Application of Rhamnolipid in Treating Different Forms of Bacillus cereus

Abstract

The present invention belongs to the technical field of food safety, and discloses the application of rhamnolipid in treating Bacillus cereus in different forms, wherein the different forms of Bacillus cereus refer to vegetative cell state Bacillus cereus, spore state Bacillus cereus or biofilm state Bacillus cereus. Taking three forms of Bacillus cereus as the research object, the present invention finds that rhamnolipid alone has inhibitory, killing or eliminating effects on different forms of Bacillus cereus; the combination of rhamnolipid and ultrasonic can significantly improve the elimination effect on biofilms and has a synergistic antibacterial effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety, and particularly relates to the application of rhamnolipids in treating Bacillus cereus in different forms. Background Art

[0002] Currently, foodborne pathogenic bacteria mainly include Bacillus cereus, Salmonella, Staphylococcus aureus, Escherichia coli, etc. On the one hand, they contaminate food and agricultural products, causing them to deteriorate, affecting food quality and safety, and bringing huge losses to social and economic development; on the other hand, they produce toxins directly or indirectly with food as a carrier, causing damage to the body, organ failure, and even clinical diseases such as cancer, increasingly threatening people's health and safety.

[0003] Bacillus cereus, also known as cactus bacillus, is a Gram-positive, β-hemolytic rod-shaped bacterium. It can cause food poisoning in adults and infants by contaminating foods such as flour products and dairy products, endangering human health. The food poisoning cases caused by this bacterium rank among the top in bacterial foods in China. Research shows that Bacillus cereus has three forms: vegetative cells, spores, and biofilms. Spore-form Bacillus cereus is a dormant body formed by its vegetative cells under conditions of insufficient nutrients or external environmental stress. It has strong resistance to high temperature, ultraviolet light, drying, ionizing radiation, and a variety of chemical toxic substances. In addition, Bacillus cereus can aggregate to form biofilms and adhere to the surfaces of various materials, causing more serious harm and being more difficult to remove. Currently, physical sterilization technologies such as pasteurization and irradiation, and chemical antibacterial methods centered on food preservatives are commonly used means for killing and inhibiting microorganisms in current food production, playing a key role in inactivating harmful microorganisms and ensuring food quality and safety. However, there are still few inactivation technologies for Bacillus cereus in different forms. Therefore, researchers are committed to finding methods that can effectively inactivate Bacillus cereus in different forms.

[0004] Rhamnolipids (RLs) are secondary metabolites synthesized by Pseudomonas aeruginosa fermentation. They are a natural anionic glycolipid surfactant with various physicochemical properties and biological activities such as antibacterial and emulsifying properties. The safety evaluation of biodegradation and other aspects was also completed in 2004. The antibacterial activity of RLs was discovered as early as 1971, and the speculated bactericidal mechanism is that it inserts into the biological membrane and destroys the biological membrane through osmosis. However, there are few reports on the research of antibacterial methods of RLs against Bacillus cereus in different forms. Therefore, developing an antibacterial agent that can inhibit Bacillus cereus in different forms is a problem that needs to be solved. In addition, developing an antibacterial agent that inhibits Bacillus cereus in different forms based on rhamnolipids is of great significance for promoting its industrial application. Summary of the Invention

[0005] In view of the technical problem that there is currently no research on the antibacterial method for different morphological Bacillus cereus, the present invention provides the application of rhamnolipid in inhibiting or killing different morphological Bacillus cereus, and determines the concentration of rhamnolipid for inhibiting, killing or removing different morphological Bacillus cereus, which has guiding significance for the development of food antibacterial agents, preservatives, antibacterial films and other products.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides the application of rhamnolipid in treating different morphological Bacillus cereus, and the different morphological Bacillus cereus refers to vegetative cell state Bacillus cereus, spore state Bacillus cereus or biofilm state Bacillus cereus.

[0008] In one technical solution, the specific method for the rhamnolipid of the present invention to inhibit or kill vegetative cells of Bacillus cereus is as follows: Add rhamnolipid with a purity of not less than 90% and a concentration of 2.0 - 64.0 mg / L to the vegetative cell broth of Bacillus cereus to inhibit or kill the growth and reproduction of vegetative cells of Bacillus cereus.

[0009] In a preferred technical solution, the concentration of rhamnolipid for inhibiting vegetative cells of Bacillus cereus is not less than 16.0 mg / L, and the concentration of rhamnolipid for killing vegetative cells of Bacillus cereus is not less than 32.0 mg / L.

[0010] In one technical solution, the specific method for the rhamnolipid of the present invention to inhibit or kill spores of Bacillus cereus is as follows: Add rhamnolipid with a purity of not less than 90% and a concentration of 2.0 - 512.0 mg / L to the spore broth of Bacillus cereus to inhibit or kill the growth and reproduction of spores of Bacillus cereus.

[0011] In a preferred technical solution, the concentration of rhamnolipid for inhibiting spores of Bacillus cereus is not less than 80.0 mg / L, and the concentration of rhamnolipid for killing spores of Bacillus cereus is not less than 160.0 mg / L.

[0012] In one technical solution, the specific method for the rhamnolipid of the present invention to inhibit the formation of Bacillus cereus biofilm is as follows: Add rhamnolipid with a purity of not less than 90% and a concentration of 2.0 - 256.0 mg / L to the broth of Bacillus cereus to inhibit the formation of Bacillus cereus biofilm.

[0013] In a preferred technical solution, the concentration of rhamnolipid for inhibiting the formation of Bacillus cereus biofilm is not less than 32.0 mg / L.

[0014] In one technical solution, the specific method for the rhamnolipid of the present invention to remove the biofilm of Bacillus cereus is as follows: Add rhamnolipid with a purity of not less than 90% and a concentration of 2.0 - 256.0 mg / L to the biofilm bacterial solution of Bacillus cereus to remove the biofilm of Bacillus cereus.

[0015] In a preferred technical solution, the specific method for the rhamnolipid of the present invention to remove the biofilm of Bacillus cereus is as follows: Add rhamnolipid with a purity of not less than 90% and a concentration of 2.0 - 256.0 mg / L to the biofilm bacterial solution of Bacillus cereus, and under ultrasonic conditions, remove the biofilm of Bacillus cereus.

[0016] In a preferred technical solution, the ultrasonic conditions are as follows: the power is 200 W, the frequency is 40 kHz, and the treatment time is 10 - 60 min.

[0017] In a preferred technical solution, the concentration of the rhamnolipid for removing the biofilm of Bacillus cereus is above 32.0 mg / L, and the ultrasonic treatment time is 20 - 60 min.

[0018] The present invention also provides the application of rhamnolipid in the preparation of antibacterial products for treating Bacillus cereus in different forms.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention takes three forms of Bacillus cereus as the research object, and finds that rhamnolipid alone has inhibitory, killing or removing effects on different forms of Bacillus cereus; the combination of rhamnolipid and ultrasound can significantly enhance the removal effect on biofilms and has a synergistic antibacterial effect; the present invention specifically measures the concentration of rhamnolipid for inhibiting, killing or removing different forms of Bacillus cereus, which has guiding significance for the development of products such as food antibacterial agents, preservatives, and antibacterial films. Description of the Drawings

[0021] Figure 1 This is the MIC determination of RLs of the present invention on the vegetative cells of Bacillus cereus.

[0022] Figure 2 This is the MBC determination of RLs of the present invention on the vegetative cells of Bacillus cereus.

[0023] Figure 3 This is the change in membrane potential of the vegetative cells of Bacillus cereus treated with RLs of the present invention.

[0024] Figure 4 This is the change in the reactive oxygen species content of the vegetative cells of Bacillus cereus treated with RLs of the present invention.

[0025] Figure 5For the present invention, the changes in the contents of catalase and superoxide dismutase in the vegetative cells of Bacillus cereus treated with RLs.

[0026] Figure 6 For the present invention, the changes in the inhibition rate of RLs on the vegetative cells of Bacillus cereus in fresh wet noodles.

[0027] Figure 7 For the present invention, the MIC determination of RLs on the spore growth of Bacillus cereus.

[0028] Figure 8 For the present invention, the MBC determination of RLs on the spores of Bacillus cereus.

[0029] Figure 9 For the present invention, the changes in the DPA content in the spores of Bacillus cereus treated with RLs.

[0030] Figure 10 For the present invention, the ultraviolet spectrum changes of the spore DNA of Bacillus cereus after treatment with RLs.

[0031] Figure 11 For the present invention, the MBIC determination of RLs on Bacillus cereus.

[0032] Figure 12 For the present invention, the elimination effect of RLs on the biofilm of Bacillus cereus.

[0033] Figure 13 For the present invention, the elimination effect of RLs + US treatment on the biofilm of Bacillus cereus.

[0034] Figure 14 For the present invention, the scanning electron microscope image of the biofilm of Bacillus cereus treated with RLs + US.

[0035] Figure 15 For the present invention, the changes in the exopolysaccharide of the biofilm of Bacillus cereus under different treatments.

[0036] Figure 16 For the present invention, the changes in the extracellular protein of the biofilm of Bacillus cereus under different treatments.

[0037] Figure 17 For the present invention, the changes in the eDNA of the biofilm of Bacillus cereus under different treatments. Detailed implementation manners

[0038] The following examples are used to illustrate the present invention, but are not used to limit the protection scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are all conventional methods unless otherwise specified.

[0039] In the embodiments of the present invention, the Bacillus cereus adopted has the strain name Bacillus cereus, the preservation number is CMCC(B)63301, and it is from Beijing Baocang Biotechnology Co., Ltd.; rhamnolipid, paste-like, with a purity of ≥90%, purchased from Huzhou Zijin Biotechnology Co., Ltd.; nutrient agar medium: peptone 5.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L, pH adjusted to 7.0; nutrient broth medium: peptone 5.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, pH adjusted to 7.0; nutrient agar medium with manganese sulfate tetrahydrate: manganese sulfate tetrahydrate 0.05 g / L, peptone 5.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L, pH adjusted to 7.0; superoxide dismutase (SOD) kit, reactive oxygen species (ROS) test kit, bacterial genomic DNA extraction kit, catalase (CAT) kit, from Beijing Solarbio Science & Technology Co., Ltd.; propidium iodide (PI), from Anhui Coolaber Biotechnology Co., Ltd.; rhodamine 123, n-hexadecane, and other reagents are all of analytical grade, from Shanghai Aladdin Biochemical Technology Co., Ltd.; wheat flour, from Wudeli Flour Group. Table salt, from Lesaffre Management (Shanghai) Co., Ltd.; resazurin, from Anhui Coolaber Biotechnology Co., Ltd.; glacial acetic acid, toluene, crystal violet, methanol, absolute ethanol, rhodamine 123, tert-butanol, glucose, glutaraldehyde, sulfuric acid, phenol, the above reagents are all of analytical grade, from Tianjin Hengxing Chemical Reagent Co., Ltd. Cell culture plates, from Haimen Huangjie Experimental Equipment Co., Ltd.

[0040] Preparation of main reagents:

[0041] (1) 0.2 mol / L PBS buffer: Take 21.72 g of Na2HPO4·2H2O, 10.76 g of NaH2PO4·H2O, and 58.44 g of NaCl, add water to dissolve to 1.0 L, and that's it.

[0042] (2) 1.0 mg / mL glucose standard solution: Take 0.1000 g of the standard product, add water to dissolve to 100 mL, and that's it.

[0043] Test instruments: analytical balance, AB204-S, manufactured by Metterler Toledo, Switzerland; laminar flow hood, ZHJH-C1109C, manufactured by Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; vertical autoclave, YXQ-LS, manufactured by Shanghai Boxun Industry Co., Ltd.; vortex oscillator, Vortex-2 Genie, manufactured by Scientific Industries, USA; pH meter, SevenEasyS20, manufactured by Mettler Toledo Instruments (Shanghai) Co., Ltd.; high-speed centrifuge, 5424, manufactured by Eppendorf, Germany; ultraviolet spectrophotometer, UV-2550, manufactured by Shimadzu, Japan; digital display constant temperature water bath, HH-4, manufactured by Shanghai Huayan Medical Instrument Co., Ltd.; shaking incubator, ZQZY-C8, manufactured by Shanghai Zhichu Instrument Co., Ltd.; microplate reader, SPARK, manufactured by Tecan, Australia.

[0044] Determination of antibacterial activity of RLs against vegetative Bacillus cereus

[0045] 1.1 Preparation of vegetative Bacillus cereus

[0046] The Bacillus cereus stored at -80°C was inoculated into nutrient broth medium at 2% (v / v) for activation culture for 24 h, then transferred to a new nutrient broth medium and cultured for 24 h to obtain vegetative Bacillus cereus for standby.

[0047] 1.2 Determination of minimum inhibitory concentration (MIC)

[0048] Nutrient broth media containing RLs at concentrations of 0.0, 2.0, 4.0, 8.0, 16.0, 32.0, 48.0, and 64.0 mg / L were prepared respectively, and each shake flask was filled with 50.0 mL. The one without RLs was used as the control group.

[0049] Inoculate with 2% (v / v) bacterial suspension (OD 600 about 1.2), and culture at 37°C and 180 r / min for 24 h. Measure OD 600 , and the RLs concentration in the experimental group with a change less than 5% compared with the initial OD 600 is the minimum inhibitory concentration of RLs against Bacillus cereus spores.

[0050] 1.3 Determination of minimum bactericidal concentration (MBC)

[0051] Prepare nutrient agar medium, take 100.0 μL from each experimental group in the determination of minimum inhibitory concentration and spread it evenly on the solid plate. Compared with the control group without RLs, the minimum RLs concentration in the experimental group with a 99.9% reduction in the number of colonies is used as the minimum bactericidal concentration.

[0052] 1.4 Determination of the Oxidative Stress Response of Bacillus cereus

[0053] 1.4.1 Determination of Reactive Oxygen Species (ROS)

[0054] Take 50.0 mL of the bacterial suspension (OD 600 is approximately 1.2), add the RLs of MIC and MBC, and add the same volume of normal saline to the control group. Incubate at 37 °C and 180 rpm. Take out 200.0 μL of the bacterial solution at 0, 30, 60, 90, and 120 min respectively. According to the operation steps of the reactive oxygen species detection kit, use a microplate reader to detect the fluorescence intensity of the filtrate of each group of samples at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0055] 1.4.2 Determination of the Enzyme Activities of Catalase (CAT) and Superoxide Dismutase (SOD)

[0056] The pretreatment is the same as 1.4.1. Sampling is carried out at 0, 30, 60, 90, and 120 min. Centrifuge at 8000 rpm for 10 min at 4 °C, discard the culture medium, wash 3 times with PBS, and measure the absorbance values at 240 nm and 560 nm respectively according to the instructions of the CAT and SOD activity determination kits.

[0057] 1.5 Production of Fresh Wet Noodles and Evaluation of Antibacterial Effect

[0058] 1.5.1 Production and Inoculation of Fresh Wet Noodles

[0059] The production of fresh wet noodles includes processes such as weighing, adding water, kneading, rolling, ripening, secondary rolling, and cutting. Kneading: Weigh 100.0 g of wheat flour respectively, add 30.0 mL of brine (1.5 g of salt), and knead with a needle-type kneader for 5 min to form noodle flocs. Rolling: Roll the noodle flocs, and roll the noodles 5 times repeatedly. Ripening: Place the made noodle sheets in a self-sealing bag and ripen for 30 min. Secondary rolling: Roll once without folding at rolling distances of 3, 2, and 1 mm respectively. Cutting: Place the noodle sheets between the cutters and cut into noodles with a thickness of 1.0 mm, a width of 2.0 mm, and a length of 20 cm. Spray the Bacillus cereus bacterial suspension onto the noodles so that the initial total number of Bacillus cereus colonies in the fresh wet noodles reaches 4 - 5 (lg(CFU / g)). Use the group without adding RLs as the control group, and make fresh wet noodles by adding RLs of MIC, 2MIC, 4MIC, 8MIC, and 64MIC (i.e., the RLs concentrations are 16, 32, 64, 128, and 1024 mg / L) as the experimental groups. Seal and store them separately in fresh-keeping bags, 25 g per bag. After storing at 25 °C for 60 h, analyze.

[0060] 1.5.2 Determination of the Inhibition Rate of Bacillus cereus in Fresh Wet Noodles

[0061] The total number of Bacillus cereus in the samples was determined at 0 h, 12 h, 24 h, 36 h, 48 h, and 60 h of storage according to the method of GB / T 4789.2-2010 "National Food Safety Standard Microbiological Examination of Foods - Determination of Total Number of Colonies", and the inhibition rate of Bacillus cereus was calculated using the formula:

[0062]

[0063] In the formula, Q is the number of colonies of Bacillus cereus (CFU / g) at each storage time, and Q0 is the number of colonies of Bacillus cereus (CFU / g) at 0 h of storage.

[0064] 1.6 Result Analysis

[0065] The MIC and MBC of RLs against vegetative Bacillus cereus were determined, and the results are as Figure 1 and Figure 2 shown. It can be seen from Figure 1 that after culturing with 0 - 8.0 mg / L of RLs for 24 h, the absorbance increased significantly, indicating that this concentration had no inhibitory effect on the growth of Bacillus cereus; when the concentration of RLs reached 16.0 mg / L, after culturing for 24 h, OD 600 showed no obvious difference from the initial OD 600 , and the MIC of RLs against vegetative Bacillus cereus was determined to be 16.0 mg / L. It can be seen from Figure 2 that after diluting the bacterial solutions of the control group and each experimental group 1000 times and then performing plate coating, when the concentration of RLs was 32.0 mg / L, no colonies grew on the plate, indicating that the killing rate of 32.0 mg / L RLs against vegetative Bacillus cereus reached 99.9%, so the MBC of RLs against Bacillus cereus was determined to be 32.0 mg / L.

[0066] It was known from Figure 3 that the membrane potential of Bacillus cereus after treatment with RLs decreased significantly (P < 0.05), and the fluorescence intensity of the MBC group was significantly lower than that of the MIC group. It was speculated that the integrity and permeability of the cell membrane of Bacillus cereus were damaged, resulting in the continuous outflow of intracellular potassium ions, hyperpolarization of the cell membrane, and a decrease in intracellular fluorescence intensity.

[0067] As Figure 4 shown, the fluorescence value of the RLs treatment group was significantly higher than that of the control group, and the fluorescence intensity showed an upward trend over time, indicating that RLs induced the generation of a large amount of ROS in the cells. It was known from Figure 5It can be seen that after the RLs treatment of MIC and MBC for different times, the activities of CAT and SOD in Bacillus cereus both showed an upward trend, indicating that the bacteria would produce an oxidative stress response after being stimulated by RLs. At this time, chemical modification reactions would occur in proteins, lipids, nucleic acids, etc., affecting their own structures and functions, and then having an adverse effect on cell activity.

[0068] Bacillus cereus is one of the main microorganisms causing the spoilage of fresh wet noodles. As Figure 6 shown, RLs at concentrations of 16 mg / L and 32 mg / L had an inhibitory effect on Bacillus cereus within the first 24 h, and there was no obvious inhibitory effect after 36 - 60 h of placement. While the inhibition rates of 64 mg / L, 128 mg / L, and 1024 mg / L treatments on Bacillus cereus in fresh wet noodles were close to 100% at the 0 - 12 h stage, indicating that RLs added to fresh wet noodles at this concentration had a bactericidal effect on Bacillus cereus. Among them, the inhibition rates of RLs at concentrations of 128 mg / L and 1024 mg / L on Bacillus cereus remained at about 90% throughout the storage period, indicating that high - concentration RLs could play a bactericidal role in fresh wet noodles and could also maintain an inhibitory effect for a long time.

[0069] Determination of the inhibitory activity of RLs against spore - state Bacillus cereus in Example 2

[0070] 2.1 Preparation of spore - state Bacillus cereus

[0071] Prepare a nutrient cell suspension of Bacillus cereus with an OD 600 of about 1.2. Take 100.0 μL and spread it on a nutrient agar medium containing 0.05 g / L manganese sulfate tetrahydrate, and culture it at 37 °C for 7 d to induce spore formation. Then gently scrape the spores with a sterilized glass slide and suspend them in sterile water. Centrifuge at 4 °C and 6000 r / min for 10 min, discard the supernatant, wash 3 times with sterile water, and resuspend the spores in sterile water. The OD 600 of the spore suspension is about 1.2. Treat it in a water bath at 80 °C for 20 min to kill the vegetative cells. The prepared spore suspension is stored at - 20 °C for later use.

[0072] 2.2 Determination of the minimum inhibitory concentration (MIC)

[0073] Prepare nutrient broth media with RLs concentrations of 0.0, 16.0, 32.0, 64.0, 80.0, 96.0, 112.0, 128.0, 144.0, 160.0, 256.0, 512.0 mg / L respectively, and aliquot 50.0 mL into each shake flask. The one without RLs serves as the control group.

[0074] According to 2% (v / v) spore suspension (OD 600Inoculate (about 1.2), culture at 37 °C and 180 r / min for 48 h to obtain. Measure OD 600 , compared with the initial OD 600 , the RLs concentration in the experimental group with a change of less than 5% is the minimum inhibitory concentration against Bacillus cereus spores.

[0075] 2.3 Determination of minimum bactericidal concentration (MBC)

[0076] Same as 1.3 in Example 1.

[0077] 2.4 Determination of DPA release amount of spores

[0078] Add RLs to the prepared spore suspension, with final concentrations of MIC and MBC respectively, and the group without adding RLs is the control group. Prepare a color reagent by adding 1% (w / v) ascorbic acid and 1% (w / v) ammonium ferrous sulfate to 0.5 M acetic acid - sodium acetate buffer (pH 5.5). Centrifuge the samples at 4 °C and 6000 rpm for 15 min, take 4.00 mL of the supernatant and mix it with 1.00 mL of the color reagent, and measure OD 440 , and calculate the DPA content in each group of samples using the standard curve (the standard curve is y = 0.0019x + 0.0488, R 2 = 0.9911).

[0079] Measure the total amount of DPA in untreated spores. Take 5.00 mL of the untreated spore suspension, treat it at 121 °C for 30 min under high temperature and high pressure, add 0.10 mL of 1 mol / L acetic acid solution after cooling, and let it stand for 1 h. Centrifuge and take 4.00 mL of the supernatant to measure the DPA content, which is the positive control group.

[0080] 2.5 Determination of spore surface hydrophobicity

[0081] Use the affinity of spores for n - hexadecane to measure their hydrophobicity. Take 3.00 mL of the spore suspension of the control group and the RLs - treated group respectively and mix them with 0.60 mL of n - hexadecane, vortex for 20 s and then let it stand for 10 min. The OD 600 of the spore suspension in the control group is A0. Measure the OD 600 of the aqueous phase again, and the calculation formula for the proportion of hydrophobic spores (RHS) is:

[0082]

[0083] 2.6 Interaction between RLs and spore DNA

[0084] Extract the genomic DNA of Bacillus spores according to the extraction method of the bacterial genomic DNA kit, dilute it to 1.00 mL with Tris-HCl (0.1 mol / L, pH 8.0), add the RLs of MIC and MBC to the DNA solution respectively, and use the treatment without adding RLs as the control group. React at 37 °C for 30 min and perform ultraviolet spectral scanning in the wavelength range of 220 - 400 nm.

[0085] 2.7 Result analysis

[0086] Measure the MIC and MBC of RLs against Bacillus cereus spores respectively, and the results are as Figure 7 and Figure 8 shown. When the concentration of RLs reaches 80.0 mg / L, after inoculating Bacillus cereus spores and culturing for 48 h, the OD 600 of the culture solution changes significantly, while adding RLs at 0 - 64.0 mg / L has almost no inhibitory effect on the growth of Bacillus cereus spores. Determine the MIC of RLs against Bacillus cereus spores to be 80.0 mg / L. As can be seen from Figure 8 , after spreading the control group and the bacterial solutions cultured with different concentrations of RLs on the plate, it is found that when the concentration of RLs reaches 160.0 mg / L, the killing rate of Bacillus cereus spores reaches 99.9%. Therefore, determine the MBC of RLs against Bacillus cereus spores to be 160.0 mg / L.

[0087] After the untreated bacterial solution is heated at 121 °C for 20 min, all of the dipicolinic acid (DPA) in the spores is released, and the total amount of DPA in the spores measured at this time is 166.83 ± 1.44 μg / mL. As can be seen from Figure 9 , the treatment with RLs will cause the release of DPA, and the high concentration of RLs promotes the release amount of DPA to reach 86.11 ± 2.01 μg / mL, accounting for about 51.61% of the total amount, indicating that RLs can effectively reduce the heat resistance of spores, thereby accelerating spore damage and inactivation.

[0088] The changes in the surface hydrophobicity of Bacillus cereus spores treated with RLs are shown in Table 1. According to Table 1, the surface hydrophobicity of untreated Bacillus cereus spores is 59.938%, showing strong hydrophobicity. After treatment with RLs, the surface hydrophobicity of the spores decreases significantly (P < 0.05), the MIC group decreases to 12.310%, and the MBC group decreases to 10.792%. The proportion of spores distributed in n-hexadecane decreases significantly (P < 0.05), and the surface of the spores changes from hydrophobic to hydrophilic. It shows that RLs can reduce the adhesion ability of spores to hydrophobic surfaces and interfaces, providing a potential possibility for reducing the contamination of Bacillus cereus spores in food processing.

[0089] Table 1 Changes in the surface hydrophobicity of Bacillus cereus spores treated with RLs

[0090]

[0091]

[0092] The changes in the ultraviolet spectrum after the interaction between DNA and RLs are as Figure 10 shown. It was found that the maximum ultraviolet absorption peak of Bacillus cereus spore DNA decreased with the increase in the treatment concentration of RLs, showing a hypochromic effect, indicating that RLs can interact with the DNA molecule of Bacillus cereus spores.

[0093] Example 3 Determination of the inhibitory and clearance effects of RLs on biofilm-forming Bacillus cereus

[0094] 3.1 Determination of the minimum biofilm inhibitory concentration of RLs against Bacillus cereus

[0095] 50.0 mL of sterile medium and 2% (v / v) bacterial suspension (OD 600 about 1.2) were added to the shake flasks respectively. Subsequently, RLs were added to make their final concentrations 2.0 mg / L, 4.0 mg / L, 8.0 mg / L, 16.0 mg / L, 32.0 mg / L, 64.0 mg / L, 128.0 mg / L, and 256.0 mg / L respectively. Then, 200 μL was added to each well of a 96-well plate and cultured at 37 °C for 36 h. The medium was discarded, and the planktonic bacteria were removed by washing with PBS. 200 μL of methanol was added to each well, and after fixing for 15 min, the methanol was discarded and the wells were washed with PBS; then 1% crystal violet was added to each well, and after 5 min, the excess stain was removed by washing with PBS; after air-drying naturally, 200 μL of glacial acetic acid solution (33%) was added to each well and incubated at 37 °C for 30 min, and the absorbance was measured at 570 nm.

[0096] Taking the sterile medium as the blank control group, the minimum concentration with a significant change in the OD value compared with the blank group was defined as the minimum biofilm inhibitory concentration (MBIC).

[0097] 3.2 Clearance effect of RLs on the biofilm of Bacillus cereus

[0098] 200 μL of bacterial suspension (OD 600Cultivate at 37 °C for 36 h (about 1.2) to form a mature biofilm, discard the culture medium and wash 2 - 3 times with sterile PBS. Subsequently, add sterile culture medium containing RLs, where the RLs concentrations are 2.0 mg / L, 4.0 mg / L, 8.0 mg / L, 16.0 mg / L, 32.0 mg / L, 64.0 mg / L, 128.0 mg / L, and 256.0 mg / L respectively. Use the group without adding RLs as the control group, and treat at 37 °C for 24 h. Refer to the crystal violet staining method in 3.1 to measure the OD570 of each group of samples. Calculate the clearance rate of different concentrations of RLs on the Bacillus cereus biofilm. The calculation formula is:

[0099]

[0100] In the formula, OD0 is the absorbance measured at 0 h, and OD1 is the absorbance measured after 24 h.

[0101] 3.3 Result analysis

[0102] Use the spectrophotometric method to determine the MBIC of RLs on biofilm - state Bacillus cereus. The results are as Figure 11 shown. It can be seen that adding RLs at 0 - 16.0 mg / L has almost no inhibitory effect on the growth of the Bacillus cereus biofilm. When the RLs concentration is at 32.0 - 256.0 mg / L, after the biofilm is cultured for 36 h and stained with crystal violet, OD 570 significantly decreases. It is determined that the MBIC of RLs on the Bacillus cereus biofilm is 32.0 mg / L.

[0103] Cultivate Bacillus cereus for 36 h to form a mature biofilm, add different concentrations of RLs for treatment, and evaluate its clearance effect on the biofilm. The results are shown in Figure 12 . Compared with the control group (clearance rate is 0%), RLs at 2.0 mg / L can show a clearance effect on the Bacillus cereus biofilm, and the clearance rate gradually increases with the increase of the RLs concentration. Among them, the clearance rates of RLs at 16 - 64.0 mg / L on the biofilm are all about 40%, and there is no positive correlation with the concentration. It is speculated by analysis that this concentration range has exceeded the critical micelle concentration of the used RLs, causing RLs to aggregate and form a micelle state with hydrophobic groups inside and hydrophilic groups on the surface, resulting in the amount of RLs in contact with the biofilm not increasing with the increase of the concentration. The clearance rates of RLs at 128 and 256.0 mg / L on the biofilm increase. It is analyzed that the RLs aggregates at this time transform into smaller particles in distribution, and can better embed and penetrate the biofilm, promoting the further increase of the clearance rate. However, after treating the Bacillus cereus biofilm with RLs at 256.0 mg / L for 24 h, the clearance rate only reaches 63.18%.

[0104] Example 4: Clearing effect of RLs combined with ultrasound on Bacillus cereus biofilm

[0105] 4.1 Clearing effect of RLs + US on Bacillus cereus biofilm

[0106] The pretreatment was the same as 3.2 in Example 3. After culturing to form mature biofilm, RLs with concentrations of 16.0 mg / L, 32.0 mg / L, and 64.0 mg / L were added, and the blank medium was used as the control group. In addition, the ultrasound (US) power was set at 200 W, the ultrasound frequency was 40 kHz, and the time placed in the ultrasound was 0 min, 10 min, 20 min, 30 min, and 60 min respectively. The clearance rate was calculated according to formula (3-1).

[0107] 4.2 Observation of biofilm microscopic morphology

[0108] Bacillus cereus biofilm was cultured, and samples of the control group, RLs, and ultrasound treatment group were prepared with reference to 4.1. Among them, the final concentrations of RLs were 16.0, 32.0 (MBIC), and 64.0 mg / L respectively, the ultrasound time was determined according to the results of 4.1, and it was left standing at 37 °C for 24 h. The 96-well plate was taken out, the culture medium was removed, and it was washed 2-3 times with sterile PBS buffer. 2.5% glutaraldehyde was added and fixed overnight at 4 °C. It was dehydrated successively with 30%, 50%, 70%, and 90% ethanol, dehydrated twice with 100% ethanol, and then replaced with ethanol:tert-butanol (2:1, 1:1) and 100% tert-butanol to make a tert-butanol biofilm bacterial solution, which was dropped onto aluminum foil paper, pre-cooled at -80 °C and then freeze-dried, and observed and photographed by scanning electron microscope (SEM).

[0109] 4.3 Determination of extracellular polymeric substances of biofilm

[0110] 4.3.1 Extraction and determination of extracellular polymeric substances

[0111] Bacillus cereus biofilm was cultured, and samples of the control group, RLs, and ultrasound treatment group were prepared with reference to 4.1. Among them, the final concentrations of RLs were 16.0, 32.0 (MBIC), and 64.0 mg / L respectively, the ultrasound time was determined according to the results of 4.1, and it was left standing at 37 °C for 24 h. The culture medium was removed and washed with sterile PBS buffer, and then an appropriate amount of sterile PBS buffer was added. The biofilm was oscillated and dispersed into the solution, and the bacterial suspension was centrifuged at 10000 rpm for 10 min at 25 °C, and the supernatant was collected.

[0112] Determination of the standard curve of glucose solution: Pipette 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 mL of the glucose standard solution into a volumetric flask and make up to 10.0 mL. Take 2.0 mL of the standard solution in a test tube, add 1.0 mL of 6% phenol and 5.0 mL of concentrated sulfuric acid to each test tube, let it stand at 100 °C for 15 min, and measure the OD with an enzyme-linked immunosorbent assay (ELISA) reader. 490 And draw the standard curve (standard curve: y = 45.631x + 0.0329, R2 = 0.9948).

[0113] Take 1.0 mL of the supernatant and mix it with 1.0 mL of phenol by shaking for 30 s, then add 15.0 mL of concentrated sulfuric acid, let it stand in the dark for 15 min. After taking it out, vortex for 10 s, place it in a water bath at 25 °C for 20 min, and measure the OD with an ELISA reader. 490 .

[0114] Prepare the standard curve with reference to the Bradford protein concentration assay kit. Take an appropriate amount of 25 mg / mL protein standard solution and dilute it to a final concentration of 0.5 mg / mL. Add 0.0, 1.0, 2.0, 4.0, 8.0, 16.0, 20.0 μL to a 96-well plate, and make up the volume to 20.0 μL with the standard diluent if it is less than 20.0 μL. Add 200.0 μL of Coomassie Brilliant Blue G250 solution to each well, let it stand at 25 °C for 3 - 5 min, and measure the OD with an ELISA reader. 595 (Standard curve: y = 1.657x + 0.6187, R2 = 0.9994).

[0115] Take 50.0 μL of the supernatant and 0.5 mL of Coomassie Brilliant Blue G250 solution, mix well, let it stand at room temperature for 3 - 5 min, and measure the OD with an ELISA reader. 595 .

[0116] Extract the DNA in the bacterial suspension using a bacterial genomic DNA extraction kit and measure the optical density value at 260 nm using a micro ultraviolet spectrophotometer.

[0117] 4.4 Results and analysis

[0118] The results of the removal effect of RLs + US on the biofilm of Bacillus cereus are as Figure 13 shown. It can be seen that when RLs with concentrations of 16.0 mg / L, 32.0 mg / L (MBIC), and 64.0 mg / L are added alone, the biofilm removal rates are 39.61%, 42.64%, and 44.19% respectively; when US is treated alone for 20 min, 30 min, and 60 min, the biofilm removal rates of Bacillus cereus are 22.35%, 31.63%, and 33.42% respectively; it shows that RLs or ultrasound alone can effectively remove the biofilm of Bacillus cereus, but the removal rates are relatively low.

[0119] Further analysis revealed that at the same ultrasonic treatment time, the biofilm clearance rate increased with the increase in the RLs concentration, indicating that there was still a positive correlation between the clearance rate of the combined ultrasonic treatment on the biofilm and the RLs concentration. When the same concentration of RLs was added, the clearance rate of the biofilm by ultrasonic treatment for 10 min decreased slightly, while ultrasonic treatment for more than 20 min could significantly improve the biofilm clearance rate, but no higher clearance efficiency was observed for treatment for 30 min and 60 min. Considering the energy consumption cost and the clearance effect, ultrasonic treatment for 20 min was selected as the treatment condition for the subsequent experiments.

[0120] Comprehensive analysis of the treatment effect of the combination of RLs and ultrasound showed that the clearance rate of the combined treatment of 32.0 mg / L (MBIC) + ultrasound for 20 min was 62.54%, which was close to the biofilm clearance rate (63.18%) of 256.0 mg / L of RLs alone. At the same time, the clearance rate of the biofilm by the treatment of 256.0 mg / L + 60 min of ultrasound reached the maximum value of 78.67%. These results indicate that compared with the single treatment, the combined treatment of RLs and US has an obvious synergistic antibacterial effect, and the clearance effect of the combination of RLs at a lower concentration and ultrasound can reach the effect of the single treatment of RLs at a higher concentration.

[0121] The microscopic morphological changes of the Bacillus cereus biofilm were observed by scanning electron microscopy, and the results are shown in Figure 14 . It can be seen that the untreated Bacillus cereus (control group) showed clear and relatively plump rod shapes, with a smooth surface and intact cells. After treatment with 32.0 mg / L (MBIC) of RLs, pores appeared on the surface of the bacteria, and the structure showed concave deformation. After 20 min of US treatment alone, the adhesion substances between the bacteria decreased, the biofilm was lysed, and the bacteria were more dispersed. After the combined treatment of RLs and US, the number of adhered bacteria decreased significantly, most of the bacteria showed severe deformation and shrinkage on the surface, and the extracellular polymeric substances were largely eliminated. The above results indicate that RLs and ultrasonic treatment have a synergistic effect on the destruction of the bacterial cells and extracellular matrix of the Bacillus cereus biofilm, and RLs play a major destructive role.

[0122] The effects of different treatments on the exopolysaccharides of the Bacillus cereus biofilm were measured and calculated, and the results are shown in Figure 15 . The content of exopolysaccharides in the untreated control group was 12.28 μg / mL, while after treatment with the RLs group, the US group, and the combined treatment group of both, the content of exopolysaccharides in the Bacillus cereus biofilm showed different degrees of decrease. Among them, the inhibitory effects of the RLs group at 1 / 2MBIC concentration and the US single treatment group on exopolysaccharides were relatively weak, and their contents were 11.24 μg / mL and 11.38 μg / mL, respectively. After the combined treatment, the content of exopolysaccharides could be reduced to 8.04 μg / mL.

[0123] The effects of different treatments on the extracellular proteins of Bacillus cereus biofilms were determined and calculated, and the results are shown in Figure 16 . The extracellular protein content in the untreated biofilm was approximately 400 μg / g. From this, it can be seen that the most abundant component in the entire extracellular matrix is extracellular protein. After treatment with different concentrations of RLs, the extracellular protein content decreased by 25.71%, 32.81%, and 40.77% respectively, while treatment with US alone did not significantly inhibit extracellular proteins (P < 0.05). After the two were combined, extracellular proteins were significantly inhibited (P < 0.05), and the content decreased to 181.19 μg / g, indicating that the synthesis and secretion of extracellular proteins were inhibited to a large extent. Among them, the extracellular protein content in the US + MBIC group was the same as that in the treatment with 2MBIC alone, indicating that the combined treatment of US and low-concentration RLs can achieve the same inhibitory effect as that of high-concentration RLs alone.

[0124] The effects of different treatments on eDNA are presented in Figure 17 . The OD 260 of the control group was 0.231. Whether treated alone or in combination, the OD values decreased to varying degrees, indicating that both RLs and US treatments can damage the extracellular DNA of Bacillus cereus biofilms. The OD value of the combined treatment group was always lower than that of the RLs alone treatment group, which further confirmed the synergistic bactericidal effect of RLs and US. It is analyzed that ultrasound destroys the permeability of the biofilm, promoting RLs to enter the interior of the biofilm and embed with DNA, thereby inhibiting the synthesis and secretion of DNA.

[0125] The above-described embodiments are only the preferred embodiments of the present invention, and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art of this technology, of course, according to the technical content disclosed in this specification, other implementation manners can be easily made by means of substitution or change. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. Use of rhamnolipid in treating biofilm Bacillus cereus, characterized in that The specific treatment method is as follows: Add rhamnolipid with a purity of not less than 90% and a concentration of 32 - 256 mg / L to the biofilm bacterial solution of Bacillus cereus, and under ultrasonic conditions, the ultrasonic treatment time is 20 - 60 min to remove the biofilm of Bacillus cereus.

2. The application according to claim 1, wherein The ultrasonic conditions are as follows: the power is 200W and the frequency is 40kHz.

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