Clostridium praeparatum cell-free supernatant and application thereof in anti-Listeria monocytogenes biological membrane

Abstract

The invention discloses a clostridium praeparatum cell-free supernatant and application thereof in anti-Listeria monocytogenes biofilm, and belongs to the technical field of microorganisms. The invention provides the new application of the clostridium praeparatum DSM 17677 cell-free supernatant, and the clostridium praeparatum DSM 17677 cell-free supernatant can inhibit the formation of a listeria monocytogenes biological membrane and destroy the listeria monocytogenes biological membrane, so that the colonization of the listeria monocytogenes is reduced, and a new guarantee is provided for human health.

Description

Technical Field

[0001] The invention relates to a Faecalibacterium prausnitzii cell-free supernatant and application thereof in anti-monocytogenic Listeria biofilm, belonging to the technical field of microorganisms. Background Art

[0002] Listeria monocytogenes, also known as Listeria monocytogenes, is a small Gram-positive rod belonging to the genus Listeria. It is the main pathogen of listeriosis and can cause listeriosis in humans and animals. Listeria monocytogenes is widely present in nature, especially in soil, sewage, feces, plants, feed and raw milk. This bacterium can survive in the range of 0℃ to 45℃ and can grow and reproduce even at the refrigeration temperature of the refrigerator (4-6℃), which is an important difference from other foodborne pathogens. Because Listeria monocytogenes is an intracellular parasite, susceptible populations include newborns with underdeveloped or defective immune systems, pregnant women, chronic disease patients and the elderly. Pregnant women infected with Listeria may only show mild cold symptoms, but may cause fetal infection, leading to serious consequences such as fetal arrest or miscarriage.

[0003] Listeria monocytogenes is able to form biofilms, which are resistant to adverse environmental conditions. Isolation of bacteria from biofilms may lead to persistent contamination of food, increasing the risk of foodborne illness. Biofilm formation increases the likelihood of persistent spread and contamination of Listeria monocytogenes in factory and kitchen environments, potentially leading to outbreaks of related foodborne illnesses, resulting in health and economic losses.

[0004] Faecalibacterium prausnitzii, also known as Faecalibacterium prausnitzii, is one of the most important bacteria in the human gut microbiota. It accounts for 5-15% of the total bacteria in fecal samples of healthy people. Faecalibacterium prausnitzii is one of the important producers of butyrate, has anti-inflammatory effects, can maintain the activity of bacterial enzymes, and protect the digestive system from intestinal pathogens. Faecalibacterium prausnitzii is associated with a variety of health problems. For example, the number of this microorganism is reduced in individuals with chronic constipation, celiac disease, irritable bowel syndrome, and inflammatory bowel diseases (including Crohn's disease and ulcerative colitis). In addition, lower levels of Faecalibacterium prausnitzii have also been observed in patients with type 2 diabetes, colorectal cancer, etc. Changes in the number of Faecalibacterium prausnitzii may be a sign of intestinal flora imbalance, so scientists regard it as a candidate for the next generation of probiotics.

[0005] The effects of Faecalibacterium prausnitzii cell-free solution (CFS) in the intestine may include regulating immune responses, affecting the balance of intestinal microorganisms, and participating in the metabolism of nutrients, etc. These effects together affect the overall health of the host.

[0006] In summary, L. monocytogenes biofilm formation increases the risk of foodborne illness and poses a major threat to food safety. Therefore, studying and controlling the formation of this biofilm is essential to protect public health. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a new use of Faecalibacterium prausnitzii supernatant in inhibiting the formation of Listeria monocytogenes biofilm.

[0008] The present invention provides application of Faecalibacterium prausnitzii metabolites in inhibiting Listeria monocytogenes.

[0009] In one embodiment, the Faecalibacterium prausnitzii is Faecalibacterium prausnitzii DSM 17677.

[0010] In one embodiment, the metabolite is the supernatant of Faecalibacterium prausnitzii DSM 17677 cultured without cell solids.

[0011] In one embodiment, the metabolite is prepared by culturing the Faecalibacterium prausnitzii DSM 17677 in a culture medium for a period of time, centrifuging and discarding the supernatant after precipitation.

[0012] In one embodiment, the metabolite is prepared by culturing the Faecalibacterium prausnitzii DSM 17677 in a culture medium for a period of time, centrifuging and discarding the precipitate, and then filtering the supernatant with a 0.22 μm membrane.

[0013] In one embodiment, the method for preparing the metabolite is: culturing Faecalibacterium prausnitzii DSM 17677 in a modified fortified Clostridium culture medium under an anaerobic environment of 35-37° C. for at least 48 hours, centrifuging the culture solution, discarding the bacterial precipitate, and filtering the supernatant with a 0.22 μm membrane to obtain Faecalibacterium prausnitzii metabolites.

[0014] In one embodiment, the improved fortified Clostridium culture medium contains: 10.0 g of peptone, 10.0 g of beef extract powder, 3.0 g of yeast extract, 5.0 g of glucose, 5.0 g of NaCl, 1.0 g of soluble starch, 0.5 g of L-cysteine ​​hydrochloride, 3.0 g of sodium acetate, and 0.001 g of resazurin.

[0015] In one embodiment, the use includes but is not limited to inhibiting Listeria monocytogenes biofilm and inhibiting Listeria biofilm metabolic activity.

[0016] In one embodiment, the application is to spray the Faecalibacterium prausnitzii metabolite on the surface of food to prevent or delay the colonization of Listeria on the food surface.

[0017] The present invention also provides the use of the metabolites of Faecalibacterium prausnitzii in the preparation of a medicine for preventing or assisting in the treatment of Listeria monocytogenes biofilm infection.

[0018] In one embodiment, the medicament includes but is not limited to a liquid formulation.

[0019] Beneficial effects:

[0020] The present invention provides a new use of the cell-free supernatant of Faecalibacterium prausnitzii DSM 17677, which can inhibit the formation of Listeria monocytogenes biofilm, destroy the Listeria monocytogenes biofilm, and further reduce the colonization of Listeria monocytogenes, thereby providing a new guarantee for human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The staining of Listeria monocytogenes biofilm. A: Representative image of crystal violet staining of Listeria monocytogenes biofilm; B: Quantitative analysis of crystal violet staining of Listeria monocytogenes biofilm.

[0022] Figure 2 The effect of CFS on the motility of Listeria monocytogenes. A: Representative image of Listeria monocytogenes swimming experiment; B: Quantitative analysis of Listeria monocytogenes swimming experiment; C: Representative image of Listeria monocytogenes clustering experiment; D: Quantitative analysis of Listeria monocytogenes clustering experiment.

[0023] Figure 3 The effect of CFS on biofilm formation of Listeria monocytogenes.

[0024] Figure 4 The effect of CFS on the metabolic activity of Listeria monocytogenes. A: Representative image of CCK-8 staining of Listeria monocytogenes biofilm, B: Quantitative analysis of CCK-8 staining of Listeria monocytogenes, C: Representative image of live and dead cell staining of Listeria monocytogenes biofilm.

[0025] Figure 5Biofilm formation of Listeria monocytogenes on different surfaces, including A: stainless steel, B: plastic, C: lettuce, D: beef and E: milk. DETAILED DESCRIPTION

[0026] Improved fortified Clostridium medium (per L): peptone 10.0 g, beef extract powder 10.0 g, yeast extract 3.0 g, glucose 5.0 g, NaCl 5.0 g, soluble starch 1.0 g, L-cysteine ​​hydrochloride 0.5 g, sodium acetate 3.0 g, resazurin 0.001 g.

[0027] Definition and calculation method of biofilm metabolic activity: The CCK-8 kit is used for detection. The CCK-8 kit is a rapid cell proliferation and cytotoxicity sensitivity test kit based on 2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonybenzene)2H-tetrazolium monosodium salt (WST-8). WST-8 can be reduced by dehydrogenases in living cells to form orange-yellow formazan.

[0028] Cell survival rate = (A s -A b ) / (A C -A b ]×100%;

[0029] Inhibition rate = I(A c -A s ) / (A c -A b )]×100%;

[0030] A s : Absorbance of experimental wells (containing cells, culture medium, CCK-8 solution and drug solution);

[0031] A c Absorbance of control wells (containing cells, culture medium, CCK-8 solution, but no drugs);

[0032] A b : Absorbance of blank well (containing culture medium, CCK-8 solution, but no cell drug).

[0033] Example 1 Preparation of Faecalibacterium prausnitzii Cell-free Supernatant (CFS)

[0034] Experimental method: Clostridium prausnitzii DSM 17677 was cultured in a modified fortified Clostridium medium at 37°C in an anaerobic incubator (10% H2, 10% CO2, 80% N2) for 48 h. After reaching the optimal growth condition (bacterial concentration of 1×10 8CFU / mL), the culture medium of Faecalibacterium prausnitzii was centrifuged (12000rmp / min, 10min, 4°C), the bacterial precipitate was discarded, and the cell-free supernatant was obtained by passing through a 0.22μm sterile membrane. The supernatant was stored at -80°C before use.

[0035] Experimental results: Through the above method, a cell-free supernatant of Faecalibacterium prausnitzii was obtained. After testing, it was found that the supernatant contained butyric acid, salicylic acid and other components.

[0036] Example 2 Effect of CFS on Crystal Violet Staining of Listeria monocytogenes Biofilm

[0037] Experimental method: Listeria monocytogenes was cultured continuously, and an appropriate amount of Listeria monocytogenes liquid was pipetted into a 96-well plate containing different proportions of supernatant (0%, 25%, 50%, 75%, 100%, v / v). The plates were cultured at 37°C for 24h and 48h, respectively, and the plates were rinsed with sterile distilled water, repeated twice, and the 96-well plates were placed in a 60°C incubator for drying for 20min. After drying, 200μL of 0.1% crystal violet solution was added to each well of the 96-well plate to stain the biofilm, and the staining was carried out at room temperature for 20min. After that, the plates were rinsed with sterile distilled water, repeated twice, to remove the unbound colorant crystal violet, and the 96-well plates were placed in a 60°C incubator for drying for 20min. After drying, 200μL of 33% glacial acetic acid was added to each well of the 96-well plate, and the crystal violet was dissolved at room temperature for 30min, and the absorbance was measured at 570nm.

[0038] Experimental results: Figure 1 Shown is the staining of Listeria monocytogenes biofilm. It can be seen that CFS can significantly inhibit the formation of Listeria monocytogenes biofilm.

[0039] Example 3 Effect of CFS on the motility of Listeria monocytogenes

[0040] LB broth medium: 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride.

[0041] Experimental method: LB broth medium and CFS were mixed in different proportions (0%, 50%, 100%, v / v), and agar was added at 0.3% and 0.5% of the total volume according to the mixed volume. The mixture was sterilized at 121℃ for 20min. After the 0.3% and 0.5% LB agar was cooled to about 60℃, the plates were poured. The concentration of Listeria monocytogenes was adjusted to 1×10 7CFU / mL, use a pipette to draw 2 μL of Listeria monocytogenes liquid and drop it in the center of the plate, incubate the plate in a 37°C incubator for 12 h, take pictures using the Bio-Rad gel imaging system, and use a vernier caliper to record the movement radius of the bacteria to determine its motility.

[0042] Experimental results: The effect of CFS on the motility of Listeria monocytogenes is shown in Figure 2 As shown in the figure, the motility and clustering of Listeria monocytogenes treated with 50% CFS were 53.56% and 44.8% of those in the control group, and the motility and clustering of Listeria monocytogenes treated with 100% CFS were 10.04% and 10.08% of those in the control group, showing a significant decrease in motility (p<0.0001) in a concentration-dependent manner.

[0043] Example 4 Effect of CFS on Listeria monocytogenes biofilm formation

[0044] Listeria monocytogenes was cultured continuously. An appropriate amount of Listeria monocytogenes solution was pipetted into a 24-well plate containing different proportions of supernatant (0%, 50%, 100%, v / v) using a pipette. Each well contained a high-temperature sterilized round glass slide and was cultured at 37°C for 48h. After incubation, the floating bacteria were removed, and the round glass slide was washed with PBS to remove unbound bacteria, and the process was repeated twice. Then, the slide was fixed with 2.5% (v / v) glutaraldehyde at 4°C overnight, and then the round glass slide was washed and rinsed with PBS to remove the glutaraldehyde fixative, and the process was repeated twice. The slide was then dehydrated with a series of ethanol (10%, 30%, 50%, 70%, 80%, 90%, 100%, v / v). The sample was dried at 60°C for 4h, and then the gold coating was sprayed under vacuum conditions. The microscopic surface of the biofilm was observed using a scanning electron microscope.

[0045] Experimental results: Figure 3 As shown, CFS had a significant inhibitory effect on the biofilm formation of Listeria monocytogenes. Scanning electron microscopy showed that the biofilm density of Listeria monocytogenes without any treatment was higher, while the biofilm density formed by the treated bacteria was lower and in a concentration-dependent manner.

[0046] Example 5 Effect of CFS on the metabolic activity of Listeria monocytogenes biofilm

[0047] Experimental method: Listeria monocytogenes was cultured continuously. The appropriate amount of Listeria monocytogenes liquid was pipetted into 96-well plates containing different proportions of CFS (0%, 25%, 50%, 75%, 100%, v / v) using a pipette. The plates were cultured at 37°C for 48 hours. After incubation, the floating bacteria were removed and the biofilm was washed with PBS. The process was repeated twice. Then, 200 μL of 10% CKK-8 solution was added to each well. The plates were cultured in the dark at 37°C for 1 hour. The OD450 nm was read to reflect the metabolic activity of the biofilm. After washing, SYTO9 (green fluorescence, live bacteria staining) and propidium iodide PI (red fluorescence, dead bacteria staining) were added for live and dead cell staining. The plates were stained at 37°C for 20 minutes. The unbound stain was washed with PBS and the process was repeated twice.

[0048] Experimental results: After 24 hours of CFS treatment of Listeria monocytogenes, the metabolic activity of its biofilm decreased by 58.11%, 71.48%, 82.04% and 87.15% respectively (p<0.001). The formed biofilm was stained with PI / SYTO9 staining, and the results showed that there were fewer live bacteria in the biofilm, but there was no significant change in dead bacteria. These results indicate that the supernatant significantly inhibits the formation of Listeria monocytogenes biofilm in vitro.

[0049] Example 6 Effect of CFS on Listeria monocytogenes colonization

[0050] Biofilm formation on various foods and related surfaces (beef, raw milk, lettuce, stainless steel, and food-grade plastic) was evaluated. Beef, raw milk, lettuce, stainless steel, and food-grade plastic were completely immersed in LB broth containing different proportions of CFS. At the same time, 1 ml of a bacterial concentration of 1×10 8 CFU / mL bacterial suspension. After 48 h of incubation, all samples were transferred and washed twice with PBS. The samples were then transferred to PBS containing sterile glass beads and vortexed and shaken for 2 min. Finally, the number of biofilm cells was determined by serial dilution.

[0051] like Figure 5 As shown, CFS was able to significantly reduce the number of L. monocytogenes in each sample.

[0052] Based on Examples 1-6, it can be concluded that the cell-free supernatant of Faecalibacterium prausnitzii can inhibit the formation of Listeria monocytogenes biofilm, destroy the biofilm of Listeria monocytogenes, thereby reducing the colonization of Listeria monocytogenes and alleviating health diseases induced by Listeria monocytogenes biofilm.

[0053] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of metabolites of Faecalibacterium prausnitzii in inhibiting Listeria monocytogenes.

2. The use according to claim 1, characterized in that: The Faecalibacterium prausnitzii is Faecalibacterium prausnitzii DSM 17677.

3. The use according to claim 1 or 2, characterized in that: The preparation method of the metabolite is as follows: culturing the Faecalibacterium prausnitzii DSM 17677 in a culture medium for a period of time, centrifuging and discarding the supernatant after precipitation.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation method of the metabolite is as follows: the Faecalibacterium prausnitzii DSM 17677 is cultured in a culture medium for a period of time, centrifuged and the precipitate is discarded, and then the supernatant is filtered using a 0.22 μm membrane.

5. The use according to any one of claims 1 to 4, characterized in that: The applications include but are not limited to inhibiting Listeria monocytogenes biofilm and inhibiting the metabolic activity of Listeria biofilm.

6. The use according to any one of claims 3 to 5, characterized in that: The application is to spray the metabolites of Faecalibacterium prausnitzii DSM 17677 on the surface of food to prevent or delay the colonization of Listeria on the surface of food.

7. Application of metabolites of Faecalibacterium prausnitzii in the preparation of drugs for preventing or assisting in the treatment of Listeria monocytogenes biofilm infections.

8. The use according to claim 7, characterized in that: The Faecalibacterium prausnitzii is Faecalibacterium prausnitzii.

9. The use according to claim 7 or 8, characterized in that: The preparation method of the metabolite is as follows: culturing the Faecalibacterium prausnitzii DSM 17677 in a culture medium for a period of time, centrifuging and discarding the supernatant after precipitation.

10. The use according to any one of claims 7 to 9, characterized in that: The medicine includes but is not limited to liquid preparations.

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