Application of Lactobacillus johnsonii cell-free supernatant in inhibiting Salmonella biofilm
By using cell-free supernatant of Lactobacillus johnsonii BNCC186110 as an antibacterial agent, the stability and contamination problems of live microorganism technology on food contact surfaces were solved, and the effective inhibition and reduction of Salmonella typhimurium biofilm were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DALIAN POLYTECHNIC UNIVERSITY
- Filing Date
- 2026-04-17
- Publication Date
- 2026-06-02
AI Technical Summary
Existing live microbial technology cannot function stably on food contact surfaces and is prone to causing secondary contamination.
Cell-free supernatant of Lactobacillus johnsonii BNCC186110 was used as an antibacterial agent and sprayed on the surface of food to inhibit the formation of biofilm by Salmonella typhimurium.
This provides a safe and stable method that can effectively inhibit the formation of Salmonella typhimurium biofilm, reduce its colonization, lower the risk of Salmonella in food, and avoid live bacteria contamination.
Smart Images
Figure CN122123386A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the application of cell-free supernatant of Lactobacillus johnsonii in inhibiting Salmonella biofilms, and belongs to the field of microbial technology. Background Technology
[0002] Salmonella typhimurium ( Salmonella Typhimurium Salmonella (Salmonella) is a Gram-negative bacterium and one of the major foodborne pathogens. Salmonella-related food poisoning incidents constitute a significant portion of bacterial foodborne illnesses. Salmonella is widely found in beef, pork, fruits, vegetables, and seafood. Salmonella infection occurs through the ingestion of contaminated food. Salmonella infection can cause a variety of illnesses, including gastroenteritis, sepsis, and localized infections, and in severe cases, death, posing a serious threat to public food safety and health.
[0003] Salmonella typhimurium can form biofilms that are resistant to adverse environmental conditions. Isolation of the bacteria from these biofilms can lead to persistent food contamination, increasing the risk of foodborne illnesses. When attached to food surfaces, it can also act as a source of cross-contamination, spreading pathogens between different food items and potentially triggering more serious outbreaks. It is estimated that over one million people in the United States alone seek medical attention annually for Salmonella infections. Therefore, with increasing public concern about food health, the development of novel antimicrobial products has become an urgent need.
[0004] Lactobacillus johnsonii ( Lactobacillus johnsonii Lactobacillus johnsonii is one of the most important bacteria in the human gut microbiota. As a beneficial gut microbiome, it is often used to regulate the gut microbiota, improve intestinal barrier function, prevent diseases such as high uric acid and kidney damage, and reduce the risk of acute myocardial infarction. Changes in the number of Lactobacillus johnsonii may be an indication of gut microbiota dysbiosis, therefore scientists consider it a candidate for the next generation of probiotics.
[0005] Lactobacillus johnsonii cell-free supernatant ( Lactobacillus johnsonii Cell-free solutions (CFS) may play roles in the gut, including regulating immune responses, influencing gut microbiota balance, and participating in nutrient metabolism. These roles collectively affect the overall health of the host.
[0006] In conclusion, the formation of Salmonella typhimurium biofilms increases the risk of foodborne illnesses and poses a significant threat to food safety. While there are reports of using whole-cell cultures of Lactobacillus johnsonii to inhibit Salmonella on metal or other material surfaces, whole-cell cultures cannot be used in food, as this would introduce new microbial contamination. Summary of the Invention
[0007] Technical issues The technical problem that this invention aims to solve is that existing live microbial technology cannot function stably on food contact surfaces and is prone to causing secondary contamination.
[0008] Technical solution This invention provides Lactobacillus johnsonii ( Lactobacillus johnsonii Cell-free supernatant from BNCC186110 inhibits Salmonella typhimurium. SL1344 and Salmonella 14028 Applications related to biofilm formation. These applications include the use of *Lactobacillus johnsonii* (…). Lactobacillus johnsonii The cell-free supernatant of BNCC186110 is used to make an antibacterial agent, which can be sprayed on the surface of food to prevent or delay the colonization of Salmonella typhimurium on the food surface.
[0009] The cell-free supernatant is the supernatant obtained after liquid culture of Lactobacillus johnsonii BNCC186110 to remove cells and solids.
[0010] The cell-free supernatant is prepared by culturing *Lactobacillus johnsonii* BNCC186110 in MRS liquid medium for a period of time, centrifuging, and discarding the precipitate to obtain the supernatant. In one embodiment, the supernatant obtained after centrifugation and precipitate discarding is further filtered through a 0.22 μm membrane. In one embodiment, the culture conditions are 35–37°C, anaerobic, for at least 24 h.
[0011] In one embodiment, the inhibition includes, but is not limited to, inhibiting Salmonella typhimurium biofilm and inhibiting the metabolic activity of Salmonella typhimurium biofilm.
[0012] This invention also provides Lactobacillus johnsonii ( Lactobacillus johnsonii The application of cell-free supernatant of BNCC186110 in the preparation of drugs for the prevention or adjunctive treatment of Salmonella typhimurium biofilm infection.
[0013] In one embodiment, the drug includes, but is not limited to, a liquid formulation.
[0014] Beneficial effects: This invention provides a safe, stable, and cell-free supernatant of Lactobacillus johnsonii BNCC186110, which can inhibit the formation of Salmonella typhimurium biofilm and destroy Salmonella typhimurium biofilm, thereby reducing the colonization of Salmonella typhimurium in food and providing a new guarantee for human health. Attached Figure Description
[0015] Figure 1The biofilm formation capabilities of Salmonella Typhimurium SL1344 and 14028 are shown. A: Biofilm staining and biomass formation of Salmonella Typhimurium SL1344; B: Biofilm staining and biomass formation of Salmonella Typhimurium 14028.
[0016] Figure 2 The image shows the effect of CFS on the swimming motility of Salmonella Typhimurium SL1344 and 14028. A: Swimming image of Salmonella Typhimurium SL1344; B: Swimming image of Salmonella Typhimurium 14028; C: Swimming diameter of Salmonella Typhimurium SL1344; D: Swimming diameter of Salmonella Typhimurium 14028.
[0017] Figure 3 The image shows the effect of CFS on the swarming motility of Salmonella Typhimurium SL1344 and 14028. A: Image of swarming motility of Salmonella Typhimurium SL1344; B: Image of swarming motility of Salmonella Typhimurium 14028; C: Diameter of swarming motility of Salmonella Typhimurium SL1344; D: Diameter of swarming motility of Salmonella Typhimurium 14028.
[0018] Figure 4 The effect of different concentrations of CFS on the biofilm structure of Salmonella Typhimurium SL1344 and 14028. Wherein, A: Salmonella Typhimurium SL1344; B: Salmonella Typhimurium 14028.
[0019] Figure 5 The effects of different CFS concentrations on the metabolic activities of Salmonella Typhimurium SL1344 and 14028 are shown. A: Salmonella Typhimurium SL1344; B: Salmonella Typhimurium 14028.
[0020] Figure 6 Biofilm formation of Salmonella Typhimurium SL1344 and 14028 on various food and food contact surfaces. A: Glass; B: Silica gel; C: Stainless steel; D: Beef; E: Blueberry. Detailed Implementation
[0021] How Lactobacillus johnsonii BNCC186110 was obtained / sourced: Lactobacillus johnsonii BNCC186110 was purchased from Beina Biotechnology Co., Ltd. (Langfang, China).
[0022] How Salmonella Typhimurium SL1344 and 14028 were obtained / sourced: Salmonella Typhimurium SL1344 and Salmonella Typhimurium 14028 were purchased from American Culture Library.
[0023] MRS liquid culture medium (per liter): peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, dipotassium hydrogen phosphate 2 g, diammonium citrate 2 g, sodium acetate 5 g, Tween 80 1 mL, magnesium sulfate 0.5 g, manganese sulfate 0.25 g.
[0024] Example 1: Preparation of cell-free supernatant (CFS) of Lactobacillus johnsonii Experimental method: Lactobacillus johnsonii BNCC186110 was cultured in MRS liquid medium in an anaerobic incubator at 37°C for 24 h until the optimal growth conditions were reached (bacterial concentration of 1×10⁻⁶). 8 The *Lactobacillus johnsonii* culture was centrifuged at 12000 rpm for 10 min at 4°C. The bacterial pellet was discarded, and the culture was passed through a 0.22 μm sterile membrane to obtain a cell-free supernatant. The supernatant was stored at -80°C before use. The cell-free supernatant was prepared into different volume concentrations (v / v) using MRS liquid medium: 0%, 25%, 50%, 75%, and 100%. The 0% cell-free supernatant served as the control group.
[0025] Example 2: Effect of CFS on crystal violet staining of Salmonella Typhimurium SL1344 and 14028 biofilms The reason why crystal violet staining can determine the formation of biofilms is that positive and negative charges attract each other. Crystal violet is a basic dye with positively charged molecules. However, the main component of biofilms—extracellular polymers—contains a large number of negatively charged substances.
[0026] Experimental methods: Continuous culture of Salmonella Typhimurium, OD before use. 600 The value was adjusted to 0.5. Two Salmonella strains and supernatants of different concentrations were added in equal volumes to 96-well plates. The plates were incubated at 37°C for 24 h. The plates were then washed with PBS twice. 200 μL of 0.1% crystal violet solution was added to each well to stain the biofilm, and staining was performed at room temperature for 20 min. The plates were then washed with PBS twice to remove unbound crystal violet. The 96-well plates were then dried in a 60°C incubator for 20 min. After drying, 200 μL of 33% glacial acetic acid was added to each well of the 96-well plate to dissolve the crystal violet at room temperature for 30 min. The absorbance was measured at 570 nm (Table 1).
[0027] Table 1. Staining of Salmonella Typhimurium SL1344 and 14028 biofilms after treatment with different concentrations of CFS.
[0028] Experimental results: such as Figure 1 As shown in Table 1 above, the biofilm staining of Salmonella Typhimurium SL1344 and 14028 significantly inhibited the formation of biofilms from both Salmonella Typhimurium strains.
[0029] Example 3: Effects of CFS on the motility of Salmonella Typhimurium SL1344 and 14028 LB broth medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride.
[0030] Experimental Methods: Semi-solid culture media containing 0.3% and 0.5% LB broth were prepared as motile and clustering plates, respectively. The media were sterilized at 121 °C for 20 min. After the 0.3% and 0.5% LB agar media cooled to approximately 60 °C, the plates were inverted. Two Salmonella strains were mixed with equal volumes of supernatant of different concentrations. Using a pipette, 5 μL of Salmonella SL1344 and 14028 bacterial suspensions were added to the center of the plates. The plates were incubated at 37 °C for 24 h. Images were taken using a Bio-Rad gel imaging system to record the bacterial motility radius, thus determining their motility.
[0031] Experimental results: The effect of CFS on the motility of Salmonella Typhimurium SL1344 and 14028 is shown in [the table below]. Figure 2 .like Figure 2 As shown, compared with the control group (group with a supernatant volume ratio of 0%), the motility of Salmonella treated with supernatant was significantly reduced, and the diameter was significantly smaller. Furthermore, the higher the supernatant concentration, the more pronounced the decrease in motility. The trends were consistent between the two strains. The results indicate that the supernatant of *Lactobacillus johnsonii* significantly inhibited the motility of both Salmonella strains in a dose-dependent manner.
[0032] Example 4: Effect of CFS on biofilm formation of Salmonella Typhimurium SL1344 and 14028 Continuous culture of Salmonella Typhimurium, OD before use 600The value was adjusted to 0.5. Two Salmonella strains and supernatants of different concentrations prepared in Example 1 were added in equal volumes to 24-well plates. Each well contained a sterilized circular glass slide, and the plates were incubated at 37 °C for 24 h. After incubation, airborne bacteria were removed, and the circular glass slides were washed with PBS to remove unbound bacteria; this process was repeated twice. The slides were then fixed overnight at 4 °C with 2.5% (v / v) glutaraldehyde, followed by washing with PBS to remove the glutaraldehyde fixative; this process was repeated twice. The slides were then dehydrated with a series of ethanol solutions (10%, 30%, 50%, 70%, 80%, 90%, 100%, v / v). The samples were dried at 60 °C for 4 h, then coated with gold under vacuum, and the microscopic surface of the biofilm was observed using a scanning electron microscope.
[0033] Experimental results: such as Figure 3 As shown, Figure 3 A and 3B show the biofilm structure of Salmonella Typhimurium SL1344 and 14028 after treatment with different concentrations of Lactobacillus johnsonii supernatant, observed using scanning electron microscopy. It is clearly visible that the biofilm structure of the two Salmonella control groups (with a supernatant volume ratio of 0%) is intact and dense. In contrast, the two Salmonella strains treated with Lactobacillus johnsonii supernatant showed shrinkage and indentation, with the biofilm density gradually decreasing. In some cases, even after treatment with high concentrations of CFS, the cells ruptured.
[0034] Example 5: Effects of CFS on the metabolic activity of Salmonella Typhimurium SL1344 and 14028 biofilms Experimental methods: Continuous culture of Salmonella Typhimurium, OD before use. 600 The value was adjusted to 0.5. Equal volumes of the two Salmonella strains and the supernatants of different concentrations prepared in Example 1 were added to 96-well plates. The plates were incubated at 37 °C for 24 h. After incubation, planktonic bacteria were removed, and the biofilm was washed with PBS, repeated twice. The metabolic activity of Salmonella present in the biofilm was then measured using XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), with XTT reagent (1 mg, ml PBS) and menadione solution (1 mM acetone) added at a ratio of 12.5:1. The plates were incubated in the dark for 5 hours, and the absorbance was measured at OD=490 nm.
[0035] Experimental Results: After 24 h of treatment with CFS, Salmonella Typhimurium SL1344 and 14028 inhibited the metabolic activity of Salmonella Typhimurium. Compared with the control group, the metabolic activity of both Salmonella strains was significantly reduced in a dose-dependent manner. These results indicate that Lactobacillus johnsonii supernatant has a significant inhibitory effect on the metabolic activity of Salmonella Typhimurium SL1344 and 14028.
[0036] Example 6: CFS inhibits the formation of Salmonella on food contact surfaces and food surface biofilms. Biofilm formation of two Salmonella strains on different food contact surfaces (1×1 cm), including stainless steel sheets, food-grade silicone sheets, and glass sheets, as well as on the surfaces of foods such as beef and blueberries, was quantitatively investigated.
[0037] Two Salmonella bacterial cultures (OD) 600 After mixing equal amounts of CFS (0%, 25%, 50%, and 100%) prepared in Example 1 with a value adjusted to 0.5, stainless steel sheets, silica gel sheets, and glass slides were completely immersed in the sample and incubated at 37°C for 48 hours to form a biofilm. The biofilm was then transferred using sterile glass beads after washing with PBS, and the formation of the biofilm was quantified using a serial dilution method. Beef and blueberries were purchased from a supermarket in Dalian, Liaoning Province. The beef was cut into 1×1×1cm pieces using a sterile knife. The beef and blueberries were washed with PBS and treated with UV-C on both sides for 1 hour each. The experimental procedure for the stainless steel sheets was then performed as described above.
[0038] like Figure 6 As shown, CFS can significantly reduce the biofilm biomass of Salmonella Typhimurium SL1344 and 14028 in various samples.
[0039] Based on Examples 1-6, it can be concluded that Lactobacillus johnsonii cell-free supernatant can inhibit the formation of biofilms of Salmonella Typhimurium SL1344 and 14028, destroy the biofilms of Salmonella Typhimurium SL1344 and 14028, thereby reducing the colonization of Salmonella Typhimurium SL1344 and 14028 and alleviating the health diseases induced by Salmonella Typhimurium SL1344 and 14028 biofilms.
[0040] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. Lactobacillus johnsonii ( Lactobacillus johnsonii Cell-free supernatant of BNCC186110 was used in the preparation of a solution for inhibiting Salmonella typhimurium. SL1344 and / or Salmonella 14028 Applications in products that form biofilms.
2. The application according to claim 1, characterized in that, The cell-free supernatant is the supernatant obtained after liquid culture of Lactobacillus johnsonii BNCC186110 to remove cells and solids.
3. The application according to claim 1 or 2, characterized in that, The product is an antibacterial agent.
4. The application according to claim 1 or 2, characterized in that, The method for preparing the cell-free supernatant is as follows: Lactobacillus johnsonii BNCC186110 is cultured in MRS liquid medium for a period of time, and the supernatant is obtained by centrifugation and discarding the precipitate.
5. The application according to claim 4, characterized in that, The supernatant obtained after centrifugation and precipitate removal is further filtered through a 0.22 μm membrane.
6. The application according to claim 4, characterized in that, The culture conditions are 35-37°C, anaerobic, and at least 24 hours.
7. Lactobacillus johnsonii ( Lactobacillus johnsonii The application of cell-free supernatant of BNCC186110 in the preparation of drugs for the prevention or adjunctive treatment of Salmonella typhimurium biofilm infection.
8. The application according to claim 7, characterized in that, The drug includes, but is not limited to, liquid formulations.