Application of a new slackia strain D-W1 in degrading vomitoxin

Abstract

The application isolates a strain of Slackia bacteria D-W1 from goose intestinal tract, which is named Slackia sp. D-W1. The average nucleotide identity analysis (ANI) of the strain and other strains in the Slackia genus is less than 80.5%, which belongs to a new species of the Slackia genus. The strain has been preserved in the Guangdong Microbial Culture Collection Center on March 9, 2023, and the preservation number is GDMCC NO: 63251. The strain can metabolize vomitoxin (Deoxynivalenol, DON) into almost non-toxic product DOM-1. The temperature for growth and metabolism of the strain is 32-42 DEG C, and the pH value is 3-10. The strain can be used for preparing DON detoxification metabolic enzyme, DON detoxification enzyme preparation and DON detoxification engineering bacteria, and is used for feed processing, food processing and livestock and poultry breeding.

Description

Technical Field

[0001] This invention belongs to the field of microbial screening and mycotoxin degradation technology, and relates to the application of Slackia bacteria D-W1 in the degradation of vomitoxin. Background Technology

[0002] Trichothecene toxins, a large group of fungal toxins, are mainly produced by fungi such as *Fusarium graminearum* and belong to a large class of sesquiterpenes with similar chemical structures. Deoxynivalenol (DON) belongs to type B trichothecene toxins. While DON has relatively weak toxicity, it is widespread and has a high detection rate, exhibiting acute and chronic toxic effects, such as intestinal toxicity, cytotoxicity, immunotoxicity, and neurotoxicity. In humans and animals, after ingesting food and feed contaminated with DON, dopamine receptors in the vomiting center of the brainstem interact with DON, leading to vomiting; therefore, DON is also known as a vomiting toxin. The molecular formula of deoxynivalenol is C0.05. 15 H 20 O6, chemically named 3α,7α,15-trihydroxy-12,13-epoxytrichosporon-9-en-8-one, is readily soluble in water, unstable in methanol, and can be stored for a long time in acetonitrile and ethyl acetate. Its pure form is a colorless needle-like crystal, stable under high pressure, high temperature, and acidic conditions, while its toxicity decreases under alkaline conditions.

[0003] Although DON has relatively low toxicity, it is widespread and its contamination of food crops is extensive worldwide. Statistics show that from 2015 to 2020, the contamination of fumonisin (FB1) and DON in complete animal feed and cereal raw materials in my country showed an increasing trend, with overall mycotoxin contamination levels in complete animal feed being high and at dangerous levels. A survey on mycotoxin contamination in animal feed raw materials in my country in 2021 showed that DON was widely present in samples from various regions, with pig feed being the most severely contaminated. In summary, in most surveys and studies on mycotoxin contamination of food crops and animal feed raw materials, DON contamination is the most widespread among type B trichothecene toxins. Although its toxicity is relatively low, concentrations exceeding the standard are still found in some positive samples.

[0004] Currently, the main methods for removing or reducing the DON content in grains include physical processing, chemical methods, and biodegradation. The most commonly used physical methods include washing, grinding, high-temperature heating, radiation treatment, and adsorption. Experiments have shown that activated carbon and diatomaceous earth are the most commonly used physical adsorbents for trichothecene toxins, with their interaction occurring through hydrophobic bonds, hydrogen bonds, coordination bonds, or electrostatic attraction. However, this method cannot effectively degrade or remove toxins from food and may also reduce or destroy the nutritional components of grains, affecting their nutritional value and taste. Chemical detoxification methods for DON involve using various chemical reagents such as oxidants (ozone, sodium hypochlorite), reducing agents (sodium bisulfite, sodium metabisulfite), and alkaline reagents (ammonia, calcium hydroxide) to alter the molecular structure of DON. However, using acids, alkalis, and oxidants to treat trichothecene toxins can alter their structure, leading to the formation of modified trichothecenes in food and feed, causing secondary pollution and resulting in other harmful side effects and further public health problems. While existing physical and chemical methods can effectively remove DON, they also have limitations. Biological detoxification is considered an effective and reliable alternative for controlling mycotoxin contamination in food. Biological detoxification utilizes detoxifying bacteria and enzymes to convert DON into less toxic metabolites, thereby reducing the harm of toxins to human and animal health. Because biological detoxification can efficiently reduce or remove DON under mild conditions and has little impact on the sensory characteristics and palatability of grains and feed, it is currently a promising method for DON detoxification.

[0005] Bacteria are the most studied microorganisms in DON detoxification, mostly derived from soil and animal digestive tracts. Several degradation products have been identified, primarily 3-keto-DON, 3-epi-DON, and DOM-1. Pure cultures capable of de-epoxygenating DON to DOM-1 include BBSH797, LS100, Slackia sp.D-G6, Raoultibacter sp.DII-9, and PGC-3-9. Strains capable of metabolizing DON to 3-keto-DON or 3-epi-DON include Agrobacterium Rhizobium.E3-39, Nocardioides sp.WSN05-2, Devosia.17-2-E-8, Devosia sp.DDS-1, Devosia insulae A16, and Nocardioides sp.ZHH-013. Although many microorganisms capable of degrading DON have been isolated, only BBSH797 has been assessed as safe and approved for application; this strain has already been developed and is used as a feed additive. The main factors hindering the application of DON-degrading microorganisms are the questionable safety of the strains themselves and the toxicity of the degradation products after DON metabolism. Developing microbial preparations or degrading enzymes for DON de-epoxygenation would undoubtedly promote the efficient development of my country's livestock industry. While there are reports on microorganisms capable of DON de-epoxygenation metabolism, the mechanism of this metabolism remains unclear. The limited resources of DON-degrading strains make the exploration of related enzymes and the study of the DON de-epoxygenation mechanism even more challenging for researchers.

[0006] Therefore, it is necessary to find strains capable of de-epoxygenating DON to provide detoxifying strains or degrading enzymes for DON detoxification in the food and feed industries, and to provide a theoretical basis for their application. Summary of the Invention

[0007] The first objective of this invention is to provide a new species of slackia bacteria, D-W1, capable of efficiently degrading DON.

[0008] A second objective of this invention is to provide the application of D-W1 in the degradation of DON.

[0009] A third objective of this invention is to provide conditions for D-W1 to degrade DON.

[0010] A fourth objective of this invention is to provide the application of D-W1 in the preparation of DON deoxygenase preparations.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] The present invention isolates and screens a strain of Slackia sp. D-W1 (hereinafter referred to as D-W1) from the contents of goose intestines, which is capable of de-epoxy metabolism of DON. The characteristic of this invention is that the strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 9, 2023, with the accession number GDMCC NO: 63251.

[0013] This invention also provides the application of D-W1 in the degradation of DON.

[0014] This invention also provides the application of D-W1 in the preparation of feed additives or food additives.

[0015] This invention also provides the application of D-W1 in the preparation of DON deoxygenase preparations.

[0016] In some embodiments of the present invention, the strain is Slackia sp. D-W1 (D-W1).

[0017] In some embodiments of the present invention, when used to metabolize DON, the culture medium for D-W1 is Wilkins-Chalgren anaerobic broth (WCA).

[0018] In some embodiments of the present invention, the concentration of D-W1 is 10 when used to metabolize DON. 4 -10 8 CFU / mL.

[0019] In some embodiments of the present invention, when used to metabolize DON, the culture temperature of D-W1 is 37-42°C and the pH is 7-10.

[0020] In some embodiments of the present invention, when used for the metabolism of DON, the culture gas conditions of D-W1 are a mixture of 80% N2, 10% H2 and 10% CO2.

[0021] In some embodiments of the present invention, a preferred culture system is a bacterial strain concentration of 10. 4 With a concentration of CFU / mL and a system volume of 200 μL, 5 μg of DON can be completely degraded into DOM-1 within 48-72 h.

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

[0023] This invention provides a Slackia bacterium, D-W1, capable of de-epoxygenating vomitoxin. This strain was isolated and screened from goose gut microbes and can efficiently degrade DON into DOM-1. Moreover, the metabolic activity of strain D-W1 for DON is independent of the continuous presence of DON; that is, D-W1 can still perform the function of de-epoxygenating DON in culture media that do not use DON as the sole carbon source.

[0024] This strain exhibits stable activity and strong metabolic capacity, demonstrating its potential for application in the preparation of DON detoxification agents in feed and food processing. It also provides new insights into the localization of genes related to DON deoxygenation metabolism, the isolation of DON deoxygenation metabolic enzymes, and the development of engineered DON deoxygenation metabolic bacteria. Attached Figure Description

[0025] Figure 1 The results are HPLC detection results of DON deoxygenation metabolism by goose gut microbe D-W1; a is the negative control of DON in WCA medium, and b is the HPLC detection results of strain D-W1, WCA medium and DON incubated together.

[0026] Figure 2 A phylogenetic tree for strain D-W1 based on its 16S rDNA sequence.

[0027] Figure 3 This study analyzed the average nucleic acid identity of the genomes of strain D-W1 with those of strains of the genus Slackia.

[0028] Figure 4 The metabolism of DON by strain D-W1 under different culture media.

[0029] Figure 5 The metabolism of DON by strain D-W1 at different temperatures.

[0030] Figure 6 The metabolism of DON by strain D-W1 at different pH values.

[0031] Figure 7 The relationship between the growth of strain D-W1 and its metabolism of DON under optimal metabolic conditions. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments 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 this technical field.

[0033] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0034] Example 1: Analysis of the DON deoxygenation metabolic capacity of mixed gut microbiota in goose

[0035] 1. Experimental Methods

[0036] (1) Purchase a Magang goose randomly from the market (Jiahe Agricultural Trade Market, Baiyun District, Guangzhou), dissect its abdomen and remove the goose intestines. The intestinal contents are resuspended in sterile water.

[0037] (2) After the intestinal contents suspension has been allowed to stand for 3-5 minutes, 20 μL of the intestinal contents suspension is added to 175 μL of LB, NB, BHI, MSM and WCA culture media respectively. Three experimental groups are set up for each culture medium, and 5 μL of 1 mg / mL DON stock solution is added together for incubation to make the final DON concentration 25 μg / mL.

[0038] (3) Place the inoculated sample and anaerobic bag into the anaerobic box and anaerobic culture at 37°C for 3 days. Detect the DON metabolism by HPLC.

[0039] Example 2: Separation of Slackia sp. D-W1

[0040] 1. Experimental Methods

[0041] First, the goose gut microbiota described in Example 1 was serially diluted to obtain an enriched microbiota with DON de-epoxygenation metabolism capability. The specific method is as follows:

[0042] (1) Gradual dilution screening: Viable bacterial groups were used as inoculum and serially diluted 10^6 times. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 The highest activity gradient was selected, and then subcultured and diluted. This process was repeated several times to enrich the degrading bacteria.

[0043] (2) Dilute the sample to the highest factor with metabolic activity, and then spread 200 μL of bacterial solution at this dilution gradient onto plates. Spread 20 μL of bacterial solution onto one WCA solid culture dish, and spread a total of 10 plates. Incubate the plates anaerobically for three days and observe the growth of single clones on the plates during this period.

[0044] (3) Randomly select single clones that grow on the plate after the 2nd and 3rd day and culture them in fresh liquid culture medium. At the same time, DON is added and incubated together. The final concentration of DON in the system is 25 μg / mL. Five days later, the degradation of DON in the sample is detected by HPLC.

[0045] (4) The monoclonal samples that were found to have degradation activity were plated and anaerobically cultured at 37°C for three days.

[0046] (5) Randomly select 5-10 single clones from the plate described in (4) and culture them in fresh liquid culture medium. At the same time, add DON and incubate together. The final concentration of DON toxin in the system is 25 μg / mL. Five days later, HPLC is used to detect the DON toxin metabolism.

[0047] (6) If all 5-10 monoclonal samples selected in (5) have DON metabolic activity, then it is preliminarily determined that the monoclonal samples obtained in (4) are pure strains capable of de-epoxygenation and DON degradation.

[0048] 2. Experimental Results

[0049] The experimental results are attached. Figure 1 As shown, the obtained monoclonal strain was able to deepoxidize DON to DOM-1.

[0050] Example 3: Identification of Slackia sp. D-W1

[0051] 1. Experimental Methods

[0052] (1) The obtained viable single clone strains were subjected to 16S rDNA gene sequencing, and a phylogenetic tree was constructed (see attached). Figure 4 (As shown). Sequence alignment revealed that the strain belongs to the genus Slackia. Therefore, the strain was named Slackia sp.D-W1 and deposited on March 9, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 63251. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0053] (2) The whole genome sequence of D-W1 was analyzed and its average nucleotide identification analysis (ANI) was performed with other strains of Slackia.

[0054] 2. Experimental Results

[0055] (1) The results of the 16S rDNA sequence analysis are attached. Figure 2 As shown, D-W1 belongs to the genus Slackia.

[0056] (2) The ANI value analysis results among Slackia strains are attached. Figure 3 As shown, the ANI value of D-W1 and other strains in the genus is less than or equal to 80.5%, therefore D-W1 is determined to be a new species of the genus Slackia.

[0057] Example 4: Optimal conditions for the degradation of DON by Slackia bacteria D-W1

[0058] I. Effects of culture medium on the DON metabolic activity of strain D-W1

[0059] 1. Experimental Methods

[0060] (1) Set up several different culture media, namely WCA, NB, BHI, MSM and LB liquid culture media, and autoclave them at 121℃ for later use.

[0061] (2) Mix 180 μL of liquid culture medium with 15 μL of bacterial solution, and then add 5 μL of DON (1 mg / mL) to each well. Incubate the mixture in a 96-well plate under anaerobic conditions at 37°C for 3 days. After 3 days, take samples for HPLC analysis to detect the degradation of DON in the samples.

[0062] 2. Experimental Results

[0063] The experimental results are attached. Figure 4 When used to metabolize DON, the optimal culture medium for D-W1 is WCA liquid medium.

[0064] II. Effect of pH on the DON metabolic activity of strain D-W1

[0065] 1. Experimental Methods

[0066] (1) Select the most suitable culture medium, namely WCA liquid culture medium, and set up a series of WCA liquid culture media with different pH values, namely pH 3-10, which are sterilized at 121℃ for later use.

[0067] (2) Mix 180 μL of liquid culture medium with 15 μL of bacterial solution, and then add 5 μL of DON (1 mg / mL) to each well. Incubate the mixture in a 96-well plate at 37°C for anaerobic incubation. Take samples for HPLC detection of DON degradation at 48 h and 72 h respectively.

[0068] 2. Experimental Results

[0069] The experimental results are attached. Figure 5 When used to metabolize DON, the optimal pH for D-W1 is 9.

[0070] III. Effects of Temperature on the DON Metabolic Activity of Strains D-W1

[0071] 1. Experimental Methods

[0072] (1) Set a series of temperature gradients, namely 27℃, 32℃, 37℃, 42℃ and 47℃.

[0073] (2) Mix 180 μL of liquid culture medium with 15 μL of bacterial solution, and then add 5 μL of DON (1 mg / mL) to each well. Incubate the mixture together in a 96-well plate at 37°C under anaerobic conditions for three days. After three days, detect the degradation of DON in the sample by HPLC.

[0074] 2. Experimental Results

[0075] The experimental results are attached. Figure 6 When used to metabolize DON, the optimal temperature for D-W1 is 37℃.

[0076] IV. Study on the optimal growth and metabolic conditions of strain D-W1

[0077] 1. Experimental Methods

[0078] (1) Adjust the initial bacterial suspension concentration to a cell density of approximately 10. 2 The system was incubated with CFU / mL DON, with a final DON concentration of 25 μg / mL. The incubation conditions were anaerobic at 37°C.

[0079] (2) Every 12 hours, pipette the sample to ensure even suspension and distribution of bacterial cells. Take 10 μL to 90 μL of fresh WCA liquid culture medium and perform a serial dilution method to dilute the 10 μL sample stock solution to 10 μL. 9 The sample was placed under anaerobic conditions for 3 days. After 3 days, the highest dilution with bacterial growth was observed to confirm the bacterial density of the original sample solution. Data were collected to plot the growth curve of strain D-W1.

[0080] (3) Samples were collected every 12 hours for HPLC analysis to detect the degradation of DON in the samples, and the data were used to plot the vomitoxin metabolism curve of strain D-W1.

[0081] 2. Experimental Results

[0082] The experimental results are attached. Figure 7 When used to metabolize DON, D-W1 exhibits a state of simultaneous growth and DON metabolism. When the cell concentration reaches 10... 3 -10 4 At CFU / mL, the deepoxidation degradation reaction of DON begins, and the 5 μg of DON contained in the system is completely metabolized within 48 h.

Claims

1. A plant Slackia Bacteria ( Slackia sp.)D-W1, characterized in that, This strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 9, 2023, with accession number GDMCC NO: 63251.

2. The application of the D-W1 of claim 1 in the metabolism of DON.

3. The application according to claim 2, characterized in that, When used to metabolize DON, D-W1 is cultured in Wilkins-Chalgren anaerobic broth medium.

4. The application according to claim 2, characterized in that, When used to metabolize DON, the concentration of D-W1 is 10. 5 -10 8 CFU / mL.

5. The application according to claim 2, characterized in that, When used for DON metabolism, D-W1 is cultured at a temperature of 37-42℃ and a pH of 7-10.

6. The application according to claim 2, characterized in that, When used for the metabolism of DON, the culture gas conditions for D-W1 are a mixture of 80% N2, 10% H2 and 10% CO2.

7. The application according to claim 2, characterized in that, The application involves using D-W1 to degrade DON in animal feed processing, livestock and poultry farming, and food processing.

8. The application according to claim 2, characterized in that, D-W1 was used to prepare feed additives, DON deoxygenating metabolic enzymes, DON detoxification enzyme preparations, and DON detoxification engineered bacteria.

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