figure Description

[0018] Figure 1 The graphs show mouse survival rate, weight change, and DAI score, where (A) mouse survival rate, (B) mouse weight change, and (C) Disease Activity Index (DAI) score results on day 6 after infection.

[0019] Figure 2The content of Salmonella in the intestinal contents of mice is shown in the figures: (A) S. Typhimurium SL1344 content in the cecal contents and (B) S. Typhimurium SL1344 content in the colon contents.

[0020] Figure 3 The length of the mouse colon;

[0021] Figure 4 Pathological changes in mouse intestinal tissue;

[0022] Figure 5 This refers to the organ index in mice;

[0023] Figure 6 The images show the histopathological changes in mouse liver tissue, including (A) control group, (B) model group, (C) L. lactis KLDS 4.0325 + heme group, (D) L. lactis KLDS 4.0325 + PBS group, and (E) LGG group. Red arrows represent focal hepatocyte necrosis, yellow arrows represent inflammatory cell infiltration, and blue arrows represent lipid vacuoles.

[0024] Figure 7 The expression levels of Salmonella virulence genes in mice are: (A) sipB expression level, (B) sipC expression level, and (C) sopE2 expression level.

[0025] Figure 8 The expression levels of colonic barrier-related genes in mice are shown in Figure 1, where (A) colonic ZO-1 level, (B) colonic Occludin level, and (C) colonic Muc2 level.

[0026] Figure 9 This represents the expression level of colonic barrier-related proteins in mice.

[0027] Figure 10 The values ​​represent the concentrations of cytokines in the mouse colon, including (A) colonic TNF-α level, (B) colonic IFN-γ level, (C) colonic IL-1β level, (D) colonic IL-10 level, and (E) colonic sIgA level.

[0028] Figure 11 The concentrations of LPS and CRP in mouse serum are shown in Figure 1. (A) Serum LPS level, (B) Serum CRP level.

[0029] Figure 12 The concentrations of cytokines in mouse serum include (A) serum TNF-α level, (B) serum IL-1β level, and (C) serum IL-10 level.

[0030] Figure 13The expression levels of autophagy and NF-κB-related genes in the mouse colon are shown in Figure 1. (A) Colonic LC3-α level, (B) Colonic NF-κB p65 level.

[0031] Figure 14 The expression levels of autophagy and NF-κB-related proteins in the mouse colon are shown in Figure 1. (A) Colonic LC3 level, (B) Colonic NF-κB p65 level.

[0032] Figure 15 For the diversity analysis of mouse gut microbiota, (A) Chao 1 index; (B) Principal component analysis (PCA) plot;

[0033] Figure 16 The composition of the mouse gut microbiota at the phylum level;

[0034] Figure 17 The composition of the mouse gut microbiota at the genus level;

[0035] Figure 18 LEfSe analysis for gut microbiota;

[0036] Figure 19 This is a Spearman correlation analysis heatmap, where red represents a positive correlation, green represents a negative correlation, and the darker the color, the stronger the correlation. * indicates a significant difference (P<0.05), and ** indicates an extremely significant difference (P<0.01).

[0037] Figure 20 For comparison of differences in metabolic pathways;

[0038] Figure 21 The content of short-chain fatty acids in the intestinal contents of mice;

[0039] Figure 22 This is a volcano plot of differentially expressed genes, where (A) is the model group vs. the control group, and (B) is the L. lactis KLDS4.0325+ heme group vs. the model group. Red dots represent upregulated differentially expressed genes, blue dots represent downregulated differentially expressed genes, and gray dots represent indifferentially expressed genes.

[0040] Figure 23 Differentially expressed genes were classified into GO groups, including (A) model group vs. control group, and (B) L. lactis KLDS4.0325+ heme group vs. model group.

[0041] Figure 24 The KEGG enrichment bubble plot shows the differentially expressed genes, where (A) is the model group vs. the control group, and (B) is the L. lactis KLDS 4.0325+ heme group vs. the model group. The bubble color represents the change in P-value, and the bubble size represents the number of differentially expressed genes. Detailed Implementation

[0042] The invention will now be further described with reference to the accompanying drawings.

[0043] Lactococcus lactis subsp. lactis KLDS 4.0325 is deposited at the Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University. This strain was collected from fermented mare's milk made by herdsmen in Xinjiang, China. The existing technical source is: Liu Fei, Li Bailiang, Du Jincheng, Yu Shangfu, Ding Xiuyun, Xu Min, Huo Guicheng. Study on aerobic respiratory metabolism of Lactococcus lactis based on gene level [J]. Modern Food Science and Technology, 2016, 32(09): 56-61+215. It is available to the public from Northeast Agricultural University.

[0044] Example 1

[0045] 1. The role of aerobic lactococci in preventing Salmonella infection in mice.

[0046] 1.1 Effects of aerobic lactococcus on Salmonella-infected mice

[0047] To investigate whether aerobic respiration of *Lactococcus lactis* could alleviate damage caused by *Salmonella typhimurium* in mice, 100 seven-week-old male C57BL / 6J mice were used as experimental animals. Mice had free access to water and feed. After one week of acclimatization, they were randomly divided into five groups (Table 1): control group (N), model group (M), aerobic respiration group (H), fermentation metabolism group (C), and LGG treatment group (G), with 20 mice in each group. The experimental period was 25 days, with days 1-14 as the prevention period, day 15 as the modeling period, and days 16-25 as the observation period. During the preventative period, each mouse was administered 0.2 mL / day via gavage. The experimental group mice received additional heme or PBS added to the bacterial suspension before gavage, at a concentration of 2% of the original culture medium volume. This gavage was stopped after 14 days. On day 15, all mice were deprived of water and food for 2 hours. The control group mice were administered 0.1 mL of PBS solution via gavage, while the other groups were administered 0.1 mL of *S. typhimurium* SL1344 bacterial suspension via gavage. Water was provided immediately after gavage, and the mice were fed 2 hours later. On day 22, some mice were anesthetized and euthanized, and relevant tissues were collected for subsequent testing. Starting from the administration of *Salmonella typhimurium* SL1344 via gavage, the mice's activity was observed and recorded daily.

[0048] Table 1. In vivo inhibitory effects of aerobic respiration Lactococcus lactis on Salmonella. Experimental animal grouping. (Tab. 1 Animal experimental design of inhibitory effects of aerobic respiration L. lactis KLDS 4.0325 on mice)

[0049] infected with S.Typhimurium SL1344

[0050]

[0051] Changes in the survival curve ( Figure 1 A) It can be seen that mouse mortality first began in group G on day 4 post-infection, mainly concentrated after day 6. On day 10 post-infection, no deaths occurred in group N; groups M (3 / 9) and G (3 / 9) had the lowest survival rates, at 33.33%; while groups H (5 / 9) and C (5 / 9) had survival rates of 55.56%.

[0052] After mice were infected with Salmonella typhimurium, their body weight generally showed a trend of first increasing and then decreasing, while the body weight of uninfected mice in group N consistently showed an increasing trend. Figure 1 B). Mouse body weight was measured on the day of gavage administration of S. Typhimurium SL1344, and there were no significant differences among the groups (P>0.05). Mouse body weight began to decrease on day 3 post-infection, and by day 6 post-infection, the body weight of group M mice was significantly lower than the other four groups (P<0.05), indicating that L. lactis KLDS 4.0325 and LGG can alleviate the weight loss induced by S. Typhimurium SL1344 infection.

[0053] Observations of mouse activity revealed that, compared to group N, mice infected with Salmonella Typhimurium began exhibiting lethargy, loss of appetite, and huddling together on day 3; mild diarrhea began on day 4, although the stool remained formed; and convulsions and even death occurred after day 6. The DAI score was calculated the day before sampling (day 6 post-infection). Figure 1C) Compared with normal mice in group N, mice in group M infected with Salmonella typhimurium showed significantly higher DAI scores (P<0.05). Both L. lactis KLDS 4.0325 and LGG intervention significantly reduced the upregulation of DAI caused by Salmonella typhimurium infection (P<0.05), and group H recovered to levels close to those of group N (P>0.05). However, the DAI scores of mice in group C were significantly higher than those in group N (P<0.05), indicating that L. lactis KLDS 4.0325 combined with heme was better at improving the disease state caused by Salmonella typhimurium infection than L. lactis KLDS 4.0325 alone by gavage.

[0054] 1.2 Salmonella load in mouse intestinal contents

[0055] This study used streptomycin-resistant *S. typhimurium* SL1344 to infect mice. By selecting MacConkey agar medium, which is capable of isolating intestinal pathogens, and adding streptomycin, specific screening for *Salmonella* was performed, thereby detecting the *S. typhimurium* SL1344 load in the intestinal contents of each group of mice. Figure 2 The results showed that all three treatment groups (H, C, and G) significantly reduced the Salmonella typhimurium load in the colonic and cecal contents (P<0.05). In the colonic contents, the load in groups H and C was significantly lower than that in group G (P<0.05), indicating that L. lactis KLDS 4.0325 was more effective in reducing the colonization of Salmonella typhimurium in the host colon.

[0056] 1.3 Pathological changes in the mouse intestine

[0057] The length of the colon can reflect the severity of intestinal inflammation in mice to some extent; the shorter the colon, the higher the severity. Therefore, it can be used as a biological indicator to characterize the degree of inflammation. During the experiment, the colon length of mice in each group was measured using a ruler. Figure 3 The N group of mice had the longest colon. The colon length of mice infected with Salmonella Typhimurium was significantly reduced (P<0.05). Compared with the M group, the H, C and G groups all had significantly increased colon length (P<0.05), with the H group being slightly higher than the C and G groups (P>0.05).

[0058] Sections of fixed cecum and colon were prepared and stained with hematoxylin and eosin (HE) to observe pathological changes in the intestinal tissues. Figure 4In the N group of normal mice, the intestinal tissue structure was normal, with neat and compact mucosal structure, clear and intact crypt structure, and no cell infiltration. In contrast, the intestinal tissue structure of mice infected with Salmonella typhimurium was abnormal. Epithelial cells of the cecal mucosa were sloughed off, the lamina propria was exposed, the interstitial space was enlarged, and inflammatory cell infiltration was observed. Edema was present in the submucosa of the colon, the interstitial space was enlarged, and focal infiltration of inflammatory cells was visible. The intestinal tissue damage in all three treatment groups (H group, C group, and G group) was improved, the epithelial cells were neatly arranged, there was no edema or only mild edema in the submucosa, and the inflammatory cell infiltration was reduced. The improvement effect was most significant in the H group. Figure 4 In the diagram, red arrows represent epithelial cell shedding, blue arrows represent increased interstitial spaces, and yellow arrows represent inflammatory cell infiltration.

[0059] 1.4 Pathological changes in mouse organs

[0060] Organ indices can reflect the strength of immune function, and their calculation formula is as follows.

[0061]

[0062] In this study, the spleen and liver were weighed during the experimental sampling, and the organ index was calculated. Figure 5 Compared to group N, mice in group M infected with Salmonella Typhimurium showed significantly increased spleen and liver indices (P<0.05). All three treatment groups (H, C, and G) significantly reduced the spleen index after Salmonella Typhimurium infection, with group H showing the best effect (P<0.05). Regarding the liver index, group H mice were significantly lower than group M mice (P<0.05), while groups C and G were slightly lower than group M, but the difference was not significant (P>0.05). These results indicate that L. lactis KLDS 4.0325 combined with heme is more effective in alleviating splenomegaly and hepatomegaly caused by Salmonella Typhimurium infection.

[0063] Pathological changes in mouse liver were observed by HE staining. Figure 6 In group N, normal mice showed normal liver tissue structure, rounded nuclei, uniform sinusoidal size, and no inflammatory cell infiltration. However, mice infected with Salmonella typhimurium showed obvious lesions in their livers, abnormal tissue structure, focal necrosis of numerous hepatocytes, and extensive inflammatory cell infiltration. All three treatment groups showed varying degrees of relief from liver damage: group H showed no significant inflammatory infiltration, only mild hepatocyte degeneration, and a small number of fat vacuoles; group C showed reduced inflammatory infiltration; and group G showed intact stem cell structure, but still exhibited inflammatory infiltration. These results indicate that both L. lactis KLDS 4.0325 and LGG can alleviate liver damage caused by Salmonella typhimurium infection to some extent, with the best effect observed when L. lactis KLDS 4.0325 was combined with heme.

[0064] 1.5. Effects of aerobic lactococci on Salmonella virulence genes in the host.

[0065] Existing technologies show that, in vitro, *L. lactis* KLDS 4.0325 can inhibit the expression of *Salmonella typhimurium* sipB, sipC, and sopE2 virulence genes through aerobic respiration. Therefore, this study used RT-qPCR to detect the expression of virulence genes in *Salmonella typhimurium* colonized in the host. Figure 7 Compared with normal mice in group N, virulence genes in group M were significantly upregulated (P<0.05). All three treatment groups (group H, group C, and group G) significantly inhibited the expression of virulence genes of Salmonella typhimurium (P<0.05), and L. lactis KLDS 4.0325 combined with heme was more effective than the single treatment.

[0066] 1.6 Effects of Aerobic Lactococcus lactis on the intestinal barrier in mice

[0067] 1.6.1. mRNA expression of intestinal barrier genes

[0068] The gut is the body's first line of defense. Changes in intestinal barrier permeability can lead to the entry of various antigens into the systemic circulation, thereby inducing inflammation and damage to extraintestinal organs. This study used RT-qPCR to detect the expression of mouse colon tight junction protein genes ZO-1 and Occludin, as well as the mucin gene Muc2.

[0069] Transcriptional expression results of tight junction proteins showed that, compared with group N, colonic ZO-1 expression in group M mice infected with Salmonella typhimurium was significantly downregulated (P<0.05), and colonic ZO-1 expression in group G mice was also significantly downregulated (P<0.05), while colonic ZO-1 expression in group H mice recovered to the level of normal mice in group N (P>0.05). Figure 8 A); Regarding colonic Occludin expression, the M group infected with Salmonella Typhimurium was significantly upregulated (P<0.05), and all three treatment groups (H, C, and G) significantly reduced the upregulation of Occludin expression induced by Salmonella Typhimurium infection (P<0.05). Figure 8 B).

[0070] The transcriptional expression results of mucin Muc2 showed that the expression of Muc2 in the colon of mice in group M after infection with Salmonella typhimurium was significantly upregulated (P<0.05). All three treatment groups showed some recovery after intervention, with group H recovering to near the level of normal mice in group N (P>0.05), while the expression of Muc2 in group G was significantly downregulated compared to group N (P<0.05). Figure 8 C).

[0071] 1.6.2 Expression of intestinal barrier proteins

[0072] The expression of colonic barrier proteins in mice was re-examined using Western blot, and the changes in the expression levels of the three proteins were consistent with the changes in their mRNA expression.

[0073] The results of tight junction protein expression analysis showed that, compared with normal mice in group N, the expression of ZO-1 in the colon of mice in group M was significantly decreased (P<0.05), and compared with group M, the expression of ZO-1 in the colon of mice in group H was significantly increased (P<0.05), while the expression of ZO-1 in the colon of mice in group G was the lowest. Figure 9 Regarding colonic occludin expression, group M was significantly higher than group N (P<0.05), while compared to group M, all three treatment groups (group H, group C, and group G) significantly reduced colonic occludin expression (P<0.05). Figure 9 ).

[0074] Muc2 expression results showed that, compared with group N, colonic Muc2 expression was significantly increased in group M mice (P<0.05). Compared with group M, colonic Muc2 expression was significantly decreased in all three treatment groups (group H, group C, and group G) (P<0.05), with group H recovering to near the level of normal mice in group N (P>0.05). Figure 9 ).

[0075] The above results indicate that aerobic respiration metabolism of *L. lactis* KLDS 4.0325 can restore intestinal barrier alterations induced by *Salmonella typhimurium* infection, stabilize tight junction ZO-1, and reduce the elevated levels of Occludin and Muc2 caused by *Salmonella typhimurium*. Different letter representations showed significant differences (P<0.05).

[0076] 1.7 Effects of Aerobic Lactococcus lactis on intestinal immunity in mice

[0077] Inflammatory cytokines produced in the inflamed gut play a crucial role in its pathological process, and regulating the balance of these factors can help prevent inflammatory responses. This study used an ELISA kit to detect the levels of TNF-α, INF-γ, IL-1β, IL-10, and sIgA in mouse colon tissue. Figure 10Compared with normal mice in group N, mice in group M infected with Salmonella Typhimurium showed significantly increased levels of pro-inflammatory cytokines TNF-α, IFN-γ, and IL-1β in the colon (P<0.05), while significantly decreased levels of anti-inflammatory cytokines IL-10 and immunoglobulin sIgA (P<0.05). This indicates that Salmonella Typhimurium infection can induce colonic inflammation in mice. Compared with group M, all three treatment groups (group H, group C, and group G) significantly reduced colonic levels of TNF-α, IFN-γ, and IL-1β (P<0.05) and significantly increased colonic levels of IL-10 (P<0.05). However, the sIgA levels in the three treatment groups (group H, group C, and group G) were only slightly higher than those in group M, with no significant difference (P>0.05). The above results indicate that aerobic respiration metabolism of L. lactis KLDS 4.0325 can regulate colonic immunity, inhibit the level of pro-inflammatory cytokines, and increase the level of anti-inflammatory cytokines, thereby alleviating the inflammatory response caused by Salmonella typhimurium infection.

[0078] 1.8 Effects of Aerobic Lactococcus lactis on the resistance of mice to systemic infection

[0079] Lipopolysaccharide (LPS) is a component of the cell walls of many pathogenic bacteria and is pathogenic to the host. Compared with group N, mice in group M had significantly higher serum LPS concentrations after infection with Salmonella Typhimurium (P<0.05), while the serum LPS levels in the three treatment groups (groups H, C, and G) were significantly lower than those in group M (P<0.05). Figure 11 A).

[0080] CRP levels in the blood are an indicator for diagnosing bacterial infections in clinical practice. Serum CRP concentrations were significantly elevated in mice infected with Salmonella Typhimurium (P<0.05), while all three treatment groups (H, C, and G) showed significantly reduced serum CRP levels (P<0.05). Figure 11 B).

[0081] During inflammation, cytokines are also secreted into the bloodstream; therefore, the levels of TNF-α, IL-1β, and IL-10 in mouse serum were measured. Figure 12Compared with normal mice in group N, mice in group M infected with Salmonella Typhimurium showed significantly elevated serum levels of pro-inflammatory cytokines TNF-α and IL-1β (P<0.05). All three treatment groups (H, C, and G) significantly reduced TNF-α and IL-1β levels (P<0.05), with IL-1β recovering to near-normal levels (P>0.05). Compared with normal mice in group N, group M showed significantly reduced levels of the anti-inflammatory cytokine IL-10 (P<0.05), while groups H and C significantly increased IL-10 levels after Salmonella Typhimurium infection (P<0.05). Group G showed a slight increase, but the difference was not statistically significant (P>0.05). These results indicate that aerobic respiration metabolism of L. lactis KLDS 4.0325 can reduce serum levels of pro-inflammatory cytokines induced by Salmonella Typhimurium, increase levels of anti-inflammatory cytokines, and alleviate systemic infection caused by Salmonella Typhimurium.

[0082] 1.9 Effects of aerobic-respiratory Lactococcus lactis on autophagy and the NF-κB pathway in mice

[0083] 1.9.1 mRNA expression of pathway-related genes

[0084] To elucidate the mechanism by which aerobic lactococci resist Salmonella typhimurium invasion, this study used RT-qPCR to detect the expression levels of key genes in the colonic autophagy and NF-κB signaling pathways.

[0085] Microtubule-associated protein light chain 3 (LC3) is an important regulator of autophagy. It binds to the membrane by interacting with autophagy effectors, thereby initiating autophagosome formation. LC3-α is a subtype of LC3 and an important biomarker for autophagy activation. Compared with group M, L. lactis KLDS4.0325 intervention significantly upregulated LC3-α expression (P<0.05), and the LC3-α level in group H was significantly higher than in other groups (P<0.05), while there was no significant difference between group C and group M (P>0.05). Figure 13 A).

[0086] The NF-κB signaling pathway participates in the body's immune and inflammatory responses, and its transcriptional activity is regulated by complete activation through post-translational modifications. Compared with normal mice in group N, p65 expression was significantly upregulated in the colon of mice in group M (P<0.05), and p65 expression was also significantly upregulated in all three treatment groups (groups H, C, and G) (P<0.05). However, compared with group M, p65 expression levels in groups H and G were significantly decreased (P<0.05). Figure 13 B).

[0087] 1.9.2 Expression of pathway-related proteins

[0088] The results were consistent with the changes in its mRNA expression.

[0089] LC3-I and LC3-II are two forms of LC3. During autophagy, the cytoplasmic form of LC3-I is converted into the membrane-bound form of LC3-II. Therefore, the LC3-II / LC3-I ratio can be used as a biomarker for autophagy. Results on the expression of key proteins in the autophagy pathway showed that, compared with group M mice, the LC3-II / LC3-I levels in groups H and C were significantly increased (P<0.05). Figure 14 A).

[0090] The expression results of key proteins in the NF-κB signaling pathway showed that, compared with normal mice in group N, the NF-κB p65 level in the colon of mice in group M was significantly increased (P<0.05). All three treatment groups (group H, group C, and group G) could reduce the p65 level after Salmonella typhimurium infection (P<0.05), and groups H and G had better inhibitory effects on NF-κB activity. Figure 14 B).

[0091] The above results indicate that aerobic respiration metabolism of L. lactis KLDS 4.0325 can activate autophagy and inhibit the NF-κB signaling pathway, thereby clearing Salmonella typhimurium and inhibiting intestinal inflammation.

[0092] 1.10. Microbial structure of mouse colon contents

[0093] 1.10.1 Changes in microbial diversity

[0094] To investigate the effects of different interventions on the gut microbiota of mice, 16S rDNA sequencing technology was used for detection. First, changes in α- and β-diversity of the mouse gut microbiota were compared.

[0095] Alpha diversity focuses on the diversity of the microbial community within a sample. This study used the Chao 1 index to assess alpha diversity; a higher Chao 1 index indicates a greater total number of species. Results showed that the Chao 1 index of the microbial community in group M mice infected with *Salmonella typhimurium* was slightly lower than that in group N. After intervention, the Chao 1 index of the microbial community in groups H and G was slightly higher than that in group M, with group H having the highest Chao 1 index. However, there were no significant differences among the groups (P>0.05). Figure 15 A).

[0096] β-diversity focuses on the differences in microbial community composition among different samples. This study used principal component analysis (PCA) to measure β-diversity. The results showed that the distance between group N and group M was greater than the distance between group N and group H. Figure 15B) This indicates that Salmonella typhimurium interferes with the composition of the intestinal flora in mice, while L. lactis KLDS 4.0325 combined with heme can restore the composition of the intestinal flora in mice to a certain extent.

[0097] 1.10.2 Changes in microbial community structure at the phylum level

[0098] After analyzing the diversity of gut microbiota, this study compared the composition of the mouse gut microbiota at the phylum level. Figure 16 The results showed that Bacteroides, Firmicutes, Proteobacteria, and Actinobacteriota were the main constituents of the intestinal flora in mice. The dominant flora in the control group were Firmicutes (50.54%), Bacteroides (41.37%), Desulfobacterota (5.05%), and Actinobacteria (2.37%). In contrast, the abundance of Firmicutes (24.20%), Actinobacteria (1.76%), and Desulfobacterota (0.46%) decreased in group M mice infected with Salmonella typhimurium, while the abundance of Proteobacteria increased significantly, from 0.07% in group N to 18.08%. All three treatment groups (H, C, and G) were able to restore this disorder to varying degrees, but group H showed the closest recovery to group N. Intervention in mice using L. lactis KLDS 4.0325 in combination with heme increased the abundance of Firmicutes (32.79%), Actinobacteria (2.19%), and Dethiobacteria (2.12%), while decreasing the abundance of Proteobacteria (0.29%). Furthermore, in group M mice infected with Salmonella typhimurium, the abundance of Campylobacterota (which can cause bacterial gastroenteritis) increased, while the abundance of Patescibacteria and Verrucomicrobiota decreased; this change was improved in group H.

[0099] 1.10.3 Changes in bacterial community structure at the genus level

[0100] After analyzing the phylum level, a genus-level heatmap analysis was performed on the relative abundance of each species in each sample group. Figure 17Compared to group N, mice in group M infected with Salmonella typhimurium showed increased relative abundance of Enterobacter, Escherichia-Shigella, Parasutterella, Mucispirillum, Alloprevotella, Bacteroides, and Helicobacter, while the relative abundance of Eubacterium siraeum, Eubacterium xylanophilum, Coridextribacter, Marvinbryantia, Lachnospiraceae UCG-006, and Desulfovibrio decreased. Intervention with L. lactis KLDS 4.0325 combined with heme restored these changes. In addition, compared with group H, the relative abundance of genera such as Anaerorotruncus, Blautia, and Muribauculum, which produce short-chain fatty acids, increased in group G.

[0101] 1.10.4 Species Difference Analysis

[0102] This study further analyzed the species differences in gut microbiota between group M and group H mice using LEfSe software. Figure 18 Compared to group H, group M mice showed a significantly increased abundance of Proteobacteria in their gut microbiota, including Enterobacteriaceae, Escherichiae-Shigella, and Enhydrobacter (LDA>2), all of which are facultative anaerobes. Conversely, compared to group M, group H mice treated with L. lactis KLDS 4.0325 combined with heme showed a significantly increased abundance of Firmicutes in their gut microbiota, including Eubacterium coprostanoligenes, Eubacterium xylanophilum, and Lachnospiraceae UCG-006, all of which are obligate anaerobes. These results are consistent with studies on changes in gut microbiota at both the phylum and genus levels.

[0103] 1.10.5 Correlation analysis between physiological indicators and gut microbiota levels

[0104] To investigate the role of L. lactis KLDS 4.0325 combined with heme intervention in the prevention and alleviation of Salmonella typhimurium infection in mice, this study analyzed the Spearman correlation between the gut microbiota of mice in groups M and H and mouse organ indices (Spleen index and Liver index), colonic cytokines (TNF-α, IFN-γ, IL-1β and IL-10), LPS, CRP, and signaling pathways (Autophagy and NF-κB). Figure 19 The results showed that the spleen index was significantly negatively correlated with the *Eubacterium*_coprostanoligenes_group; the liver index was significantly negatively correlated with both the *Eubacterium*_coprostanoligenes_group and the *Eubacterium*_siraeum_group; colonic TNF-α was significantly positively correlated with *Alloprevotella*, *Enterobacter*, and *Faecalibaculum*, and significantly negatively correlated with *Muribaculaceae*; colonic IFN-γ was significantly positively correlated with *Alloprevotella*, and significantly negatively correlated with both the *Eubacterium*_coprostanoligenes_group and *Muribaculaceae*; and colonic IL-1β was significantly positively correlated with *Bacteroides* and *Enterobacter*. r showed a significant positive correlation with Desulfovibrio and Muribaculaceae; colonic IL-10 showed a significant negative correlation with Alloprevotella; serum CRP showed a significant positive correlation with Enterobacter and Faecalibaculum, and a significant negative correlation with [Eubacterium]_coprostanoligenes_group and Muribaculaceae; the autophagy pathway showed a significant positive correlation with Desulfovibrio; the NF-κB signaling pathway showed a significant positive correlation with Enterobacter and Faecalibaculum, and a significant negative correlation with Clostridia_UCG-014. These results indicate a correlation between gut microbiota and the effects of Salmonella typhimurium infection on host physiological parameters.

[0105] 1.10.6 Analysis of differences in functional abundance

[0106] To investigate potential functional differences between group M (infected with Salmonella typhimurium) and group H (treated with L. lactis KLDS 4.0325 combined with heme), PICRUSt was used to infer the composition of functional genes in the samples based on the gut microbiota composition, thereby comparing the functional abundance differences between groups M and H. Figure 20 The results showed that, compared with group M, group H had increased abundance in pathways related to galactose metabolism, amino sugar and nucleotide sugar metabolism, glycolysis / gluconeogenesis, primary bile acid biosynthesis, secondary bile acid biosynthesis, and starch and sucrose metabolism; and increased abundance in lipopolysaccharide biosynthesis proteins, Vibrio cholerae infection, cell motility and secretion (Unclassified), lipopolysaccharide biosynthesis, bacterial secretion system, flagellar assembly, Vibrio cholerae pathogenic cycle, and bacterial motility. The abundance of pathways related to proteins and Shigella disease is reduced.

[0107] 1.11 Effects of Aerobic Lactococcus lactis on short-chain fatty acids in mouse intestinal contents

[0108] Short-chain fatty acids (SCFAs) are important metabolites of gut microbiota and have immunomodulatory effects on humans. Therefore, this study used GC-MS to detect the SCFA content in mouse intestinal contents. Figure 21Compared to group N, the levels of propionic acid and butyric acid in the intestinal contents of mice infected with Salmonella typhimurium in group M were decreased, but the difference was not statistically significant (P>0.05). After intervention with L. lactis KLDS 4.0325 combined with heme, the levels of acetic acid, propionic acid, and butyric acid were slightly increased (P>0.05). LGG treatment significantly increased the levels of acetic acid, propionic acid, and butyric acid (P<0.05).

[0109] 1.12 Transcriptomic analysis of mouse colon tissue

[0110] 1.12.1 Differentially expressed genes

[0111] This study analyzed gene distribution using colonic transcriptome data. The results showed that L. lactis KLDS 4.0325 combined with heme intervention could affect gene expression distribution. Group M had 1884 differentially expressed genes compared to group N, of which 807 were upregulated and 1077 were downregulated. Figure 22 A); Group H had 1455 differentially expressed genes compared to Group M, of which 891 were upregulated and 564 were downregulated. Figure 22 B).

[0112] 1.12.2 GO enrichment analysis of differentially expressed genes

[0113] To further explore the impact of these differentially expressed genes, this study performed GO functional enrichment analysis on these genes. GO comprises three ontology categories: biological processes, cellular components, and molecular functions.

[0114] Differentially expressed genes in groups M and N are mainly involved in biological processes such as cellular processes, single-organism processes, and biological regulation; the cellular components mainly regulated by differentially expressed genes are cells, cell parts, and organelles; binding, catalytic activity, and molecular transducer activity are the molecular functions with the most enriched genes. Figure 23 A).

[0115] Differentially expressed genes in group H, relative to group M, are mainly involved in biological processes such as cellular processes, single-organism processes, and biological regulation; the cellular components mainly regulated by differentially expressed genes are cells, cellular parts, and membranes; binding, catalytic activity, and molecular transduction activity are the molecular functions with the most enriched genes. Figure 23 B).

[0116] 1.12.3 KEGG enrichment analysis of differentially expressed genes

[0117] This study used KEGG analysis to investigate the metabolic pathways in the colon tissue of mice in each group. Figure 24 The differentially expressed genes between groups M and N are mainly enriched in metabolic pathways such as cytokine-cytokine receptor interaction. Figure 24 A) Differentially expressed genes in groups H and M are involved in metabolic pathways such as cytokine-cytokine receptor interactions, chemokine signaling pathways, and NF-κB. Figure 24 B).

[0118] Example 2

[0119] The only difference between this embodiment and Embodiment 1 is that:

[0120] A lactococcal composition for alleviating intestinal damage in mice caused by Salmonella includes the following components: a viable count of 1.0 × 10⁻⁶ bacteria. 8 CFU / g of Lactococcus lactis subsp. lactis KLDS 4.0325 and heme; the mass ratio of heme to KLDS 4.0325 was 1:50.

[0121] A type of lactococcus lactis that can alleviate intestinal damage caused by Salmonella in mice, comprising 1.0 × 10⁻⁶ live bacteria. 8 CFU / g of Lactococcus lactis subsp. lactis KLDS 4.0325.

[0122] Example 3

[0123] The only difference between this embodiment and Embodiment 1 is that:

[0124] A lactococcal composition for alleviating intestinal damage in mice caused by Salmonella includes the following components: a viable count of 1.0 × 10⁻⁶ bacteria. 10 CFU / g of Lactococcus lactis subsp. lactis KLDS 4.0325 and heme; the mass ratio of heme to KLDS 4.0325 was 1:150.

[0125] A type of lactococcus lactis that can alleviate intestinal damage caused by Salmonella in mice, comprising 1.0 × 10⁻⁶ live bacteria. 10CFU / g of Lactococcus lactis subsp. lactis KLDS 4.0325.

[0126] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a lactococcus lactis composition in the preparation of a drug for preventing colitis caused by Salmonella infection, characterized in that, The composition comprises the following components: a viable count of 1.0 × 10⁻⁶ cells / day. 8 -1.0×10 10 cfu / g of Lactococcus lactis subsp. ( Lactococcus lactis subsp. lactis KLDS 4.0325 and heme; the mass ratio of heme to KLDS 4.0325 is 1:(50-150).

2. The use of a lactococcus lactis composition in the preparation of a drug to alleviate colitis caused by Salmonella infection, characterized in that, The composition comprises the following components: a viable count of 1.0 × 10⁻⁶ cells / day. 8 -1.0×10 10 cfu / g of Lactococcus lactis subsp. ( Lactococcus lactis subsp. lactis KLDS 4.0325 and heme; the mass ratio of heme to KLDS 4.0325 is 1:(50-150).

3. The use of a lactococcal composition in the preparation of a drug to alleviate intestinal damage caused by Salmonella infection, characterized in that, The composition comprises the following components: a viable count of 1.0 × 10⁻⁶ cells / day. 8 -1.0×10 10 cfu / g of Lactococcus lactis subsp. ( Lactococcus lactis subsp. lactis KLDS 4.0325 and heme; the mass ratio of heme to KLDS 4.0325 is 1:(50-150).

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