Non-lactose fermenting streptococcus and application thereof in preparation of anti-salmonella infection preparation
By providing non-lactolytic streptococcus alactolyticus for preparation of formulations, the lack of research on the anti-Salmonella infection effect of non-lactolytic streptococcus in the prior art has been addressed, and the effects of improving host resistance and alleviating infection symptoms have been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-05-07
- Publication Date
- 2026-06-02
AI Technical Summary
There is a lack of research on the effects of non-lactose-degrading streptococci on Salmonella infection, especially their effects on reducing the systemic infection burden of the host and alleviating weight loss, which have not been clearly reported.
A strain of non-lactolytic streptococcus (CCTCC NO: M 2026565) is provided for use in the preparation of probiotic preparations, feed additives and veterinary preparations, which enhance the host's resistance to Salmonella through indirect immunomodulation.
It effectively alleviates weight loss caused by Salmonella infection, reduces tissue damage, lowers the pathogen load in the liver and spleen, and enhances the host's resistance to Salmonella.
Smart Images
Figure CN122128190A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a non-lactolytic streptococcus and its application in the preparation of anti-Salmonella infection agents. Background Technology
[0002] Salmonella is a foodborne zoonotic pathogen. In livestock and poultry farming, infection with this bacterium can cause symptoms such as diarrhea, stunted growth, and even death in animals.
[0003] In recent years, relevant studies have confirmed that specific probiotics or symbiotic microorganisms can protect the host from Salmonella invasion through multiple mechanisms. These mechanisms can be mainly divided into two categories: the first is direct competitive inhibition, specifically manifested in competing for nutrients, occupying host intestinal colonization sites, and secreting antibacterial active substances; the second is indirect immunomodulatory effects, namely, reducing tissue damage caused by Salmonella infection by strengthening the host's intestinal epithelial barrier function and regulating mucosal immune response levels. For example, the symbiotic bacterium Clostridium perfringens (Clostridium perfringens) Enterocloster clostridioformis This study found that Salmonella infection can effectively alleviate histopathological damage caused by cecal epithelial cells and improve the survival rate of infected mice by upregulating the expression levels of tolerance-related genes and increasing the number of local regulatory T cells. Furthermore, this protective effect is not solely achieved by reducing the viral load. Other studies have shown that the core gut microbiota of mice, *Schaedler* (a type of spirochete),... Mucispirillum schaedleri By competing for anaerobic electron acceptors and other pathways, Salmonella virulence genes can be interfered with, thereby exerting a protective effect on the host. These research findings fully demonstrate that regulating the gut microbiota structure, especially by utilizing strains with specific immunomodulatory functions to enhance the host's own defense capabilities, has broad application prospects.
[0004] To date, information regarding non-lactolytic streptococci (Non-lactolytic streptococci) has been published Streptococcus alactolyticus Research on its effectiveness against Salmonella infection is still relatively scarce, especially regarding whether this strain can reduce the systemic infection load of the host (such as bacterial load in organs like the liver and spleen) and whether it can alleviate weight loss caused by infection. There are currently no clear research reports on these aspects. Summary of the Invention
[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a non-lactolytic streptococcus strain, which aims to reduce the use of antibiotics in the prevention and treatment of Salmonella infection in the prior art.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a strain of non-lactolytic streptococcus, wherein the non-lactolytic streptococcus ( Streptococcus alactolyticus It was deposited on March 30, 2026 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026565.
[0007] A second aspect of the present invention provides the use of the aforementioned non-lactose-degrading Streptococcus in the preparation of probiotic formulations for the prevention or adjunctive treatment of Salmonella typhimurium infection.
[0008] The third aspect of the present invention provides the use of the aforementioned non-lactose-degrading Streptococcus in the preparation of feed additives or veterinary preparations for improving the resistance of livestock and poultry to Salmonella typhimurium infection.
[0009] The fourth aspect of the present invention provides the use of the aforementioned non-lactose-degrading Streptococcus in the preparation of feed additives or veterinary preparations for reducing the Salmonella typhimurium load in the liver and spleen of livestock and poultry after infection with Salmonella typhimurium.
[0010] The fifth aspect of the present invention provides the use of the aforementioned non-lactose-degrading Streptococcus in the preparation of feed additives or veterinary preparations for alleviating weight loss in poultry and livestock caused by Salmonella typhimurium infection.
[0011] In the applications described above, the formulation is a lyophilized powder, capsule, tablet, oral liquid, or feed premix.
[0012] Beneficial effects: This invention provides a non-lactose-degrading Streptococcus ( Streptococcus alactolyticus The preservation number of the non-lactolytic streptococcus is CCTCC NO: M 2026565. Pretreatment of mice with the non-lactolytic streptococcus can effectively enhance their resistance to Salmonella typhimurium. Specifically, it can effectively alleviate the weight loss in mice infected with Salmonella typhimurium, reduce tissue damage, and decrease the pathogen load. Attached Figure Description
[0013] Figure 1 This shows the sequence homology comparison results of the strains.
[0014] Figure 2 A schematic diagram of the design for Salmonella infection experiments in mice.
[0015] Figure 3 The effect of Zhao F227 pretreatment on body weight in mice challenged with Salmonella.
[0016] Figure 4 The effect of Zhao F227 pretreatment on organ indices in mice challenged with Salmonella.
[0017] Figure 5The effect of Zhao F227 pretreatment on Salmonella load in organs of mice challenged with Salmonella.
[0018] Figure 6 The in vitro antibacterial effect of Zhao F227 against Salmonella choleraesuis was measured.
[0019] Figure 7 The in vitro antibacterial effect of Zhao F227 on Salmonella Typhimurium was measured. Detailed Implementation
[0020] This invention provides a non-lactolytic streptococcus and its application in the preparation of anti-Salmonella infection agents. 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 examples. 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] Example 1 1.1 Strains Isolation 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 environment (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, designated Zhao F227.
[0022] 1.2 Molecular biological identification Genomic DNA was extracted from strain Zhao F227 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 1462 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 a 99.93% similarity to *Streptococcus alactolyticus* strain ATCC 43077. Figure 1 ).
[0023] The following is the 16S sequence, SEQ ID NO:1: This strain was deposited on March 30, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026565.
[0024] Example 2 Investigation on the function of strain Zhao F228 in enhancing mouse resistance to Salmonella typhimurium infection By establishing a mouse model of Salmonella infection, the oral prophylactic intervention effect of strain Zhao F227 was systematically evaluated.
[0025] 2.1 Experimental Materials and Animal Grouping Experimental strain: strain Zhao F227; challenge strain: Salmonella Typhimurium ATCC 14028 (SL1344, streptomycin-resistant).
[0026] Experimental animals: 24 SPF-grade, 6-week-old female C57BL / 6 mice were randomly divided into 3 groups of 8 mice each. Blank control group (Group A): PBS was administered by gavage; no infection was observed.
[0027] Infection model group (Group B): PBS was administered by gavage, and infection occurred 14 days later.
[0028] Non-lactolytic streptococcal strain Zhao F227 pretreatment group (Group C): 2×10 gavage 7 CFU / day, infection occurred 14 days later.
[0029] 2.2 Experimental Procedure ( Figure 2 ) Pretreatment: For 14 consecutive days, mice in group C were administered the corresponding dose of Zhao F227 live bacteria (suspended in 0.2 mL PBS) by gavage, while mice in groups A and B were administered the same volume of PBS by gavage.
[0030] Infection: The day after the pretreatment, mice in groups B and C were administered 5×10⁻⁵ gavage orally. 7 CFU of Salmonella Typhimurium SL1344 (dissolved in 0.2 mL PBS) was administered to Group A, who received an equal volume of PBS.
[0031] 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.
[0032] 2.3 Experimental Results and Analysis (1) Weight changes: After infection with Salmonella, the weight of mice gradually decreased, significantly lower than that of the control group, while the weight of mice in the Zhao F227 intervention group decreased slowly, and the percentage of body weight was significantly higher than that in the Salmonella challenge group. Figure 3 ).
[0033] (2) Organ index: On day 5 after infection, intact mouse livers and spleens were weighed, and the organ index was calculated as a percentage of body weight. The results showed that Zhao F227 could alleviate the increase in liver and spleen organ index caused by Salmonella infection to some extent. Figure 4 ).
[0034] (3) Salmonella load: On day 5 of infection, liver, spleen, and mesenteric lymph nodes of mice were homogenized and spread on SS agar plates for counting. The results showed that Zhao F227 could significantly reduce the Salmonella load in the liver and spleen ( Figure 5 ).
[0035] Example 3 In vitro antibacterial effect of strain Zhao F227 against Salmonella choleraesuis and Salmonella typhimurium 3.1 Experimental Procedure Take Salmonella choleraesuis colonies from the agar plate and adjust the bacterial count to 1×10⁻⁶. 8 Using a sterile cotton swab, collect bacterial suspension at a density of 1 / mL and spread it evenly onto NB medium. Punch up wells in the medium and take 50 μL of Zhao F227 bacterial suspension (2.5 × 10⁻⁶ cells / mL). 8 Add the cells / mL to the wells of the prepared culture medium (for a negative control, add 50 μL of sterile liquid culture medium; for a positive control, add 50 μL of 10 μg / mL gentamicin solution).
[0036] Take Salmonella typhimurium colonies from the agar plate and adjust the bacterial count to 1×10⁻⁶. 8 Using a sterile cotton swab, collect bacterial suspension at a density of 10 cells / mL and spread it evenly onto LB medium. Punch a well in the medium and take 50 μL of Zhao F227 bacterial suspension (2.5 × 10⁻⁶ cells / mL). 8 Add the cells / mL to the wells of the prepared culture medium (for a negative control, add 50 μL of sterile liquid culture medium; for a positive control, add 50 μL of 10 μg / mL gentamicin solution).
[0037] Incubate upright at 37°C for 24 h, then observe and measure the size of the transparent inhibition zone.
[0038] 3.2 Experimental Results and Analysis In vitro inhibition experiment of Salmonella choleraesuis: A clear inhibition zone with a diameter of 32 mm was observed in the positive control group, while no inhibition zone was observed in the sterile liquid culture group and the Zhao F227 control group. Figure 6 ).
[0039] In vitro inhibition experiment of Salmonella typhimurium: A clear inhibition zone with a diameter of 30 mm was observed in the positive control group, while no inhibition zone was observed in the sterile liquid culture group and the Zhao F227 control group. Figure 7 ).
[0040] The above results indicate that the non-lactolytic streptococcus Zhao F227 provided by this invention does not enhance the host's resistance to Salmonella through direct competitive inhibition, but rather reduces tissue damage caused by Salmonella infection through indirect immunomodulation.
[0041] 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 non-lactolytic streptococcus, characterized in that, The non-lactolytic streptococci ( Streptococcus alactolyticus It was deposited on March 30, 2026 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026565.
2. The use of the non-lactolytic streptococcus 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 the non-lactose-degrading Streptococcus 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 non-lactose-degrading Streptococcus as described in claim 1 in the preparation of feed additives or veterinary preparations for reducing the Salmonella typhimurium load in the liver and spleen of livestock and poultry after infection with Salmonella typhimurium.
5. The use of the non-lactose-degrading Streptococcus as described in claim 1 in the preparation of feed additives or veterinary preparations for alleviating weight loss in poultry and livestock caused by 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.