A strain of bacteria Raoultibacter massiliensis D-G5 capable of efficiently degrading vomitoxin and its application

Abstract

The present invention isolated a strain of Raoultibacter massiliensis bacteria from the intestinal contents of ducks and named it Raoultibacter massiliensis D-G5 (D-G5). The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 1, 2024, and its deposit number is GDMCC NO: 64485. Strain D-G5 can de-epoxidize vomitoxin (DON) into low-toxic DOM-1, which is beneficial to the degradation of DON by the strain within the range of pH 5-11 and temperature 30-42°C. D-G5 has stable degradation activity and strong metabolic capacity, and can be used for the preparation of DON detoxification preparations, and is applied in the fields of feed processing and food processing.

Description

Technical Field

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

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a secondary metabolite produced by fungi such as Fusarium graminearum and belongs to the type B trichothecenes family of toxins. DON is primarily found in cereals such as wheat, barley, oats, rye, and corn. It is one of the most commonly detected mycotoxins in grains and feed, causing significant economic losses to the food and feed industries.

[0003] DON has both chronic and acute toxicity. Long-term consumption of low-dose DON-contaminated feed can cause animals to experience problems such as anorexia, growth retardation, and immunotoxicity. Acute toxicity manifests itself in symptoms such as abdominal pain, diarrhea, and vomiting. Currently, detoxification strategies for DON mainly include physical, chemical, and biological methods. Physical methods include sorting and separation, heat treatment, radiation, and adsorption. Chemical methods include the use of alkali, acid, and oxidants to treat contaminated feed. Although physical and chemical methods can achieve detoxification effects to a certain extent, their specificity is poor, affecting the nutritional value and taste of food, and may introduce harmful chemicals to cause secondary pollution. In contrast, biological detoxification methods based on microorganisms and enzymes are considered to be an ideal detoxification method. This method has the advantages of high specificity, mild action conditions, no effect on palatability, and safety and environmental protection.

[0004] In summary, the widespread contamination of DON in feed and feed ingredients poses a significant threat to livestock and poultry farming. Biological detoxification using microbial degradation is an ideal method for DON detoxification and has broad application prospects. Summary of the Invention

[0005] The first object of the present invention is to provide a strain of Raoultibacter massiliensis bacteria D-G5 that can efficiently degrade DON.

[0006] The second object of the present invention is to provide the use of D-G5 in degrading DON.

[0007] The third object of the present invention is to provide the use of D-G5 in preparing DON detoxification preparations.

[0008] The fourth object of the present invention is to provide the use of D-G5 in constructing DON-detoxifying engineered bacteria or cultivating DON-tolerant transgenic plants.

[0009] The present invention is achieved through the following technical solutions:

[0010] The present invention screened and isolated a strain capable of degrading DON from the intestinal contents of ducks. After 16S rRNA comparative analysis, the strain was identified as belonging to the species Raoultibacter massiliensis and named Raoultibactermassiliensis D-G5 (hereinafter referred to as D-G5). The strain was deposited in the Guangdong Provincial Microbial Culture Collection on April 1, 2024, and its deposit number is GDMCC NO: 64485.

[0011] The present invention also provides the use of D-G5 in degrading DON.

[0012] The present invention also provides the use of D-G5 in preparing feed additives or food additives.

[0013] The present invention also provides the use of D-G5 in preparing a DON-degrading enzyme preparation.

[0014] In some embodiments of the present invention, the strain is Raoultibacter massiliensis D-G5 (D-G5).

[0015] In some embodiments of the present invention, when used to degrade DON, the culture medium of D-G5 is WCA medium.

[0016] In some embodiments of the present invention, when used to degrade DON, the concentration of D-G5 is 10 4 -10 8 CFU / mL.

[0017] In some embodiments of the present invention, when used to degrade DON, the culture temperature of D-G5 is 20-42° C., and the pH of the culture medium is 4-11.

[0018] The present invention has the following beneficial effects:

[0019] The present invention provides a strain of Raoultibacter massiliensis bacteria, D-G5, capable of degrading DON. This strain was isolated from duck intestine. D-G5 can degrade DON into DOM-1. Its degradation activity is independent of the continued presence of DON and persists even after passage in DON-free culture medium. D-G5 exhibits stable degradation activity and strong metabolic capacity, potentially enabling its application in the preparation of DON detoxification preparations in feed and food processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Figure 1 is the HPLC and mass spectrometry results of strain D-G5 degrading DON to DOM-1; Figure a is the negative control with DON added to the culture medium, Figure b is the HPLC detection result of strain D-G5 degrading DON to DOM-1, and Figure c is the mass spectrometry result of the degradation product DOM-1;

[0021] Figure 2 is a transmission electron micrograph of the monoclonal strain D-G5;

[0022] Figure 3 is the phylogenetic tree of strain D-G5 based on the 16S rRNA sequence;

[0023] Figure 4 is the degradation of DON by D-G5 in different culture media;

[0024] Figure 5 is the degradation of DON by strain D-G5 at different pH;

[0025] Figure 6 is the degradation of DON by strain D-G5 at different temperatures;

[0026] Figure 7 This is the relationship between the growth of strain D-G5 and DON degradation under the optimal degradation conditions. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional in the art.

[0028] Example 1 Enrichment and Isolation of DON-Degrading Bacteria

[0029] 1. Experimental methods

[0030] (1) Nine cecae of freshly slaughtered ducks were randomly collected from Shangyuangang Farmers' Market in Guangzhou. The cecae were immediately returned to the laboratory. The intestinal contents were squeezed out and resuspended in 5 mL of PBS per 1 g of contents.

[0031] (2) After the intestinal content suspension was allowed to stand at room temperature for 5 min, 10 μL of the intestinal content suspension was inoculated into 190 μL of LB, BHI, TSB, and WCA culture media containing 25 μg / mL DON. Samples inoculated into 10 μL of sterile water were set as negative controls.

[0032] (3) The inoculated sample was transferred to an anaerobic workstation and cultured anaerobically at 37°C for 3 days.

[0033] (4) After the incubation, take the remaining 100 μL of each sample and add 300 μL of ethyl acetate to vortex and mix thoroughly for extraction. Let it stand for 1 min, then draw the upper organic phase into a clean EP tube. Use a nitrogen blower to blow dry the organic phase in the EP tube, add 200 μL of 25% methanol, and vortex and shake to dissolve the precipitate.

[0034] (5) HPLC was used to detect the degradation of DON in the samples. The samples with DON degradation activity were diluted in a gradient manner. 20 μL of bacterial solution was taken each time and added to 180 μL of fresh DON-containing culture medium and mixed. The samples were gradually diluted to 10 8 times, and then cultured under anaerobic conditions for 3 days, and this operation was repeated to detect the activity gradient of the samples.

[0035] (6) Dilute the sample to the highest metabolic activity multiple, and spread the bacterial solution of this gradient and the above two gradients on the plate, with 20 μL of bacterial solution spread on each plate, and culture anaerobically for 3 days.

[0036] (7) Randomly pick single clones from the plate and culture them in fresh liquid culture medium containing DON. Continue to culture them anaerobically at 37°C for 3 days to detect DON degradation.

[0037] (8) Streak the single clones with degradation activity onto a plate and incubate anaerobically at 37°C for 3 days before observation. If the clones that grow have uniform morphology, select 5-10 of them for activity verification.

[0038] (9) If all the monoclones selected in (8) have DON degradation activity, they are preliminarily judged to be pure culture strains.

[0039] (10) The monoclonal strain was incubated with DON, and the DON degradation products of the monoclonal strain were identified using UPLC-MS.

[0040] 2. Experimental results

[0041] The experimental results are as follows Figure 1 As shown, the monoclonal strain D-G5 finally isolated was able to de-epoxidize DON to DOM-1.

[0042] Example 2 Identification of strain D-G5

[0043] 1. Experimental methods

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

[0045] (2) The 16S rRNA gene of D-G5 was sequenced and a phylogenetic tree was constructed.

[0046] 2. Experimental results

[0047] (1) The morphology of D-G5 was detected and analyzed by transmission electron microscopy. Figure 2 As shown, the cell morphology of D-G5 is rod-shaped, with a size of approximately 0.8-1.3 × 0.3-0.6 μm, and no flagella.

[0048] (2) The results of 16S rRNA sequence analysis are shown in the attached Figure 3 As shown in the results, D-G5 was most closely related to the strain of Raoultibacter massiliensis, so it was named Raoultibacter massiliensis D-G5.

[0049] Example 3 Optimal conditions for DON degradation by strain D-G5

[0050] 1. Ability of D-G5 to degrade DON in different culture media

[0051] 1. Experimental methods

[0052] (1) Use hydrochloric acid and sodium hydroxide to adjust the pH value of WCA medium to 4-11, sterilize it by high pressure at 121℃ and set aside.

[0053] (2) Adjust the strain concentration in the logarithmic growth phase to 10 6 -10 8 After centrifugation and removal of the supernatant, the cells were resuspended in an equal volume of the above-mentioned culture medium with different pH values. DON was added to make the final concentration of DON in the system 30 μg / mL. The cells were cultured anaerobically at 37°C for 3 days, with three replicates for each pH value.

[0054] (3) After the incubation, each sample was extracted and tested, and the degradation rate of DON was calculated.

[0055] 2. Experimental results

[0056] The experimental results are as follows Figure 5 , D-G5 showed good DON degradation activity in the pH range of 5-11, and even at pH 4, the degradation activity remained above 50%.

[0057] 2. Effects of different pH values ​​on the ability of D-G5 to degrade DON

[0058] 1. Experimental methods

[0059] (1) Use hydrochloric acid and sodium hydroxide to adjust the pH value of WCA medium to 4-11, sterilize it by high pressure at 121℃ and set aside.

[0060] (2) Adjust the strain concentration in the logarithmic growth phase to 10 6 -10 8After centrifugation and removal of the supernatant, the cells were resuspended in an equal volume of the above-mentioned culture medium with different pH values. DON was added to make the final concentration of DON in the system 30 μg / mL. The cells were cultured anaerobically at 37°C for 3 days, with three replicates for each pH value.

[0061] (3) After the incubation, each sample was extracted and tested, and the degradation rate of DON was calculated.

[0062] 2. Experimental results

[0063] The experimental results are as follows Figure 5 , D-G5 showed good DON degradation activity in the pH range of 5-11, and even at pH 4, the degradation activity remained above 50%.

[0064] 3. Effects of different temperatures on the ability of D-G5 to degrade DON

[0065] 1. Experimental methods

[0066] (1) Adjust the concentration of the strain in the logarithmic growth phase to 10 6 -10 8 Within the CFU / mL range, the cells were cultured at 20°C, 25°C, 30°C, 37°C, and 42°C after inoculation, with three replicates set for each temperature.

[0067] (2) After the incubation, each sample was extracted and tested, and the degradation rate of DON was calculated.

[0068] 2. Experimental results

[0069] The experimental results are as follows Figure 6 , D-G5 showed good DON degradation activity in the temperature range of 30-42 °C, with the highest activity at 37 °C.

[0070] Example 4 Growth and degradation curves of strain D-G5

[0071] 1. Experimental methods

[0072] (1) Adjust the logarithmic growth phase of the bacteria to 10 3 CFU / mL, cultured anaerobically in the medium at the optimal pH and temperature, with three replicates.

[0073] (2) Samples were taken every 12 hours to detect the degradation of DON. At the same time, the bacterial solution was diluted to observe the highest dilution multiple of bacterial growth and determine the bacterial order of magnitude.

[0074] (3) Cultivate the strain under optimal conditions and monitor the degradation of DON every 12 hours.

[0075] 2. Experimental results

[0076] The experimental results are as follows Figure 7 The logarithmic growth phase of D-G5 occurred between 24 and 60 hours of culture. During the 48-60 hours of culture, the degradation rate of DON by D-G5 increased significantly, and by 72 hours of culture, DON was completely degraded by D-G5.

Claims

1. A strain of bacteria (Raoultibacter massiliensis) D-G5 that efficiently degrades vomitoxin, characterized by: This strain was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 1, 2024, and its deposit number is GDMCC NO: 64485.

2. Use of the D-G5 strain as claimed in claim 1 in degrading DON.

3. The use according to claim 2, characterized in that The application is to prepare a DON detoxifying agent, a DON detoxifying enzyme or a DON detoxifying engineered bacterium.

4. The use according to claim 2, characterized in that The conditions for degrading DON are: culture temperature 30-42°C, pH 5-11.

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

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