Application of violacein in preparation of product for inhibiting or killing bacillus cereus

Abstract

The invention relates to an application of violacein in preparation of a product for inhibiting or killing bacillus cereus, the violacein not only has inhibiting and killing effects on nutritional and spore bacillus cereus, but also can inhibit the formation of a bacillus cereus biofilm and remove the biofilm formed by the bacillus cereus; the bacillus cereus toxin gene expression can be inhibited, and the bacillus cereus is inhibited from generating toxin; therefore, the bacillus cereus can be used for preparing a product for preventing and removing bacillus cereus pollution, preparing a medicine for preventing and treating bacillus cereus infection and / or preparing a medicine for preventing and treating diseases caused by bacillus cereus infection.

Description

Technical Field

[0001] The invention belongs to the field of hazard control of Bacillus cereus, and particularly relates to application of violacein in the preparation of a product for inhibiting or killing Bacillus cereus. Background Art

[0002] Bacillus cereus is a type of Gram-positive bacteria with flagella, which can move, form spores, and easily form biofilms. It has strong adaptability to growth environments and pathogenic biofilms and is widely found in soil, air, water, and various raw and cooked foods. As a typical foodborne pathogen, pathogenic Bacillus cereus can produce a variety of toxins, such as hemolysin (Hbl), non-hemolysin enterotoxin (Nhe), vomitoxin (Ces), and cytotoxin K (CytK). These toxins attack host cells and cause food poisoning phenomena such as nausea, vomiting, abdominal pain, and diarrhea. In severe cases, they can also cause meningitis, sepsis, and even death. According to statistics from the World Health Organization, about one in ten consumers worldwide will become ill every year due to eating spoiled food, among which infection incidents of Bacillus cereus rank third, accounting for 11.4% of bacterial poisoning incidents. Therefore, how to inhibit and remove Bacillus cereus in food and control the production of its toxins has attracted widespread attention.

[0003] Bacillus cereus has three different forms, namely, vegetative state, spore state and biofilm state. Among them, vegetative Bacillus cereus is the most sensitive to antibacterial agents, high temperature and other antibacterial sterilization methods, and is the form that is most easily killed. When Bacillus cereus produces spores, the spore metabolism is extremely inactive, and it has strong resistance to physical sterilization methods such as heat, salt, acid, radiation and various disinfection preservatives. Therefore, conventional sterilization methods are extremely difficult to remove the spore-state bacteria. Moreover, Bacillus cereus is also prone to form biofilms, which reduces the penetration of preservatives, can protect the vegetative and spore-state bacteria in the film, enhance the resistance of bacteria to external physical, chemical and biological stress factors, and reduce the actual effect of physical heat sterilization and the addition of chemical preservatives. Therefore, biofilm is also an important way for Bacillus cereus to contaminate food. In summary, Bacillus cereus is one of the most difficult pathogenic microorganisms to inhibit or kill due to the existence of spores and biofilm forms. Studies have found that drugs that can inhibit or kill vegetative Bacillus cereus, such as antibiotics, have extremely unsatisfactory inhibitory effects on spore and biofilm forms of Bacillus cereus. Some require antibiotic concentrations that are dozens to dozens of times higher than those in the vegetative state to kill spore and biofilm forms of Bacillus cereus. Therefore, how to eliminate all forms of Bacillus cereus contamination has become a major challenge in the field of food safety and is of great significance.

[0004] Because Bacillus cereus has the above three different forms, and the currently known drugs for preventing and treating Bacillus cereus contamination or infection are only effective against a certain form, but have no effect or very poor effect on other forms. Therefore, it is often necessary to use a combination of multiple drugs to effectively control Bacillus cereus contamination or infection and avoid its repeated occurrence. The combination of multiple drugs not only increases the difficulty of drug administration, but also undoubtedly increases the probability of drug side effects. Therefore, screening a drug or product that is effective against different states of Bacillus cereus is crucial for preventing and controlling Bacillus cereus contamination and preventing and treating Bacillus cereus infection. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects in the prior art, and provide an application of violacein in the preparation of a product for inhibiting or killing Bacillus cereus. The violacein can not only inhibit or remove Bacillus cereus in the vegetative state, spore state, and full-stage biofilm state, but also the sub-inhibitory concentration can reduce the production of Bacillus cereus toxins, inhibit its motility, thereby reducing its pathogenicity and transmissibility. The violacein can be used to prepare antibacterial agents and preservatives for preventing and treating Bacillus cereus contamination and infection, and can completely solve the problem of Bacillus cereus contamination and infection.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The invention discloses an application of violacein in the preparation of products for preventing and removing Bacillus cereus contamination, the preparation of drugs for preventing and treating Bacillus cereus infection and / or the preparation of drugs for preventing and treating diseases caused by Bacillus cereus infection.

[0008] As a further technical solution, the diseases caused by Bacillus cereus infection are prevented and treated, including food poisoning diseases such as nausea, vomiting, abdominal pain and diarrhea caused by Bacillus cereus infection.

[0009] As a further technical solution, the Bacillus cereus includes one or more of Bacillus cereus in the vegetative cell state, Bacillus cereus in the spore state, Bacillus cereus in the biofilm state, and Bacillus cereus in the toxin-producing stage.

[0010] As a further technical solution, the product includes one or more of an antibacterial agent, a bactericidal agent, a preservative, a medicine, and a food additive.

[0011] As a further technical solution, purple bacteriocin has one or more of the following effects;

[0012] 1) Violet bacteriocin can inhibit the growth and reproduction of vegetative cell-state Bacillus cereus;

[0013] 2) Violet bacteriocin can kill vegetative cell-state Bacillus cereus;

[0014] 3) Violet bacteriocin can inhibit the formation of Bacillus cereus biofilm;

[0015] 4) Violet bacteriocin can remove the biofilm of Bacillus cereus;

[0016] 5) Violatin can inhibit the germination of Bacillus cereus spores;

[0017] 6) Violet bacteriocin can inhibit the expression of Bacillus cereus toxin genes and inhibit the production of toxins by Bacillus cereus;

[0018] As a further technical solution, the concentration of violacein inhibiting the germination of Bacillus cereus spores is greater than or equal to 3.125 mg / L;

[0019] As a further technical solution, purple bacteriocin can inhibit the formation of Bacillus cereus biofilm in the adhesion stage, pre-formation stage, growth stage, late formation stage and mature stage of Bacillus cereus biofilm;

[0020] Purple bacteriocin can remove the biofilm of Bacillus cereus in the adhesion stage, early formation stage, growth stage, late formation stage and mature stage of the biofilm of Bacillus cereus.

[0021] The concentration of violacein that inhibits the formation of Bacillus cereus biofilm or eliminates Bacillus cereus biofilm is greater than or equal to 0.5 mg / L.

[0022] As a further technical solution, the concentration of violacein that inhibits the expression of Bacillus cereus toxin gene is greater than or equal to 0.781 mg / L.

[0023] The invention discloses an application of a composition containing violacein in preparing products for preventing and removing Bacillus cereus contamination, preparing medicines for preventing and treating Bacillus cereus infection and / or preparing medicines for preventing and treating diseases caused by Bacillus cereus infection.

[0024] As a further technical solution, the composition further comprises a second antibacterial agent or bactericidal agent;

[0025] As a further technical solution, the second antibacterial agent or bactericidal agent uses an antibacterial agent or bactericidal agent for vegetative cell-state Bacillus cereus. Since violacein has a blue-purple color, and the concentration used when violacein is used to prevent or treat vegetative cell-state Bacillus cereus is high, its color is more obvious. When its color is not liked in certain applications, violacein can be compounded with other antibacterial agents or bactericidal agents for vegetative cell-state Bacillus cereus. Violet bactericidal agent only needs to prevent and control spore-state Bacillus cereus, biofilm-state Bacillus cereus, and toxin-producing Bacillus cereus. At this time, the dosage of violacein can be greatly reduced, so that the color in the product is invisible or slightly visible.

[0026] As a further technical solution, the composition also includes a diluent or other auxiliary materials.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The purple bacteriocin screened out by the present invention is a natural blue-purple pigment synthesized by bacteria. Therefore, compared with other drugs for preventing and controlling Bacillus cereus, it is environmentally friendly and safe.

[0029] 2. The purple bacteriocin screened out by the present invention is formed by the oxidation condensation of two molecules of tryptophan molecules. It is an indole derivative and has multiple physical and chemical properties and biological activities such as antibacterial, antioxidant, and anti-ultraviolet. It not only has antibacterial and sterilizing effects on vegetative Bacillus cereus, but also can inhibit the formation of Bacillus cereus biofilm and remove the biofilm already formed by Bacillus cereus; it can inhibit the expression of Bacillus cereus toxin genes and inhibit Bacillus cereus from producing toxins; thereby, it can be used to prepare products for preventing and removing Bacillus cereus contamination, prepare drugs for preventing and treating Bacillus cereus infection, and / or prepare drugs for preventing and treating diseases caused by Bacillus cereus infection.

[0030] 3. The purple bacteriocin screened out in the present invention has prevention, control and treatment effects on different forms of Bacillus cereus. Compared with the prior art, it avoids a series of defects caused by the compounding of multiple drugs.

[0031] 4. When violacein is used to control or treat different forms of Bacillus cereus, it shows a certain dosage relationship within a certain concentration range. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The effect of the purple bacteriocin of the present invention on the growth curve of vegetative Bacillus cereus;

[0033] Figure 2 The effect of the purple bacteriocin of the present invention on the viability of vegetative Bacillus cereus cells;

[0034] Figure 3 The effect of the purple bacteriocin of the present invention on the cell membrane integrity of vegetative Bacillus cereus;

[0035] exist Figure 3 A:OD 260 nm; B:OD 280 nm;

[0036] Figure 4 The effect of the purple bacteriocin of the present invention on the cell wall integrity of vegetative Bacillus cereus;

[0037] Figure 5 The effect of the purple bacteriocin of the present invention on the microscopic morphology of vegetative Bacillus cereus;

[0038] Figure 6 The inhibitory and killing effects of the purple bacteriocin of the present invention on Bacillus cereus in fish meat;

[0039] Figure 7 The antibacterial effect of the purple bacteriocin of the present invention on spore-forming Bacillus cereus;

[0040] Figure 8 The inhibitory effect of the purple bacteriocin of the present invention on the biofilm of Bacillus cereus;

[0041] Fig. 9 The effect of the purple bacteriocin of the present invention on the initial adhesion ability of Bacillus cereus biofilm;

[0042] Fig.10 The effect of the purple bacteriocin of the present invention on the biofilm formation of Bacillus cereus;

[0043] Fig.11 The present invention is the effect of purple bacteriocin on the removal of mature biofilm of Bacillus cereus;

[0044] Fig.12 The effect of the purple bacteriocin of the present invention on the exopolysaccharide content of Bacillus cereus;

[0045] Fig.13 The effect of the sub-inhibitory concentration of violacein on the production of Bacillus cereus toxins of the present invention;

[0046] Fig.14 The effect of the sub-inhibitory concentration of violacein on the swimming motion of Bacillus cereus of the present invention;

[0047] Fig.15 The figure shows the effect of the sub-inhibitory concentration of violacein on the swarming movement of Bacillus cereus. DETAILED DESCRIPTION

[0048] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] 1. Purity of violacein ≥95%.

[0050] 2. LB medium: 10g / L tryptone, 10g / L sodium chloride, 5g / L yeast powder; MSgg medium: 5mmol / L potassium phosphate, 100mmol / L 3-(N-morpholino)propanesulfonic acid sodium salt, 2mmol / L magnesium chloride, 0.7mmol / L calcium chloride, 0.05mmol / L ferric chloride, 0.05mmol / L manganese chloride, 1μmol / L zinc chloride, 1μmol / L thiamine, 0.5% glycerol, 0.5% glutamic acid, 50mg / L tryptophan, 50mg / L phenylalanine; swimming motility medium: 10g / L tryptone, 5g / L sodium chloride, 3g / L agar powder; swarming motility medium: 10g / L peptone, 5g / L sodium chloride, 5g / L glucose, 5g / L agar powder.

[0051] 3. The raw materials used in the present invention are all commercially available unless otherwise specified.

[0052] Experiment 1: Study on the antibacterial effect of violacein on vegetative Bacillus cereus

[0053] Since LB medium is a medium that promotes bacterial growth, this experiment used LB medium to culture Bacillus cereus to keep it in a nutrient state.

[0054] The test materials used in Examples 1-7 are as follows:

[0055] Preparation of Bacillus cereus: Pick Bacillus cereus with good growth from 4℃ LB plate, inoculate into 20mL LB liquid medium, and culture at 37℃ with shaking for 12h until it reaches the logarithmic phase. After counting, the concentration of Bacillus cereus is 2×10 8 CFU / mL, and diluted with fresh LB liquid culture medium to the concentration of the bacterial suspension used in Example 1-6 for later use.

[0056] Bacillus cereus ATCC 14579 is a model strain of Bacillus cereus, so Bacillus cereus ATCC 14579 was selected in Examples 2-7.

[0057] Example 1: Minimum inhibitory concentration and minimum bactericidal concentration of violacein against vegetative Bacillus cereus

[0058] 1. Test methods:

[0059] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the doubling dilution method. The purple bacteriocin was diluted into 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, and 0.391 mg / L solutions using LB medium in a sterile 96-well plate. 100 μL of purple bacteriocin of the corresponding concentration was added to each well. At the same time, 100 μL of 2×10 6 CFU / mL of Bacillus cereus, with 100μL of saline as negative control, shake well, and culture at 37℃ for 24h, then check each well to determine the MIC and MBC. MIC is defined as the minimum sample concentration at which no visible growth is observed after culture as the minimum inhibitory concentration of violacein. 100μL of bacterial solution is taken from the wells with a concentration higher than the minimum inhibitory concentration and spread on the LB plate, cultured at 37℃ for 24 hours, and the minimum sample concentration at which no colonies grow on the plate is determined as the MBC. The MIC and MBC of violacein against Bacillus cereus are shown in Table 1.

[0060] 2. Test results and analysis

[0061] Table 1 Minimum inhibitory concentration and minimum bactericidal concentration of violacein against Bacillus cereus

[0062]

[0063] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of violacein against the growth of three Bacillus cereus were determined by the microbroth dilution method. The results showed that the MIC of violacein against the three vegetative Bacillus cereus in LB medium was 3.125 mg / L, and the MBC was 12.5 mg / L.

[0064] Example 2: Effect of violacein on the growth curve of vegetative Bacillus cereus

[0065] 1. Test methods

[0066] Take 2mL 2×10 6 CFU / mL of Bacillus cereus suspension, add 2mL of violacein prepared in LB to make the final concentration of 1.563, 3.125, and 6.25mg / L, that is, 0.5MIC, 1MIC, and 2MIC concentrations, with 2mL LB medium as the control, culture at 37℃, sample at 0, 2, 4, 6, 8, 10, 12, and 24h, and use a microplate reader to measure 600nm to monitor the bacterial concentration. 600The relationship between the values ​​is plotted as a time-growth curve, and the results are shown in Figure 1 .

[0067] 2. Test results and analysis

[0068] The time-growth curve of purple bacteriocin on vegetative Bacillus cereus is as follows Figure 1 As shown, with the increase of the concentration of violacein, the bactericidal effect on vegetative Bacillus cereus became more obvious, that is, the inhibitory effect of violacein on the growth of vegetative Bacillus cereus showed concentration dependence. Among them, the control group began to enter the logarithmic growth phase after 2 hours. When 0.5MIC of violacein was added to the culture medium, the bacteria grew slowly and the lag phase increased. After 6 hours, the vegetative Bacillus cereus began to enter the logarithmic phase, indicating that low concentrations of violacein may interfere with the anabolism of vegetative Bacillus cereus; while the culture medium with 1MIC and 2MIC of violacein did not show turbidity and no bacteria grew, indicating that the growth of vegetative Bacillus cereus cells was completely inhibited.

[0069] Example 3: Effect of violacein on the viability of vegetative Bacillus cereus cells

[0070] 1. Test methods

[0071] Take 2mL 2×10 6 CFU / mL Bacillus cereus, 2mL of purple bacteriocin prepared with liquid LB was added to make the final concentrations of 0, 1.563, 3.125, and 6.25mg / L, i.e., 0, 0.5MIC, 1MIC, and 2MIC concentrations, and the samples were cultured at 37℃ for 0, 2, 4, 6, 8, 10, 12, and 14h, respectively. According to the instructions of the CCK-8 kit, the samples were diluted and the reagents were added. The absorbance at 450nm and 650nm was measured by a microplate reader to calculate the cell viability. The results are shown in the table. Figure 2 The formula is as follows:

[0072] Bacterial activity = (OD 450 -OD 650 )×dilution factor

[0073] 2. Test results and analysis

[0074] CCK-8 is a compound that is not overtly toxic to cells and can be reduced by some dehydrogenases in mitochondria to form an orange formazan in the presence of electron coupling. Therefore, it can be used to assess the number of viable cells treated with antimicrobial agents due to the linear relationship between color intensity and cell number. Figure 2The results showed that the activity of vegetative Bacillus cereus in each group of samples with added violacein was significantly lower than that in the control group, and the higher the concentration of added violacein, the lower the cell activity, indicating that the concentration of violacein was negatively correlated with cell metabolic activity.

[0075] Example 4: Effect of violacein on the cell membrane integrity of vegetative Bacillus cereus

[0076] 1. Test methods

[0077] Take 3 ml of the solution with a concentration of 1×10 8 The suspension of Bacillus cereus with a CFU / mL was washed three times with normal saline, and then 3 mL of violacein (0, 0.5 MIC, MIC, 2 MIC) was added with normal saline at final concentrations of 0, 1.563, 3.125, and 6.25 mg / L, respectively, and cultured at 37°C. Samples were taken at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, and 5 h, and centrifuged at 4°C and 12000 r / min for 10 min. The supernatant was taken and filtered through a 0.22 μm filter membrane, and the OD was determined by a UV microplate reader. 260nm and OD 280nm The changes in absorbance at the 240 nm position were used to determine the leakage of nucleic acids and proteins in the cells. Figure 3 .

[0078] 2. Test results and analysis

[0079] The cell membrane is the natural protective barrier of bacteria. Under normal conditions, macromolecular substances such as proteins and nucleotides (including DNA and RNA) in the cell cannot penetrate the membrane. However, when bacteria are exposed to certain antimicrobial agents, the integrity of the cell membrane will be damaged, resulting in the release of intracellular substances into the external environment, leading to an increase in its ultraviolet absorption at 260nm and 280nm. Therefore, the antibacterial mechanism of violacein against vegetative Bacillus cereus can be determined by measuring the content of bacterial proteins and nucleotides that pass through the cell membrane of vegetative Bacillus cereus.

[0080] Figure 3 The results showed that the absorbance of nucleic acid at 260nm and protein at 280nm in the experimental group were higher than those in the control group, and the absorbance value increased more significantly with the increase of the action time and concentration of violacein, indicating that the presence of violacein changed the permeability of the cell membrane, leading to the leakage of intracellular macromolecules (proteins and nucleic acids), and the leakage of cell contents gradually increased with the increase of action time and concentration. In summary, violacein can cause a certain degree of damage to the cell membrane of vegetative Bacillus cereus, and it can change the integrity of the cell membrane, thereby causing the leakage of its contents and causing bacterial death.

[0081] Example 5: Effect of violacein on the cell wall integrity of vegetative Bacillus cereus

[0082] 1. Test methods

[0083] The effect of violacein on cell wall integrity was determined by measuring the change in the activity of alkaline phosphatase (AKP), an enzyme located between the cell wall and the cell membrane, in the extracellular supernatant. 8 CFU / mL, aspirate 1.5mL of bacterial suspension, add 1.5mL of violacein with final concentrations of 0, 1.563, 3.125, and 6.25mg / L (i.e., 0, 0.5MIC, MIC, and 2MIC), mix well, and culture at 37℃. Samples are taken at 0, 6, 12, and 24h, and centrifuged at 4℃, 120000r / min for 10min. The supernatant is taken, and the reagent is added according to the instructions of the intracellular alkaline phosphatase kit. The absorbance at 510nm is detected by a microplate reader, and the AKP enzyme activity is calculated by the formula. The results are shown in Figure 4 .

[0084] The enzyme activity formula is as follows:

[0085] ΔA assay = A assay tube - A control tube; ΔA standard = A standard tube - A blank tube

[0086] AKP activity (U / mL) = C standard × ΔA measurement × 10 μL / ΔA standard × V sample × T

[0087] =0.167×ΔA determination / ΔA standard

[0088] C standard: 0.25 μmol / mL standard tyrosine solution; T: catalytic reaction time, 10 min.

[0089] 2. Test results and analysis

[0090] Usually, AKP cannot be monitored in the supernatant of cells under normal conditions because it is an intracellular enzyme located between the cell wall and the cell membrane. Therefore, this assay proposes a method to assess cell wall integrity by measuring AKP activity in the extracellular supernatant.

[0091] like Figure 4As shown in the figure, the AKP activity of vegetative Bacillus cereus treated with 0.5MIC, 1MIC and 2MIC violacein at the same time was significantly enhanced compared with the control group, among which the AKP activity of 1MIC and 2MIC reached the maximum at 12h and decreased slightly at 24h, and the AKP enzyme activity of 0.5MIC violacein reached the peak at 24h. The reason why the AKP enzyme activity of 0.5MIC violacein reached the peak later may be that the concentration was not enough, and it took a longer time to act on the cells. The slight decrease in enzyme activity of 2MIC at 24h may be due to the degradation of AKP enzyme caused by the leakage of other substances in the cell, such as proteases. In summary, the inhibitory effect of violacein on vegetative Bacillus cereus can be achieved by destroying the cell wall.

[0092] Example 6: Effect of violacein on the microscopic morphology of vegetative Bacillus cereus

[0093] 1. Test methods:

[0094] To 1×10 8 A final concentration of 3.125 mg / L (1 MIC) of violacein was added to a suspension of CFU / mL of Bacillus cereus, and the cells were cultured at 37°C for 1 hour, centrifuged for 10 minutes, washed twice with PBS buffer, the supernatant was discarded, and the bacterial cells were collected and used as a control under the same treatment conditions of physiological saline. The bacterial cells were fixed with 2.5% glutaraldehyde at 4°C for 3 hours, washed three times with PBS buffer, and dehydrated for 10 minutes using a concentration gradient of 50%, 70%, 90% and 100% ethanol. After the dehydration reaction was completed, it was replaced with a mixed solution of ethanol-tert-butanol with a volume ratio of 1:1 for 20 minutes, and finally replaced with 100% tert-butanol for 20 minutes, and the operation was repeated once. The final sample was freeze-dried and photographed with a scanning electron microscope and a transmission electron microscope. The results are shown in Figure 2. Figure 5 .

[0095] 2. Test results and analysis

[0096] To further investigate the mechanism of damage of violacein to vegetative Bacillus cereus, the treated cells were evaluated by scanning electron microscopy and transmission electron microscopy. Figure 5As shown in the figure, scanning electron microscopy showed that the cell morphology of Bacillus cereus without purple bacteriocin was intact, long rod-shaped, with clear cell outline, smooth surface and neat edges. However, after 1 MIC purple bacteriocin was applied for 1 hour, the cell surface became rough and uneven, and even some holes and fractures were observed, as shown by the blue arrows. Transmission electron microscopy images showed that the cells in the control group were long rod-shaped, retaining intact cell walls, clear cell membranes and dense cytoplasm with uniform electron density; while the vegetative Bacillus cereus cells treated with purple bacteriocin had irregular shapes, concave surfaces, uneven cell walls and plasma membranes, and some cells had low electron density and cavities (the white area inside the bacteria indicated by the arrow in the figure). Low electron density or vacuoles symbolize the damage of intracellular DNA and the imbalance of intracellular homeostasis caused by cytoplasmic leakage.

[0097] Example 7: Inhibitory and killing effects of violacein on Bacillus cereus in fish

[0098] 1. Test methods

[0099] Grass carp (1.5 ± 0.2 kg) was stunned, and the head, tail, scales and internal organs were removed. The whole back muscle was taken and cut into uniform fish fillets (1 × 1 × 0.5 cm) of uniform size. The fish fillets were rinsed with sterile water, drained at room temperature, and then inoculated with 4 mL of Bacillus cereus. After mixing, the content of Bacillus cereus in the fish fillets was determined. The initial concentration was 4.42 × 10 4 CFU / g, and then randomly divided into 4 groups, each with 3 parallels, the fish fillets were placed in sterile fresh-keeping bags, among which the blank control group added 1mL of distilled water, the Vio-1MIC group added 1mL of purple bacteriocin prepared with distilled water to make its concentration in the fish fillets 3.125mg / kg, the Vio-2MIC group added 1mL of purple bacteriocin prepared with distilled water to make its final concentration in the fish fillets 6.25mg / kg, the positive control added 1mL of Nisin prepared with distilled water to make its final concentration in the fish fillets 0.5g / kg, mixed and transferred to a 4℃ refrigerator for refrigeration, and samples were taken on days 1, 2, 3, 4 and 5 to test the number of Bacillus cereus in the fish fillets. The results are shown in Figure 6 .

[0100] 2. Test results and analysis

[0101] Biofilm With the rise of cold chain distribution and pre-prepared seafood dishes (such as pre-prepared pickled fish), pathogenic Bacillus cereus has been gradually isolated from fish and fish dishes, causing food poisoning in consumers. Figure 6 It can be seen that the number of Bacillus cereus in the distilled water group increased with the increase of refrigeration days, from the initial 4.42Log 10 CFU / g increased to 5.91Log on the fifth day 10The number of Bacillus cereus in the 3.125mg / kg and 6.25mg / kg violacein-treated groups and the nisin-treated groups decreased with the extension of storage time. On the fifth day, the number of Bacillus cereus in the Vio-1MIC group, Vio-2MIC group and Nisin group was 3.00, 2.62 and 2.10Log, respectively. 10 CFU / g. This indicates that 3.125 mg / kg of violacein added to fish meat has a bactericidal effect on vegetative Bacillus cereus, and a high concentration (6.25 mg / kg) of violacein has a better bactericidal effect.

[0102] In summary, the minimum inhibitory concentration of violacein against vegetative Bacillus cereus in LB medium is 3.125 mg / L, and the minimum bactericidal concentration is 12.5 mg / L. Violet violacein at or above 3.125 mg / L can significantly inhibit the growth of vegetative Bacillus cereus, reduce cell viability, and cause death of vegetative Bacillus cereus by damaging the cell wall and cell membrane of bacterial cells. Violet violacein at or above 6.25 mg / L has a good bactericidal effect on vegetative Bacillus cereus in fish meat.

[0103] Experiment 2: Study on the antibacterial effect of violacein on spore-forming Bacillus cereus

[0104] In this experiment, LB solid medium with 100 mg / L CaCl2 and 40 mg / L MnSO4 was used to culture Bacillus cereus to stimulate its spore production.

[0105] Bacillus cereus ATCC 14579 is the model strain of Bacillus cereus, so Bacillus cereus ATCC 14579 was selected in this test.

[0106] The test materials used in Example 8 are as follows:

[0107] Preparation of Bacillus cereus: Pick Bacillus cereus with good growth from a 4°C LB plate, inoculate it into 20 mL LB liquid culture medium, and culture it at 37°C with shaking for 12 hours until it reaches the logarithmic phase, and set aside.

[0108] Example 8: Effect of violacein on spore germination of Bacillus cereus

[0109] 1. Test methods

[0110] The liquid cultured cereus spore bacillus was spread on LB solid medium, and 100 mg / L CaCl2and40 mg / L MnSO4 were added to the LB medium to stimulate spore formation. After culturing at 30°C for 7 days, the single colony on the plate was scraped, resuspended and washed with 20 mL of sterile PBS, and centrifuged at 12000 r / min for 10 min until milk white. The precipitate was resuspended in 20 mL of 50% ethanol solution and allowed to stand at 4°C for 12 h to remove the vegetative cereus Bacillus, then washed with sterile water 3 times, and the precipitate was resuspended with 5 mL of physiological saline, which was the spore (also called spore-state cereus Bacillus). The spore concentration after counting was 10 9 Pieces / mL.

[0111] Take 100 μL of spores and add them to 9.9 mL of LB medium, i.e., spore working solution. Take 1.5 mL of spore working solution and add 1.5 mL of violacein with a final concentration of 1.563, 3.125, and 6.25 mg / L (0.5 MIC, 1 MIC, and 2 MIC), respectively, mix well, and use 1.5 mL of sterile water as the control group (CK). After standing at 37 ° C for 12 hours, take 100 uL of the mixed solution and dilute it to an appropriate multiple for plate colony counting.

[0112] The spore germination rate was calculated according to the formula. The results are shown in Figure 7 ;

[0113] The calculation formula of germination rate G is as follows:

[0114]

[0115] G: germination rate; M: number of spore colonies treated with purple bacteriocin; C: number of spore colonies in the control group

[0116] 2. Test results and analysis

[0117] from Figure 7 It can be seen that after the spore-forming Bacillus cereus was treated with violacein, the spore germination rate showed a very significant difference (P<0.001). Compared with the control group (CK), when the concentration of violacein was 0.5MIC (1.563mg / L), 16.33% of the spores successfully germinated and grew into vegetative cells. In addition, when the concentration rose to 1MIC (3.125mg / L), the germination rate was only 0.33%. When the concentration reached 2MIC (6.25mg / L), only 0.082% germinated, which was considered to be non-germination. Therefore, the above data show that a concentration of 3.125mg / L of violacein can almost completely inhibit the germination of Bacillus cereus spores. Experiment 3: Study on the inhibitory and removal effects of violacein on biofilm-forming Bacillus cereus

[0118] Bacillus cereus has peritrichous flagella, which can gather on the surface of living or non-living organisms by swimming, adhere to and aggregate with each other, and secrete polysaccharides, proteins and other extracellular matrices to wrap themselves, forming a highly organized membrane polymer, i.e., biofilm. After Bacillus cereus forms a biofilm, it will be different from the vegetative state of Bacillus cereus. Its extracellular matrix plays a protective role on the bacteria inside, reducing the penetration of preservatives, enhancing the resistance of bacteria to external physical, chemical and biological stress factors, and reducing the actual effect of sterilization methods such as physical heat sterilization and adding chemical preservatives.

[0119] The test materials used in Examples 9-14 are as follows:

[0120] Preparation of Bacillus cereus biofilm: Bacillus cereus with good growth was picked from the 4°C LB plate, inoculated into 20 mL of MSgg liquid culture medium, and placed in 37°C shaking culture for 24 hours, i.e., Bacillus cereus seed solution.

[0121] Since MSgg medium can promote the formation of biofilms by most Bacillus species, it can be used to characterize or quantify the formation of biofilms. Therefore, in this experiment, MSgg medium was used to culture Bacillus cereus to form biofilms.

[0122] Bacillus cereus ATCC 14579 is the model strain of Bacillus cereus and is prone to form biofilm in MSgg medium. Therefore, Bacillus cereus ATCC 14579 was selected in this experiment.

[0123] Example 9: Minimum inhibitory concentration of violacein against Bacillus cereus biofilm

[0124] 1. Test methods

[0125] Take 0.5mL of Bacillus cereus seed solution and add 10mL of MSgg medium as Bacillus cereus working solution. Add 100μL of Bacillus cereus working solution to each well of the 96-well plate, culture at 37℃ for 18h, add 100μL of violacein diluted by two-fold method of sterile culture medium to make its concentration 8.00, 4.00, 2.00, 1.00, 0.50, 0.25, 0.125mg / L solution, add 100μL of sterile culture medium as control, shake well, continue to culture at 37℃ for 24h, and use crystal violet staining method to determine the content of biofilm formed in each well. Calculate the inhibition rate of different concentrations of violacein on Bacillus cereus biofilm, the calculation formula is:

[0126] Inhibition rate (%) = (A 对照 -A 处理 ) / A 对照 ×100

[0127] Where: A 对照 A is the absorbance of the control group measured at 595 nm; 处理 It is the absorbance of the purple bacteriocin group measured at 595 nm.

[0128] The minimum inhibitory concentration (MBIC) of violacein against Bacillus cereus biofilm refers to the lowest concentration at which the biofilm inhibition rate is greater than 50%.

[0129] Among them, the crystal violet staining method: After the above-mentioned 96-well cell culture plate is cultured at 37°C for 24 hours, the liquid culture medium is discarded, leaving the surface biofilm. Add 200 μL of sterile saline to each well for washing twice, then add 200 μL of methanol to fix for 20 minutes, remove the methanol, and place it at 60°C for drying for 20 minutes. Then add 2mL of 1% crystal violet to dye at room temperature for 10 minutes, wash with sterile saline to remove excess dye, add anhydrous ethanol after drying at 60°C for 20 minutes, incubate at 37°C for 30 minutes to dissolve the crystal violet, and measure the absorbance value at 595nm of each well, that is, the content of the biofilm.

[0130] At the same time, the MIC of violacein against vegetative Bacillus cereus in MSgg medium was determined according to the method of Example 1, except that the LB medium in Example 1 was replaced with MSgg medium, and the other contents remained unchanged.

[0131] 2. Test results and analysis

[0132] Table 2 Minimum inhibitory concentration of violacein against Bacillus cereus and minimum bactericidal concentration of biofilm

[0133]

[0134] The results are shown in Table 2. The MIC of violacein against vegetative Bacillus cereus in MSgg medium is 0.5 mg / L, and the MBIC against biofilm Bacillus cereus is 0.5 mg / L. Biofilms are often resistant to antibiotics, and usually antibiotics with a concentration of more than ten to several dozen times the MIC are required to inhibit biofilms. The MBIC value of violacein is the same as the MIC, indicating that the addition of violacein equal to or higher than the MIC (0.5 mg / L) also has an antibacterial effect on biofilm Bacillus cereus.

[0135] Example 10: Inhibitory effect of violacein on Bacillus cereus biofilm

[0136] 1. Test methods

[0137] Take 0.5mL of Bacillus cereus seed solution and add 10mL of MSgg culture medium as the Bacillus cereus working solution. Place sterile round glass coverslips (9mm in diameter) in the 24-well cell culture plate in turn, add 0.3mL of Bacillus cereus working solution to each well, and after culturing for 6 hours, add 0.3mL of violacein prepared in sterile culture medium to make the final concentration of 0.5mg / L (MBIC). Add 0.3mL of sterile culture medium to the control group (CK group), shake well, and culture at 37°C. Take out the round glass coverslips in the wells at 24h, 36h, and 48h, respectively, rinse the coverslips with sterile saline twice to remove the floating bacteria in the sample, dry them, stain them with 0.5% crystal ammonium lithospermate staining solution for 1min, rinse them with distilled water, dry them, and observe the amount of bacteria on the coverslips under a microscope. Bacteria appear blue under an optical microscope. The ability of bacteria to form biofilms is judged based on the degree of blue on the coverslips observed under an optical microscope. The results are shown in Figure 8 ;

[0138] 2. Test results and analysis

[0139] The results of biofilm staining under an optical microscope are shown in Figure 8 In the control group, fewer bacteria adhered at 24h, biofilms began to form at 36h, and the biofilm formed at 48h covered the entire coverslip. However, the biofilm formation ability of Bacillus cereus with the addition of 1MIC violacein was significantly inhibited. At 24h, fewer Bacillus cereus adhered than the control group, and the amount of biofilm formed at 24h was also less. At 36h, the amount of biofilm formed only covered half of the coverslip. In summary, the addition of 1MBIC (0.5mg / L) violacein can effectively inhibit the formation of Bacillus cereus biofilm.

[0140] Example 11: Effect of violacein on the initial adhesion ability of Bacillus cereus biofilm

[0141] 1. Test methods

[0142] Take 1 mL of Bacillus cereus seed solution and add 50 mL of MSgg medium as Bacillus cereus working solution. Add 0.6 mL of Bacillus cereus working solution to each well of the 24-well cell culture plate, and add 0.6 mL of violacein prepared in sterile medium to make the final concentrations of 0.25, 0.5, and 1.0 mg / L (i.e., 0.5 MBIC, 1 MBIC, and 2 MBIC). Add 0.6 mL of sterile medium to the control group (CK group), shake well, and put sterile round stainless steel sheets (12 mm in diameter and 1 mm in thickness) in each well in turn, incubate at 37°C in the dark for 2 h to allow initial adhesion. The stainless steel sheet was removed and rinsed three times with sterile saline to remove excess bacterial solution. The sheet was placed in 10 mL of sterile saline containing glass beads and shaken thoroughly to shake off the bacteria adhering to the sheet (the purpose of this operation is to remove the non-adherent Bacillus cereus cells and retain only the adherent Bacillus cereus cells). The plate colony count was performed. The initial adhesion capacity was calculated as 100 μg / cm 2 The number of bacteria adhered to the stainless steel sheet was expressed as lgCFU / cm 2 The results are shown in Fig. 9 .

[0143] 2. Test results and analysis

[0144] The formation of biofilm can be divided into two major stages: first, the hydrophobic proteins or polysaccharide adhesins on the surface of bacteria initially adhere to the material to form a bacterial community, and then, through the mediation of the extracellular matrix, they aggregate with each other to form a biofilm. Initial adhesion is the primary condition for the formation of biofilm.

[0145] The initial adhesion ability of violacein to Bacillus cereus on stainless steel sheets was studied. Fig. 9 The results showed that the initial adhesion of Bacillus cereus on the stainless steel sheet in the control group reached 5.71±0.02lgCFU / cm 2 , the adhesion after treatment with 2MBIC (1.0mg / L) purple bacteriocin concentration was 2.01±0.03lgCFU / cm 2 , the adhesion after treatment with 1MBIC (0.5mg / L) purple bacteriocin concentration was 3.31±0.03lgCFU / cm 2 , the adhesion after treatment with 0.5MBIC (0.25mg / L) purple bacteriocin concentration was 4.58±0.09lgCFU / cm 2 ; This indicates that adding violacein at a concentration equal to or higher than MBIC (0.5 mg / L) inhibits the adhesion of Bacillus cereus, and as the concentration of violacein added increases, the adhesion ability of Bacillus cereus gradually decreases.

[0146] Example 12: Effect of violacein on biofilm formation of Bacillus cereus

[0147] 1. Test methods

[0148] Take 1.0mL of Bacillus cereus seed liquid and add 20mL of MSgg medium as Bacillus cereus working liquid. Add 100μL of Bacillus cereus working liquid to each well of the 96-well cell culture plate and culture at 37℃. Add 100μL of violacein prepared in sterile culture medium at 0h, 6h, 12h, and 24h of culture, respectively, to make the final concentration of 0.25, 0.5, and 1.0mg / L (i.e. 0.5MBIC, 1MBIC, and 2MBIC). Add 100μL of sterile culture medium to the control group (CK group), shake well, and continue to culture at 37℃ for 24h. Then use crystal violet staining to determine the relative amount of biofilm formation. The results are shown in Fig.10 .

[0149] Relative amount of biofilm formation (%) = A 处理 / A 对照 ×100

[0150] Where: A 对照 is the absorbance of the control group at 595 nm; A 处理 is the absorbance of the purple bacteriocin group at 595 nm.

[0151] Among them, crystal violet staining method: see Example 9.

[0152] 2. Test results and analysis

[0153] Depend on Fig.10 It can be seen that compared with the control group, the amount of biofilm formation by violaceous bacteriocin on Bacillus cereus adhesion (0h, the first stage of biofilm formation, i.e. the initial stage) at 0.5MBIC, 1MBIC and 2MBIC was 8.48+3.35%, 24.42+0.86% and 20.14+2.56%, and the adhesion inhibition rate was higher than 50%, indicating that violaceous bacteriocin can exert an inhibitory effect in the initial stage of biofilm formation (bacterial adhesion).

[0154] When 0.5MBIC, 1MBIC and 2MBIC violacein were added after 6h of attachment (early stage of biofilm growth), the relative contents of biofilm were 50.50+2.19%, 30.61+2.09% and 29.22+0.76%, respectively, indicating that violacein can play an inhibitory role in the early stage of biofilm formation.

[0155] When 0.5MBIC, 1MBIC and 2MBIC violacein were added after the biofilm had grown for 12h (late stage of biofilm growth), the relative contents of biofilm were 51.62+2.38%, 39.77+1.57% and 35.95+1.25%, respectively, indicating that violacein had a greater destructive effect on the biofilm formed by Bacillus cereus.

[0156] When 0.5MBIC, 1MBIC and 2MBIC violacein were added after the biofilm had grown for 24h (biofilm maturity stage), the relative contents of biofilm were 67.60+2.39%, 58.74+0.93% and 45.04+1.68%, respectively, indicating that violacein played a certain destructive role on the maturation of Bacillus cereus biofilm at high concentrations.

[0157] In conclusion, the addition of violacein at a concentration equal to or higher than MBIC (0.5 mg / L) can inhibit the formation of Bacillus cereus biofilm at all stages.

[0158] Example 13: Effect of violacein on the removal of mature biofilm of Bacillus cereus

[0159] 1. Test methods

[0160] Take 0.5mL of Bacillus cereus seed liquid and add 10mL of MSgg medium as Bacillus cereus working liquid. Add 100μL of Bacillus cereus working liquid to each well of the 96-well cell culture plate and culture at 37℃ for 24h, then gently discard the bacterial suspension. The bacteria attached to the wall are the biofilm. Gently add 100μL of violacein prepared in sterile culture medium to make the final concentrations 0.25, 0.5, and 1.0mg / L (i.e. 0.5MBIC, 1MBIC, and 2MBIC), respectively. Gently add 100μL of sterile culture medium to the control group (CK group), without shaking, and continue to culture at 37℃ for 24h. The amount of biofilm formation was determined by crystal violet staining. The results are shown in Fig.11 ;

[0161] Relative amount of biofilm formation (%) = A 处理 / A 对照 ×100

[0162] Where: A 对照 is the absorbance of the control group at 595 nm; A 处理 is the absorbance of the purple bacteriocin group at 595 nm.

[0163] The crystal violet staining method is shown in Example 9.

[0164] 2. Test results and analysis

[0165] In order to explore the effect of violacein on mature biofilm of Bacillus cereus, the experiment of removing mature biofilm of Bacillus cereus was carried out;

[0166] Depend on Fig.11 It can be seen that the addition of 0.5MBIC, 1MBIC and 2MBIC concentrations of violacein made the amount of Bacillus cereus biofilm formation 53.18±5.33%, 47.16±1.19% and 28.05±0.58% of the control group, that is, the removal rates of MBIC and 2MBIC were 52.85±1.18% and 72.95±0.58%, both higher than 50%. With the increase of the concentration of violacein, the relative content of Bacillus cereus biofilm decreased, indicating that the addition of violacein equal to or higher than MBIC (0.5mg / L) can effectively remove the biofilm of Bacillus cereus.

[0167] Example 14: Effect of violacein on the exopolysaccharide content of Bacillus cereus

[0168] 1. Test methods

[0169] Take 5mL of Bacillus cereus seed solution and add 45mL of MSgg medium as Bacillus cereus working solution. Add 1.5mL of Bacillus cereus working solution to a 6-well cell culture plate, and add 1.5mL of violacein prepared in sterile medium to make the final concentrations 0.25, 0.5, and 1.0mg / L (i.e. 0.5MBIC, 1MBIC, and 2MBIC), respectively. Gently add 1.5mL of sterile culture medium to the control group (CK group), and culture at 37℃ for 36h in the dark. Gently pour out the culture medium, add 3ml of sterile saline and glass beads, shake, break up the biofilm on the pore wall and surface, and then transfer to a 15mL centrifuge tube. Repeat this process twice (a total of 9mL of biofilm bacterial solution), continue to ultrasonically shake the biofilm in the centrifuge tube for 30min, further break up the biofilm, and then centrifuge at 4℃ and 12000r / min for 10min, take the supernatant and add anhydrous ethanol at 4℃ overnight to precipitate the extracellular polysaccharide. After precipitation, centrifuge at 4°C and 12000r / min for 20min, discard the supernatant, and the precipitate is the extracellular polysaccharide. Add 1mL of sterile water to dissolve the polysaccharide. Take 100uL of the polysaccharide solution, add 100uL of 5% phenol and 500uL of concentrated sulfuric acid to react in the dark for 20min, transfer to a 96-well cell culture plate, and use an ELISA reader to measure the absorbance OD at 492nm 492 , calculate the relative content of extracellular polysaccharides, the results are shown in Fig.12 .

[0170] Relative content of extracellular polysaccharide (%) = A 处理 / A 对照 ×100

[0171] Where: A对照 is the absorbance of the control group at 492 nm; A 处理 is the absorbance of the purple bacteriocin group at 492 nm.

[0172] 2. Test results and analysis

[0173] Exopolysaccharides in Bacillus biofilms account for about 50%-90% of the total organic matter. Exopolysaccharides play an important role in the initial adhesion and aggregation of cells, the maturation of biofilms, and the stability of their structure. They are an important component of bacterial biofilms and play an important role in maintaining their cohesion, obtaining nutrients, and preventing antibiotics from entering cells. Fig.12 It can be seen that the addition of 0.5MBIC, 1MBIC and 2MBIC concentrations of violacein made the exopolysaccharide content of Bacillus cereus 82.3%, 60.4% and 40.7% of that of the control group, respectively, and with the increase of the concentration of added violacein, the relative content of exopolysaccharides of Bacillus cereus decreased, indicating that the addition of violacein equal to or higher than MBIC can effectively inhibit the formation of exopolysaccharides of Bacillus cereus, thereby reducing its adhesion, thereby inhibiting biofilm formation and maturation.

[0174] Experiment 4: Effect of violacein on the pathogenicity of Bacillus cereus

[0175] The test materials used in Examples 15-16 are as follows:

[0176] Preparation of Bacillus cereus (preparation of indicator bacteria): Pick Bacillus cereus with good growth from 4°C LB plate, inoculate it into 20mL LB liquid culture medium, and place it at 37°C for shaking culture for 24h, i.e. Bacillus cereus seed solution. Take 1mL of Bacillus cereus seed solution and add 50mL of LB culture medium as Bacillus cereus working solution.

[0177] Bacillus cereus ATCC 14579 is a model strain of Bacillus cereus. It can rapidly produce a variety of pathogenic toxins in a nutrient-rich culture medium (such as LB culture medium), has flagella, and can move. Therefore, Bacillus cereus ATCC 14579 was selected in this test.

[0178] Example 15: Effect of sub-inhibitory concentration of violacein on toxin production by Bacillus cereus

[0179] 1. Test methods

[0180] Add 2mL of Bacillus cereus working solution to each test tube, and add 2mL of purple bacteriocin prepared with LB medium to make the final concentration 0.781mg / L (1 / 4MIC). Add 2mL of LB medium to the control group (CK group), shake well, and culture at 37℃ for 10-14h. Then take 3mL of culture solution and centrifuge at 4℃, 12000r / min for 10min, discard the supernatant, wash the precipitate with enzyme-free water (i.e. DEPC water) twice, extract RNA with bacterial RNA extraction kit, and measure the concentration of extracted RNA with ultra-micro nucleic acid analyzer. The extracted Bacillus cereus RNA was reverse transcribed and quantitatively analyzed for virulence genes using a one-step reverse transcription real-time fluorescence quantitative PCR kit. Using 16S rRNA as the internal reference gene, 2 -ΔΔCT The relative content of each virulence gene was analyzed by the method, and the results are shown in Fig.13 .

[0181] Table 3 Primers used in RT-qPCR (reverse transcription real-time fluorescence quantitative PCR)

[0182]

[0183] 2. Test results and analysis

[0184] Bacillus cereus has a variety of virulence genes, including hemolysin BL genes (hblC, hblD), enterotoxin T genes (bceT), cytotoxin K genes (cytK), non-hemolytic enterotoxin (nheA, nheB), and vomitoxin genes (ces), etc. These virulence genes play an important role in the bacterial pathogenicity. The RT-qPCR method was used to detect the effect of 1 / 4MIC purple bacillus on the expression of key toxin-related genes of Bacillus cereus. Fig.13 The results showed that sub-inhibitory concentrations of violacein can inhibit the metabolism of Bacillus cereus and significantly down-regulate the expression of six virulence genes, nheA, nheB, bceT, cytK, hblC, and hblD. After treatment with violacein, the toxins of Bacillus cereus were only 76.48%, 0.95%, 61.06%, 63.36%, 14.70%, and 48.62% of those without violacein. This indicates that sub-inhibitory concentrations of violacein (1 / 4MIC, 0.781 mg / L) can inhibit the production of toxins and reduce their pathogenicity.

[0185] Example 16: Effect of sub-inhibitory concentration of violacein on the motility of Bacillus cereus

[0186] 1. Test methods

[0187] 1. Swimming ability test: Prepare swimming motility culture medium, sterilize at 121℃ for 15min, and when the temperature drops to 50-60℃, add violaceous bacteriocin prepared with sterile water to make the final concentration of violaceous bacteriocin in the culture medium 1.563, 3.125, and 6.25mg / L, i.e. 1 / 2MIC, 1MIC, and 2MIC concentrations, respectively, and pour the plate. The plate with sterile water is used as a control. Pipette 5μL of the cereus spore working solution and drop it in the center of the plate. After it penetrates into the culture medium, place it at 37℃ for 24h and observe the migration of the colonies. The results are shown in the table below. Fig.14 .

[0188] Swimming ability inhibition rate (%) = (A1-A2) / A1×100

[0189] Where: A1 is the colony area of ​​the control group plate; A2 is the colony area of ​​the plate with purple bacteriocin added.

[0190] 2. Clustering ability test: Prepare clustering movement culture medium, sterilize at 121℃ for 15min, and when the temperature drops to 50-60℃, add violet bacteriocin prepared with sterile water to make the final concentration of violet bacteriocin in the culture medium 1.563, 3.125, and 6.25mg / L, i.e. 1 / 2MIC, 1MIC, and 2MIC concentrations, respectively, and pour the plates. Use the plate with sterile water as a control. Pipette 5μL of the cereus spore working solution and drop it in the center of the plate. After it penetrates into the culture medium, place it at 37℃ for 24h and observe the colony migration. The results are shown in the table below. Fig.15 .

[0191] Swarming ability suppression rate (%) = (A1-A2) / A1×100

[0192] Where: A1 is the colony area of ​​the control group plate; A2 is the colony area of ​​the plate with purple bacteriocin added.

[0193] 2. Test results and analysis

[0194] The strong motility of pathogenic bacteria can help them quickly colonize the host and expand their spread. The results of the motility of Bacillus cereus under different concentrations of purple bacteriocin are shown in Figure 14-15 When the concentration of violacein reached 1MIC and 2MIC, the growth of Bacillus cereus was completely inhibited. At the sub-inhibitory concentration (1 / 2MIC, 1.563 mg / L), the addition of violacein reduced the migration distances of the two movements, and the inhibition rates of swimming and swarming movement reached 25.5% and 41.7%, respectively. This indicates that under the premise of not affecting the normal growth of bacteria, sub-inhibitory concentrations of violacein can inhibit the swimming and swarming movement of Bacillus cereus, thereby inhibiting the colonization of bacteria in the host and weakening its spread in the environment.

[0195] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. Use of violacein in the preparation of products for preventing and removing Bacillus cereus contamination, the preparation of drugs for preventing and treating Bacillus cereus infection, and / or the preparation of drugs for preventing and treating diseases caused by Bacillus cereus infection.

2. The use according to claim 1, characterized in that: The Bacillus cereus includes one or more of Bacillus cereus in the vegetative cell state, Bacillus cereus in the spore state, Bacillus cereus in the biofilm state, and Bacillus cereus in the toxin-producing stage.

3. The use according to claim 1, characterized in that: The product includes one or more of antibacterial agents, bactericides, preservatives, medicines, and food additives.

4. The use according to claim 1, characterized in that: Violet bacterin has one or more of the following effects; 1) Violet bacteriocin can inhibit the growth and reproduction of vegetative cell-state Bacillus cereus; 2) Violet bacteriocin can kill vegetative cell-state Bacillus cereus; 3) Violet bacteriocin can inhibit the formation of Bacillus cereus biofilm; 4) Violet bacteriocin can remove the biofilm of Bacillus cereus; 5) Violatin can inhibit the germination of Bacillus cereus spores; 6) Violet bacteriocin can inhibit the expression of toxin genes of Bacillus cereus and inhibit the production of toxins by Bacillus cereus.

5. The use according to claim 4, characterized in that: The concentration of violacein that inhibits the germination of Bacillus cereus spores is greater than or equal to 3.125 mg / L.

6. The use according to claim 4, characterized in that: Violet bacterin can inhibit the formation of Bacillus cereus biofilm in the adhesion stage, early formation stage, growth stage, late formation stage and mature stage of Bacillus cereus biofilm. Purple bacteriocin can remove the biofilm of Bacillus cereus in the adhesion stage, early formation stage, growth stage, late formation stage and mature stage of the biofilm of Bacillus cereus. The concentration of violacein that inhibits the formation of Bacillus cereus biofilm or eliminates Bacillus cereus biofilm is greater than or equal to 0.5 mg / L.

7. The use according to claim 4, characterized in that: The concentration of violacein that inhibits the expression of Bacillus cereus toxin gene is greater than or equal to 0.781 mg / L.

8. Use of a composition containing violacein in the preparation of a product for preventing and removing contamination by Bacillus cereus, a drug for preventing and treating infection by Bacillus cereus, and / or a drug for preventing and treating diseases caused by infection by Bacillus cereus.

9. The use according to claim 8, wherein the composition further comprises an antibacterial agent or a sterilizing agent for vegetative cell state Bacillus cereus.

10. The use according to claim 8, wherein the composition further comprises a diluent or other auxiliary materials.

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