A method for degrading aflatoxins using bacillus

Abstract

The application discloses a method for degrading aflatoxin by using bacillus, 5ml of bacterial liquid is transferred into a sterilized glass tube, aflatoxin standard B1 and G1 are added, and the final concentration is adjusted, meanwhile, a blank medium with standard samples is used as a control, and culture is carried out at 37 DEG C on a shaking table; centrifugal and filtration treatment are carried out on the sample, and the treated sample is detected on a machine; S2: the degradation product of bacillus 21-1-2 for degrading aflatoxin is identified by using high performance liquid chromatography-high resolution mass spectrometry combined with UNIFI 1.8.2 / Masslynx V 4.1 software, and the final identification result is obtained; the method adopts real-time external standard method mass number correction technology, ensures high accuracy of the mass number, and has small mass number deviation; the MSE full information tandem mass spectrometry data acquisition mode has no content discrimination, ensures full acquisition of information, acquisition of fragment ions of low response compounds, and simultaneously ensures accuracy of the mass number of parent ions and fragment ions; the metabolite identification result is that bacillus 21-1-2 can degrade Alfatoxin G1 into Aflatoxin G1+CH2CH2-C3H2O.

Description

[0001] This application is a divisional application. The original application was entitled "A Method for Identifying Aflatoxin Degradation Products by Bacillus," application number 202310911982.9, and application date July 24, 2023. Technical Field

[0002] This invention relates to the field of food testing technology, and specifically to a method for identifying degradation products of aflatoxin by Bacillus subtilis. Background Technology

[0003] Aflatoxin (AFT) is a difuran ring toxin produced by certain strains of Aspergillus flavus and Aspergillus parasiticus. This toxin is a potent biotoxic chemical substance classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization and is widely found in various foods. Grains such as corn, peanuts, rice, and wheat, and their byproducts, are major sources of aflatoxin. Aflatoxin is one of the three major causes of liver cancer and a leading cause of stomach and intestinal cancers, posing a significant threat to human health. Countries worldwide have strict limits on aflatoxin levels in food and animal feed.

[0004] The prevention and control of aflatoxin contamination mainly involves chemical control, biological control, and strengthened post-harvest storage management. The use of chemical reagents can lead to strain resistance, and improper use can even increase toxin production. Biological control is the most promising and effective method for reducing or completely eliminating fungal toxin contamination. Currently, strains that can antagonize Aspergillus flavus or degrade aflatoxin have been screened from lactic acid bacteria, Bacillus, yeasts, and Aspergillus.

[0005] This study collected soil samples from peanut-growing areas and screened out Bacillus 21-1-2, an antagonistic bacterium that can strongly degrade aflatoxin. The degradation products of aflatoxin by Bacillus 21-1-2 were identified using high-performance liquid chromatography-high-resolution mass spectrometry combined with UNIFI 1.8.2 / Masslynx V 4.1 software. A preliminary analysis of the degradation mechanism was conducted, providing a new approach for basic research on aflatoxin pollution control. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a novel identification method for aflatoxin degradation products using Bacillus subtilis, providing a new direction for basic research on aflatoxin pollution control.

[0007] The specific technical solution of the present invention is as follows: A method for identifying degradation products of aflatoxin by Bacillus subtilis, the method comprising the following steps: S1: Inoculate Bacillus 21-1-2 into nutrient broth medium and incubate at 37°C for 24 hours to revive. Transfer the revived Bacillus to a new medium and incubate at 37°C for 48 hours. Take 5 ml of the bacterial culture and transfer it to a sterilized glass tube. Add aflatoxin standards B1 and G1 and adjust to the final concentration. Use a blank medium with standards as a control. Incubate at 37°C. Centrifuge and filter the samples, and then perform instrumental analysis on the treated samples. S2: The degradation products of aflatoxin from Bacillus 21-1-2 were identified by high-performance liquid chromatography-high-resolution mass spectrometry combined with UNIFI 1.8.2 / Masslynx V 4.1 software, and the final identification results were obtained. It should be noted that the specific structural formula of aflatoxin G1 studied in this application is as follows: Aflatoxin G1: . In one specific embodiment, in step S1, the standard is AFG with a concentration of 20 ppb.

[0008] In one specific embodiment, step S2 involves identifying the degradation products of aflatoxin by Bacillus 21-1-2 using high-performance liquid chromatography-high-resolution mass spectrometry combined with UNIFI 1.8.2 / Masslynx V 4.1 software. The identification results show that Bacillus 21-1-2 can degrade Alfatoxin G1 into Aflatoxin G1+CH2CH2-C3H2O.

[0009] In one specific embodiment, in step S1, Bacillus 21-1-2 is used for aflatoxin degradation, while a blank culture medium with added standards is used as a control.

[0010] In one specific embodiment, the conditions for high-performance liquid chromatography in step S2 are as follows: Chromatographic column: HSS T3 2.1*100mm, 1.8m Column temperature: 40℃ Mobile phase A: H2O Mobile phase B: ACN.

[0011] In one specific embodiment, the conditions for high-performance liquid chromatography in step S2 are as follows: Chromatographic column: HSS T3 2.1*100mm, 1.8m Column temperature: 40℃ Mobile phase A: H2O Mobile phase B: ACN.

[0012] In one specific embodiment, the gradient table of the high-performance liquid chromatography is shown below:

[0013] In one specific embodiment, the analytical conditions for mass spectrometry in step S2 are as follows: Ionization mode: ESI+ Capillary voltage: 3.0kV Source temperature: 120℃ Atomizing gas temperature: 550℃ Atomizing airflow rate: 1000L / h Acquisition mode: MSE.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. Bacillus 21-1-2 can completely degrade aflatoxin G1, and the degradation products were identified for the first time. The results showed that Bacillus 21-1-2 can degrade Alfatoxin G1 (molecular formula C17H12O7) into Aflatoxin G1+CH2CH2-C3H2O (molecular formula C16H14O6).

[0015] 2. The Xevo G2-XS QTOF system combines LockSprary real-time external standard method mass number correction technology to ensure high accuracy of mass numbers and small mass number deviation; 3. The MSE full-information tandem mass spectrometry data acquisition method has no content discrimination and ensures the full acquisition of information, especially the acquisition of fragment ions of low-response compounds, while ensuring the accuracy of parent ions, fragment ions, and mass numbers. Attached Figure Description

[0016] Figure 1 This is the chromatogram of aflatoxin G1.

[0017] Figure 2 This is the mass spectrum of aflatoxin G1.

[0018] Figure 3 This is a graph showing the data analysis in the identification of known metabolites. Detailed Implementation

[0019] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0020] The overall scheme of this invention is as follows: Bacillus 21-1-2 is inoculated into nutrient broth medium and cultured in a shaker at 37°C for 24 hours to recover; the recovered Bacillus is then transferred to a new culture medium and cultured in a shaker at 37°C for 48 hours. 5 ml of bacterial culture was transferred to a sterilized glass tube, and aflatoxin standards B1 and G1 were added and adjusted to the final concentration. A blank culture medium with standards was used as a control. The samples were incubated at 37°C in a shaker. The samples were centrifuged and filtered, and then analyzed. The degradation products of aflatoxin by Bacillus 21-1-2 were identified using high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-MS / MS) combined with UNIFI 1.8.2 / Masslynx V 4.1 software. The final identification results were obtained. The method employed real-time external standard mass number correction technology to ensure high accuracy and small deviation of the mass numbers. MSE (Metabolic Sequencing) tandem mass spectrometry data acquisition mode was used, without content discrimination, ensuring full information acquisition, acquisition of fragment ions of low-response compounds, and simultaneously ensuring the accuracy of the mass numbers of the parent ion and fragment ions. Metabolite identification results: Bacillus 21-1-2 can degrade Alfatoxin G1 (molecular formula C17H12O7) into Aflatoxin. G1+CH2CH2-C3H2O (molecular formula C16H14O6).

[0021] The specific operating steps of this invention are as follows: Refer to Figure 1-2 As shown 1. Inoculate Bacillus 21-1-2 into nutrient broth medium and incubate at 37°C in a shaker for 24 hours to revive.

[0022] 2. Transfer the revived Bacillus to a new culture medium at an inoculation rate of 10% and incubate at 37°C in a shaker for 48 hours.

[0023] 3. Take 5 ml of bacterial culture and transfer it to a sterilized glass tube. Add aflatoxin standard G1 to a final concentration of 80 ppb. At the same time, use blank culture medium with the standard as a control. Incubate at 37°C in a shaker.

[0024] 4. Sample processing: Take 1 ml of the sample after 4 days of culture, centrifuge at 10,000 rpm for 10 min, and filter through a 0.22 μm filter membrane.

[0025] Example 1: 1) Experimental Objective Identification of degradation products of aflatoxin G1 by Bacillus 21-1-2; Sample information: – Control group reference: Blank culture medium + aflatoxin G1 standard –Experimental group sample: Blank culture medium + aflatoxin G1 standard + Bacillus subtilis 21-1-2 Each group was repeated 10 times. Sample processing method: –Injection volume: 5 l.

[0026] 2) Experimental apparatus ACQUITY UPLC I class System; Xevo G2-XS QTOF; Analysis software: UNIFI 1.8.2 / Masslynx V 4.1.

[0027] 3) Experimental methods 31) UPLC conditions Chromatographic column: HSS T3 2.1*100mm, 1.8m Column temperature: 40℃ Mobile phase A: H2O Mobile phase B: ACN.

[0028] gradient table

[0029] 32)MS conditions Ionization mode: ESI+ Capillary voltage: 3.0kV Source temperature: 120℃ Atomizing gas temperature: 550℃ Atomizing airflow rate: 1000L / h Acquisition mode: MS.

[0030] 33) Software Masslynx V4.1 / UNIFI.

[0031] 4) The identification process for metabolites is as follows: Figure 1-3 As shown, 41) Chromatogram and mass spectrum of aflatoxin G1; 42) Data acquisition and analysis process; 43) Specific process of UNIFI metabolite data processing Edit the list of Expected metabolites οDealkylaiton metabolic pathways The collected data was checked and matched against the edited list of metabolites. οm / z, parent ion οm / z, fragment ions Additionally, the software can predict and match fragments based on their structure (Massfragment function). Example 2: Identification process of known metabolites 1. Metabolite Identification Process - Identification Results: UNIFI matches metabolite lists based on m / z, parent ion / fragment ion, etc., and the matched components are marked as Identified.

[0032] 2. Metabolite Identification Process - Data Analysis: Based on the chromatographic peak shape / retention time, mass number accuracy, fragmentation information, binary comparison, etc., further confirm whether it is a drug metabolite, and if possible, further identify the structure; Taking AlfatoxinG1+C2H4-C3H2O as an example, refer to Figure 3 .

[0033] The present invention has been described above with reference to specific embodiments. However, those skilled in the art should understand that these descriptions are exemplary and not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications and variations to the present invention based on its spirit and principles, and these modifications and variations are also within the scope of the present invention.

Claims

1. A method for degrading aflatoxin using Bacillus subtilis, characterized in that, Includes the following steps: Bacillus 21-1-2 was inoculated into nutrient broth medium and cultured at 37°C for 24 hours to recover. The recovered Bacillus was then transferred to a new medium and cultured at 37°C for 48 hours. 5 ml of the bacterial culture was transferred to a sterilized glass tube, and aflatoxin standard G1 was added to adjust to the final concentration. A blank medium with the standard was used as a control. The samples were then cultured at 37°C. The samples were centrifuged and filtered to obtain the aflatoxin degradation products from Bacillus.

2. A formulation for degrading aflatoxin, characterized in that, Including Bacillus 21-1-2.

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