Method for inhibiting salmonella virulence factors by sodium propionate and application thereof

Abstract

The application discloses a method and application of sodium propionate for inhibiting Salmonella virulence factors, and comprises the following steps: inoculating Salmonella liquid into modified Martin culture medium for culture to obtain first bacterial liquid; inoculating the first bacterial liquid into new modified Martin culture medium for culture to obtain second bacterial liquid; centrifuging the second bacterial liquid, washing, and diluting by using modified Martin solution to obtain bacterial suspension; culturing the bacterial suspension and sodium propionate under certain conditions to obtain third bacterial liquid; and detecting the expression of virulence factors of Salmonella in the third bacterial liquid. It is found through detection that different concentrations of sodium propionate can significantly reduce the expression of Salmonella virulence factors. The method provided by the application can weaken the infection toxicity of Salmonella, so that the infection and incidence probability of poultry Salmonella can be reduced, and the infection symptoms can be alleviated.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method and application of sodium propionate for inhibiting Salmonella virulence factors. Background Technology

[0002] Salmonella is a group of zoonotic facultative intracellular Gram-negative bacteria, and one of the world's most important foodborne pathogens, with poultry being its largest reservoir host. In poultry, Salmonella enteritidis infection primarily causes gastroenteritis, infecting chicks via the fecal-oral route, leading to acute infection and high morbidity and mortality rates, causing significant economic losses to the poultry industry. Furthermore, contaminated poultry and poultry products are major sources of Salmonella enteritidis infection in humans, capable of transmitting the pathogen to humans through the food chain, seriously threatening human food safety. Therefore, controlling Salmonella infection rates is of great significance to both the poultry industry and human health.

[0003] Virulence factors are specific characteristics or mechanisms possessed by bacteria, viruses, or other pathogens that enable them to cause disease within a host. These factors include adhesins, secretion systems, toxins, invasion mechanisms, and biofilms. They can increase a pathogen's ability to attack its host, making it easier for the pathogen to invade, multiply, and damage host tissues. The effects of virulence factors on animal infections are diverse and can influence the host's immune system, cellular structure, and function, leading to severe disease.

[0004] Virulence factors influence Salmonella infection in animals through multiple mechanisms. Furthermore, inhibiting the expression of Salmonella virulence factors can alleviate infection symptoms in animals. Virulence factors may affect the reproduction and spread rates of Salmonella, thereby influencing infection and disease progression. Therefore, research on Salmonella virulence factors can help reduce Salmonella infection and disease incidence in poultry. The invention with application number 2023110909429 provides a method for inhibiting Salmonella virulence factors. This method found that different concentrations of acetic acid can significantly reduce the expression of different virulence factors. For example, acetic acid at concentrations of 0.04, 0.08, 0.16, 0.31, and 0.63 mM reduces the expression of luxS and invA; acetic acid at concentrations of 0.08, 0.16, 0.31, 0.63, and 1.25 mM reduces the expression of sopB and csgD; acetic acid at concentrations of 0.08, 0.16, 0.31, and 0.63 mM reduces the expression of hilA; and acetic acid at concentrations of 0.04, 0.08, 0.16, 0.31, 0.63, and 1.25 mM reduces the expression of sipA. The research direction of this application tends to study methods for inhibiting Salmonella virulence factors in vitro, without conducting research at the cellular level and in animal experiments. Although in vitro experiments can provide some reference value, the in vitro environment is different from the intracellular environment and the influencing factors are more complex. Therefore, research at the cellular level and in animal experiments is more valuable for application and is of great significance for reducing Salmonella infection and disease in birds. Summary of the Invention

[0005] This invention provides a method and application for inhibiting Salmonella virulence factors with sodium propionate, aiming to solve the aforementioned problems existing in the background art.

[0006] The technical solution provided by this invention is as follows:

[0007] The first aspect of this invention provides a method for inhibiting Salmonella virulence factors with sodium propionate, comprising the following steps:

[0008] Salmonella bacterial suspension was inoculated into the first culture medium at a ratio of 1:8 to 12. The culture was incubated in a constant temperature shaker at 37°C and 160 rpm for 16 hours to obtain the first bacterial suspension. The first culture medium was a modified Martin medium.

[0009] The first bacterial culture was inoculated into the second culture medium at a ratio of 1:20 to 30. The culture was incubated in a constant temperature shaker at 37°C and 160 rpm for 3 hours to obtain the second bacterial culture. The second culture medium was a modified Martin medium.

[0010] The second bacterial culture was collected and centrifuged for the first time. The supernatant was removed to obtain the first precipitate. The first precipitate was washed with sterile PBS, followed by a second centrifugation. The second precipitate was collected and diluted with modified Martin solution to obtain a Salmonella count of 1 × 10⁻⁶. 7 CFU / mL bacterial suspension;

[0011] Take 5 mL of the bacterial suspension and add it into 6 test tubes respectively. Add 5-10 mL of sodium propionate of different concentrations to each test tube to make the final concentrations of sodium propionate 0, 25, 50, 100, 200 and 400 mmol / L respectively. Incubate overnight at 37℃ to obtain the third bacterial suspension of different concentrations.

[0012] The expression of virulence factors of Salmonella in the third bacterial culture was detected. The results showed that 100 mM sodium propionate reduced the expression of flgA, flicC, motA, csgB and luxS, and 200 mM sodium propionate reduced the expression of flgA, flicC, motA, motB and csgD.

[0013] When the OD600 of the third bacterial culture was 1.0, the third bacterial culture was diluted 100-200 times and inoculated into 96-well plates. A negative control group, a Salmonella group, and a sodium propionate + Salmonella group were set up. After static incubation at 37°C for 4, 8, and 18 hours, the supernatant was discarded, the culture was washed with PBS and dried, fixed with methanol for 15 minutes, stained with 200 μL of 1% crystal violet solution for 1-5 minutes, and then 180-200 μL of 33% glacial acetic acid was added. The OD570 absorbance was measured using an ELISA reader. The results showed that 50, 100, and 200 mM sodium propionate significantly reduced Salmonella biofilm formation, and 100 and 200 mM sodium propionate significantly reduced AI-2 activity.

[0014] The Salmonella strain in question has the accession number CMCC50041.

[0015] Furthermore, it also includes:

[0016] Prepare a modified Martin medium by dividing the prepared Martin medium into two portions, namely the first medium and the second medium.

[0017] Furthermore, the preparation of the modified Martin medium specifically involves:

[0018] Weigh 28.5g of modified Martin powder, heat and stir to dissolve in 1000mL of pure water, and then autoclave at 121℃ for 15min to obtain modified Martin medium.

[0019] Furthermore, the conditions for performing the first centrifugation and the second centrifugation are both: centrifugation at 8000 rpm for 5 minutes.

[0020] Further, the detection of the expression of Salmonella virulence factors in the third bacterial culture includes:

[0021] Primers were designed for Salmonella CMCC50041 based on the complete genome sequence and virulence factor gene sequences in the VFDB database.

[0022] Different third bacterial cultures were taken and RNA extraction, reverse transcription, and mRNA expression level detection were performed sequentially to detect the expression results of Salmonella virulence factors.

[0023] Further, the RNA extraction specifically involves: collecting the third bacterial culture, centrifuging, discarding the supernatant, resuspending the precipitate in PBS, adding RNA-easy, and repeatedly pipetting until fully lysed; adding RNase-free ddH2O to the lysate, inverting to mix, and incubating at room temperature for 5 min; centrifuging at 12000 rpm at room temperature for 15 min, adding an equal volume of isopropanol to the aqueous phase, inverting to mix, and incubating at room temperature for 10 min; centrifuging at 12000 rpm at room temperature for 10 min until a white precipitate appears at the bottom of the centrifuge tube, and discarding the supernatant; adding 500 μL of 75% ethanol, suspending the precipitate, and inverting several times; centrifuging at 8000 rpm at room temperature for 3 min, and discarding the supernatant; repeating the 75% ethanol washing step; air-drying at room temperature, adding 50 μL of RNase-free ddH2O to dissolve the precipitate, and repeatedly pipetting to fully dissolve the RNA.

[0024] A second aspect of the present invention provides the application of sodium propionate in reducing the expression of virulence factors after cell infection with Salmonella.

[0025] Furthermore, the following steps are adopted:

[0026] Add 10-20% fetal bovine serum and 1% penicillin-streptomycin to MEM medium;

[0027] Resuscitate Caco-2 cells in liquid nitrogen, thaw them, transfer them to 10 mL centrifuge tubes, centrifuge at 1000 rpm for 3–5 min, discard the supernatant and resuspend them in 1–3 mL of complete culture medium, then transfer them to 100 mm Petri dishes, add complete culture medium to 10 mL, and incubate at 37 °C in a 5% CO2 incubator. Passage the Caco-2 cells when they have proliferated to 80%–90%.

[0028] Caco-2 cells were seeded in 24-well plates at a density of 1–5 × 10⁶ cells / well. 5Each well was treated with 20 mM sodium propionate for 4 hours, then revived Salmonella was added and cultured for 4 hours.

[0029] After culture, Caco-2 cells were treated with gentamicin for 30 min, washed three times with PBS, and then treated with 300-500 μL of 0.5% Triton X-100 for 5-10 min. Caco-2 cells were then collected for Salmonella plate counting.

[0030] The third aspect of this invention provides the application of sodium propionate in reducing the expression of Salmonella virulence factors in chicks after Salmonella enteritidis challenge.

[0031] Furthermore, the following steps are adopted:

[0032] One-day-old SPF chicks were randomly divided into a Salmonella group and a 50mM sodium propionate + Salmonella group. All chicks were fed a basal diet, with sodium propionate added via drinking water. At 8 days of age, all SPF chicks were administered 10 mM sodium propionate orally. 10 CFU / mL Salmonella suspension was sampled and tested at 9 and 11 days of age.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] This invention provides a method for inhibiting Salmonella virulence factors using sodium propionate. Salmonella is inoculated twice into modified Martin medium at different inoculation amounts and cultured. The culture is then centrifuged to obtain bacterial pellets, which are prepared into bacterial suspensions. Finally, the bacterial suspensions are cultured in vitro with different concentrations of sodium propionate. Detection showed that different concentrations of sodium propionate significantly reduced the expression of Salmonella virulence factors. This method can weaken the virulence of Salmonella infection, thereby reducing the probability of Salmonella infection and disease in poultry and alleviating infection symptoms. This invention also provides the application of sodium propionate in reducing the expression of virulence factors after cellular infection with Salmonella and in reducing the expression of Salmonella virulence factors in chicks after challenge with Salmonella enteritidis. The results verified that sodium propionate significantly reduced the expression of virulence factors after cellular infection with Salmonella. 50 mM sodium propionate significantly reduced the relative expression levels of motA in cecal tissue, LuxS in cecal digesta, and csgD. This invention, through research on Salmonella virulence factors, helps reduce Salmonella infection and disease in poultry. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the method for inhibiting Salmonella virulence factors with sodium propionate in an embodiment of the present invention;

[0036] Figure 2 The effect of sodium propionate on the in vitro virulence factors of Salmonella enteritidis in this embodiment of the invention. Figure 1 ;

[0037] Figure 3 The effect of sodium propionate on the in vitro virulence factors of Salmonella enteritidis in this embodiment of the invention. Figure 2 ;

[0038] Figure 4 The effect of sodium propionate on the in vitro virulence factors of Salmonella enteritidis in this embodiment of the invention. Figure 3 ;

[0039] Figure 5 The effect of sodium propionate on the in vitro virulence factors of Salmonella enteritidis in this embodiment of the invention. Figure 4 ;

[0040] Figure 6 The effect of sodium propionate on the in vitro virulence factors of Salmonella enteritidis in this embodiment of the invention. Figure 5 ;

[0041] Figure 7 The effect of sodium propionate on the in vitro virulence factors of Salmonella enteritidis in this embodiment of the invention. Figure 6 ;

[0042] Figure 8 The effect of sodium propionate on the in vitro virulence factors of Salmonella enteritidis in this embodiment of the invention. Figure 7 ;

[0043] Figure 9 This is a diagram illustrating the effect of sodium propionate on the formation of Salmonella enteritidis biofilm in vitro, as described in this invention.

[0044] Figure 10 This is a diagram showing the effect of sodium propionate on AI-2 activity in an embodiment of the present invention;

[0045] Figure 11 The effect of sodium propionate on cytotoxic factors of Salmonella infection in this embodiment of the invention. Figure 1 ;

[0046] Figure 12 The effect of sodium propionate on cytotoxic factors of Salmonella infection in this embodiment of the invention. Figure 2 ;

[0047] Figure 13 The effect of sodium propionate on cytotoxic factors of Salmonella infection in this embodiment of the invention. Figure 3 ;

[0048] Figure 14 The effect of sodium propionate on cytotoxic factors of Salmonella infection in this embodiment of the invention. Figure 4 ;

[0049] Figure 15This invention illustrates the effect of sodium propionate on the expression of Salmonella virulence factors in chicks after challenge with Salmonella enteritidis. Figure 1 ;

[0050] Figure 16 This invention illustrates the effect of sodium propionate on the expression of Salmonella virulence factors in chicks after challenge with Salmonella enteritidis. Figure 2 ;

[0051] Figure 17 This invention illustrates the effect of sodium propionate on the expression of Salmonella virulence factors in chicks after challenge with Salmonella enteritidis. Figure 3 . Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of this application, not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0053] Therefore, the detailed description of the embodiments of this application provided below with reference to the accompanying drawings is intended merely to illustrate selected embodiments of this application and is not intended to limit the scope of protection claimed by this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0054] See Figure 1 This invention provides a method for inhibiting Salmonella virulence factors with sodium propionate, comprising the following steps:

[0055] Step 1: Inoculate the Salmonella bacterial suspension into the first culture medium at a ratio of 1:8 to 12. Incubate the culture medium at 160 rpm for 16 hours in a constant temperature shaker at 37°C to obtain the first bacterial suspension. The first culture medium is a modified Martin medium.

[0056] Step 2: Inoculate the first bacterial culture into the second culture medium at a ratio of 1:20 to 30. Incubate the culture in a constant temperature shaker at 37°C and 160 rpm for 3 hours to obtain the second bacterial culture. The second culture medium is a modified Martin medium.

[0057] Step 3: Collect the second bacterial culture, perform a first centrifugation, remove the supernatant to obtain the first precipitate, wash the first precipitate with sterile PBS, then perform a second centrifugation, collect the second precipitate, and dilute the second precipitate with modified Martin solution to obtain a Salmonella count of 1×10⁻⁶. 7CFU / mL bacterial suspension.

[0058] Step 4: Take 5 mL of bacterial suspension and add it to 6 test tubes. Add 5-10 mL of sodium propionate of different concentrations to each test tube to make the final concentrations of sodium propionate 0, 25, 50, 100, 200 and 400 mmol / L respectively. Incubate overnight at 37℃ to obtain the third bacterial suspension of different concentrations.

[0059] Step 5: The expression of Salmonella virulence factors in the third bacterial culture was detected. The results showed that 100 mM sodium propionate reduced the expression of flgA, fliC, motA, csgB and luxS, and 200 mM sodium propionate reduced the expression of flgA, fliC, motA, motB and csgD.

[0060] Step 6: When the OD600 of the third bacterial culture is 1.0, dilute the third bacterial culture 100-200 times and inoculate it into a 96-well plate. Set up a negative control group, a Salmonella group, and a sodium propionate + Salmonella group. After incubating at 37℃ in a biochemical incubator for 4, 8, and 18 hours, the supernatant was discarded, washed with PBS and dried, fixed with methanol for 15 minutes, stained with 200 μL of 1% crystal violet solution for 1-5 minutes, and then added 180-200 μL of 33% glacial acetic acid. The OD570 absorbance was measured using an ELISA reader. The results showed that 50, 100, and 200 mM sodium propionate significantly reduced Salmonella biofilm formation, and 100 and 200 mM sodium propionate significantly reduced AI-2 activity.

[0061] The preservation number for Salmonella is CMCC50041.

[0062] Optionally, the method further includes:

[0063] Prepare a modified Martin medium by dividing the prepared Martin medium into two portions, namely the first medium and the second medium.

[0064] Optionally, a modified Martin medium is prepared, specifically as follows:

[0065] Weigh 28.5g of modified Martin powder, heat and stir to dissolve in 1000mL of pure water, and then autoclave at 121℃ for 15min to obtain modified Martin medium.

[0066] Optionally, the conditions for the first and second centrifugation processes are both: centrifugation at 8000 rpm for 5 minutes.

[0067] Optionally, the expression of Salmonella virulence factors in the third bacterial culture can be detected, including:

[0068] Primers were designed for Salmonella based on the whole genome sequence of Salmonella CMCC50041 and the virulence factor gene sequences in the VFDB database.

[0069] Different third bacterial cultures were taken, and RNA extraction, reverse transcription, and mRNA expression level detection were performed sequentially to detect the expression results of Salmonella virulence factors.

[0070] Optionally, RNA extraction is performed as follows: Collect the third bacterial culture, centrifuge, discard the supernatant, resuspend the precipitate in PBS, add RNA-easy, and repeatedly pipette until fully lysed; add RNase-free ddH2O to the lysate, invert and mix well, and let stand at room temperature for 5 min; centrifuge at 12000 rpm at room temperature for 15 min, add an equal volume of isopropanol to the aqueous phase, invert and mix well, and let stand at room temperature for 10 min; centrifuge at 12000 rpm at room temperature for 10 min until a white precipitate appears at the bottom of the centrifuge tube, and discard the supernatant; add 500 μL of 75% ethanol, suspend the precipitate, and invert several times; centrifuge at 8000 rpm at room temperature for 3 min, and discard the supernatant; repeat the 75% ethanol washing step for the precipitate; let it air dry at room temperature, add 50 μL of RNase-free ddH2O to dissolve the precipitate, and repeatedly pipette to fully dissolve the RNA.

[0071] Example 1

[0072] This embodiment provides a method for inhibiting Salmonella virulence factors using sodium propionate. The Salmonella strain used is a standard strain (CMCC50041), and the method includes the following steps:

[0073] 1. Co-culturing sodium propionate with Salmonella

[0074] (1) Preparation of modified Martin medium

[0075] Weigh 42.5g of modified Martin powder, heat and stir to dissolve in 1000mL of pure water, autoclave at 121℃ for 15min, and set aside.

[0076] (2) Co-culture of sodium propionate and Salmonella

[0077] 1) Take Salmonella out of the -80℃ freezer and inoculate it into modified Martin (bacterial solution: modified Martin = 1:10). Incubate it in a constant temperature shaker at 37℃ for 16 hours to obtain the first bacterial solution.

[0078] 2) On the second day, the first bacterial culture was taken out and inoculated into a new modified Martin (first bacterial culture: modified Martin = 1:25). It was incubated in a constant temperature shaker at 37°C and 160 rpm for 3 hours to obtain the second bacterial culture.

[0079] 3) Collect the second bacterial culture, centrifuge at 8000 rpm for 5 min, discard the supernatant, wash once with sterile PBS, centrifuge at 8000 rpm for 5 min, and collect the bacterial pellet. Dilute the bacterial pellet with a modified Martin dilution to a concentration of 1×10⁻⁶. 7 CFU / mL, store at 4℃ for later use.

[0080] 4) Take 12 test tubes and add 5 mL of bacterial suspension to each. Add 5-10 mL of acetic acid of different concentrations to each test tube to make the final concentrations of acetic acid 0, 25, 50, 100, 200 and 400 mmol / L respectively. Incubate at 37℃ overnight to obtain the third bacterial suspension of different concentrations.

[0081] 2. Effects of sodium propionate on Salmonella virulence factors

[0082] (1) Primer design

[0083] The primers used for detecting Salmonella virulence factors were designed and synthesized based on the whole genome sequence of Salmonella CMCC50041 and the virulence factor gene sequences in the VFDB database, as shown in Table 1.

[0084] Table 1 Primers for Salmonella virulence factors

[0085]

[0086]

[0087] (2) RNA extraction

[0088] Collect the third bacterial culture, centrifuge at 12000 rpm for 5 min, discard the supernatant, collect the precipitate, resuspend the bacterial precipitate in 1×PBS, centrifuge again, discard the supernatant; resuspend the precipitate in 2 mL PBS, add 500 μl of RNA-easy, and repeatedly pipette until complete lysis; add 2 / 5 volume of RNase-free dd H2O to the lysis mixture (use 200 μl per 500 μl of RNA-easy), mix by inverting, and incubate at room temperature for 5 min; centrifuge at 12000 rpm for 15 min at room temperature, remove the centrifuge tube, carefully aspirate the upper aqueous phase into a new centrifuge tube; add an equal volume of isopropanol, mix by inverting, and incubate at room temperature for 10 min; centrifuge at 12000 rpm for 10 min at room temperature, a white precipitate appears at the bottom of the centrifuge tube, discard the supernatant; add 500 μl of isopropanol... Resuspend the precipitate in 75% ethanol, inverting it several times; centrifuge at 8000 rpm for 3 min at room temperature, discarding the supernatant; repeat the 75% ethanol washing step; allow to air dry at room temperature, add an appropriate amount of RNase-free ddH2O to dissolve the precipitate, and use a pipette to repeatedly blow and agitate to fully dissolve the RNA.

[0089] (3) RNA reverse transcription

[0090] 1) Genomic DNA removal

[0091] Preparation of mixture

[0092]

[0093] 2) Preparation of reverse transcription reaction system

[0094] Add 5×HiScript III qRT SuperMix directly to reaction tube A.

[0095]

[0096] 3) Reverse transcription reaction

[0097]

[0098] (4) mRNA expression level detection

[0099] The cDNA obtained in (3) was used to detect the mRNA level. The reaction system is shown in the table below:

[0100] 1) Preparation of the mixture

[0101]

[0102] 2) qPCR reaction

[0103]

[0104] 3. Effects of sodium propionate on Salmonella biofilm formation

[0105] Salmonella was cultured in modified Martin medium. When OD600 = 1.0, the medium was diluted 100–200 times, mixed well, and 200 μL was inoculated into 96-well plates. A negative control group, a Salmonella group, and a sodium propionate + Salmonella group were set up and incubated statically at 37°C for 4, 8, and 18 h. After incubation, the supernatant was discarded, the plates were washed three times with PBS and air-dried, fixed with methanol for 15 min, the methanol was discarded and the plates were air-dried, stained with 200 μL of 1% crystal violet solution for 1–5 min, the crystal violet was discarded and the plates were air-dried, and finally 180–200 μL of 33% glacial acetic acid was added, and the absorbance (OD570) was measured using a microplate reader.

[0106] qPCR results showed that... Figures 2-8As shown, 50, 100, and 200 mM sodium propionate all reduced the expression of Salmonella virulence factors (P<0.05). Among them, 100 mM sodium propionate significantly reduced the expression of flgA, fliC, motA, csgB, and luxS, while 200 mM sodium propionate significantly reduced the expression of flgA, fliC, motA, motB, and csgD.

[0107] Depend on Figure 9 , Figure 10 It can be seen that 50, 100 and 200 mM sodium propionate significantly reduced Salmonella biofilm formation (P<0.05) and significantly reduced AI-2 activity (P<0.05).

[0108] Example 2: Effects of sodium propionate on cytotoxic factors in Salmonella infection

[0109] This embodiment provides an application of sodium propionate in reducing the expression of virulence factors after Salmonella infection, specifically including the following steps:

[0110] (1) Preparation of MEM complete culture medium

[0111] Add 10-20% fetal bovine serum and 1% penicillin to MEM medium.

[0112] (2) Caco-2 cell culture

[0113] Resuscitate Caco-2 cells in liquid nitrogen, thaw, and transfer to 10 mL centrifuge tubes. Centrifuge at 1000 rpm for 3–5 min. Discard the supernatant and resuspend the cells in 1–3 mL of complete culture medium. Transfer the resuspended cells to 100 mm Petri dishes and bring the total culture medium volume to 10 mL. Incubate at 37°C in a 5% CO2 incubator. Passage the Caco-2 cells when they reach 80%–90% confluence.

[0114] (3) Treatment of Caco-2 cells with sodium propionate and Salmonella

[0115] Caco-2 cells from step 4(2) were seeded into 24-well plates at a density of 1–5 × 10⁶ cells / well. 5 Cells / well. Treat with sodium propionate (20 mM) for 4 h, then add revived Salmonella (MOI = 10) and incubate for 4 h.

[0116] (4) Salmonella invasiveness test

[0117] After culture, Caco-2 cells were treated with gentamicin for 30 min, then washed three times with PBS, and treated with 300-500 μL of 0.5% Triton X-100 for 5-10 min. Caco-2 cells were then collected for Salmonella plate counting.

[0118] Depend on Figures 11-14 It is evident that sodium propionate significantly reduced the expression of virulence factors in cells infected with Salmonella (P<0.05).

[0119] Example 3

[0120] This embodiment provides an application of sodium propionate in reducing the expression of Salmonella virulence factors in chicks after Salmonella enteritidis challenge, including the following steps:

[0121] Seventy 1-day-old SPF chicks were randomly divided into a Salmonella group and a sodium propionate (50 mM) + Salmonella group, with 35 chicks in each group. All chicks were fed a basal diet. Sodium propionate was added via drinking water. At 8 days of age, all chicks were orally administered a Salmonella suspension (10 mM). 10 (CFU / mL). Samples were collected and tested at 9 and 11 days of age.

[0122] Depend on Figures 15-17 It is evident that 50 mM sodium propionate significantly reduced the relative expression levels of motA, LuxS, and csgD in cecal tissue.

[0123] In summary, this invention provides a method for inhibiting Salmonella virulence factors with sodium propionate. By co-culturing sodium propionate with Salmonella, the expression of Salmonella virulence factors can be effectively inhibited. This method weakens the virulence of Salmonella infection, thereby reducing the probability of Salmonella infection and disease in poultry and alleviating infection symptoms.

[0124] The above description is merely the preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for inhibiting Salmonella virulence factors with sodium propionate, characterized in that, Includes the following steps: Salmonella bacterial suspension was inoculated into a first culture medium at a ratio of 1:8 to 12. The suspension was incubated in a constant temperature shaker at 37°C and 160 rpm for 16 h to obtain the first bacterial suspension. The first culture medium was a modified Martin medium. The first bacterial culture was inoculated into the second culture medium at a ratio of 1:20 to 30. The culture was incubated in a constant temperature shaker at 37°C and 160 rpm for 3 h to obtain the second bacterial culture. The second culture medium was a modified Martin medium. The second bacterial culture was collected and centrifuged for the first time. The supernatant was removed to obtain the first precipitate. The first precipitate was washed with sterile PBS, followed by a second centrifugation. The second precipitate was collected and diluted with modified Martin solution to obtain a Salmonella count of 1 × 10⁻⁶. 7 CFU / mL bacterial suspension; Take 5 mL of the bacterial suspension and add it to 6 test tubes respectively. Add 5-10 mL of sodium propionate of different concentrations to each test tube to make the final concentrations of sodium propionate 0, 25, 50, 100, 200 and 400 mmol / L respectively. Incubate overnight at 37℃ to obtain the third bacterial suspension of different concentrations. Primers were designed for Salmonella CMCC50041 based on its whole genome sequence and virulence factor gene sequences in the VFDB database. The virulence factor genes of Salmonella include flgA, fliC, motA, csgB, luxS, motB, and csgD. Different third bacterial cultures were used for RNA extraction, reverse transcription, and mRNA expression level detection, respectively, to detect the expression of Salmonella virulence factors. The results showed that 100 mM sodium propionate reduced the expression of flgA, flicC, motA, csgB, and luxS, while 200 mM sodium propionate reduced the expression of flgA, flicC, motA, motB, and csgD. When the OD600 of the third bacterial culture was 1.0, the third bacterial culture was diluted 100-200 times and inoculated into 96-well plates. A negative control group, a Salmonella group, and a sodium propionate + Salmonella group were set up. After static incubation at 37°C for 4, 8, and 18 h in a biochemical incubator, the supernatant was discarded, the plates were washed with PBS and air-dried, fixed with methanol for 15 min, stained with 200 μL of 1% crystal violet solution for 1-5 min, and then 180-200 μL of 33% glacial acetic acid was added. The OD570 absorbance was measured using an ELISA reader. The results showed that 50, 100, and 200 mM sodium propionate significantly reduced Salmonella biofilm formation, and 100 and 200 mM sodium propionate significantly reduced AI-2 activity. The Salmonella strain in question has the accession number CMCC50041.

2. The method for inhibiting Salmonella virulence factors with sodium propionate according to claim 1, characterized in that, Also includes: Prepare a modified Martin medium by dividing the prepared Martin medium into two portions, namely the first medium and the second medium.

3. The method for inhibiting Salmonella virulence factors with sodium propionate according to claim 2, characterized in that, The preparation of the modified Martin medium specifically involves: Weigh 28.5 g of modified Martin powder, heat and stir to dissolve in 1000 mL of pure water, and then autoclave at 121℃ for 15 min to obtain modified Martin medium.

4. The method for inhibiting Salmonella virulence factors with sodium propionate according to claim 1, characterized in that: The conditions for both the first and second centrifugation processes were: centrifugation at 8000 rpm for 5 minutes.

5. The method for inhibiting Salmonella virulence factors with sodium propionate according to claim 1, characterized in that: The RNA extraction process is as follows: The third bacterial culture is collected and centrifuged. The supernatant is discarded, and the precipitate is resuspended in PBS. RNA-easy is added, and the mixture is repeatedly pipetted until fully lysed. RNase-free ddH2O is added to the lysate, and the mixture is inverted and allowed to stand at room temperature for 5 min. The mixture is centrifuged at 12000 rpm for 15 min at room temperature. An equal volume of isopropanol is added to the aqueous phase, and the mixture is inverted and allowed to stand at room temperature for 10 min. The mixture is centrifuged at 12000 rpm for 10 min at room temperature until a white precipitate appears at the bottom of the centrifuge tube. The supernatant is discarded. 500 μL of 75% ethanol is added to suspend the precipitate, and the mixture is inverted several times. The mixture is centrifuged at 8000 rpm for 3 min at room temperature, and the supernatant is discarded. The precipitate is washed with 75% ethanol again. The mixture is allowed to air dry at room temperature. 50 μL of RNase-free ddH2O is added to dissolve the precipitate, and the mixture is repeatedly pipetted until the RNA is fully dissolved.

6. An application of sodium propionate in reducing the expression of virulence factors after cellular infection with Salmonella, characterized in that, The sodium propionate as described in any one of claims 1-5 is used, wherein the cells are Caco-2 cells, and the specific steps are as follows: (1) Preparation of MEM complete culture medium Add 10-20% fetal bovine serum and 1% penicillin-streptomycin to MEM medium; (2) Caco-2 cell culture Resuscitate Caco-2 cells in liquid nitrogen, thaw them, transfer them to 10 mL centrifuge tubes, centrifuge at 1000 rpm for 3–5 min, discard the supernatant and resuspend them in 1–3 mL of complete culture medium, then transfer them to 100 mm Petri dishes, add complete culture medium to 10 mL, and incubate at 37°C in a 5% CO2 incubator. Passage the Caco-2 cells when they have proliferated to 80%–90%. (3) Treatment of Caco-2 cells with sodium propionate and Salmonella The Caco-2 cells from (2) were seeded into 24-well plates at a density of 1–5 × 10⁶ cells / well. 5 Each well was treated with 20 mM sodium propionate for 4 h, followed by the addition of revived Salmonella and incubation for 4 h. (4) Salmonella invasiveness test After culture, Caco-2 cells were treated with gentamicin for 30 min, then washed three times with PBS, and treated with 300-500 μL of 0.5% Triton X-100 for 5-10 min. Caco-2 cells were then collected for Salmonella plate counting. The results showed that sodium propionate significantly reduced the expression of virulence factors flgA, luxS, csgB, and csgD after cell infection with Salmonella.

7. An application of sodium propionate in reducing the expression of Salmonella virulence factors in chicks after Salmonella enteritidis challenge, characterized in that, Using sodium propionate as described in any one of claims 1-5, the following steps are performed: Seventy 1-day-old SPF chicks were randomly divided into a Salmonella group and a 50 mM sodium propionate + Salmonella group, with 35 chicks in each group. All chicks were fed a basal diet, and sodium propionate was added via drinking water. At 8 days of age, all chicks were administered 10 mM sodium propionate orally. 10 CFU / mL Salmonella suspension was sampled and tested at 9 and 11 days of age, respectively; The results showed that 50 mM sodium propionate significantly reduced the relative expression levels of motA, LuxS, and csgD in cecal tissue.

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