Application of a Sphingosine Bacterium in Degrading Fungal Toxins

Abstract

The present invention discloses a sphingomyelinase and its application in the degradation of fungal toxins. The present invention screened and obtained a sphingomyelinase (Sphingopyxis sp.) D-L6, which is a new species of the genus Sphingopyxis. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 18, 2024, with a deposit number of GDMCC NO: 64434. The strain can efficiently degrade DON and T-2 toxins, and metabolize these toxins into low-toxic or non-toxic products. Sphingomyelinase D-L6 has stable metabolic activity and mild reaction conditions. It can be used in the detoxification of fungi in the field of food and feed, the preparation of fungal toxin detoxification enzymes and fungal toxin detoxification engineered bacteria, 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 Sphingobacterium bacterium D-L6 in the degradation of mycotoxins DON and T-2 toxin. 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. Common mycotoxins in food and feed include trichothecenes, aflatoxins, fumonisins, ochratoxins, and zearalenone (ZEN). Trichothecenes are a large class of chemically similar sesquiterpenes, sharing a common molecular structure consisting of an olefinic bond substituted with various side groups and a cyclohexene ring composed of four rings. Trichothecenes are stable and remain structurally unaffected even under high temperatures and boiling. Their structure can only be disrupted and inactivated by extreme physical and chemical conditions, such as strong acids and bases, treatment at 482°C for 10 minutes, or treatment at 260°C for 30 minutes. Therefore, they are not easily degraded during storage, grinding, and processing of food crops. Types A and B of the trichothecenes have attracted considerable attention and research due to their high toxicity and widespread distribution.

[0003] Deoxynivalenol, also known as vomitoxin (DON), is a type B trichothecene toxin first isolated from barley infected with Fusarium spp. in Japan. Acute DON poisoning can cause vomiting, while chronic low-dose exposure can lead to anorexia, growth retardation, immunotoxicity, and reproductive impairment caused by maternal toxicity. The 2022 China Survey Report on Mycotoxin Contamination of Feed Ingredients showed that the detection rate of DON in corn exceeded 97%, while the detection rate in corn by-products, wheat and bran, and compound feeds was 100%, with the rate exceeding the standard exceeding 17%. T-2 toxin is a type A trichothecene toxin with hepatotoxic, nephrotoxic, immunotoxic, neurotoxic, and reproductive toxicity, making it the most toxic of the trichothecenes. In animals fed a diet contaminated with T-2 toxin, the proximal tubular epithelial cells in the kidney tissue of nearly all animals showed severe degeneration, with naked cells and proteinaceous material appearing within the tubular lumen, leading to the disappearance of the tubular lumen. In addition, nuclear enlargement and binucleation of the epithelial cells were observed. Even low levels of T-2 toxin can alter the activation of Toll-like receptors, disrupting the initiation of inflammatory immune responses against viruses and bacteria. T-2 toxin contamination is a global phenomenon. A 2019 Scottish oat survey revealed that contamination rates for T-2 toxin and HT-2 in organic and conventional oats reached 100% and 83% respectively.

[0004] Currently, methods for detoxifying mycotoxins such as DON and T-2 toxin primarily include physical, chemical, and biological methods. Physical and chemical detoxification methods, such as heat treatment, adsorption, strong acids and alkalis, ozone, and electromagnetic radiation, are relatively simple to apply, but generally have certain side effects. These include: impacts on the nutritional value and taste of the product; the potential for secondary contamination; expensive equipment; high energy consumption; and weak penetration. In contrast, biological detoxification methods primarily utilize microorganisms or enzymes to react with mycotoxins. These methods offer mild, efficient, and complete reaction conditions, minimal impact on food and feed, and possess significant potential for development and application.

[0005] The genus Sphingopyxis exists in different environments and has the ability to degrade environmental pollutants such as microcystins, fumonisins, styrene and herbicides. This genus has not been reported to have DON and T-2 toxin metabolic activity. Mycotoxin pollution is usually not composed of just one toxin alone, but rather a complex pollution of multiple mycotoxins. Among them, DON and ZEN pollution are the most widespread, while T-2 toxin is the most toxic. When dealing with mycotoxin pollution, it is often necessary to deal with different toxins one by one. Therefore, screening microorganisms that have the ability to metabolize both DON and T-2 toxins is of great significance for controlling mycotoxin pollution, developing mycotoxin detoxification preparations, and screening mycotoxin detoxification genes. Summary of the Invention

[0006] The first object of the present invention is to provide a new species of bacteria D-L6 of the genus Sphingomyelia that can efficiently degrade DON and T-2 toxins.

[0007] The second object of the present invention is to provide the use of D-L6 in degrading DON and T-2 toxin.

[0008] The third object of the present invention is to provide the use of D-L6 in the preparation of DON and T-2 toxin detoxification preparations.

[0009] The fourth object of the present invention is to provide the use of D-L6 in the preparation of DON and T-2 toxin detoxification metabolic enzymes.

[0010] The fifth object of the present invention is to provide the use of D-L6 in constructing DON and T-2 toxin-detoxifying engineered 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-L6 from the soil that can efficiently metabolize DON and T-2 toxins. After identification, the strain belongs to the genus Sphingopyxis. 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: 64434. Through 16s rDNA phylogenetic tree and average nucleotide consistency analysis, D-L6 was determined to be a new species of the genus Sphingopyxis.

[0013] The present invention also provides the use of D-L6 in degrading fungal toxins.

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

[0015] The application is to apply D-L6 to the detoxification of fungi in the field of food or feed processing.

[0016] The present invention also provides the use of D-L6 in preparing DON and T-2 toxin detoxification preparations.

[0017] The present invention also provides the use of D-L6 in preparing DON and T-2 toxin detoxification metabolic enzymes.

[0018] The present invention also provides the use of D-L6 in constructing DON and T-2 toxin-detoxifying engineering bacteria.

[0019] In some embodiments of the present invention, the strain is Sphingopyxis sp. D-L6 (D-L6).

[0020] In some embodiments of the present invention, when used to metabolize DON and T-2 toxin, the metabolic medium of D-L6 is MSM medium, and the culture temperature is 30°C.

[0021] The formula of the MSM culture medium is as follows: 0.2g of magnesium sulfate heptahydrate, 0.5g of ammonium sulfate, 0.05g of calcium chloride, 6.15g of disodium hydrogen phosphate dihydrate, and 1.52g of potassium dihydrogen phosphate. Add purified water to 1L to dissolve, the pH value is 7.2, and sterilize at 121°C for 20min.

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

[0023] The present invention provides a strain of Sphingopyxis bacteria, D-L6, capable of mycotoxin metabolism. This strain was isolated from soil and is capable of efficiently metabolizing DON and T-2 toxins. Based on ANI analysis of D-L6 compared with other strains of the genus Sphingopyxis, D-L6 was determined to be a new species of the genus Sphingopyxis. D-L6's metabolic activity against DON and T-2 toxins is maintained independently of the presence of DON or T-2 toxins. Continuous culture and subculture in culture medium devoid of DON or T-2 toxins reveals no impairment of DON and T-2 toxin degradation, demonstrating stable activity and strong metabolic capacity.

[0024] The D-L6 strain provided by the present invention can grow using DON as its sole carbon source, converting DON into its own components. The reaction is irreversible, under mild conditions, and without secondary contamination. This strain can be used for mycotoxin detoxification in feed and food processing, specifically for detoxification of DON and T-2 toxin, preparation of mycotoxin detoxification preparations and detoxification enzymes, and construction of engineered mycotoxin-detoxifying 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 DON metabolism by Sphingobacterium sphingosine D-L6 of the present invention;

[0026] Figure 2 This is a UPLC-MS / MS test result of the metabolism of T-2 toxin by Sphingobacterium D-L6 of the present invention;

[0027] Figure 3 This is a Gram staining result of Sphingobacterium sphingosine D-L6 of the present invention;

[0028] Figure 4 This is a phylogenetic tree constructed based on the 16s rDNA sequence of the sphingomyelinase D-L6 of the present invention;

[0029] Figure 5 The average nucleotide identity (ANI) analysis results of the Sphingobacterium D-L6 of the present invention and other strains in the genus Sphingobacterium are shown. DETAILED DESCRIPTION

[0030] 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.

[0031] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0032] Example 1 Screening and Identification of Soil Mycotoxin-Degrading Microorganisms

[0033] 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.

[0034] 1. Experimental methods

[0035] (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 MSM medium, and DON was added to a final concentration of 7 μg / mL. The culture was shaken at 30°C for 7 days. After the culture was completed, DON metabolism was detected by HPLC.

[0036] (2) The active samples were diluted in a gradient 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 morphology were picked and incubated in MSM medium with 10 μg / mL DON. The metabolic status was detected by HPLC.

[0037] (3) After repeated screening, several pure strains with DON metabolic activity were obtained. These strains were incubated with 20 μg / mL T-2 toxin in MSM liquid medium under shaking conditions at 30°C, and the metabolism of T-2 toxin was detected by UPLC-MS / MS.

[0038] 2. Experimental results

[0039] Finally, a pure strain with DON and T-2 toxin metabolism activity was obtained, designated D-L6. D-L6 grew slowly in MSM medium, so the bacteria were inoculated and amplified in R2A medium. An appropriate amount of the bacterial suspension was mixed with an equal volume of 80% glycerol and stored at -80°C.

[0040] Example 2: Use of pure strain D-L6 to degrade DON and T-2 toxins

[0041] 1. Experimental methods

[0042] D-L6 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 and 20 μg / mL T-2 toxin in MSM medium with shaking at 30°C. After the incubation, the metabolism of DON was detected by HPLC, and the metabolism of T-2 toxin was detected by UPLC-MS / MS.

[0043] 2. Experimental results

[0044] (1) The results of incubation of D-L6 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-L6 incubated with DON, the results show that D-L6 can degrade DON.

[0045] (2) The results of incubation of D-L6 with T-2 toxin are as follows Figure 2 As shown, (a) is the chromatographic result of a T-2 toxin blank control, (b) is the chromatographic result of D-L6 incubated with T-2 toxin, with a T-2 toxin retention time of 9.5 minutes, and (c) is the mass spectrometry result of T-2 toxin. The results show that D-L6 can completely degrade T-2 toxin.

[0046] Example 3 Identification of pure strain D-L6

[0047] 1. Experimental methods

[0048] (1) Spread an appropriate amount of D-L6 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-L6 colonies for Gram staining and observe the staining results under a microscope.

[0049] (2) An appropriate amount of D-L6 bacterial culture was used as a template, and the 16s rDNA gene of D-L6 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;

[0050] (3) The whole genome of D-L6 was sequenced, and the whole genome sequence was compared with other Nocardia-like strains for average nucleic acid identification analysis (ANI).

[0051] 2. Experimental results

[0052] (1) Gram staining results are as follows Figure 3 The results showed that D-L6 was red in Gram staining and was a Gram-negative bacterium.

[0053] (2) Based on the sequence alignment of the 16s rDNA gene of D-L6, the phylogenetic tree was constructed as follows Figure 4 The results showed that the bacterium belongs to the genus Sphingopyxis and was named Sphingopyxis sp. D-L6. The bacterium was deposited in the Guangdong Provincial Microbiological Culture Collection on March 18, 2024, with the deposit number GDMCC NO: 64434. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0054] (3) The ANI value analysis results of the strains in the genus Sphingosine are shown in the figure below. Figure 5 The results showed that the ANI values ​​of D-L6 and other strains in the genus Sphingobacterium were all less than 83.4%. In the identification of closely related species, ANI values ​​greater than 95% are considered to be the same species. Therefore, we determined that D-L6 is a new species in the genus Sphingobacterium.

[0055] 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 Sphingopyxis sp. D-L6, 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: 64434.

2. Use of the D-L6 strain as claimed in claim 1 in degrading the fungal toxins DON and T-2 toxin.

3. Use of the D-L6 strain as claimed in claim 1 in the preparation of detoxified preparations of mycotoxins DON and T-2 toxin.

4. Use of the D-L6 strain as claimed in claim 1 in the preparation of mycotoxin DON and T-2 toxin detoxification enzymes.

5. Use of the D-L6 strain as claimed in claim 1 in the preparation of mycotoxin DON and T-2 toxin detoxification engineered bacteria.

6. The use according to claim 2, characterized in that When used to degrade the fungal toxins DON and T-2 toxin, the culture medium is a mineral salt medium (MSM). The formula of the culture medium is: 0.2g magnesium sulfate heptahydrate, 0.5g ammonium sulfate, 0.05g calcium chloride, 6.15g disodium hydrogen phosphate dihydrate, and 1.52g potassium dihydrogen phosphate. Add purified water to 1L to dissolve, the pH value is 7.2, and sterilize at 121°C for 20 minutes.

7. The use according to claim 2, characterized in that When used to degrade fungal toxins DON and T-2 toxin, the strain concentration is: 10 5 -10 8 CFU / mL.

8. The use according to claim 2, characterized in that The culture conditions for degrading fungal toxins DON and T-2 toxin are: culture temperature 30°C, pH 6-7.

9. The use according to claim 2, characterized in that The application is for the degradation of mycotoxins DON and T-2 toxin in the fields of animal feed processing, livestock and poultry breeding, and food processing.

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