Application of a Nocardia-like bacterium D-L4 in the degradation of mycotoxins

Abstract

The present invention discloses a strain of Nocardioides sp. and its application in the degradation of fungi toxins. The present invention screened and obtained a strain of Nocardioides sp. D-L4, which is a new species of the genus Nocardioides. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 18, 2024, with a deposit number of GDMCC NO: 64435. The strain can efficiently degrade fungi such as DON, T-2 toxin and ZEN, and metabolize these toxins into low-toxic or non-toxic products. The metabolic activity of Nocardioides sp. D-L4 is stable and the reaction conditions are mild. It can be used in the preparation of fungal toxin detoxification, fungal toxin detoxification enzymes and fungal toxin detoxification engineered bacteria in the field of food and feed, and has good application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of mycotoxin degradation, and more specifically relates to the application of a Nocardia-like bacterium D-L4 in the degradation of mycotoxins DON, T-2 toxin, and ZEN. Background Art

[0002] Mycotoxins, also known as mycotoxins, are toxic secondary metabolites produced by fungi such as Fusarium graminearum when they infect food crops under conditions of high temperature and humidity. Mycotoxin poisoning increases vulnerability to microbial diseases in humans and animals, exacerbates the effects of malnutrition, and can interact synergistically with other toxins. Common mycotoxins in food and feed include trichothecenes, aflatoxins, fumonisins, ochratoxins, and zearalenone (ZEN). DON and T-2 toxin belong to the type B and type A trichothecenes, respectively. Studies have shown that DON is found in over 90% of mycotoxin-contaminated samples. In addition to its widespread distribution, T-2 toxin is also the most toxic of the trichothecenes. ZEN is a non-steroidal estrogenic mycotoxin that can cause multiple toxic effects, including reproductive toxicity. A study of 1,160 samples sampled from my country found a ZEN detection rate of over 93%, with the highest rate exceeding the standard being 22%. The detection rate of mycotoxins in food and feed raw materials and their products remains high. They are naturally occurring and unavoidable pollutants in grain crops, posing a serious threat to human and animal health, causing huge economic losses to the food and feed industry, and have attracted widespread attention in the field of food and feed safety.

[0003] At present, the detoxification methods for mycotoxins such as DON, T-2 toxin and ZEN mainly include physical, chemical and biological methods. Physical detoxification methods such as heat treatment, adsorption, electromagnetic radiation, etc. are relatively simple to apply, but generally have limitations. For example: they affect the nutritional value and taste of the product, the equipment is expensive, the energy consumption is high, and the penetration capacity is weak. Chemical methods such as strong acids, strong bases, ozone, etc. usually require the introduction of chemical reagents and have requirements for reaction conditions. During the detoxification process, the chemical reagents will be in direct contact with the product, affecting the nutritional value, taste and appearance of the product, etc., and are likely to cause secondary pollution and increase the subsequent processing costs. In contrast, biological detoxification methods mainly use microorganisms or enzymes to react with mycotoxins. The reaction conditions are mild, efficient, and thorough, with less impact on food and feed, and have great development and application potential.

[0004] Nocardia-like bacteria, belonging to the genus Actinomycetes, have been reported to utilize a variety of organic environmental pollutants as carbon sources, including hydrocarbons, aromatic compounds, nitrogen heterocyclic compounds, halogenated alkanes, and polyesters, offering broad potential for pollutant control and degradation. Currently, two Nocardia-like strains, WSN05-2 and ZHH-013, have been found to possess DON metabolic activity, completely converting and utilizing DON as a carbon source. The intermediates metabolized by these strains include 3-keto-DON and 3-epi-DO, which are further converted. However, the complete DON metabolic pathway and the enzymes responsible for its metabolic activity have not yet been described. No Nocardia-like strains have been reported to degrade T-2 toxin and ZEN.

[0005] Mycotoxin contamination is typically not caused by a single toxin, but rather by a combination of multiple toxins. DON and ZEN are the most widespread, while T-2 toxin is the most toxic. Therefore, treating mycotoxin contamination often requires addressing each toxin individually. Therefore, screening for microorganisms that can simultaneously degrade DON, T-2 toxin, and ZEN is crucial for controlling mycotoxin contamination, developing mycotoxin detoxification agents, and screening for mycotoxin detoxification genes. Summary of the Invention

[0006] The first object of the present invention is to provide a new Nocardia-like bacterium D-L4 that can efficiently degrade DON, T-2 toxin and ZEN.

[0007] The second object of the present invention is to provide the use of D-L4 in degrading DON, T-2 toxin and ZEN.

[0008] The third object of the present invention is to provide the use of D-L4 in the preparation of DON, T-2 toxin and ZEN detoxification preparations.

[0009] The fourth object of the present invention is to provide the use of D-L4 in the preparation of DON, T-2 toxin and ZEN detoxification metabolic enzymes.

[0010] The fifth object of the present invention is to provide the use of D-L4 in constructing DON, T-2 toxin and ZEN detoxification engineering bacteria.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] After a long period of screening and separation, the present invention successfully isolated a strain D-L4 from the soil that can efficiently metabolize DON, T-2 toxin and ZEN. After identification, the strain belongs to the genus Nocardioides. It is characterized in that the strain has been deposited in the Guangdong Provincial Microbial Culture Collection Center on March 18, 2024, and its deposit number is GDMCC NO: 64435. Through 16s rDNA phylogenetic tree and average nucleotide consistency analysis, D-L4 was determined to be a new species of Nocardioides.

[0013] The present invention also provides the use of D-L4 in degrading fungal toxins.

[0014] Specifically, the application degrades DON, T-2 toxin and ZEN among fungal toxins.

[0015] The application is to apply D-L4 to the detoxification of fungi in the field of food or feed processing.

[0016] The present invention also provides the use of D-L4 in preparing DON, T-2 toxin and ZEN detoxification preparations.

[0017] The present invention also provides the use of D-L4 in preparing DON, T-2 toxin and ZEN detoxification metabolic enzymes.

[0018] The present invention also provides the use of D-L4 in constructing DON, T-2 toxin and ZEN detoxification engineering bacteria.

[0019] In some embodiments of the present invention, the strain is Nocardioides sp. D-L4 (D-L4).

[0020] In some embodiments of the present invention, when used to metabolize DON, T-2 toxin, and ZEN, the metabolic medium of D-L4 is R2A medium, and the culture temperature is 30°C.

[0021] The formula of the R2A culture medium is as follows: 0.5 g yeast extract powder, 0.5 g peptone, 0.5 g casein hydrolyzate, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.024 g anhydrous magnesium sulfate, and 0.3 g sodium pyruvate. Add purified water to 1 L to dissolve, the pH value is 7.2, and sterilize at 121° C. for 15 min.

[0022] The present invention has the following beneficial effects:

[0023] The present invention provides a Nocardia-like bacterium D-L4 capable of mycotoxin metabolism. This strain was isolated and screened from soil and is capable of efficiently degrading DON, T-2 toxin, and ZEN among the mycotoxins. Its ANI values ​​compared to other strains within the genus Nocardia are all lower than 82%, thus determining that it belongs to a new species of the genus. D-L4's metabolic activity for DON, T-2 toxin, and ZEN is maintained independently of the continued presence of these substances. D-L4's metabolic activity for DON, T-2 toxin, and ZEN does not decrease or disappear during continuous culture in a medium devoid of DON, T-2 toxin, or ZEN, demonstrating stable activity and strong metabolic capacity.

[0024] The D-L4 strain provided by the present invention can grow using DON as its sole carbon source and convert DON into its own components. The reaction is irreversible, under mild conditions, and without secondary pollution. This strain can be used for mycotoxin detoxification in feed and food processing, specifically for the detoxification of DON, T-2 toxin, and ZEN, the preparation of mycotoxin detoxification preparations and mycotoxin detoxification enzymes, and the construction of mycotoxin-engineered bacteria. It has great potential for controlling mycotoxin contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the HPLC detection results of the metabolism of DON by the Nocardia-like bacteria D-L4 of the present invention;

[0026] Figure 2 This is a graph showing the UPLC-MS / MS detection results of T-2 toxin metabolism by Nocardia-like bacteria D-L4 of the present invention;

[0027] Figure 3 This is a graph showing the HPLC detection results of the metabolism of ZEN by Nocardia-like bacteria D-L4 of the present invention;

[0028] Figure 4 This is a Gram staining result of Nocardia-like bacteria D-L4 of the present invention;

[0029] Figure 5 This is a phylogenetic tree constructed based on the 16s rDNA sequence of the Nocardia-like bacteria D-L4 of the present invention;

[0030] Figure 6 The average nucleotide identity (ANI) analysis results of the Nocardia-like strain D-L4 of the present invention and other strains in the genus Nocardia. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0032] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0033] Example 1 Screening and Identification of Soil Mycotoxin-Degrading Microorganisms

[0034] Since DON is the most serious mycotoxin contamination, we first screened for DON-metabolizing microorganisms, obtained pure strains, and then tested the metabolic activity of the strains on other mycotoxins.

[0035] 1. Experimental methods

[0036] (1) Several soil samples were randomly selected from a wheat field in Henan Province. The soil samples were resuspended in sterile water, mixed, and allowed to stand. 200 μL of the supernatant was added to 800 μL of R2A medium, and DON was added to a final concentration of 7 μg / mL. The medium was shaken and cultured at 30°C for 7 days. After the culture, DON metabolism was detected by HPLC.

[0037] (2) The active samples were diluted in a gradient manner and incubated with DON. The highest dilution gradient sample that retained DON metabolic activity was selected and spread on R2A plates. After incubation at 30°C for 3 days, single colonies with different colony morphologies were picked and incubated in R2A medium with 10 μg / mL DON. The metabolic status was detected by HPLC.

[0038] (3) After repeated screening, several pure strains with DON metabolic activity were obtained. These strains were incubated with 20 μg / mL T-2 toxin, 15 μg / mL ZEN, 10 μg / mL FB1, 20 μg / mL OTA, and 5 μg / mL AFB1 in R2A liquid medium under shaking conditions at 30°C. The metabolism of ZEN, AFB1, and OTA was detected by HPLC, and the metabolism of T-2 toxin and FB1 was detected by UPLC-MS / MS.

[0039] 2. Experimental results

[0040] Finally, a pure strain with DON, T-2 toxin, and ZEN metabolic activity was obtained, designated D-L4. D-L4 was inoculated into R2A medium and cultured until the logarithmic growth phase. An appropriate amount of the bacterial broth was mixed with an equal volume of 80% glycerol and stored at -80°C.

[0041] Example 2: Use of pure strain D-L4 to degrade DON, T-2 toxin and ZEN

[0042] 1. Experimental methods

[0043] D-L4 was inoculated into R2A medium and cultured at 30℃ with shaking until the growth phase. The bacterial concentration was adjusted to 10 5 -10 6 CFU / mL, and incubated with 10 μg / mL DON, 20 μg / mL T-2 toxin, and 15 μg / mL ZEN in R2A medium with shaking at 30°C. After the incubation, the metabolism of DON and ZEN was detected by HPLC, and the metabolism of T-2 toxin was detected by UPLC-MS / MS.

[0044] 2. Experimental results

[0045] (1) The results of incubation of D-L4 and DON are as follows Figure 1 As shown, a is the HPLC result of DON blank control, the retention time of DON is 11.9min, b is the HPLC result of D-L4 incubated with DON, the results show that D-L4 can completely degrade DON.

[0046] (2) The results of incubation of D-L4 with T-2 toxin are as follows Figure 2 As shown, a is the chromatographic result of the T-2 toxin blank control, b is the chromatographic result of D-L4 incubated with T-2 toxin, the retention time of T-2 toxin is 9.1min, and c is the primary mass spectrometry result of T-2 toxin. The results show that D-L4 can completely degrade T-2 toxin.

[0047] (3) The results of incubation of D-L4 and ZEN are as follows Figure 3 As shown, a is the HPLC result of ZEN blank control, the retention time of ZEN is 10.5min, b is the HPLC result of D-L4 incubated with ZEN, the results show that D-L4 can completely degrade ZEN.

[0048] Example 3 Identification of pure strain D-L4

[0049] 1. Experimental methods

[0050] (1) Spread an appropriate amount of D-L4 bacterial solution on an R2A plate. After culturing to the vigorous growth stage, use a clean cotton swab to pick up an appropriate amount of D-L4 colonies for Gram staining and observe the staining results under a microscope.

[0051] (2) An appropriate amount of D-L4 bacterial culture was used as a template, and the 16s rDNA gene of D-L4 was amplified by PCR using the 16s rDNA universal primers 27F / 1492R. After sequencing, the sequences were aligned in the NCBI database, and a phylogenetic tree was constructed;

[0052] (3) The whole genome of D-L4 was sequenced, and the whole genome sequence was compared with other Nocardia-like strains for average nucleic acid identification analysis (ANI).

[0053] 2. Experimental results

[0054] (1) Gram staining results Figure 4 The results showed that D-L4 was blue-purple in Gram staining and was a Gram-positive bacterium.

[0055] (2) Based on the sequence alignment of the 16s rDNA gene of D-L4, the phylogenetic tree was constructed as follows Figure 5 The results showed that the bacterium belongs to the genus Nocardioides and was named Nocardioides sp. D-L4. The bacterium was deposited with the Guangdong Provincial Microbiological Culture Collection on March 18, 2024, with the deposit number GDMCC NO: 64435. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0056] (3) The ANI value analysis results of Nocardia-like strains are as follows Figure 6 The results showed that the ANI values ​​of D-L4 and other strains in the genus Nocardia were all less than 81.8%. In the identification of closely related species, ANI values ​​greater than 95% are considered to be the same species. Therefore, we determined that D-L4 is a new species in the genus Nocardia.

[0057] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A strain of Nocardioides sp. D-L4, characterized in that: This strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on March 18, 2024, and its deposit number is GDMCC NO: 64435.

2. Use of the D-L4 strain as claimed in claim 1 in degrading the fungal toxins DON, I-2 toxin and ZEN.

3. Use of the D-L4 strain as claimed in claim 1 in the preparation of detoxified preparations of mycotoxins DON, T-2 toxin and ZEN.

4. Use of the D-L4 strain as claimed in claim 1 in the preparation of mycotoxin DON, T-2 toxin and ZEN detoxification metabolizing enzymes.

5. Use of the D-L4 strain as claimed in claim 1 in constructing detoxification engineering bacteria for mycotoxins DON, T-2 toxin and ZEN.

6. The use according to claim 2, characterized in that When used to metabolize the mycotoxins DON, T-2 toxin, and ZEN, the culture medium for D-L4 is R2A medium.

7. The use according to claim 2, characterized in that When used to metabolize the mycotoxins DON, T-2 toxin and ZEN, the concentration of D-L4 is 10 5 -10 8 CFU / mL.

8. The use according to claim 2, characterized in that When used to metabolize the mycotoxins DON, T-2 toxin, and ZEN, D-L4 is cultured at a temperature of 25-37°C and a pH of 4-10.

9. The use according to claim 2, characterized in that The application is to apply D-L4 to the degradation of fungal toxins DON, T-2 toxin and ZEN in the fields of animal feed processing, livestock and poultry breeding and food processing.

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