Strains for decomposing deoxynivalenol and their use

Abstract

The present invention relates to the technical fields of microorganisms, feed, food and ecological remediation, in particular to a strain capable of decomposing deoxynivalenol (DON) and its use. The storage number of the strain is CCTCC No. M 2020565. The strain grows with the toxic compound DON as the only carbon source and converts DON into its own chemical components. This reaction is irreversible, the reaction conditions are mild, and it does not cause secondary pollution. The strain provided in the present invention can be used to prepare a biological detoxifier for DON. The strain provided in the present invention can decompose DON in feed and food raw materials, primary processed products, deep processed products and related processing by-products. The strain provided in the present invention can be applied to various ecosystems such as soil and water bodies contaminated by DON to decompose DON and achieve the purpose of remediating the ecosystem.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to strains that decompose deoxynivalenol and their use.

Background Art

[0002] Deoxynivalenol (DON) is also called vomitoxin because it can cause vomiting in humans and animals. Deoxynivalenol is a common mycotoxin derived from food and widely exists in food raw materials such as wheat, barley, oats, and corn. DON is very stable chemically and physically, and it is difficult to remove by general cooking and processing techniques. It may be transformed into other compounds with unknown toxicity (such as nor-DON, DON lactone, etc.) during high-temperature baking processes. Furthermore, DON can exist in a "latent" form within plants, such as 3-β-D-glucose-DON. This "latent" form of derivative is considered to be a product of plant self-defense. Unfortunately, there is still a possibility that latent DON can return to DON due to the action of intestinal microorganisms, further increasing the exposure risk for humans and animals.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The biodegradation technology of DON has characteristics such as being efficient, environmentally friendly, having mild reaction conditions, and being easily applicable on a large scale, and has been widely noticed in recent years. However, the biological resources used in research and use are very limited. The acquisition of DON-decomposing strains by pure culture and the elucidation of the decomposition mechanism of the strains against DON are considered to provide important hints for the development of DON decomposition products.

[0004] Devosia is a type of DON-degrading strain that has been widely studied. Multiple species and subspecies in the genus Devosia have been shown to degrade DON into 3-keto-DON and 3-epi-DON. In 2018, the Zhou Ting group in Canada confirmed that two enzymes, DepA and DepB, are involved in the two-step degradation process respectively. Since then, multiple teams have successively confirmed that independently isolated Devosia strains can transform DON. However, not all Devosia strains can achieve the above two-step transformation.

[0005] Nocardioides was found to have a DON-degrading function almost synchronously with Devosia. In the published literature, only the Tsushima group in Japan reported that it can completely transform and utilize DON as a carbon source. During the degradation of DON by Nocardioides, 3-epi-DON is generated and further transformed, but the complete degradation mechanism has not been studied in detail yet. One of the main reasons is that the strains of Nocardioides with DON-degrading function are very rare, and it is difficult to obtain pure cultures by enrichment isolation.

[0006] The present invention has been made in view of the above problems. The object of the present invention is to provide a Nocardioides strain for degrading deoxynivalenol.

[0007] Another object of the present invention is to provide a biological detoxifying agent made using the Nocardioides strain.

[0008] Still another object of the present invention is to provide a method for preparing the biological detoxifying agent.

Means for Solving the Problems

[0009] The present invention provides a novel strain of Nocardioides sp. with the strain number ZHH-013, which was deposited at the China Center for Type Culture Collection (CCTCC) on September 30, 2020, and the deposit number is CCTCC No. M 2020565. Although the function of this strain is similar to that of Nocardioides sp. WSN05-2, the identity of its 16S rDNA is less than 97%. Through the comparison of the morphological, molecular biological, and physiological and biochemical characteristics of related strains, it was confirmed that it is a new species of the Nocardioides genus.

[0010] As the fermentation medium, add 10 g of casein tryptone, 5 g of yeast fermentation extract, 10 g of sodium chloride, and 1.5% of agar, add distilled water to 1 L, adjust the pH to 7.0 - 7.2, and sterilize at 121 °C for 20 minutes.

[0011] The biological detoxifying agent according to the present invention contains Nocardioides sp. ZHH-013 or its intracellular lysate (for example, intracellular protein) as an active ingredient. The Nocardioides sp. ZHH-013 has the deposit number CCTCC No. M 2020565. The biological detoxifying agent is prepared by a conventional preparation method and may be in a liquid dosage form or a solid dosage form.

[0012] The present invention provides a method for preparing the biological detoxifying agent. The preparation method includes activating Nocardioides sp. ZHH-013 with the deposit number CCTCC No. M 2020565, performing enlarged culture in multiple stages, and when the bacterial cells are in the stationary phase, collecting the fermentation broth to prepare a liquid biological detoxifying agent.

[0013] The method for preparing a biological detoxifying agent according to the present invention is to concentrate its fermentation broth by methods that do not affect the activity of the bacterial cells, such as natural sedimentation, centrifugation, and filtration, to prepare a high-concentration bacterial cell suspension. Preferably, a nutrient solution or a mixture of a nutrient solution and a protective agent is further added to the bacterial cell suspension to prepare a liquid biological detoxifying agent. Furthermore, a solid biological detoxifying agent can be prepared from the liquid biological detoxifying agent by adding, for example, an adsorbent, a protective agent, etc. according to conventional methods in the art.

[0014] The method for preparing a biological detoxifying agent according to the present invention may further include preparing a liquid biological detoxifying agent by pulverizing the obtained high-concentration bacterial cell suspension or bacterial cells by conventional methods such as homogenization and ultrasonic waves, removing impurities such as bacterial cell fragments, and then concentrating to obtain a high-concentration protein solution. A solid biological detoxifying agent can be prepared from the liquid biological detoxifying agent by adding, for example, an adsorbent, a protective agent, etc. according to conventional methods in the art.

[0015] The above-mentioned biological detoxifying agent is packaged by general packaging techniques in the art and can be stored according to specific environmental conditions.

[0016] The present invention has the following advantages.

[0017] (1) The present invention provides a strain Nocardioides sp. ZHH-013 that is isolated from soil within the territory of China and has the function of degrading DON. The DON-degrading function of this strain is similar to that of Nocardioides strains isolated within the territory of Japan that have already been reported, but the identity of 16S rDNA is less than 97%.

[0018] (2) The strain provided by the present invention has been classified and identified by classification methods such as morphology, physiological biochemistry, and 16S rDNA sequence analysis. By comparing the morphological and physiological biochemical characteristics of related strains, it has been confirmed that it is a new species of the genus Nocardioides.

[0019] (3) The strain provided by the present invention grows using the toxic compound DON as the sole carbon source and converts DON into its own chemical components. This reaction is irreversible, the reaction conditions are mild, and it does not cause secondary pollution.

[0020] (4) The strain provided by the present invention can be used for the preparation of various DON biological detoxifying agents.

[0021] (5) The strain provided by the present invention and various DON biological detoxifying agents prepared therefrom can be used for the decomposition of DON in feeds and food raw materials, preliminary processed products, deep processed products and related processing by-products.

[0022] (6) The strain provided by the present invention and various DON biological detoxifying agents prepared therefrom can be applied to various ecosystems such as soil and water areas contaminated with DON to decompose DON and achieve the purpose of restoring the ecosystem.

Brief Description of the Drawings

[0023]

Figure 1

Figure 2

Examples

[0024] Example 1 Isolation and Identification of Deoxynivalenol-Degrading Strains Soil samples were collected from Zhangjiakou City, Hebei Province. Using the enrichment flask shaking method, the samples were first prepared into bacterial suspensions in sterile water. Inoculated into LB liquid medium at an inoculation amount of 10%, with the final concentration of DON in the medium being 50 μg / ml, after culturing for 7 days, transplanted at an inoculation amount of 10%, and the DON concentration in the medium remained unchanged. After continuously performing 5 transplantations, the DON content was detected, and the uninoculated DON-LB medium with the same DON concentration was used as the negative control. The bacterial suspension with DON-degrading function was spread on an LB agar plate at an appropriate dilution by the dilution plating method and cultured at 30 °C for 72 hours. Then, single colonies with good separation and different colony morphologies were selected, and a detoxification test was carried out in an LB medium with a DON concentration of 50 μg / ml. The DON content was measured according to Example 1, and the screening was repeated. Finally, 1 strain of bacteria capable of degrading DON was screened and numbered ZHH-013. The ZHH-013 single colony was transferred to an LB liquid medium and cultured until the mid-logarithmic phase. Then, its culture and 50% glycerin were mixed in the same volume and stored at -80 °C.

[0025] Classification and identification of the degrading strain were carried out by classification methods such as morphology, physiology and biochemistry, and 16S rDNA sequence analysis. The ZHH-013 strain of the present invention was cultured at 30 °C on an LB agar medium. On the 7th day, circular white colonies with a diameter of 1 mm were confirmed, with smooth edges, a shiny surface, and a transparent ring was confirmed around the colonies. When cultured at 30 °C on a TSB agar medium, on the 7th day, circular white colonies were confirmed, with smooth edges, a shiny surface, and a transparent ring was confirmed around the colonies. On the 14th day, the diameter of the colonies reached 3 mm. The result of Gram staining was positive.

[0026] Here, as the LB medium, 10 g of casein tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 1.5% agar were added, distilled water was added to 1 L, adjusted to pH 7.0 - 7.2, and sterilized at 121 °C for 20 minutes.

[0027] The TSB medium was purchased from BD, and the brand was Difco.

[0028] From all the results, it was revealed that the ZHH-013 strain of the present invention should be classified as Nocardioides sp. This classification is based on direct comparison in the laboratory and search for descriptions of similar species that have already been published.

[0029] Example 2 Degradation of Deoxynivalenol by the Strain 1, Detection Method of DON Regarding the preparation of the DON stock solution, 5.0 mg of the DON standard was accurately weighed and dissolved in 1 mL of sterile water to prepare a DON stock solution with a final concentration of 5 mg / mL. It was sterilized by filtration using a 1 mL syringe filter. The DON stock solution was stored at -20°C and was valid for 3 months.

[0030] Regarding sample preparation, 500 μL of the sample solution was taken, the same volume of pure methanol was added, and after shaking and mixing uniformly, it was centrifuged (12,000 rpm, 4°C, 10 minutes), and 500 μL of the supernatant was taken for HPLC detection. The content of DON was measured by the HPLC method. The measurement conditions were that the column was an Agilent 5 TC-C18(2) reverse-phase column (250×4.6 mm, 5 μm), the mobile phase was methanol:water (15:85), the elution method was isocratic elution, the flow rate was 1 mL / min, the injection volume was 20 μL, the column temperature was 30°C, the detection wavelength of the ultraviolet detector was 220 nm, and the retention time of DON was 14.9 minutes.

[0031] 2. Monoclonal strains of Nocardioides sp. ZHH-013 were selected from the plate, inoculated into 3 mL of LB medium, cultured at 30 °C for 7 days, and then transferred to 3 mL of LB medium at an inoculation amount of 1%. Here, the final concentration of DON was 50 μg / ml. Shake culture was carried out at 30 °C for 3 days, and the DON content was measured by the above method. The test group without DON added was used as a negative control. The chromatographic analysis results are shown in Figure 1. a shows the control inoculated with only the bacterial solution. e shows the control with only DON added. b and c show the two treatment groups after overnight culture. Compared with the control, it is obvious that DON was completely decomposed.

[0032] 3. Degradation of DON by crude enzyme solution As in step 2 above, the bacterial cells were prepared, resuspended in PBS buffer (pH 6.9), and the OD 600 was adjusted to about 1.0. After disrupting the bacterial cells using an ultrasonic disruptor, centrifugation (12,000 rpm, 4 °C, 10 minutes) was performed to take out 490 μL of the supernatant. 10 μL of the DON stock solution was added to the supernatant to make the final concentration 50 μg / mL. After overnight culture, the DON content was measured by the above method. The chromatographic analysis results are shown in d of Figure 1. After overnight culture, it is obvious that DON was completely decomposed.

[0033] 4. Degradation of DON in corn steep liquor by the strain Monoclonal strains of Nocardioides sp. ZHH-013 were selected from the plate, inoculated into 3 mL of LB medium, cultured at 30 °C for 7 days, and then transferred to 300 mL of LB medium at an inoculation amount of 1% and shake cultured at 30 °C for 5 days to collect the bacterial cells. The bacterial cells were washed thoroughly with sterile water at least 3 times to completely remove the medium. The bacterial cells were resuspended thoroughly in PBS buffer (pH 6.9), and the OD 600It was adjusted to approximately 1.0. An aqueous solution of 10% of 10% corn steep liquor was added to each, and a sample without the addition of the aqueous solution of 10% corn steep liquor was used as a control. After overnight culture, the DON content was measured by the above method. The chromatographic analysis results are shown in Figure 2. a shows the treatment group to which the aqueous solution of 10% corn steep liquor was added. b shows the treatment group to which the aqueous solution of 10% corn steep liquor was not added. c shows the control group to which neither corn steep liquor nor the degrading strain was added. When compared with the control group c, the DON degradation rate after overnight culture in the treatment group a was 80% or more, and the DON content in the treatment group b also decreased significantly. Therefore, regardless of the addition of corn steep liquor, DON can be degraded. The degrading function of the strain is stable and does not change even when the substrate composition changes.

[0034] 5, Growth of the strain using DON as the sole carbon source Monoclonal of Nocardioides sp. ZHH-013 was selected from the plate, inoculated into 1 mL of M9D medium, cultured at 30 °C for 7 days, and then transferred once every 7 days by the transfer method for a total of 3 transfers. The fermentation broth after each 7-day culture was collected, 150 μL of the fermentation broth was spread on an LB agar plate for culture, and the DON content of the remaining fermentation broth was measured by the above method. As the M9D medium, M9 medium (Difco) with 50 mg of DON added, adjusted to pH 7.0, and distilled water was added to 1 L. The above transfer method refers to the method of transferring to 1 mL of M9D medium at an inoculation amount of 0.1% and culturing at 30 °C for 7 days.

[0035] In this experiment, three consecutive transfers were performed, and in each transfer, the bacterial solution was diluted 1000-fold. When DON cannot be utilized as a carbon source, DON is not decomposed and cannot be decomposed in any of the three consecutive experiments. Regarding the number of colonies on the LB agar plate, there is a regular pattern that the greater the number of dilutions, the fewer the number of colonies. When DON can be utilized as a carbon source, since the number of colonies increases after each culture, in the three experiments, DON is decomposed in each case, and the number of colonies on the LB agar plate is relatively large and almost the same.

[0036] From the experimental results, it was shown that in the culture fermentation broth collected three times, the degradation rate of DON was 80% or more in each case, and the bacterial flora of Nocardioides sp. ZHH-013 grew vigorously on the corresponding LB agar plate. That is, it was shown that Nocardioides sp. ZHH-013 can grow using DON as the sole carbon source.

[0037] It is obvious that the above description of the embodiments is merely an example for clearly explaining the present invention and does not limit the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes and modifications can be made. Here, it is impossible to list all the embodiments. Needless to say, obvious changes and modifications from the idea of the present invention are still included in the protection scope of the present invention.

Claims

1. A strain ZHH-013 of the genus Nocardiopsis, characterized in that the deposit number is CCTCC No. M 2020565.

2. A biological detoxifying agent, characterized in that it contains the strain ZHH-013 of the genus Nocardiopsis according to Claim 1 as an active ingredient.

3. The biological detoxifying agent according to Claim 2, characterized in that the dosage form of the biological detoxifying agent is liquid or solid.

4. Inoculating a medium with the strain ZHH-013 of the genus Nocardiopsis; The step of enlarging and culturing the strain ZHH-013 in multiple stages; and Collecting the fermentation broth and preparing the biological detoxifying agent, A method for preparing a biological detoxifying agent, characterized by comprising the above steps.

5. Preparing the fermentation broth of the strain ZHH-013 of the genus Nocardiopsis into a biological detoxifying agent in a liquid dosage form or a solid dosage form, The method for preparing a biological detoxifying agent according to Claim 4.

6. Further preparing the biological detoxifying agent in a liquid dosage form into a solid dosage form, The method for preparing a biological detoxifying agent according to Claim 5.

7. Use of the strain ZHH-013 of the genus Nocardiopsis according to Claim 1, characterized in that it decomposes DON in feed, food raw materials, preliminary processed products, deep processed products and related processing by-products.

8. Use of the biological detoxifying agent according to Claim 2, characterized in that it decomposes DON in an ecosystem contaminated with DON.

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