Nitrate reduction oceanobacter sp. And application thereof

Abstract

The invention relates to nitrate reduction pelagic bacteria and application thereof. The nitrate reductive ocean bacillus can be fermented to produce pyrroloquinoline quinone (PQQ) and trichothecenes toxin degrading enzyme, the trichothecenes toxin degrading enzyme mainly exists in bacterial cell lysis buffer, and the degradation rate of the trichothecenes toxin degrading enzyme to vomitoxin is 83%. The bacterial strain fermentation liquor can be used for degrading 39.3 percent of nivalenol and 100 percent of 15-acetyl deoxynivalenol. The strain or the trichothecene toxin degrading enzyme produced by the strain can be used for complete feed, concentrated feed, premixed feed, feed raw materials such as grain, corn, peanut meal, wheat and oat, silage, grain, grain and oil, grain processing and agricultural and sideline products, aged grain, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour, corn flour and corn flour. Through biodegradation of industrial ethanol processing byproducts and trichothecenes in food, the quality safety of feed and food can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial application, and in particular relates to a nitrate-reducing Pelagibacterium nitratireducens capable of producing pyrroloquinoline quinone and trichothecene toxin degrading enzymes, a biological preparation and an application thereof. Background Art

[0002] There are many types of trichothecenes, including deoxynivalenol (DON), also known as vomitoxin (NIV), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), and T-2 toxin. These are toxic secondary metabolites produced by fungi such as Fusarium graminearum and Fusarium graminearum. Trichothecenes are common in food and feed production. They are widely found in wheat, corn, and soybeans, making them one of the most common mycotoxins found in cereal raw materials and feed. A World Health Organization survey found that the detection rate of DON in oats was 68%, and in other cereals it ranged from 27% to 59%. A 2003 European Union survey reported that the detection rate of DON in food reached 57%. When animals consume moldy feed, the toxins and their metabolites remain in livestock products and processed goods, entering the human body through the food chain and posing a serious threat to human health. The latest version of the safety standard issued by the General Administration of Quality Inspection and Quarantine of China in 2017 stipulates that the DON content in plant-based feed raw materials should not exceed 5 mg / kg, the DON content in pig compound feed in feed products should not exceed 1 mg / kg, and the DON content in other compound feeds should not exceed 3 mg / kg.

[0003] Traditional physical and chemical mycotoxin detoxification methods have problems such as poor effectiveness, high cost, secondary pollution and loss of nutrients, making them difficult to apply in agricultural production. Microbial detoxification methods mainly use microbial strains or enzymes to perform cell adsorption, degradation, transformation or enzymatic decomposition of mycotoxins. Due to its advantages of being green, efficient, safe and free of secondary pollution, it has shown good application potential and has developed rapidly in recent years. Toxicological research results show that the toxic groups of trichothecenes are mainly C 12 ,13 epoxy ring and C3 hydroxyl group, the degradation and transformation of these two groups are the key sites of biological detoxification. For example, Clostridiales sp., Anaerofilum sp. and Bacillus sp. can degrade the bacteria through C 12,13Epoxy-epoxidation ring opening generates de-epoxidized DON to achieve biological detoxification; Devosia sp., Pseudomonas sp. and Lysobacter sp. can generate 3-keto-DON or 3-epi-DON through C3 hydroxyl oxidation or isomerization to achieve biological detoxification; some microorganisms such as Devosia insulae can generate 3-keto-DON or 3-epi-DON through C3 or C 15 Acetylation of the hydroxyl group at the 3 position achieves biological detoxification.

[0004] Due to the highly stable molecular structure of trichothecenes, the species and number of microorganisms currently discovered that can degrade trichothecenes are very limited, and research reports on DON-degrading enzymes are even scarcer. Among the reported microorganisms, some strains are not probiotics. Although they have high DON-degrading activity, they cannot be directly applied to degrade DON in food or feed. To address the problem of DON contamination in feed and its raw materials, it is urgent to isolate microorganisms with the ability to efficiently degrade DON. However, there are no reports on the degradation of DON by nitrate-reducing Bacillus pelagicus.

[0005] Reports indicate that some dehydrogenases isolated from DON-degrading bacteria can degrade DON to 3-keto-DON using the natural compound pyrroloquinoline quinone as a prosthetic group. Pyrroloquinoline quinone, a reddish-brown solid at room temperature and pressure, is an organic molecule with unique biological activity. It plays a role in maintaining mitochondria and other functions, similar to vitamins. Pyrroloquinoline quinone, a novel prosthetic group, was first isolated from bacteria. Pyrroloquinoline quinone is expensive on the market, and its large-scale use in feed or feed ingredients is costly. Therefore, developing a method for producing pyrroloquinoline quinone is crucial.

[0006] The present invention screened out a strain of nitrate-reducing Pelagobacterium ZH15, which has high degradation efficiency for vomitoxin (DON), nivalenol (NIV), and 15-acetyldeoxynivalenol (15-ADON) and can produce pyrroloquinoline quinone. This strain is expected to be used as a new bacterial resource for the biological detoxification of trichothecenes in the food and feed industries. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to screen out new strains capable of degrading trichothecenes from a large number of natural samples, thereby solving the problem of trichothecenes contamination in feed and food causing harm to animal and human health.

[0008] To solve the above problems, the present invention provides a nitrate-reducing Pelagibacterium nitratireducens ZH15 that simultaneously produces pyrroloquinoline quinone and trichothecene toxin-degrading enzymes.

[0009] The nitrate-reducing Pelagibacterium nitratireducens ZH15 of the present invention was deposited in the General Microbiology Center of the China Culture Collection Administration on September 13, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 28454.

[0010] The nitrate-reducing pelagic bacterium Pelagibacterium nitratireducens ZH15 was isolated from a large number of natural samples by the inventors through complex screening work.

[0011] The nitrate-reducing pelagic bacterium has the following characteristics:

[0012] (1) Colony characteristics: When cultured on LB plates at 37°C for 24 h, the colonies are bright yellow and smooth. Under a microscope, the cells appear short rod-shaped and are 1.2-1.8 μm × 0.8-1.2 μm in size.

[0013] (2) Molecular characteristics: The 16S rDNA gene sequence of this strain was 100% homologous to the 16S rDON gene sequence of Pelagibacterium nitratireducens strain JLT2005, and it was identified as Pelagibacterium nitratireducens strain.

[0014] (3) Salt tolerance: Nitrate-reducing Pelagibacterium ZH15 can grow normally in a culture medium with a NaCl concentration of less than or equal to 3%; 6% NaCl has a certain inhibitory effect on nitrate-reducing Pelagibacterium ZH15, and its growth is relatively slow; more than 9% NaCl completely inhibits the growth of nitrate-reducing Pelagibacterium ZH15.

[0015] (4) Production of pyrroloquinoline quinone: The fermentation broth of nitrate-reducing Bacillus pelagicus ZH15 can produce more than 50 mg / L of pyrroloquinoline quinone.

[0016] (5) When the lysate of nitrate-reducing Bacillus pelagicus ZH15 cells was incubated with vomitoxin (DON) at 30°C for 24 h, the degradation rate of DON reached 83%.

[0017] (6) When the fermentation broth of nitrate-reducing Bacillus thuringiensis ZH15 was incubated with nivalenol (NIV) at 30°C for 24 h, the degradation rate of NIV was 39.3%.

[0018] (7) When the fermentation broth of nitrate-reducing Bacillus pelagicus ZH15 was incubated with 15-acetyldeoxynivalenol (15-ADON) at 30°C for 24 h, the degradation rate of 15-ADON was 100%.

[0019] The present invention also provides a composition of nitrate-reducing Pelagobacterium and fermentation broth, and a method for fermenting and producing pyrroloquinoline quinone, characterized in that:

[0020] The composition includes nitrate-reducing pelagic bacillus ZH15 and a fermentation broth, wherein the concentration of pyrroloquinoline quinone in the fermentation broth is 10.00 mg / L or more, specifically 20.00 mg / L or more, 30.00 mg / L or more, 40.00 mg / L or more, 50.00 mg / L or more, 60.00 mg / L or more, 70.00 mg / L or more, 80.00 mg / L or more, 90.00 mg / L or more, 100.00 mg / L or more, and preferably 50.00 mg / L or more.

[0021] The fermentation method for producing pyrroloquinoline quinone is specifically as follows: placing the nitrate-reducing Pelagobacterium ZH15 on a slant culture medium and culturing it at 25-30° C. for 20-30 hours for activation; then picking a single colony of the nitrate-reducing Pelagobacterium from the slant culture medium and inoculating it into 5 mL of seed culture medium, shaking and culturing it at 25-30° C. and 180-220 rpm for 20-30 hours to obtain a seed solution; inoculating the obtained seed solution into a fermentation medium at a volume ratio of 1%-10%, and fermenting and culturing it at 25-30° C. and 180-220 rpm for 40-50 hours to obtain pyrroloquinoline quinone.

[0022] Preferably, the nitrate-reducing Pelagobacterium ZH15 is placed in a slant culture medium and cultured at 28° C. for 24 hours for activation; a single colony of the nitrate-reducing Pelagobacterium is picked from the slant culture medium and inoculated into 5 mL of seed culture medium, and shaken and cultured at 28° C. and 200 rpm for 24 hours to obtain a seed liquid; the obtained seed liquid is inoculated into a fermentation medium at a volume ratio of 2%, and fermented and cultured at 28° C. and 200 rpm for 48 hours to obtain pyrroloquinoline quinone.

[0023] The seed culture medium and fermentation culture medium contain: 4-6g of peptone, 0.5-1.5g of yeast extract powder, 19-21g of sodium chloride, 5-7g of magnesium chloride, 2-4g of sodium sulfate, 1-3g of calcium chloride, 0.5-1g of potassium chloride, 0.1-0.3g of sodium carbonate, pH 7.4-7.8, and distilled water is added to 800-1200ml.

[0024] Preferably, the seed culture medium and fermentation culture medium contain: 5g peptone, 1g yeast extract powder, 20g sodium chloride, 6g magnesium chloride, 3.5g sodium sulfate, 2g calcium chloride, 0.55g potassium chloride, 0.16g sodium carbonate, pH 7.6, and distilled water is added to 1000mL.

[0025] The present invention also provides a method for producing a trichothecene toxin degrading enzyme by fermenting Bacillus nitrate-reducing Pelagobacterium, which is characterized in that the fermentation broth of Bacillus nitrate-reducing Pelagobacterium ZH15 is centrifuged to obtain bacterial cells, the collected bacterial cells are washed, resuspended with PBS buffer, and then ultrasonically disrupted in an ice bath, the disrupted cell fluid is freeze-centrifuged for 10 minutes, and the supernatant after centrifugation is filtered through a filter membrane with a pore size of 0.22 μm to obtain the trichothecene toxin degrading enzyme.

[0026] The present invention also provides a biological preparation composed of nitrate-reducing Pelagobacterium cell bodies, characterized in that the biological preparation is obtained by centrifuging the cells obtained by fermenting nitrate-reducing Pelagobacterium ZH15, and collecting the cells after washing. Alternatively, the cells collected after centrifugation are resuspended in a protective agent to a concentration of 10 8 CFU / mL, and the suspension was freeze-dried to obtain a biological preparation composed of nitrate-reducing Pelagobacterium cells.

[0027] The protective agent can be one or more of sucrose, bran, skimmed milk powder, starch, corn gluten powder and soybean protein concentrate powder.

[0028] The present invention further provides an application of nitrate-reducing Pelagobacterium in the biodegradation of trichothecene toxins, characterized in that:

[0029] 600-800 μL of nitrate-reducing Bacillus pelagicus ZH15 cell lysate and 150-200 μL of 500 μg / mL vomitoxin were incubated at pH 7-8 and 25-30°C for 2-72 hours.

[0030] 600-800 μL of nitrate-reducing Bacillus pelagicus ZH15 fermentation broth and 150-200 μL of 100 μg / mL nivalenol were incubated at pH 7-8 and temperature 25-30°C for 2-72 hours.

[0031] 600-800 μL of nitrate-reducing Bacillus pelagicus ZH15 fermentation broth and 150-200 μL of 100 μg / mL 15-acetyldeoxynivalenol were incubated at pH 7-8 and temperature 25-30°C for 2-72 hours.

[0032] By implementing the specific invention content of the present invention, the following beneficial effects can be achieved:

[0033] 1) The nitrate-reducing pelagic bacillus or trichothecene toxin-degrading enzyme or its biological preparation of the present invention has a high degradation rate for vomitoxin, nivalenol, and 15-acetyldeoxynivalenol. After 24 hours of reaction, the degradation rate of vomitoxin exceeds 80%, the degradation rate of nivalenol exceeds 40%, and the degradation rate of 15-acetyldeoxynivalenol reaches 100%, and the reaction is an irreversible biodegradation process.

[0034] 2) The nitrate-reducing Pelagobacterium of the present invention catalyzes the enzymatic biodegradation of trichothecenes, which is characterized by high detoxification activity, specific action, mild effect, no destruction of nutrients in the feed, and no impact on the sensory quality of the feed.

[0035] 3) The method of degrading trichothecenes by using nitrate-reducing Bacillus pelagicus in the present invention is simple to operate, has no environmental pollution, and overcomes the problems existing in traditional detoxification methods.

[0036] Biological Deposit Description

[0037] ZH15, classification name: Pelagibacterium nitratireducens, was deposited on September 13, 2023 in the General Microbiology Center of China Culture Collection Administration, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 28454. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art:

[0039] Figure 1 Phylogenetic tree of strain ZH15 of the present invention;

[0040] Figure 2 Colony morphology and scanning electron microscopy images of the strain ZH15 of the present invention;

[0041] Figure 3 Electrophoresis diagram of 16S rDNA PCR amplification products of strain ZH15 of the present invention;

[0042] Figure 4 The salt-tolerant growth curve of the strain ZH15 of the present invention;

[0043] Figure 5 The active component of the strain ZH15 of the present invention in degrading vomitoxin is determined;

[0044] Figure 6 Time curve of degradation of vomitoxin by the active component of strain ZH15 of the present invention;

[0045] Figure 7 HPLC spectrum of the active component of the strain ZH15 of the present invention degrading vomitoxin;

[0046] Figure 8 LC-MS graph of the degradation of nivalenol by the fermentation broth of the strain ZH15 of the present invention;

[0047] Figure 9 LC-MS graph of the degradation of 15-acetyldeoxynivalenol by the fermentation broth of the strain ZH15 of the present invention. DETAILED DESCRIPTION

[0048] The present invention discloses a nitrate-reducing bacterium (Pelagibacterium nitratireducens) capable of degrading trichothecene toxins and a preparation method and application thereof for producing pyrroloquinoline quinone and trichothecene toxin degrading enzymes. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0049] The raw materials and reagents used in the present invention can be purchased from the market.

[0050] The present invention will be further described below in conjunction with the embodiments:

[0051] Example 1 Screening and identification of strains

[0052] The specific steps for strain screening are as follows:

[0053] 1) Strain screening: This study screened bacterial strains from soil samples taken from the Yellow Sea beach.

[0054] Preparation of seed solution: Weigh 1 g of beach soil into a sterile test tube, add 9 mL of sterile distilled water, and shake overnight at 28°C and 140 rpm. After standing for 1 hour, aspirate 100 μL of the supernatant, mix it with 900 μL of culture medium, and culture it in a 28°C incubator with shaking for 24 hours to obtain the seed solution.

[0055] The liquid culture medium has the following formula: 5 g / L peptone, 1 g / L yeast extract powder, 20 g / L sodium chloride, 6 g / L magnesium chloride, 3.5 g / L sodium sulfate, 2 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, pH 7.6; sterilize with high-pressure steam at 121°C for 20 min.

[0056] Initial strain screening: 450 μL of seed solution was taken and vortexed with 50 μL of DON (500 μg / mL) in a 2 mL centrifuge tube. The blank control was a medium without bacteria and DON was added. The culture was cultured at 28°C for 48 h, and the DON degradation rate was analyzed using high performance liquid chromatography.

[0057] Strain rescreening: Select the seed solution with toxin degradation efficiency, take 0.1mL of seed solution, and dilute it with the above culture medium by 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Take 200 μL of each diluted sample solution, spread it on a solid plate, and incubate it at 28°C for 48 hours. Observe the morphology and characteristics of the colonies, pick representative colonies, streak them on the solid plate, and then transfer them to slant culture medium for storage.

[0058] The above-mentioned preserved strains were inoculated into the above-mentioned liquid culture medium at 2% (V / V) and fermented at 28°C for 24 hours to obtain a strain fermentation broth. 900 μL of the secondary activated strain fermentation broth was taken and vortexed with 100 μL of DON standard (500 μg / mL) in a 2mL centrifuge tube. The blank control was a culture medium without bacteria plus DON, sealed with a sealing film, and cultured in a shaker at 28°C and a speed of 180 r / min for 24 hours. After the incubation, an equal volume of chromatography-grade methanol solution was used, vortexed, centrifuged to obtain the supernatant, and the supernatant was filtered using a 0.22 μm filter membrane. The DON content in the sample was detected by high performance liquid chromatography-fluorescence detection, and the DON degradation rate was calculated.

[0059] 2) Species Identification: Bacterial genomic DNA was extracted according to the instructions of the bacterial genomic DNA miniprep kit. The 16S rDNA of the strain was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). The amplified sequence was purified, recovered, and sequenced by agarose gel electrophoresis. BLAST comparison was performed against the NCBI database, and a phylogenetic tree was constructed using the nearest neighbor method to identify the strain.

[0060] Identification results: Agarose gel electrophoresis results are as follows Figure 1 As shown. After sequence alignment, the strain ZH15 belongs to nitrate-reducing Pelagobacterium, and its phylogenetic tree is shown in Figure 2 .

[0061] Morphological observation: scanning electron microscopy Figure 3 As shown, nitrate-reducing pelagic bacillus ZH15 is short rod-shaped, with a length ranging from 1-2 μm, and no flagella are seen.

[0062] Example 2 Method for producing pyrroloquinoline quinone by nitrate-reducing pelagic bacteria of the present invention

[0063] The nitrate-reducing Pelagobacterium ZH15 described in Example 1 was placed in a slant culture medium and cultured at 28° C. for 24 hours for activation; a single colony of the nitrate-reducing Pelagobacterium was picked from the slant culture medium and inoculated into 5 mL of seed culture medium, and the culture was shaken at 28° C. and 200 rpm for 24 hours to obtain a seed solution; the obtained seed solution was inoculated into a fermentation medium at a volume ratio of 2%, and fermented at 28° C. and 200 rpm for 48 hours to obtain pyrroloquinoline quinone. The content of pyrroloquinoline quinone in the fermentation broth was determined using an enzyme-linked immunosorbent assay (ELISA) kit, and the concentration was greater than 50 mg / L.

[0064] Example 3 Study on the salt tolerance of nitrate-reducing pelagic bacteria of the present invention

[0065] 2 mL of nitrate-reducing Pelagobacterium ZH15 (viable bacteria concentration of 10 8 CFU / mL), and were inoculated into 100 mL fermentation medium containing 0%, 3%, 6%, 9% and 12% NaCl, respectively. The fermentation temperature was 28 ° C, pH 7.6, the rotation speed was 200 r / min, and the fermentation time was 48 h. The OD was measured 3 h after inoculation. 600 , draw a growth curve.

[0066] The test results showed that the nitrate-reducing Pelagibacterium ZH15 could grow normally in a culture medium with a NaCl concentration of less than 3%. 6% NaCl had a certain inhibitory effect on the nitrate-reducing Pelagibacterium ZH15, and its growth was slow. 9% NaCl completely inhibited the growth of the nitrate-reducing Pelagibacterium ZH15. Figure 4 ).

[0067] Example 4 Preparation method of nitrate-reducing Pelagobacterium cells of the present invention

[0068] 20 mL of the nitrate-reducing Pelagobacterium ZH15 described in Example 3 was inoculated into 1 L of culture medium for shake flask fermentation to obtain a first-level seed solution; the first-level seed solution was then transferred to a 50 L fermentor for fermentation.

[0069] The fermentation conditions are as follows: fermentation temperature of 28° C., pH value of 7.6, rotation speed of 150 r / min, and fermentation time of 24 h.

[0070] The fermentation broth was filtered through a 0.22 μm filter membrane and freeze-dried to obtain bacterial cells.

[0071] Example 5 Determination of the active components of the nitrate-reducing bacillus of the present invention for degrading vomitoxin

[0072] Inoculate strain ZH15 at a 2% inoculation rate in a 250mL conical flask and culture for 24 hours under constant temperature. Centrifuge the cultured fermentation broth at high speed at 4°C. The supernatant after centrifugation is the fermentation broth supernatant and place it in a 4°C refrigerator for later use. Resuspend the precipitate after centrifugation with PBS buffer, and centrifuge the resuspended bacterial cell solution at 4°C. Repeat the washing of the bacterial cells three times. The resuspended bacterial cell solution after washing is the bacterial cell solution. Place a portion of the bacterial cell solution in a 4°C refrigerator for later use, and ultrasonically disrupt the other portion of the bacterial cell solution in an ice bath. After disruption, the cell lysate after centrifugal filtration is placed in a 4°C refrigerator for later use. Resuspend the crushed precipitate obtained by centrifugation with PBS buffer and place it in a 4°C refrigerator for later use.

[0073] The experimental results showed that the active components of nitrate-reducing pelagic bacillus ZH15 for degrading DON were mainly in the cell lysate, and its degradation rate for DON was 83% ( Figure 5 ), which was significantly higher than that in the fermentation broth supernatant.

[0074] Example 6 Method for producing trichothecene toxin-degrading enzyme by fermentation of nitrate-reducing Bacillus thuringiensis

[0075] The nitrate-reducing Pelagobacterium cells obtained in Example 4 were washed three times with PBS buffer at pH 7.4 and refrigerated centrifuged. The cell pellet was added with PBS buffer and ultrasonically disrupted in an ice bath. The disrupted cell fluid was then refrigerated centrifuged at 4°C and 12,000 rpm for 10 min. The supernatant obtained by centrifugation was filtered through a 0.22 μm filter membrane to obtain a crude enzyme solution of trichothecene toxin-degrading enzyme.

[0076] Example 7 Time curve of the degradation of vomitoxin by the nitrate-reducing Bacillus pelagicus cell lysate of the present invention

[0077] Take 900 μL of the cell lysate of the nitrate-reducing Bacillus pelagicus obtained in Example 5, add 100 μL of DON standard (500 μg / mL), react at 30°C for 6 h, take 100 μL of the reaction sample every 1 h, terminate with methanol, and use high performance liquid chromatography to detect the DON content in the sample.

[0078] As can be seen from the figure ( Figure 6 、 Figure 7 ), the nitrate-reducing bacillus ZH15 cell lysate can achieve good degradation of DON, and the degradation rate increases with time within 6 hours, and the degradation rate of DON in 6 hours is 74%.

[0079] Example 8 Degradation of nivalenol by the fermentation broth of the nitrate-reducing Bacillus thuringiensis of the present invention

[0080] 900 μL of the nitrate-reducing Bacillus nitrificans fermentation broth described in Example 1 was added with 100 μL of NIV standard (100 μg / mL). The reaction was incubated at 30°C for 24 h, and then an equal volume of methanol was added to terminate the reaction. The NIV content in the sample was detected by liquid chromatography-tandem mass spectrometry.

[0081] The results showed that the degradation rate of NIV by the fermentation broth of nitrate-reducing Bacillus thuringiensis ZH15 at 30℃ was 39.3% ( Figure 8 ).

[0082] Example 9 Degradation of 15-acetyldeoxynivalenol by the fermentation broth of the nitrate-reducing Bacillus thuringiensis

[0083] 900 μL of the nitrate-reducing Bacillus fermentation broth described in Example 1 was added with 100 μL of 15-ADON standard (100 μg / mL). The reaction was incubated at 30° C. for 24 h, and then an equal volume of methanol was added to terminate the reaction. The 15-ADON content in the sample was detected by liquid chromatography-tandem mass spectrometry.

[0084] The results showed that the degradation rate of 15-ADON by the fermentation broth of nitrate-reducing Bacillus thuringiensis ZH15 at 30℃ was 100% ( Figure 9 ).

[0085] Example 10 Preparation of Nitrate-Reducing Pelagibacterium Biological Preparation for Detoxification of DON

[0086] The biological preparation is a biological preparation composed of nitrate-reducing Pelagobacterium ZH15 cells obtained by centrifugation after fermentation, and the cells are collected after washing to obtain cells of nitrate-reducing Pelagobacterium. Alternatively, the cells collected after centrifugation are resuspended in a protective agent to a concentration of 10 8 CFU / mL, and the suspension was freeze-dried to obtain a biological preparation composed of nitrate-reducing Pelagobacterium cells.

[0087] Pelagobacterium nitrate-reducing ZH15 was inoculated into a fermentation medium at a 2% inoculum size and cultured at a constant temperature for 24 hours. The fermentation broth was centrifuged at high speed at 4°C, and the pellet was resuspended in PBS buffer (pH 7.4). The resuspended bacterial cell solution was centrifuged at 4°C, and the bacterial cells were washed three times repeatedly. The cells were then collected to obtain a biological preparation composed of Pelagobacterium nitrate-reducing cells.

[0088] Alternatively, resuspend the cells in a protective agent to a concentration of 1×10 8 CFU / mL. The protective agent is 150g / L skim milk powder and 100g / L sucrose. The suspension is pre-incubated at 28°C for 1 hour, then pre-frozen at -20°C for 2-5 hours, and finally freeze-dried to obtain the nitrate-reducing Pelagobacterium ZH15 bacterial powder biological preparation.

[0089] The above is a detailed introduction to the application of the trichothecene toxin degrading enzyme or biological preparation produced by the nitrate-reducing Pelagobacterium ZH15 strain provided by the present invention or the nitrate-reducing Pelagobacterium fermentation broth of the present invention in the detoxification of trichothecene toxins. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the methods and core ideas of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, the present invention can also be improved and modified in several ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A nitrate-reducing pelagic bacterium, Pelagibacterium nitratireducens ZH15, whose deposit number is CGMCC No. 28454.

2. Use of the nitrate-reducing Pelagobacterium according to claim 1 in the fermentation production of pyrroloquinoline quinone.

3. A composition comprising the nitrate-reducing Pelagobacterium ZH15 according to claim 1 and a fermentation broth, wherein the concentration of pyrroloquinoline quinone in the fermentation broth is greater than 50 mg / L.

4. A method for producing pyrroloquinoline quinone, comprising fermenting pyrroloquinoline quinone using the nitrate-reducing bacillus ZH15 of claim 1, wherein: The nitrate-reducing Pelagobacterium ZH15 is activated in a slant culture medium and a seed culture medium in sequence, and then inoculated into a fermentation culture medium for fermentation culture to obtain pyrroloquinoline quinone.

5. Use of the nitrate-reducing Pelagobacterium ZH15 according to claim 1 in the fermentation production of trichothecene toxin-degrading enzymes.

6. A method for producing a trichothecene toxin-degrading enzyme, comprising fermenting the nitrate-reducing Bacillus thuringiensis ZH15 of claim 1 to produce the trichothecene toxin-degrading enzyme, wherein: The fermentation broth of the nitrate-reducing Bacillus Pelagicus ZH15 is centrifuged to obtain bacterial cells, the collected bacterial cells are washed, resuspended with PBS buffer, and then ultrasonically disrupted in an ice bath. The disrupted cell lysate is centrifuged and filtered to obtain a trichothecene toxin-degrading enzyme.

7. A biological preparation prepared using the fermentation liquid or bacterial cells of the nitrate-reducing Bacillus pelagicus ZH15 according to claim 1, wherein the preparation method comprises: The nitrate-reducing Pelagobacterium ZH15 was activated and cultured, and then centrifuged to obtain bacterial cells. The collected bacterial cells were washed and resuspended in a protective agent solution to a cell concentration of 10 8 CFU / mL, and the suspension was further freeze-dried to obtain.

8. Use of the nitrate-reducing Pelagobacterium ZH15 strain according to claim 1, the trichothecene toxin-degrading enzyme according to claim 5, or the biological preparation according to claim 7 in the biodegradation of vomitoxin.

9. Use of the nitrate-reducing Pelagobacterium ZH15 strain according to claim 1, the trichothecene toxin-degrading enzyme according to claim 5, or the biological preparation according to claim 7 in the biodegradation of nivalenol.

10. Use of the nitrate-reducing Pelagobacterium ZH15 strain according to claim 1, the trichothecene toxin-degrading enzyme according to claim 5, or the biological preparation according to claim 7 in the biodegradation of 15-acetyldeoxynivalenol.

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