Devosia sp. D-G15 and application thereof

Abstract

The application isolates a new strain of Devosia genus bacteria from the soil of wheat field infected by Fusarium, and names it as Devosia sp. D-G15 (D-G15). The ANI value of D-G15 and other strains in the Devosia genus is less than 80%, which indicates that it is a new species of Devosia strain. The strain has been preserved in Guangdong Microbial Culture Collection Center on March 26, 2024, and the preservation number is GDMCC NO: 64453. The strain D-G15 can degrade vomitoxin (DON) into low-toxicity products, and the degradation of the strain to DON is beneficial within the range of pH value of 4-10 and temperature of 15-35 DEG C. The degradation activity of D-G15 is stable, the metabolic capacity is strong, and it is safe in the animal body, and can be used for the preparation of DON detoxification preparation and applied to the fields of feed processing and food processing.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of mycotoxin degradation, and particularly relates to a strain capable of degrading vomitoxin and application thereof. BACKGROUND

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a secondary metabolite produced by fungi such as Fusarium graminearum and Fusarium oxysporum. DON is the most common contaminant in wheat and its products, and has a particularly serious impact on grain and feed. The economic losses caused by DON contamination of grain worldwide amount to billions of dollars each year. A study analyzing product feed and corn samples from 44 countries from 2012 to 2015 found that 79% of the samples had DON levels exceeding safety standards. In a 2015-2020 survey of mycotoxin contamination in Poland, the contamination rate of DON in corn and feed samples was as high as 97.3% and 99.7%, respectively. A survey in Brazil showed that 97% of feed samples collected between 2017 and 2021 were contaminated with at least one mycotoxin, with a DON contamination rate of 87.9% in finished feed.

[0003] In China, the problem of DON contamination is also serious. A study analyzing 15,004 grain samples from various regions of China from 2010 to 2017 found that the contamination rates of DON and its derivatives in wheat flour and corn flour samples were as high as 77.5% and 80.9%, respectively. The 2018 feed inspection results in China showed that the detection rates of DON in full-price feed, corn, corn by-products, wheat and bran, and meal were all over 96%, with the highest over-standard rate of DON in wheat and bran. A 2022 survey report showed that the detection rates of DON in corn by-products, wheat and bran, and full-price feed were all 100% in 1160 samples.

[0004] DON has multiple toxic effects on the body, which can be divided into chronic toxicity and acute toxicity. Long-term consumption of low-dose DON-contaminated feed by animals often leads to anorexia, growth retardation, and immunotoxicity. Acute toxicity includes symptoms such as anorexia, diarrhea, and vomiting. DON can cross the blood-brain barrier, induce changes in hormone levels in various regions of the brain, cause brain damage, and affect animal behavior. DON can also cause immunotoxicity, increasing the risk of pathogen infection.

[0005] Currently, detoxification strategies for DON (diethyltoxin) are mainly divided into three categories: physical, chemical, and biological methods. Physical methods include sorting, heat treatment, radiation, and adsorption. Chemical methods involve using alkalis, acids, and oxidants to destroy the structure of fungal toxins. However, while physical and chemical methods can reduce DON to some extent, they also have significant limitations, such as reducing or destroying the nutritional components of grains, affecting the nutritional value and taste of food, and introducing new harmful chemicals that cause secondary pollution. In recent years, strategies utilizing microorganisms and enzymes for detoxification have attracted much attention. Their advantages, such as high specificity, mild processing conditions, minimal impact on palatability, and environmental friendliness, make them an ideal detoxification method.

[0006] In summary, DON (donalized oxygen) widely contaminates feed and feed ingredients, posing a significant threat to livestock and poultry farming. Biodetoxification based on microbial degradation is a highly efficient method for DON control and has broad application prospects. Summary of the Invention

[0007] The first objective of this invention is to provide a safe and efficient Devosia bacterium, D-G15, that degrades DON.

[0008] A second objective of this invention is to provide the application of D-G15 in the degradation of DON.

[0009] A third objective of this invention is to provide the application of D-G15 in the preparation of DON detoxification formulations.

[0010] A fourth objective of this invention is to provide the application of D-G15 in the construction of DON-free engineered bacteria or the cultivation of DON-tolerant transgenic plants.

[0011] This invention is achieved through the following technical solution:

[0012] This invention isolated a strain capable of degrading DON from wheat field soil infected with Fusarium. Through 16S rRNA and ANI comparative analysis, the strain was identified as a new species of the genus *Devosia*, named *Devosia* sp. D-G15 (hereinafter referred to as D-G15). This strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 26, 2024, with the accession number GDMCC NO: 64453.

[0013] This invention also provides the application of D-G15 in the degradation of DON.

[0014] This invention also provides the application of D-G15 in the preparation of feed additives or food additives.

[0015] This invention also provides the application of D-G15 in the preparation of DON-degrading enzyme preparations.

[0016] In some embodiments of the present invention, the strain is Devossia sp.D-G15 (D-G15).

[0017] In some embodiments of the present invention, the culture medium for D-G15 used to degrade DON is an inorganic salt culture medium.

[0018] In some embodiments of the present invention, the concentration of D-G15 is 10 when used to degrade DON. 3 -10 8 CFU / mL.

[0019] In some embodiments of the present invention, when used to degrade DON, the culture temperature of D-G15 is 15-40°C and the pH is 4-10.

[0020] The present invention has the following beneficial effects:

[0021] This invention provides a new species of Devossia bacterium, D-G15, capable of degrading vomitoxin (DON). D-G15 can grow using DON as the sole carbon source, and it can also grow and degrade DON in rich media containing other carbon sources. D-G15 exhibits stable degradation activity, strong metabolic capacity, and safety in animals, showing potential for application in the preparation of DON detoxification agents in feed and food processing. Attached Figure Description

[0022] Figure 1 Figure 1 shows the HPLC detection results of DON degradation by strain D-G15; Figure 2a shows the negative control with DON added to the culture medium, and Figure 3b shows the HPLC detection results of DON degradation by strain D-G15.

[0023] Figure 2 This is a transmission electron microscope image of the D-G15 monoclonal strain.

[0024] Figure 3 This is a phylogenetic tree constructed based on the 16S rRNA sequence of strain D-G15.

[0025] Figure 4 This is the result of comparing the average nucleotide identity (ANI) of the genomes of strain D-G15 with other strains in the genus Devosia.

[0026] Figure 5 This shows the degradation of DON by strain D-G15 at different pH levels.

[0027] Figure 6 This shows the degradation of DON by strain D-G15 at different temperatures.

[0028] Figure 7This is a comparison of the cytotoxicity of strain D-G15 against HEK293T before and after DON degradation.

[0029] Figure 8 The effect of feeding strain D-G15 on the weight of broilers.

[0030] Figure 9 This study investigates the effects of feeding strain D-G15 on broiler organs. Figure a shows a paraffin section of the heart from the control group; Figure b shows a paraffin section of the liver from the control group; Figure c shows a paraffin section of the kidney from the control group; Figure d shows a paraffin section of the heart from the D-G15 group; Figure e shows a paraffin section of the liver from the D-G15 group; and Figure f shows a paraffin section of the kidney from the D-G15 group. Image magnification: ×200, scale bar = 100 μm. Detailed Implementation

[0031] The present invention will be further described below with reference to 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.

[0032] Example 1: Enrichment and Isolation of DON-Degrading Strains

[0033] 1. Experimental Methods

[0034] (1) Forty-three soil samples were collected from wheat fields infected with Fusarium. 1 g of each sample was resuspended in 10 mL of PBS. After standing for 5 min, 20 μL of the supernatant of the resuspension was inoculated into 1 mL of inorganic salt medium containing 25 μg / mL DON. The sample inoculated with 20 μL of sterile water was used as a negative control. The samples were cultured at 30℃ and 150 rpm for 7 days.

[0035] (2) Take 20 μL of culture medium from each sample and inoculate it into an inorganic salt medium containing DON and continue culturing for 7 days. Repeat this process to eliminate contaminating bacteria that cannot utilize DON, and detect the degradation of DON in the samples by HPLC.

[0036] (3) Dilute the sample with stable degradation activity to the highest factor with degradation activity, and spread the bacterial solution of this dilution factor and the first two concentration gradients on inorganic salt sucrose plates. Spread 100 μL of bacterial solution on each solid plate and incubate upside down at 30°C for 3 days.

[0037] (4) Randomly select single clones from the plates from which colonies have grown and inoculate them into 500 μL of fresh liquid medium containing DON inorganic salts. After 7 days, detect the DON degradation in the samples.

[0038] (5) Streak the monoclonal strains that were found to have degradation activity onto plates and incubate them at 30°C for 3 days. Observe the growth of the plates. If the grown clones are uniform in size and morphology, select 5-10 monoclonal strains for activity verification.

[0039] (6) If each of the single clones selected in (5) has DON degradation activity, then it is preliminarily determined that the obtained single clone is a pure culture strain with the ability to degrade DON.

[0040] 2. Experimental Results

[0041] The experimental results are attached. Figure 1 As shown, the finally isolated monoclonal strain was able to degrade DON.

[0042] Example 2: Identification of strain D-G15

[0043] 1. Experimental Methods

[0044] (1) Observe the morphology of the monoclonal strain using transmission electron microscopy.

[0045] (2) The 16S rRNA gene of D-G15 was sequenced and a phylogenetic tree was constructed.

[0046] (3) The whole genome of strain D-G15 was sequenced, and the genome was compared with other strains in the genus Devosia using average nucleotide identification analysis (ANI).

[0047] 2. Experimental Results

[0048] (1) Morphological analysis of D-G15 was performed using transmission electron microscopy. Figure 2 As shown, D-G15 cells are rod-shaped, approximately 0.8–1.5 × 0.5–0.8 μm in size, and possess a single flagellum.

[0049] (2) The results of the 16S rRNA sequence analysis are attached. Figure 3 As shown, D-G15 has the highest sequence similarity to Devosia sp. ADB-34, reaching 99.06%, so it is named Devosia sp. D-G15.

[0050] (3) The ANI value analysis results are attached. Figure 4 As shown, D-G15 and other Devosia strains have ANI values ​​of less than 80%, therefore it is determined to be a new species belonging to the genus Devosia.

[0051] Example 3: Optimal conditions for DON degradation by strain D-G15

[0052] I. The effect of different pH values ​​on the ability of D-G15 to degrade DON

[0053] 1. Experimental Methods

[0054] (1) Adjust the pH of the inorganic salt culture medium to 3-11 using hydrochloric acid and sodium hydroxide, and autoclave at 121℃ for later use.

[0055] (2) Adjust the concentration of the strain in the logarithmic growth phase to 10. 6 -10 8 CFU / mL range, after centrifugation to remove supernatant, the cells were resuspended in the same volume of culture medium at different pH values. DON was added to make the final concentration of DON in the system 100 μg / mL. The cells were cultured at 30℃ for 3 days, with three replicates for each pH.

[0056] (3) After the culture is completed, each sample is extracted and tested to calculate the degradation rate of DON.

[0057] 2. Experimental Results

[0058] The experimental results are attached. Figure 5 D-G15 exhibits good DON degradation activity in the pH range of 4-10, with the highest degradation activity at pH 7.

[0059] II. The effect of different temperatures on the ability of D-G15 to degrade DON

[0060] 1. Experimental Methods

[0061] (1) Adjust the concentration of the strain in the logarithmic growth phase to 10. 6 -10 8 Within the CFU / mL range, after inoculation, the cells were cultured at 20℃, 25℃, 30℃, 35℃ and 40℃, with three replicates for each temperature.

[0062] (2) After the culture is completed, each sample is extracted and tested to calculate the degradation rate of DON.

[0063] 2. Experimental Results

[0064] The experimental results are attached. Figure 6 D-G15 exhibits good DON degradation activity in the temperature range of 15-35℃, with the highest activity at 30℃.

[0065] Example 4: Cytotoxicity of strain D-G15 after DON degradation

[0066] 1. Experimental Methods

[0067] HEK293T cell viability was determined using CCK-8 assay to compare cytotoxicity. DON solution and fermentation supernatant from DON degradation by the strain were sterilized by filtration through a 0.22 μm filter before use in cell experiments. HEK293T cells were seeded into 96-well plates. After cell adhesion, untreated cells were used as a control group. Different concentrations of DON and the corresponding concentration of DON-degraded product were added to each well, resulting in final concentrations of 50, 100, 250, 500, and 1500 ng / mL. Six replicates were set for each sample group. After culturing at 37°C for 24 h, CCK-8 solution was added to each well, and the 96-well plates were incubated at 37°C in the dark for 2 h. The absorbance at 450 nm was recorded using a microplate reader, and cell viability was calculated using the following formula: Cell viability (%) = (OD value of experimental wells - OD value of control group) / (OD value of negative control group - OD value of control group) × 100%.

[0068] 2. Experimental Results

[0069] The experimental results are attached. Figure 7 At a DON concentration of 50 ng / mL, the growth of HEK293T cells was significantly inhibited, with cell viability decreasing to 70.3%. At a DON concentration of 250 ng / mL, cell viability reached the IC50 threshold. 50 Value. When D-G15 was used to degrade the same concentration of DON, its inhibitory effect on the growth of HEK293T cells was significantly reduced, indicating that the product of DON degradation exhibited lower cytotoxicity compared to DON.

[0070] Example 5: Safety evaluation of strain D-G15 in animals.

[0071] Thirty one-day-old female Qingyuan Ma chickens were pre-fed a basal diet for 5 days and then randomly divided into two groups, with three cages in each group and five chickens in each cage. The groups were: (Control group: fed the basal diet) and (D-G15 group: fed the basal diet with D-G15 bacterial solution added to the drinking water at a final concentration of 10). 6 (CFU / mL). The experiment lasted 42 days, during which all animals had free access to food and water.

[0072] I. Effects of feeding strain D-G15 on broiler growth performance

[0073] 1. Experimental Methods

[0074] After the experiment began, feed intake was recorded daily and broiler weight was recorded weekly.

[0075] 2. Experimental Results

[0076] The experimental results are attached. Figure 8As shown, during the experiment, there was no significant difference in body weight between the D-G15 group and the control group, indicating that D-G15 does not affect the growth of broilers.

[0077] II. Effects of feeding strain D-G15 on routine blood parameters in broilers

[0078] 1. Experimental Methods

[0079] After the experiment, the broilers were fasted for 12 hours and blood was collected from the vein under the wing. The blood was collected in EDTA anticoagulant tubes, and blood routine indicators such as white blood cell count (WBC), red blood cell count (RBC), and hemoglobin (HGB) were measured.

[0080] 2. Experimental Results

[0081] The results are shown in Table 1. There were no significant differences in white blood cell count (WBC), red blood cell count (RBC), and hemoglobin (HGB) between the D-G15 group and the control group.

[0082] Table 1. Routine Blood Analyses

[0083]

[0084] III. Effects of feeding strain D-G15 on broiler organs

[0085] 1. Experimental Methods

[0086] After blood collection at the end of the experiment, the broiler chickens in each group were immediately dissected to observe the color and morphology of their internal organs and to check for lesions. The heart, liver and kidney organs were dissected, fixed by soaking in 4% formaldehyde solution for 24 hours, embedded in paraffin, prepared into sections, and stained with hematoxylin and eosin.

[0087] 2. Experimental Results

[0088] The results are attached. Figure 9 As shown, the heart tissue structure of broiler chickens in the control group was intact, with myocardial fibers arranged neatly and regularly; the liver tissue structure was intact, with hepatocytes tightly arranged and normal in morphology; the kidney tissue structure was normal and intact, with clear boundaries of renal tubules and nephrons. Compared with the control group, the myocardium in the D-G15 group showed no inflammatory cell infiltration, edema, or congestion; neither hepatocytes nor renal tubular epithelial cells underwent degeneration or necrosis, with only local renal tubular epithelial cells showing mild granular degeneration, consistent with the control group. These results indicate that D-G15 is safe for broiler chickens.

Claims

1. A highly efficient Devosia sp. strain D. G15, characterized in that, This strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 26, 2024, with accession number GDMCC NO: 64453.

2. The strain D according to claim 1 Application of G15 in the degradation of DON.

3. The application according to claim 2, characterized in that... D Application of G15 in the preparation of DON detoxifying agents, DON detoxifying enzymes, and DON detoxifying engineered bacteria.

4. The application according to claim 2, characterized in that, When used to degrade DON, D The culture temperature for G15 is 15°C. 35℃, pH value is 4 10.

5. The application according to claim 2, characterized in that, The application is to use D G15 is used for the degradation of DON in animal feed processing, livestock and poultry breeding and food processing.

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