Application of nitrite reductase AfNiR in simultaneous degradation of aflatoxin B1 and zearalenone
By allowing nitrite reductase AfNiR to bind with Cu2+ under specific conditions, the problems of low efficiency and poor stability in enzymatic degradation of aflatoxin B1 and zearalenone were solved, achieving highly efficient detoxification of food and feed.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HENAN UNIVERSITY OF TECHNOLOGY
- Filing Date
- 2026-02-28
- Publication Date
- 2026-05-29
AI Technical Summary
Existing enzymatic methods are difficult to effectively degrade aflatoxin B1 and zearalenone in food and feed, especially in oil systems where they have low catalytic efficiency and poor stability. Furthermore, traditional enzymes are deactivated at high temperatures, which limits their industrial application.
The nitrite reductase AfNiR was used to degrade aflatoxin B1 and zearalenone by binding with Cu2+ under conditions of 40-70℃ and pH 7-8. The unique redox catalytic ability and good structural stability of AfNiR were utilized to achieve efficient degradation of the two toxins.
It achieves highly efficient degradation of aflatoxin B1 and zearalenone in food and feed, with degradation rates of up to 99.22% and 93.53% respectively. It maintains more than 80% catalytic activity within a certain temperature range and is suitable for detoxification in the food and feed industries.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme engineering and relates to the degradation of aflatoxin B1 and zearalenone. Background Technology
[0002] Mycotoxins are a class of fungal secondary metabolites widely found in contaminated feed and food. According to data from the Food and Agriculture Organization of the United Nations (FAO) in 1985, more than 25% of global crops were contaminated with mycotoxins. This has caused enormous economic losses to the livestock and food industries and seriously threatens food safety and human and animal health. Aflatoxin B1 (AFB1), zearalenone (ZEN), ochratoxin (OTA), and deoxynivalenol (DON) are the most common mycotoxins. AFB1, mainly produced by *Aspergillus flavus* and *Aspergillus parasiticus*, has carcinogenic, teratogenic, and immunosuppressive effects and has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer. ZEN, mainly produced by *Fusarium graminearum*, *Fusarium culmorum*, *Fusarium sporotrichioides*, and *Trichoderma* spp., has reproductive toxicity, hepatotoxicity, immunotoxicity, and genotoxicity. Toxins from the same or different species can lead to comprehensive toxicity due to their interactions. Co-toxicity can be synergistic, additive, or antagonistic, depending on the combination of mycotoxins. For example, the combination of AFS with different AFS or other carcinogenic fungal toxins can lead to synergistic toxicity. The combination of AFB1 and ZEN can produce a synergistic effect in terms of cytotoxicity. Generally, symbiotic fungal toxin contamination can lead to more serious health problems compared to single fungal toxin contamination. Furthermore, mycotoxins cause significant economic losses to the food and livestock sectors annually. Aflatoxin B1 (AFB1) and zearalenone (ZEN) are widely present in the processing and production of agricultural products and by-products such as peanuts, corn, wheat, and rice, posing a serious threat to human and animal health. Due to the strong physicochemical stability of these two mycotoxins, conventional food processing and heat treatment are insufficient to effectively remove them, and they can further migrate and accumulate in highly processed products during raw material processing. Especially during the pressing and refining process of peanut oil and corn oil, AFB1 and ZEN can enter the oil system along with lipid components. Therefore, AFB1 and ZEN are the most common contaminants in peanut oil and corn oil.
[0003] Many methods, including biological, chemical, and physical methods, have been used to remove mycotoxins from grains and feed. However, physical detoxification methods suffer from poor specificity and unstable effectiveness, while chemical detoxification methods suffer from nutrient loss, implementation difficulties, and susceptibility to secondary contamination. In recent years, biological detoxification technology has become a research hotspot due to its advantages of high efficiency, short cycle, high safety, strong specificity, and environmental friendliness. Several enzymes isolated from microorganisms, including manganese peroxidase (MNP), Pseudomonas aFB1-degrading enzyme (PADE), and laccase, have been found to have biodegrading activity against AFB1. In addition, microbial-derived enzymes such as ASE, ZHD (ZHD), and ZEN hydrolase 101 (ZD101) have been reported to degrade ZEN. However, only a few types of enzymes that simultaneously degrade AFB1 and ZEN have been discovered, such as laccase, Cota laccase (BsCotA), BsDyP, and multicopper oxidase (MCO). Currently, there are relatively few enzymes available for the detoxification of symbiotic mycotoxins. Therefore, the isolation, identification, and development of novel enzymes capable of simultaneously degrading aflatoxin B1 and ZEN in food and feed are of great and urgent significance. Theoretically, enzymatic degradation is a promising strategy for removing fungal toxins from vegetable oils. However, the practical application of natural enzymes in degrading aflatoxin B1 and zearalenone in edible oils is limited by multiple factors, including low stability and low efficiency. Furthermore, the incompatibility between the oil system and the aqueous phase restricts enzyme-substrate contact, further reducing catalytic efficiency (Lu et al., 2023). In particular, enzymes with poor thermal stability struggle to withstand harsh environmental conditions and cannot maintain catalytic activity for extended periods at high temperatures (Suresh et al., 2021). Therefore, enzymes possessing both amphiphilicity and good thermal stability are crucial for meeting their application requirements under industrial conditions. It is necessary to develop effective strategies to improve the stability, lipophilicity, and catalytic efficiency of enzymes in oil systems to promote the practical application of enzymatic detoxification technology in edible oils.
[0004] Nitrite reductases (NiRs) are a class of enzymes that play a crucial role in the nitrogen cycle. Based on the type of metal ion bound to their catalytic active sites, they are generally classified into copper-dependent nitrite reductases (Cu-NiRs) and heme-dependent nitrite reductases (heme-NiRs). Copper-containing nitrite reductases (NIRs) are redox enzymes with copper ion active sites, catalyzing the reduction of nitrite to nitric oxide. They are one of the key functional enzymes in microbial denitrification. NIRs typically contain type I (T1Cu) and type II (T2Cu) copper centers, which participate in electron transfer and substrate reduction, respectively, playing a vital role in the nitrogen cycle. Therefore, they are considered core enzymes for maintaining microbial nitrogen metabolism homeostasis and environmental nitrogen balance. Due to their unique redox catalytic ability and good structural stability, NIRs show potential application value in environmental remediation, biocatalysis, and biosensing. Although the traditional functions of NIRs are mainly focused on denitrification metabolism, their metal active centers and plastic substrate-binding pockets enable them to catalyze non-classical substrates, providing a theoretical basis for their application in pollutant transformation and biodetoxification. Related studies suggest that some NIRs or NIR-like proteins may play important roles in atypical substrate transformation, but their specific functions are not yet fully elucidated. However, to date, there are no reports on the participation of copper-containing nitrite reductases in mycotoxin degradation, and their catalytic efficiency, substrate specificity, and reaction mechanisms in mycotoxin transformation lack systematic research. This research gap further highlights the necessity and importance of exploring novel biological functions of NIRs and their potential applications in mycotoxin biodetoxification. Currently, most reported degradative enzymes can only degrade specific types of mycotoxins, and this specificity limits their degradation effectiveness in practical applications. Therefore, the search and identification of new broad-spectrum enzymes for mycotoxin biodegradation is urgently needed. Summary of the Invention
[0005] To address the detoxification of mycotoxins in the food and feed industries, this invention proposes the application of a nitrite reductase, AfNiR, in the simultaneous degradation of aflatoxin B1 and zearalenone.
[0006] The technical solution of this invention is implemented as follows:
[0007] On one hand, this invention provides the application of nitrite reductase AfNiR in the simultaneous degradation of aflatoxin B1 and zearalenone, wherein the nitrite reductase AfNiR has an amino acid sequence similarity of more than 70% to that shown in SEQ ID No. 1. Amino acid sequences with an amino acid sequence similarity of more than 70% to that of this application can retain the degradation function of the nitrite reductase of this application.
[0008] Preferably, the amino acid sequence of the above-mentioned nitrite reductase AfNiR is shown in SEQ ID No. 1.
[0009] The gene encoding the aforementioned nitrite reductase AfNiR has a nucleotide sequence similarity of more than 90% with the nucleotide sequence shown in SEQ ID No. 2. A nucleotide sequence with a nucleotide sequence similarity of more than 90% with the nucleotide sequence of this application can express the amino acid sequence of this application, for example, as shown in SEQ ID No. 3, which is a nucleotide sequence optimized from E. coli codons, and its sequence has a similarity of more than 90% with the gene sequence shown in SEQ ID No. 2.
[0010] Preferably, the nucleotide sequence of the above-mentioned gene is shown in SEQ ID No. 2 or SEQ ID No. 3.
[0011] Secondly, the present invention provides a biological material comprising nitrite reductase AfNiR, a recombinant vector expressing nitrite reductase AfNiR, or engineered bacteria expressing nitrite reductase AfNiR.
[0012] Thirdly, protection is requested for the application of the aforementioned biological materials in detoxification in the food or feed industry. The aforementioned detoxification refers to the degradation of aflatoxin B1 and / or zearalenone. The detoxification temperature is 40-70℃, and the pH is 7-8; 0.5 mM Cu is also added during detoxification. 2＋ .
[0013] Fourthly, the present invention provides a method for degrading aflatoxin B1 and / or zearalenone in food or feed, wherein the above-mentioned biological material and Cu 2＋ Added to food or feed to be processed, wherein Cu 2＋ The final concentration is 0.5 mM. The food or feed is treated at 40-70℃ and pH 7-8 for 1-2 days until the aflatoxin B1 and / or zearalenone in the food or feed is reduced to the industrial required level.
[0014] Furthermore, the amount of biological material added shall be based on the total amount of food or feed, and the amount of nitrite reductase AfNiR in the biological material shall be 100 μg / mL.
[0015] The optimal temperature for nitrite reductase AfNiR to degrade aflatoxin B1 is 40℃, the optimal pH is 7.0, and the degradation rate is 99.22%.
[0016] Nitrite reductase AfNiR can maintain a degradation rate of over 80% for aflatoxin B1 within a temperature range of 40-70℃.
[0017] The optimal temperature for nitrite reductase AfNiR to degrade zearalenone in zearalenone was 50℃, the optimal pH was 8.0, and the degradation rate was 93.53%.
[0018] Nitrite reductase AfNiR can maintain a degradation rate of over 80% for zearalenone within a temperature range of 40-60℃.
[0019] The present invention has the following beneficial effects:
[0020] 1. This application discloses a fungal toxin-degrading enzyme—nitrite reductase AfNiR—capable of degrading AFB1 and ZEN. The optimal temperature for AFB1 degradation is 40°C and the optimal pH is 7.0. This enzyme achieves a degradation rate of 92.92% for AFB1. Simultaneously, nitrite reductase AfNiR can maintain a degradation rate of over 80% for aflatoxin B1 within a temperature range of 40-70°C, making it a fungal toxin-degrading enzyme that can withstand a certain degree of high temperature. This invention provides a promising candidate for AFB1 and ZEN detoxification in the food and feed industries.
[0021] 2. Grains are generally susceptible to AFB1 and ZEN contamination, especially peanuts and corn. Under the conditions described in this invention, nitrite reductase AfNiR can achieve degradation rates of up to 70.6% and 60.2% for AFB1 and ZEN in contaminated peanut flour and corn flour, respectively, showing promising application prospects in the purification of AFB1 and ZEN in food or feed.
[0022] 3. This enzyme can also effectively degrade ZEN, with an optimal degradation temperature of 50℃ and pH 8.0. The enzyme's ZEN degradation rate can reach 93.53%. Meanwhile, nitrite reductase AfNiR can maintain a ZEN degradation rate of over 80% within a temperature range of 40-60℃. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0024] Figure 1 for Figure 1 The plasmid map is for pET-28a(+).
[0025] Figure 2 The image shows the recombinant plasmid pattern of pET-28a(+)-AfNiR.
[0026] Figure 3 This is an SDS-PAGE validation image of nitrite reductase AfNiR, where M: standard protein marker; P: purified protein.
[0027] Figure 4 The degradation rates of AFB1 and ZEN by nitrite reductase AfNiR under different metal ion conditions are shown.
[0028] Figure 5 The degradation rates of AFB1 and ZEN by nitrite reductase AfNiR under different pH conditions are shown.
[0029] Figure 6 The degradation rates of AFB1 and ZEN by nitrite reductase AfNiR under different temperature conditions are shown.
[0030] Figure 7 The degradation rates of AFB1 and ZEN were measured by different amounts of nitrite reductase AfNiR.
[0031] Figure 8 The degradation rates of AFB1 and ZEN by nitrite reductase AfNiR at different reaction times are shown.
[0032] Figure 9 The degradation effect of nitrite reductase AfNiR on AFB1 contaminated peanut flour and the degradation effect of nitrite reductase AfNiR on ZEN contaminated corn flour were compared (50 μg / mL nitrite reductase AfNiR treatment group and 100 μg / mL nitrite reductase AfNiR treatment group, respectively).
[0033] Figure 10 This is a secondary mass spectrum of the degradation products of AFB1 by nitrite reductase AfNiR.
[0034] Figure 11 This is a secondary mass spectrum of the degradation products of ZEN by nitrite reductase AfNiR. Detailed Implementation
[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0036] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.
[0037] Strains: Escherichia coli DH5α, Escherichia coli BL21(DE3), purchased from Sangon Biotech (Shanghai) Co., Ltd.
[0038] Culture medium: LB medium: 10 g·L tryptone, 5 g·L yeast extract, 10 g·L sodium chloride, pH 7.0-7.2.
[0039] Experimental reagents: AFB1 and ZEN standard samples were purchased from Sigma-Aldrich; other reagents were domestically produced analytical grade.
[0040] Example 1: Preparation of recombinant Escherichia coli BL21 (DE3)
[0041] The nucleotide sequence of the AfNiR encoding gene of nitrite reductase, as shown in SEQ ID No. 2, was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and codon optimization was performed based on the preference of Escherichia coli BL21(DE3). The optimized sequence is shown in SEQ ID No. 3. A restriction enzyme site Sac1 was introduced at the 5-terminus of the encoding gene, and a restriction enzyme site Not I was introduced at the 3-terminus.
[0042] The amino acid sequence of this gene is shown in SEQ ID No. 1. The gene with the nucleotide sequence shown in SEQ ID No. 3 was ligated into the *E. coli* expression vector pET-28a(+). Figure 1 At the corresponding site, the recombinant plasmid pET-28a(+)-AfNiR (as shown in the image) was obtained. Figure 2 (As shown). The obtained recombinant plasmid was added to E. coli DH5a competent cells, heat-shocked in a 42°C water bath for 45 seconds, and then immediately placed on ice to cool for 2-3 minutes for transformation of the recombinant product. For the correctly transformed strains, the plasmid was extracted and introduced into the E. coli expression strain BL21(DE3) by heat shock. Positive clones were screened by LB agar containing kanamycin and verified by bacterial PCR to obtain recombinant E. coli that successfully expressed nitrite reductase AfNiR.
[0043] The PCR amplification system and amplification program are shown in Tables 1 and 2.
[0044] Table 1 PCR amplification system
[0045]
[0046] Table 2 PCR amplification program
[0047] .
[0048] Example 2: Induction and purification of nitrite reductase AfNiR
[0049] 1. Inducible expression of nitrite reductase AfNiR
[0050] Escherichia coli BL21(DE3) / pET-28a(+)-AfNiR was inoculated into 25 mL of LB medium and cultured at 37 °C with shaking at 200 rpm for 12 h. Then, 2% of the culture was inoculated into 300 mL of LB liquid medium and cultured at 37 °C with shaking at 200 rpm until OD600 = 0.6~0.8. Finally, IPTG was added to a final concentration of 0.1 mM and induced at 16 °C for 20 h.
[0051] 2. Purification of nitrite reductase AfNiR
[0052] Collect the induced bacterial culture, centrifuge at 8000 rpm for 10 min at 4℃, and discard the supernatant. Resuspend the bacterial cells in 10 mL of equilibration buffer, sonicate for 30 min under ice bath conditions (sonication power 300 W, sonication on for 4 s, sonication off for 4 s), centrifuge at 12000 rpm for 10 min at 4℃, collect the supernatant and filter it through a 0.45 u aqueous filter membrane. Then, purify the protein using a nickel-NTA column with a prepared 200 mM imidazole elution buffer, collect the eluent, and ultrafilter the eluent through a 10 kDa ultrafiltration tube at 4000 rpm for 30 min at 4℃ to remove imidazole, obtaining the purified protein. The purification effect of the purified protein was detected by SDS-PAGE gel electrophoresis, and the results are shown below. Figure 3 As shown.
[0053] Application Example 1: Degradation of AFB1 by the purified nitrite reductase AfNiR prepared in Example 2
[0054] The protein purified in Example 2 was analyzed for protein concentration using a Bradford protein assay kit and stored at 4°C for later use. The degradation of AFB1 by nitrite reductase AfNiR was carried out in a 500 μL system containing 5.0 μg / mL AFB1, 20 μg AfNiR protein, and 50 mM Tris-HCl buffer (containing 0.5 mM metal ions, pH 7.0). After co-incubation for 24 h, the reaction was terminated by adding 500 μL of methanol. The system was thoroughly mixed and filtered through a 0.22 μm organic filter membrane. The AFB1 content was determined by high-performance liquid chromatography (HPLC).
[0055] The chromatographic conditions for detecting AFB using high performance liquid chromatography were as follows: column: Proshe11 120-C18 column (150×4.6 mm, 4 μm); UV detector: detection wavelength 365 nm; flow rate: 1 mL / min; injection volume: 10; acquisition time: 10 min; column temperature: 35℃; mobile phase: methanol: acetonitrile: water = 1:1:3 (v / v / v).
[0056] The AFB1 degradation rate is calculated using the following formula:
[0057] Degradation rate = AFB1 content in control group - AFB1 content in experimental group - AFB1 content in control group
[0058] The pH of the reaction system was adjusted to 7.0, the temperature was controlled at 40℃, and 0.5 mM Zn was added. 2＋ Fe 3＋ Ni ＋ Mn 2＋ Mg 2＋ Cu 2＋ K ＋ Depend on Figure 4 A indicates that in Cu 2＋ In its presence, nitrite reductase AfNiR degrades AFB1.
[0059] In enzymatic reactions, the pH and temperature of the reaction system significantly affect enzyme activity. In this example, the pH and temperature of the reaction system were varied to detect the effect of nitrite reductase AfNiR on the degradation rate of AFB1. After determining the optimal pH and temperature, the amount of nitrite reductase AfNiR added was optimized accordingly. The specific steps are as follows:
[0060] Adjust the pH of the reaction system to 4.0-10.0 and control the temperature at 40℃. Figure 5 It can be seen that the degradation rate of AFB1 is the highest at pH 7.0, with a degradation rate of 98.22% after 24 hours.
[0061] The reaction temperature was adjusted to 20-80℃ at pH=7.0. Figure 6 It can be seen that the degradation rate of AFB1 reaches its highest at 40℃, and maintains a degradation rate of over 80% in the range of 40-80℃.
[0062] The pH of the reaction system was adjusted to 7.0, and the temperature was controlled at 40℃. The amounts of nitrite reductase AfNiR added were 510, 15, 20, 30, 40, and 50 μg, respectively. The results are as follows: Figure 7 As shown, the degradation rate of AFB1 by nitrite reductase AfNiR increases with the increase of the amount of nitrite reductase AfNiR, but the increasing trend gradually slows down. From an economic point of view, the optimal enzyme amount is 10 μg / mL.
[0063] The degradation rate of AFB1 at different reaction times was investigated under the conditions of pH 7.0, temperature 40℃, and enzyme addition of 10 μg. The results are as follows: Figure 8 As shown, the degradation rate of AFB1 increases with time, reaching 99.22% at 24h.
[0064] Application Example 2: Degradation of ZEN by the purified nitrite reductase AfNiR prepared in Example 2
[0065] The degradation reaction of ZEN by nitrite reductase AfNiR was carried out in a 500 L system containing 10 μg / mL ZEN, 20 μg of nitrite reductase AfNiR protein, and 50 mM Tris-HCl (containing 0.5 mM metal ions, pH 7.0) buffer solution for 24 h. The reaction was terminated by adding 500 μL of methanol. After the system was mixed thoroughly, it was filtered through a 0.22 μm organic filter membrane, and the ZEN content was determined by high performance liquid chromatography.
[0066] The chromatographic conditions for detecting ZEN using high performance liquid chromatography were as follows: column: Proshe11 120-C18 column (150×4.6 mm, 4 μm); fluorescence detector: excitation wavelength 235 nm, emission wavelength 470 nm; flow rate: 1 mL / min; injection volume: 10 L; acquisition time: 5 min; column temperature: 35 ℃; mobile phase: methanol:water = 4:1 (v / v).
[0067] The ZEN degradation rate is calculated using the following formula:
[0068] Degradation rate = ZEN content in control group - ZEN content in experimental group ZEN content in control group
[0069] The pH of the reaction system was adjusted to 7.0, the temperature was controlled at 40℃, and 0.5 mM Zn was added. 2＋ Fe 3＋ Ni＋ Mn 2＋ Mg 2＋ Cu 2＋ K ＋ .Depend on Figure 4 B indicates that in Cu 2＋ In the presence of nitrite reductase AfNiR, the degradation effect of ZEN is optimal.
[0070] Adjust the pH of the reaction system to 5.0-10.0 and control the temperature at 40℃. Figure 5 It can be seen that the degradation rate of ZEN is the highest at pH 8.0, and the degradation rate is 95% after 24 hours.
[0071] The reaction temperature was adjusted to 20-80℃ at pH=8.0. Figure 6 It can be seen that the degradation rate of ZEN reaches its highest at 60℃, and maintains a degradation rate of over 80% in the range of 50-80℃.
[0072] The pH of the reaction system was adjusted to 8.0, and the temperature was controlled at 50℃. The amounts of nitrite reductase AfNiR added were 5, 10, 15, 20, 30, 40, and 50 μg, respectively. The results are as follows. Figure 7 As shown, the degradation rate of ZEN by nitrite reductase AfNiR increases with the increase of superoxide dismutase AfSOD, but the increasing trend gradually slows down. From an economic point of view, the optimal enzyme amount is 20 μg.
[0073] The degradation rate of ZEN at different reaction times was investigated under the conditions of pH 8.0, temperature 60℃, and enzyme addition of 20 μg. The results are as follows: Figure 8 As shown, the degradation rate of ZEN increases with time, reaching 95.1% at 24 hours.
[0074] Application Example 3: Degradation of AFB1 and ZEN in peanut flour and corn flour contaminated with nitrite reductase AfNiR
[0075] Peanut and corn samples were ground and passed through an 80-mesh sieve. Then, 5 g of peanut powder or corn powder was added to 50 mL of distilled water and placed in a 100 mL Erlenmeyer flask, which was sterilized at 121 °C for 20 minutes. After cooling, AFB1 (final concentration 5 μg / mL) and ZEN (final concentration 10 μg / mL) were added to the peanut and corn samples respectively for artificial contamination. Different concentrations of AfSOD (50 and 100 μg / mL) were then added to the contaminated samples. The mixtures were incubated at 50 °C and 150 rpm for 24 h with shaking. To determine the optimal incubation time, the enzyme concentration was incubated at 100 μg / mL for 6, 12, and 24 h, respectively. After the reaction, the mixture was sonicated and centrifuged to obtain the supernatant. The toxin was then extracted using 50 mL of dichloromethane. After drying under nitrogen, the residue was redissolved in 1 mL of methanol, and the toxin residue was analyzed by HPLC. Figure 9 It can be seen that the degradation rates of AFB1 and ZEN in peanut flour contaminated with AFB1 and corn flour contaminated with ZEN were 70.6% and 60.2%, respectively.
[0076] Application Example 4: Analysis of the degradation products of AFB1 and ZEN by the purified nitrite reductase AfNiR prepared in Example 2.
[0077] AfSOD was incubated with 1 μg / mL AFB1 and 1 μg / mL ZEN under optimal degradation conditions for 24 hours. Degradation products were analyzed by UPLC-MS / MS. Chromatographic separation was performed using a reversed-phase CORTECS™ UPLC C18 column (100 × 2.1 mm, 1.6 μm) with a mobile phase of methanol and water containing 0.1% formic acid at a flow rate of 0.3 mL / min. Mass spectrometry detection was performed using positive ion electrospray ionization (ESI) mode, with a scan mass range of 100–800 m / z. The spray voltage was set to 3500 V, the nitrogen flow rate to 10 L / min, and the probe heating temperature to 350 °C. Analysis and identification of degradation products were performed using Compound Discoverer 3.3 software. Figure 10 It can be seen that the molecular formula of the AFB1 degradation product is C 17 H 12 O7, by Figure 11 It can be seen that the molecular formula of ZEN degradation products is C 18 H 22 O6.
[0078] Based on the structures of AFB1 and ZEN, it is speculated that the degradation products of the two toxins are AFQ1 / epi-AFQ1 and 15-OH-ZEN, respectively.
[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. The application of nitrite reductase AfNiR in the simultaneous degradation of aflatoxin B1 and zearalenone, characterized by: The amino acid sequence of the nitrite reductase AfNiR has more than 70% similarity to the sequence shown in SEQ ID No.
1.
2. The application of nitrite reductase AfNiR according to claim 1 in the simultaneous degradation of aflatoxin B1 and zearalenone, characterized in that: The amino acid sequence of the nitrite reductase AfNiR is shown in SEQ ID No. 1; The products of the degradation of aflatoxin B1 and zearalenone are AFQ1 / epi-AFQ1 and 15-OH-ZEN.
3. The application of nitrite reductase AfNiR according to claim 1 or 2 in the simultaneous degradation of aflatoxin B1 and zearalenone, characterized in that: The gene sequence encoding the nitrite reductase AfNiR has more than 90% similarity to the sequence shown in SEQ ID No.
2.
4. The application of nitrite reductase AfNiR according to claim 3 in the simultaneous degradation of aflatoxin B1 and zearalenone, characterized in that: The gene sequence encoding the nitrite reductase AfNiR is shown in SEQ ID No. 2 or SEQ ID No.
3.
5. A biomaterial, characterized in that: The biomaterial comprises any one of nitrite reductase AfNiR, a recombinant vector expressing nitrite reductase AfNiR, and engineered bacteria expressing nitrite reductase AfNiR, and also contains copper ions.
6. The application of the biomaterial described in claim 5 in detoxification in the food or feed industry.
7. The application according to claim 6, characterized in that: The detoxification refers to the degradation of aflatoxin B1 and / or zearalenone.
8. The application according to claim 7, characterized in that: The detoxification temperature is 40-70℃, and the pH is 7-8; 0.5mM Cu is also added during detoxification. 2＋ .
9. A method for degrading aflatoxin B1 and / or zearalenone in food or feed, characterized in that: The biomaterial described in claim 5 and 0.5 mM Cu 2＋ Add to the food or feed to be treated and treat at 40-70°C and pH 7-8 for 1-2 days until the aflatoxin B1 and / or zearalenone in the food or feed to be treated is reduced to the industrial required level.
10. The method for degrading aflatoxin B1 and / or zearalenone in food or feed according to claim 9, characterized in that: The amount of biological material added is based on the total amount of food or feed, and the amount of nitrite reductase AfNiR in the biological material is 100 μg / mL.