Method for inhibiting generation of vomitoxin from bacillus cereus

Abstract

The invention discloses a method for inhibiting production of vomitoxin from bacillus cereus, which is characterized in that isoleucine is added into a substance needing to inhibit production of vomitoxin from bacillus cereus, preferably 5-20mM of isoleucine is added. According to the present invention, the content of the toxin is detected through the liquid chromatography-mass spectrometry, and the cytotoxicity experiment verification results show that the isoleucine can significantly inhibit the synthesis of the vomitoxin of the bacillus cereus in the culture medium and the matrix, and can effectively reduce the toxic effect of the toxin on HeLa cells; the invention provides a safe and efficient biological control means which is suitable for various food systems such as starch food, dairy products, meat products and the like. Isoleucine can be used as a novel non-toxic and harmless food additive and is low in cost; the use method is simple, the output of vomitoxin can be reduced by directly adding a trace amount of isoleucine, and the food flavor is not affected.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of foodborne pathogen hazard control, and particularly relates to a method for inhibiting emetic production by Bacillus cereus. BACKGROUND

[0002] Bacillus cereus is a common foodborne pathogen, which mainly causes two types of food poisoning, diarrhea and vomiting. Emetic is a cyclic dodecapeptide composed of three tetrapeptide units, with a molecular weight of 1.2 kDa. Each tetrapeptide unit is [D-O-Leu-D-Ala-L-O-Val-L-Val], which is similar in structure to Valinomycin. Emetic has a wide tolerance range for the environment, such as temperature (still active after 60 min of treatment at 150℃) and pH, and has resistance to trypsin and pepsin. Once produced, it is difficult to be inactivated in food processing. Some food samples detected Bacillus cereus at a content of less than 10 5 CFU / g, and some even without live bacteria, but emetic was still detected, so it is crucial to prevent and control emetic.

[0003] Emetic is synthesized in food matrix and its hazard is severely underestimated. Because Bacillus cereus and Staphylococcus aureus are often found in similar food environments, and the symptoms of emetic poisoning and Staphylococcus aureus poisoning are difficult to distinguish, poisoning events caused by the former are often misdiagnosed. At the same time, due to the characteristics of self-limiting disease, patients often do not pay attention to emetic poisoning, resulting in a large number of clinical data missing caused by emetic. Generally speaking, vomiting symptoms occur within 0.5 to 6 hours after ingesting contaminated food. Emetic is absorbed and distributed throughout the body after ingestion, and part of the toxin is excreted through feces, and may also cross the blood-brain barrier, accumulating in liver, kidney, fat and muscle tissue. Emetic can damage the gastrointestinal tract and may cause gastroenteritis and even organ failure.

[0004] Different environmental factors have different effects on the synthesis of emetic toxin. The acute cases with relatively high emetic toxin content are mainly based on cereal foods such as rice or noodles, while other foods such as dairy products, meat or vegetables are rarely reported. Studies have shown that the emetic toxin production of Bacillus cereus is significantly increased in oat agar, trypsin soybean agar or blood agar, while almost no toxin is produced in milk or white rice soup medium. The addition of glucose and specific amino acids such as valine and leucine can significantly enhance the production of emetic toxin. In foods close to neutral such as mashed potatoes and noodles, the production of emetic toxin is higher, while the addition of acidic seasonings such as vinegar or ketchup can significantly inhibit the generation of toxin. Changes in oxygen concentration also affect toxin synthesis, and reducing oxygen content or replacing atmospheric oxygen with nitrogen can reduce the accumulation of toxin. In addition, water activity and salt concentration, as factors related to osmotic pressure, also affect the production of toxin. Therefore, if the exogenous addition of food raw materials can be used to prevent and control emetic toxin without affecting the flavor of food, it is of great significance for the prevention and control of the harm of Bacillus cereus. SUMMARY

[0005] A first object of the present application is to provide the use of isoleucine in inhibiting the production of emetic toxin by Bacillus cereus.

[0006] The present application finds that the addition of food raw material component isoleucine can effectively inhibit the biosynthesis of emetic toxin. Using liquid chromatography mass spectrometry analysis technology, it is found that isoleucine can significantly reduce the production of emetic toxin by Bacillus cereus; the characteristic quantitative ion pair of emetic toxin is m / z 1170.60→357.40, and the characteristic quantitative ion pair of the internal standard valinomycin is m / z 1128.70→343.10. Cell experiments further show that isoleucine can significantly reduce the cytotoxicity caused by emetic toxin, thereby improving the survival rate of HeLa cells. In addition, in food substrates such as broth and milk, isoleucine also shows obvious inhibitory effect on the production of emetic toxin by Bacillus cereus.

[0007] Based on the above findings, isoleucine can be used to inhibit the production of emetic toxin by Bacillus cereus.

[0008] Preferably, the use of isoleucine in the preparation of a preparation for inhibiting the production of emetic toxin by Bacillus cereus.

[0009] Preferably, isoleucine is added to a substrate in which the production of emetic toxin by Bacillus cereus needs to be inhibited, so as to achieve the inhibition of the production of the toxin.

[0010] Preferably, the substrate in which the production of emetic toxin by Bacillus cereus needs to be inhibited is food.

[0011] Preferably, the food is a starchy food, milk and dairy products, meat and meat products, fruits or vegetables.

[0012] Preferably, the isoleucine is added to the substrate in need of inhibiting the production of emetic toxin by Bacillus cereus at a concentration of 5-20 mM.

[0013] A second object of the present application is to provide a method for inhibiting the production of emetic toxin by Bacillus cereus, comprising the step of adding isoleucine to a substrate in need of inhibiting the production of emetic toxin by Bacillus cereus, so as to inhibit the production of emetic toxin by Bacillus cereus through isoleucine.

[0014] Preferably, the substrate in need of inhibiting the production of emetic toxin by Bacillus cereus is food.

[0015] Preferably, the food is starch food, milk and dairy products, meat and meat products, fruits or vegetables.

[0016] Preferably, the concentration of isoleucine is 5-20 mM.

[0017] The present application proposes isoleucine as an additive to significantly inhibit the synthesis of emetic toxin in Bacillus cereus from the perspective of food ingredients. This strategy is of great significance for guiding the practical application of isoleucine and preventing emetic food poisoning caused by Bacillus cereus by adjusting the proportion of food ingredients. Isoleucine has the characteristics of low cost and easy use, and can be directly added to reduce the amount of emetic toxin produced, which is a new type of potential food additive without toxicity and safety. By adding an appropriate amount of isoleucine, the amount of emetic toxin produced can be lower than the pathogenic threshold, thereby effectively controlling the biological hazards caused by Bacillus cereus. BRIEF DESCRIPTION OF DRAWINGS

[0018] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the exemplary embodiments of the present application and their descriptions are used to explain the present application, and do not constitute improper limitations on the present application.

[0019] Figure 1 Figure 1 is the growth curves of Bacillus cereus 892-1 (A) and F4810 / 72 (B) before and after the addition of isoleucine. A, the effect of the addition of isoleucine on the growth of Bacillus cereus 892-1; B, the effect of the addition of isoleucine on the growth of Bacillus cereus F4810 / 72. As can be seen from the figure, there is no obvious difference in the growth of different emetic strains before and after isoleucine treatment.

[0020] Figure 2Effect of isoleucine gradient addition on emetic toxin synthesis of B. cereus 892-1 (A) and F4810 / 72 (B). A, Effect of isoleucine gradient addition on emetic toxin of B. cereus 892-1; B, Effect of isoleucine gradient addition on emetic toxin of B. cereus F4810 / 72. As can be seen from the figure, the toxin production of different emetic strains is significantly reduced after isoleucine treatment.

[0021] Figure 3 Effect of emetic toxin extract of B. cereus 892-1 (A) and F4810 / 72 (B) before and after isoleucine addition on Hela cell activity. A, Effect of isoleucine addition on Hela cell activity induced by emetic toxin extract of B. cereus 892-1; B, Effect of isoleucine addition on Hela cell activity induced by emetic toxin extract of B. cereus F4810 / 72. As can be seen from the figure, the virulence of B. cereus decreases and the survival rate of Hela cells significantly increases after isoleucine treatment.

[0022] Figure 4 Effect of 20 mM isoleucine on emetic toxin synthesis of B. cereus F4810 / 72 (A) and 892-1 (B) in face soup and milk, 0 and 20 mM represent the concentration of isoleucine. As can be seen from the figure, isoleucine can significantly inhibit the synthesis of emetic toxin in food matrix face soup and milk. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0024] LB broth (medium) was purchased from Guangdong Huanke Microbial Technology Co., Ltd.; isoleucine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; emetic toxin cereulide standard was purchased from Japan Wako; 1% penicillin-streptomycin DMEM high-sugar culture medium was purchased from Gibco.

[0025] The following examples are further illustrations of the application and are not intended to limit the application.

[0026] Example: Method for controlling emetic toxin production of Bacillus by isoleucine

[0027] (1) Effect of isoleucine on the growth of B. cereus

[0028] First, *Bacillus cereus* 892-1 and *Bacillus cereus* F4810 / 72 (accession number: NCTC 11143), both emetic strains isolated from pasteurized milk, were inoculated into LB broth at a 1% (v / v) inoculum and cultured at 37°C and 200 rpm for 12 h for resuscitation. Subsequently, LB broth media containing isoleucine at concentrations ranging from 0 to 20 mM were prepared. The resuscitated bacterial cultures were transferred to the LB broth media containing different isoleucine concentrations at a 1% (v / v) inoculum, mixed thoroughly, and 200 μL was added to 96-well plates. Each treatment group included a blank control (containing no bacterial culture) and three replicate wells. OD values ​​at different time points were measured using a microplate reader. 600nm Measurements were taken every 0.5 hours for 24 hours. Zeroing and calibration were performed using a blank control well before each measurement. Finally, growth curves were plotted based on the obtained data. Results are as follows: Figure 1 As shown, the results indicate that there were no significant differences in the growth of the various vomit-inducing strains under different concentrations of isoleucine treatment.

[0029] (2) Effects of isoleucine on the production of vomitoxin in Bacillus cereus

[0030] Emetic strain 892-1 and emetic clinical reference strain F4810 / 72, isolated from pasteurized milk, were inoculated into 100 mL of LB broth containing 0-20 mM (0, 1, 5, 10, 20 mM) isoleucine and cultured at 30°C and 150 rpm for 24 hours with shaking. 45 mL of the bacterial culture was transferred to a 50 mL centrifuge tube and centrifuged at 4°C and 8000 g for 5 minutes, discarding the supernatant. This centrifugation step was repeated once. The mass of the centrifuge tube before and after adding the bacterial culture was recorded to calculate the wet weight of the bacteria. Then, 9.9 mL of LC-MS grade acetonitrile and 100 μL of 100 μg / mL valamicin (internal standard) were added to the centrifuge tube, and the mixture was vortexed until no obvious bacterial residue remained on the tube wall. The centrifuge tube was then placed in a shaker at 30°C and 150 rpm for 1 hour to extract the vomitoxin. After extraction, centrifuge at 4500g for 5min, discard the bacterial cells, collect the supernatant, filter the vomitoxin extract through a 0.22μm organic filter membrane, dilute the filtrate 20 times with acetonitrile, and take 200μL into a liquid chromatography-mass spectrometry (LC-MS) vial.

[0031] Using AB SCIEX Triple Quad TM 5500 + Vomitoxin detection was performed using a Ready mass spectrometry system (AB SCIEX Pte. Ltd., Framingham, USA) and a Waters C18 column (1.7 μm, 2.1 × 100 mm). Chromatographic separation employed a gradient elution program as shown in Table 1. The mass spectrometry parameters were set as follows: ion source voltage 5500 V, desolvation temperature 550 °C, ion source temperature 550 °C, collision gas pressure 9 psi, and injection volume 5 μL.

[0032] In MS1 ​​scans, vomitoxin is converted to ammonium ions [M+NH4]. + The most abundant ion was vomitoxin, with an m / z of 1170.70. The quasi-molecular ion peak of the internal standard valproicin was located at m / z 1128.60; both were used as precursor ions for quantitative analysis. Characteristic fragment ions 1170.60→357.40 (vomiting toxin) and 1128.70→343.10 (valproicin) were selected in MS2 for multiple reaction monitoring (MRM). Data acquisition and analysis were performed using software (AB SCIEX), and the standard curve was prepared and used for quantification according to the appropriate methods. The results are as follows: Figure 2 As shown, from Figure 2 It can be seen that the production of vomitoxin by Bacillus cereus was significantly reduced under isoleucine treatment.

[0033] Table 1 Liquid phase elution conditions

[0034]

[0035] Note: Mobile phase A is ultrapure water containing 0.1% formic acid and 10mM ammonium formate by volume; mobile phase B is methanol containing 0.1% formic acid by volume.

[0036] Table 2 Mass spectrometry detection parameters for vomitoxin and valine.

[0037]

[0038]

[0039] Note: * indicates quantitative ion pairs.

[0040] (3) Cytotoxicity assay to evaluate the effect of isoleucine on the virulence of Bacillus cereus.

[0041] In the cytotoxicity experiment, the extraction of vomitoxin was carried out according to step (2). The vomitoxin extract obtained in (2) was autoclaved at 121°C for 17 minutes to inactivate the enterotoxin. To avoid the toxicity of acetonitrile to cells, 10 mL of methanol was used as the extraction solvent. After vortexing and mixing, the extract was filtered through a 0.22 μm organic filter membrane. The obtained vomitoxin extract was stored at -20°C for later use.

[0042] HeLa cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C, 5% CO2, and saturated humidity, and passaged every 24 hours. Cells in the logarithmic growth phase were trypsinized, centrifuged and resuspended, then counted under a microscope and the cell concentration was adjusted to 1000 cells per well. The cells were then seeded in 96-well plates, and the outer wells were filled with complete medium (DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) to eliminate edge effects. Once the cell adhesion rate reached 80%, the original culture medium was discarded. The vomitoxin extract was diluted 500-fold with the aforementioned complete culture medium (DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin) containing 2% ethanol (vomitoxin solubilizer). 100 μL of this solution was added to each well [the positive control group received an equal volume of culture medium containing vomitoxin standard (cer), and the negative control group received an equal volume of culture medium containing methanol]. The cells were cultured for another 24 h. Afterward, the culture medium was discarded, and the cells were washed with PBS. A working solution was prepared by mixing CCK-8 reagent with DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at a volume ratio of 1:10. 100 μL of the CCK-8 working solution was added to each well, and the cells were incubated for 1 h. The mixture was then shaken to mix, and the absorbance was measured at 450 nm. Cell viability was calculated using the following formula.

[0043] Cell viability = OD of treatment group 450nm / Control group OD 450nm

[0044] The culture medium containing 1000 μg / L vomitoxin standard (cer) is prepared by diluting the 1000 μg / L vomitoxin standard 500 times with the aforementioned complete culture medium containing 2% ethanol (vomitoxin solubilizer) by volume.

[0045] The methanol-containing culture medium is prepared by diluting the methanol 500 times with the aforementioned complete culture medium containing 2% ethanol (a solubilizer for vomitoxin) by volume.

[0046] The results are as follows Figure 3 As shown, treatment with 20 mM isoleucine can effectively inhibit the synthesis of vomitoxin and improve cell survival rate.

[0047] (4) Isoleucine inhibits toxicity in food matrices

[0048] Add 50g of Golden Dragon multipurpose wheat flour to 500mL of distilled water, boil for 30min, filter through sterile gauze, and collect the filtrate as noodle soup. Commercially available Yili pure milk can be used directly without additional processing. Add the prepared noodle soup or milk to LB broth medium at a volume ratio of 4:100. Then, inoculate pasteurized milk-source vomiting Bacillus cereus strain 892-1 and vomiting clinical reference strain F4810 / 72 into 100mL of LB broth medium containing milk or noodle soup containing 0 or 20mM isoleucine, respectively, to simulate the real food matrix environment. Incubate at 28℃ and 200rpm for 24 hours. Toxin extraction and determination are as described in step (2). At the same time, the viable bacteria in the sample are counted by the dilution plating method: after the culture is completed, take 1mL of bacterial solution and perform serial dilution (10 -5 10 -6 10 -7 10 -8 Spread the culture onto LB agar plates and incubate at 30°C for 24 hours. Count the colony-forming units (CFU). Select 10... -6 and 10 -7 Gradients were used to assess bacterial growth. The results are as follows: Figure 4 As shown, from Figure 4 It can be seen that isoleucine can also significantly inhibit the synthesis of vomitoxin in food matrices. The colony count results are shown in Table 3. The addition of isoleucine had no significant effect on the final bacterial count of the two Bacillus cereus strains, indicating that isoleucine does not affect bacterial growth in the food matrices of soup and milk.

[0049] Table 3. Colony counts of Bacillus cereus 892-1 and F4810 / 72 in food matrices

[0050]

Claims

1. Application of isoleucine in inhibiting the production of vomitoxin in Bacillus cereus.

2. The application according to claim 1, characterized in that, Application of isoleucine in the preparation of agents that inhibit vomitoxin production by Bacillus cereus.

3. The application according to claim 2, characterized in that, Isoleucine is added to the matrix where it is necessary to inhibit the production of vomitoxin by Bacillus cereus.

4. The application according to claim 3, characterized in that, The substrate that needs to inhibit the production of vomitoxin by Bacillus cereus is food.

5. The application according to claim 4, characterized in that, The food products mentioned are starchy foods, milk and dairy products, meat and meat products, fruits or vegetables.

6. The application according to claim 3, characterized in that, The isoleucine is added to the matrix where it is necessary to inhibit the production of vomitoxin by Bacillus cereus, and the isoleucine concentration is 5-20 mM.

7. A method for inhibiting the production of vomitoxin by Bacillus cereus, characterized in that, The process includes the following steps: adding isoleucine to the substance that needs to inhibit Bacillus cereus from producing vomitoxin, thereby inhibiting the production of vomitoxin by Bacillus cereus.

8. The method according to claim 7, characterized in that, The substance mentioned that needs to inhibit the production of vomitoxin by Bacillus cereus is a food product.

9. The method according to claim 8, characterized in that, The food products mentioned are starchy foods, milk and dairy products, meat and meat products, fruits or vegetables.

10. The method according to claim 7, characterized in that, The concentration of isoleucine is 5-20 mM.

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