Streptococcus gallolyticus and application thereof in preparation of anti-salmonella infection preparation

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a streptococcus gallolyticus and application of the streptococcus gallolyticus in preparation of anti-salmonella infection preparation. Streptococcus gallolyticus The streptococcus gallolyticus can improve the resistance of a host to salmonella infection, and can be used for preparing probiotic preparation, functional feed additive or related health care product for preventing or assisting in treating salmonella infection.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Streptococcus gallic acid solubles and its application in the preparation of anti-Salmonella infection agents. Background Technology

[0002] Salmonella is an important foodborne zoonotic pathogen. In animal husbandry, Salmonella infection can lead to diarrhea, stunted growth, and even death, severely impacting production efficiency. Currently, antibiotics remain the primary means of preventing and controlling Salmonella infection; however, the resulting problems of antibiotic residues and bacterial resistance are becoming increasingly serious, urgently requiring the search for safe and effective alternative strategies.

[0003] The gut symbiotic microbiota plays a central role in providing "colonization resistance" against intestinal pathogens. Recent studies have revealed that specific probiotics or symbiotics can protect the host from Salmonella infection through multiple mechanisms, mainly falling into two categories: direct competitive inhibition, such as competing for nutrients, occupying space, and producing antimicrobial substances; and indirect immunomodulation, which reduces infection damage by enhancing the host's epithelial barrier function or regulating mucosal immune responses. *Streptococcus gallicolata*, a symbiotic bacterium capable of metabolizing tannins, has traditionally been studied primarily for its function in degrading dietary tannins and improving nutrient absorption in ruminants. However, whether and through what mechanisms this species can enhance the host's systemic resistance to intestinal pathogens such as Salmonella remains unclear and lacks in-depth research and definitive conclusions. In particular, no publicly reported strains of *Streptococcus gallicolata* capable of providing protective immunity by regulating the host's immune system (such as enhancing the mucosal barrier and balancing inflammatory responses) have been published. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a strain of Streptococcus gallolans and its application in the preparation of anti-Salmonella infection agents.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a strain of *Streptococcus gallolyticus*, wherein *Streptococcus gallolyticus* (… Streptococcus gallolyticus It was deposited on December 12, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252870.

[0007] The second aspect of the present invention provides the use of the above-described Streptococcus gallate or its inactivated strain in the preparation of a medicament or probiotic preparation for the prevention or adjunctive treatment of Salmonella infection.

[0008] The third aspect of the present invention provides the use of the above-described Streptococcus gallate or its inactivated strain in the preparation of feed additives or veterinary preparations for improving the resistance of livestock and poultry to Salmonella infection.

[0009] The fourth aspect of the present invention provides the use of the above-described Streptococcus gallate or its inactivated strain in the preparation of formulations for regulating intestinal flora, enhancing intestinal barrier function, or alleviating intestinal inflammation.

[0010] The fifth aspect of this invention provides the above-described Streptococcus gallate or its inactivated strain in the preparation of cells that increase B cells and CD8 in mesenteric lymph nodes. + Application in formulations that regulate T-cell ratios.

[0011] In the applications described above, the formulation is a lyophilized powder, capsule, tablet, oral liquid, or feed premix.

[0012] In the applications described above, when the formulation contains live *Streptococcus gallate*, the content of live *Streptococcus gallate* in the formulation is 10. 6 ~10 11 CFU / gram or 10 6 ~10 11 CFU / mL. The formulation may be used alone or in combination with pharmaceutically or feed-acceptable carriers, prebiotics (such as oligosaccharides) or other probiotics (such as lactobacillus).

[0013] Beneficial effects: This invention provides a strain of *Streptococcus gallate*, which can regulate the host's local and systemic immune responses (such as increasing B cells and inducing CD8). + This strain works through multiple synergistic mechanisms, including T cells, to significantly enhance the host's resistance to Salmonella infection. Derived from the gut of healthy animals, it is a symbiotic bacterium with high safety. Its application provides a novel microbial resource for the prevention and treatment of Salmonella infection, offering significant development value and broad market prospects in livestock farming, food hygiene, and public health. Attached Figure Description

[0014] Figure 1 The effects of strain Zhao F228 and its inactivated strain on the phenotype of immune cells in mouse mesenteric lymph nodes.

[0015] Figure 2 This is a flowchart of the Salmonella infection mouse experiment in Example 3.

[0016] Figure 3 The effect of strain Zhao F228 and its inactivated bacterial pretreatment on the body weight of mice challenged with Salmonella.

[0017] Figure 4The effects of strain Zhao F228 and its inactivated bacterial pretreatment on organ indices in mice challenged with Salmonella.

[0018] Figure 5 The results show the plate count of Salmonella in the liver and spleen of mice challenged with Salmonella strain Zhao F228 and its inactivated bacterial pretreatment.

[0019] Figure 6 The results show the statistical effects of Salmonella load in the liver and spleen of mice challenged with Salmonella strain Zhao F228 and its inactivated bacterial pretreatment. Detailed Implementation

[0020] This invention provides a strain of *Streptococcus gallolyticus*. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0021] In the following examples, *Streptococcus gallic acid* with accession number CCTCC NO: M 20252870 ( Streptococcus gallolyticus The strain was named Zhao F228. It was obtained from fecal samples of healthy animals (pigs) and isolated and purified using mGAM medium.

[0022] Example 1: Isolation and Identification of Streptococcus gallic acid

[0023] 1.1 Strains Isolation

[0024] Samples were taken from fresh fecal samples of healthy adult pigs. One gram of sample was weighed and added to 9 mL of sterile anaerobic phosphate buffer (0.1 M, pH 7.0, containing 0.05% L-cysteine ​​hydrochloride), and homogenized by vortexing. After a 10-fold serial dilution, 10... -4 10 -5 10 -6 Three dilutions of bacterial culture, 100 μL each, were spread onto mGAM isolation plates. The plates were incubated at 37°C in an anaerobic workstation (atmosphere: 85% N2, 10% CO2, 5% H2) for 72 hours. Single colonies with clearly defined and transparent hydrolysis zones were picked and streaked onto the same plate for three generations of purification to obtain a pure culture, named Zhao F228.

[0025] 1.2 Molecular biological identification

[0026] Genomic DNA was extracted from strain Zhao F228 using a bacterial genomic DNA extraction kit. The full-length 16S rRNA gene sequence was amplified using universal primers 27F and 1492R. The PCR product was purified and sequenced, yielding a 1455 bp sequence (as shown in SEQ ID NO:1). This sequence was submitted to the NCBI database for BLAST homology comparison, and the results showed that it was similar to *Streptococcus gallic acid* (…). Streptococcus gallolyticus The similarity to strain LT2 was as high as 99.93%. The following is the 16S sequence:

[0027]

[0028] After identification, strain Zhao F228 was deposited at the China Center for Type Culture Collection (CCTCC) on December 12, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252870.

[0029] Example 2: Investigation into the effect of strain Zhao F228 on regulating intestinal mucosal immunity

[0030] In this embodiment, the effects of live or inactivated Zhao F228 bacteria on the intestinal mucosal immune system were evaluated by administering live or inactivated Zhao F228 bacteria to mice.

[0031] 2.1 Experimental Materials and Animal Grouping

[0032] Experimental strain: Streptococcus gallate strain Zhao F228;

[0033] Experimental animals: 18 SPF-grade, 6-week-old male C57BL / 6 mice were randomly divided into 3 groups of 6 mice each;

[0034] Animal grouping and treatment methods:

[0035] Group A (Con): PBS administered by gavage, no infection;

[0036] Group B (Zhao F228): 2 x 10 gavages 7 CFU / day;

[0037] Group C (Pasteurized-Zhao F228): 2×10 gavage 7 CFU / day.

[0038] 2.2 Experimental Procedure

[0039] Treatment: For 14 consecutive days, mice in groups B and C were administered the corresponding doses of live and inactivated Zhao F228 bacteria (dissolved in 0.2 mL of PBS before gavage) by gavage, while mice in group A were administered the same volume of PBS by gavage.

[0040] Preparation method of Zhao F228 live bacteria: The preserved strain was inoculated into MRS medium for resuscitation. After 2-3 generations of subculture and verification as pure bacteria, the live bacteria from the previous day's subculture were taken daily, centrifuged, and resuspended in sterile PBS for oral administration to mice.

[0041] Preparation method of Zhao F228 inactivated bacteria: Zhao F228 live bacteria are treated at 65℃ for 30 minutes.

[0042] Observation and sampling: On day 14 after treatment, 6 mice in each group were sacrificed and lymph nodes were collected aseptically.

[0043] 2.3 Experimental Results and Analysis

[0044] Figure 1 For immune cell phenotype analysis: from Figure 1 The results showed that after 14 days of treatment with strain Zhao F228 and its inactivated bacteria, B cells and CD8+ cells in the mesenteric lymph nodes of mice were significantly reduced. + The significantly increased proportion of T cells indicates that strain Zhao F228 and its inactivated strain both activated the intestinal mucosal immunity in mice. Figure 3 In the text, "*" represents P <0.05, "**" represents P <0.01, "***" represents P <0.001, "****" represents P <0.0001.

[0045] Example 3: Functional verification of strain Zhao F228 in enhancing mouse resistance to Salmonella typhimurium infection.

[0046] This embodiment establishes a mouse Salmonella infection model to systematically evaluate the oral prophylactic intervention effect of strain Zhao F228.

[0047] 3.1 Experimental Materials and Animal Grouping

[0048] Experimental strain: Zhao F228, the strain of this invention; Challenge bacteria: Salmonella Typhimurium ( S.Typhimurium ) ATCC 14028 (SL1344, streptomycin-resistant).

[0049] Experimental animals: 32 SPF-grade, 6-week-old male C57BL / 6 mice were randomly divided into 4 groups of 8 mice each.

[0050] Group A (Con): PBS was administered by gavage; no infection was observed.

[0051] Group B ( S.Typhimurium ): PBS was administered by gavage, and infection occurred 14 days later.

[0052] Group C (Zhao F228+) S.Typhimurium ): Gavage 2×10 7 CFU / day, infection occurred 14 days later.

[0053] Group D (Pasteurized-Zhao F228+) S.Typhimurium ): Gavage 2×10 7 CFU / day, infection occurred 14 days later.

[0054] 3.2 Experimental Procedure ( Figure 2 )

[0055] Pretreatment: For 14 consecutive days, mice in groups C and D were administered the corresponding doses of live and inactivated Zhao F228 bacteria (dissolved in 0.2 mL PBS during gavage) by gavage, while mice in groups A and B were administered the same volume of PBS by gavage.

[0056] Infection: The day after pretreatment, mice in groups B, C, and D were administered 5 × 10⁵ g of the drug orally by gavage. 7 CFU of Salmonella Typhimurium SL1344 (dissolved in 0.2 mL PBS) was administered to Group A via gavage with an equal volume of PBS.

[0057] Observation and sampling: Mice were monitored continuously for 14 days post-infection, and their daily weight and survival status were recorded. On day 4 post-infection, 8 mice in each group were sacrificed, and their livers and spleens were aseptically collected.

[0058] 3.3 Experimental Results and Analysis

[0059] (1) Changes in body weight, such as Figure 3 As shown, after infection with Salmonella, the body weight of mice gradually decreased, significantly lower than that of the control group. However, the body weight of mice in group C (intervention with live Zhao F228 bacteria) and group D (intervention with inactivated bacteria) decreased slowly, and their body weight percentage was significantly higher than that of the Salmonella challenge group (group B). Figure 3 In the text, "**" represents the ratio of Group B to Group A. P <0.01, "****" represents Group B and Group A P <0.0001, "#" represents the ratio of Group C to Group A P <0.05, "##" represents the ratio of Group C to Group A P <0.01, “$$” represents the ratio of group D to group A P <0.01.

[0060] (2) Organ index such as Figure 4 As shown: On day 4 post-infection, intact mouse livers and spleens were harvested, weighed, and their organ indices were calculated as a percentage of body weight. Results showed that both live and inactivated Zhao F228 bacteria could alleviate the increase in liver and spleen organ indices caused by Salmonella infection, with the inactivated bacteria group showing better relief. Figure 4 In the text, "*" represents P <0.05, "****" represents P <0.0001.

[0061] (3) Salmonella load such as Figure 5 and Figure 6As shown: On day 4 post-infection, mouse livers and spleens were homogenized and spread on MacConkey agar plates containing streptomycin for counting. Results showed that pretreatment with live Zhao F228 bacteria tended to reduce Salmonella load in the liver, while pretreatment with inactivated bacteria significantly reduced Salmonella load in the liver. Both live Zhao F228 and inactivated bacteria pretreatment significantly reduced Salmonella load in the spleen, with the inactivated bacteria group showing better clearance. Figure 6 In the text, "*" represents P <0.05, "****" represents P <0.0001.

[0062] The above embodiments illustrate in detail the present invention *Streptococcus gallolyticus* (Gallic acid streptococcus). Streptococcus gallolyticus The isolation process of the strain, its proven efficacy against Salmonella infection, and its specific applications were investigated. Experimental data fully confirmed that administration of live or inactivated strains via gavage can effectively alleviate weight loss, reduce tissue damage, and decrease pathogen load. Its mechanism of action is related to the regulation of intestinal mucosal immunity.

[0063] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solution and inventive concept of the present invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A strain of *Streptococcus gallolyticus*, characterized in that, The Streptococcus gallus ( Streptococcus gallolyticus It was deposited on December 12, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252870.

2. The use of Streptococcus gallate as described in claim 1 in the preparation of a probiotic formulation for the prevention or adjunctive treatment of Salmonella typhimurium infection.

3. The use of Streptococcus gallate as described in claim 1 in the preparation of feed additives or veterinary preparations for improving the resistance of livestock and poultry to Salmonella typhimurium infection.

4. The use of the inactivated Streptococcus gallate as described in claim 1 in the preparation of a probiotic formulation for the prevention or adjunctive treatment of Salmonella typhimurium infection.

5. The use of the inactivated Streptococcus gallate as described in claim 1 in the preparation of feed additives or veterinary preparations for improving the resistance of livestock and poultry to Salmonella typhimurium infection.

6. The application as described in any one of claims 2-5, wherein the formulation is a lyophilized powder, capsule, tablet, oral liquid, or feed premix.

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