Marine bacillus for degrading fumonisin and its application

Abstract

The present application relates to a marine bacillus Fum‑5 (Pelagibacterium sp. Fum‑5, with a deposit number of CGMCC No. 26300) for degrading fumonisins and its application. The degradation rate of the marine bacillus for different fumonisins can reach 99.1% to 100%. The present application also relates to a bacterial agent prepared by the strain, which can be used for biological detoxification of fumonisins in food or feed.

Description

Technical Field

[0001] The present application relates to the technical field of microorganisms and their application, and to a marine bacillus, specifically a marine bacillus (Pelagibacterium sp. Fum-5) capable of degrading fumonisin and its application. Background Art

[0002] Fumonisins (FBs), also known as fumonisins, are water-soluble secondary metabolites produced by Fusarium moniliforme, Fusarium verticillioides, Fusarium proliferatum and other Fusarium fungi under suitable conditions. l , FA 2 , FA 3 , FB 1 , FB 2 , FB 3 , FB 4 There are more than 30 species, including fumonisin B 1 (FB 1 ) is the most extensively studied toxin. Fumonisins are widely contaminated by corn, wheat, sorghum and other crops, which not only reduces the nutritional value of crops, but also transmits through the food chain, seriously endangering the health of humans and animals. Fumonisins are hepatotoxic, nephrotoxic and embryotoxic, of which the liver and kidneys are the most affected by fumonisin B. 1 Target organs of attack, in addition, fumonisin B 1 It can also induce symptoms such as apoptosis, esophageal cancer and neural tube defects in humans, and has been identified as a Class 2B carcinogen by the World Health Organization. Therefore, it is of great significance to carry out research on fumonisin removal and control technology to solve the pollution of toxins to food and feed, improve animal production performance, protect people's "food safety", and ensure national food safety.

[0003] At present, the detoxification methods of mycotoxins mainly include physical, chemical and biological detoxification methods. Although physical and chemical detoxification methods have achieved a certain degree of success, they have disadvantages such as difficult operation, low detoxification efficiency, reduced nutritional quality and palatability. Compared with physical and chemical detoxification methods, biological detoxification methods have the advantages of mild action conditions, strong specificity, and minimal impact on the sensory properties and palatability of raw materials. They are considered to be the best detoxification method. However, there are few strains reported to degrade fumonisins, and the degradation efficiency is low. Therefore, it is necessary to screen microorganisms that can efficiently degrade fumonisins. Summary of the invention

[0004] The technical problem to be solved by the present application is to screen out strains capable of degrading fumonisins from a large number of natural samples, thereby solving the problem of fumonisin contamination in feed and food causing harm to animal and human health.

[0005] On the one hand, the present application relates to the marine bacillus Fum-5, whose 16S rDNA sequence is shown in SEQ NO ID: 1, and is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.26300.

[0006] In some embodiments, the marine bacillus Fum-5 grows on LB solid culture medium, and the colonies are milky white and opaque, with a smooth surface and flat edges, and single colonies are round.

[0007] In some embodiments, the components of the LB solid medium are: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar, and 1 L of distilled water.

[0008] On the other hand, the present application relates to a bacterial agent for degrading fumonisins, characterized in that the active ingredient of the bacterial agent is Marine Bacillus Fum-5 or a mutant strain derived therefrom, and the preservation number of the Marine Bacillus Fum-5 is CGMCC No.26300.

[0009] In some embodiments, the bacterial agent is a liquid bacterial agent or a solid bacterial agent.

[0010] On the other hand, the present application relates to a method for preparing a bacterial agent for degrading fumonisin, characterized in that it comprises the following steps:

[0011] (1) activating the marine bacillus Fum-5 or its derived mutant strain with a deposit number of CGMCC No. 26300 on LB solid medium;

[0012] (2) The colony activated in step (1) was inoculated into LB liquid culture medium and cultured until the concentration reached 10 8 CFU / mL, and liquid bacterial agent was prepared.

[0013] In some embodiments, the culture conditions of step (2) are 27-33°C, 180-220 rpm. In some embodiments, the culture conditions of step (2) are 30°C, 200 rpm.

[0014] In some embodiments, the components of the LB solid medium are: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar, and 1 L of distilled water.

[0015] In some embodiments, the LB liquid culture medium comprises: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 1 L of distilled water.

[0016] In some embodiments, the method further includes centrifuging the liquid bacterial agent to obtain bacterial cells, resuspending the bacterial cells with a lyophilization protectant to obtain a bacterial suspension, wherein the mass ratio of the lyophilization protectant to the bacterial cells is 1:1, mixing the bacterial suspension with a physiologically acceptable carrier, wherein the mass ratio of the bacterial suspension to the carrier is 1:1, and placing the mixture in a vacuum freeze dryer for freeze-drying using a vacuum freezing method to obtain a freeze-dried powder.

[0017] In some embodiments, the lyoprotectant is selected from one or more of trehalose, oligoxylose, skim milk powder, and sucrose, and the physiologically acceptable carrier is selected from one or more of maltodextrin, milk powder, cyclodextrin, sucrose, starch, wheat, wheat bran, rice, rice bran, limestone, talc, or montmorillonite.

[0018] In yet another aspect, the present application relates to a method for degrading fumonisin, which comprises adding the Marine Bacillus Fum-5 or bacterial agent of the present application to a sample containing fumonisin.

[0019] In some embodiments, the degradation reaction is carried out at room temperature.

[0020] Through the marine bacillus Fum-5 of the present application, a good degradation effect of fumonisins in food or feed can be achieved, and the degradation efficiency is high. Through the marine bacillus Fum-5 of the present application, fumonisins can be biodegraded at room temperature. In addition, the degradation of fumonisins by the marine bacillus Fum-5 of the present application is biodegradation, which has strong specificity and mild effect. It does not destroy the nutrients in food or feed, and has no effect on the sensory quality, palatability, etc. of food or feed. The method of degrading fumonisins by the marine bacillus Fum-5 of the present application is simple to operate, does not pollute feed, food and the environment, and overcomes at least some of the problems existing in traditional detoxification methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony morphology of Marine Bacillus Fum-5 on the LB plate;

[0022] Figure 2 is the phylogenetic tree of Marine Bacillus Fum-5;

[0023] Figure 3 The cell morphology of Marine Bacillus Fum-5 under an optical microscope; and

[0024] Figure 4 This is the liquid chromatogram of the degradation of fumonisin by Marine Bacillus Fum-5. DETAILED DESCRIPTION

[0025] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the specific implementation methods of the present application are described below in conjunction with the accompanying drawings. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0026] In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present application is not limited to the specific embodiments disclosed below.

[0027] Some embodiments of the present application are described below in conjunction with the accompanying drawings.

[0028] The present application discloses a marine bacillus (Pelagibacterium sp.Fum-5) capable of degrading fumonisin and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application 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 this application to implement and apply the technology of this application.

[0029] Unless otherwise stated, the raw materials and reagents used in this application can be purchased from the market.

[0030] Example 1 Screening of strains

[0031] The present application isolated and screened the strain from soil collected from the Hujiaying area of ​​Yanqing, Beijing, and screened the strain from soil samples around poultry farms.

[0032] (1) Take 10 g of soil sample in a 250 mL conical flask, add 100 mL of physiological saline, shake at 30 °C and 180 rpm for 30 min, and let it stand for 30-60 s;

[0033] (2) 1 mL of the upper bacterial liquid was added to an enrichment medium (MM medium) containing 10 mg / L fumonisin, and the culture was carried out at 30°C and 200 rpm for 7 days;

[0034] The formula of MM medium is: take 1.6g Na 2 HPO 4 , 1.0g KH 2 PO 4 、0.5g MgSO 4 7H 2 O, 0.5gNaNO3 , 0.5g(NH 4 ) 2 SO 4 , 0.025 g CaCl 2 7H 2 O and 1L distilled water, fully dissolved, autoclaved and then added with 2mL trace element solution and fumonisin as the only carbon source.

[0035] The formula of trace element solution is: 1.5g FeCl 2 ·4H 2 O, 0.19 gCoCl 2 6H 2 O, 0.1 gMnCl 2 ·4H 2 O, 0.07 g ZnCl 2 , 0.062 g H 3 BO 3 , 0.036g Na 2 MoO 4 ·2H 2 O, 0.024 g NiCl 2 6H 2 O, 0.017 g CuCl 2 ·2H 2 O, 0.01gAlK(SO 4 ) 2 12H 2 O and 1L distilled water, fully dissolve, sterilize and set aside.

[0036] (3) 1 mL of the enriched bacterial culture was added to MM medium containing 20 mg / L fumonisin and cultured for the second time at 30°C and 200 rpm for 7 days;

[0037] (4) Dilute the bacterial solution after the second enrichment culture to 10 -3 , 10 -4 , 10 -5 times, spread on LB solid medium, and culture at 30℃ for 24-48h;

[0038] The formula of LB solid culture medium is: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar and 1L distilled water, which are fully dissolved and sterilized for use.

[0039] (5) Select a plate with a moderate colony growth density, pick out the strains with inconsistent colony morphology on the plate and inoculate them into LB liquid culture medium, culture them at 30° C. and 200 rpm for 16 h, and then transfer them to MM medium containing 10 mg / L fumonisin at a 1% inoculum, culture them at 30° C. and 200 rpm for 12 h to obtain candidate fumonisin-degrading strains;

[0040] The formula of LB liquid culture medium is: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 1 L distilled water.

[0041] (6) All candidate fumonisin-degrading strains were screened and their fumonisin B1 degradation efficiency was tested. The strain with the highest degradation efficiency was selected and named Fum-5. The strain Fum-5 was stored in a glycerol tube at -80°C for future use.

[0042] Example 2 Identification of strains

[0043] The strain Fum-5 preserved in 15% glycerol was inoculated on LB solid medium by plate streak method for activation, and then cultured at 30℃ for 36-48h, and the morphological characteristics of the colonies were observed, such as Figure 1 As shown, the colonies of strain Fum-5 are milky white and opaque, with a smooth surface and flat edges, and single colonies are round.

[0044] The 16S rDNA gene sequence of the strain with high degradation efficiency was determined, and the amplified products were sequenced. The sequencing results were compared by BLAST, and the phylogenetic tree was constructed by the proximity method to identify the strain.

[0045] (1) Identification results: The 16S rDNA gene fragment was amplified using universal primers 27F / 1492R, and the amplified product was sequenced to obtain the 16S rDNA sequence of the strain SEQ ID NO: 1. After sequence comparison, the strain Fum-5 belonged to Pelagibacterium sp., and its phylogenetic tree is shown in Figure 2 .

[0046] (2) Morphological observation: Gram staining was used and the morphology was observed under an optical microscope (Leica). Figure 3 As shown in Figure 2, strain Fum-5 is a Gram-negative bacterium with a short rod shape.

[0047] The applicant deposited Pelagibacterium sp. Fum-5 at the General Microbiology Center of China Culture Collection Administration, with the deposit date being December 30, 2022, the deposit number being CGMCC No. 26300, and the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0048] Example 3 Degradation of Fumonisin by Marine Bacillus Fum-5

[0049] 1. Fumonisin Determination Method

[0050] Fumonisins were detected by HPLC (pre-column chemical derivatization) under the following conditions:

[0051] Mobile phase: Phase A: 0.1 mol / L sodium dihydrogen phosphate (pH 3.3); Phase B: methanol. Isocratic elution, 63% Phase B;

[0052] Chromatographic column: C18 column: 250mm×4.6mm, 5μm;

[0053] Mobile phase flow rate: 0.8 mL / min;

[0054] Detection wavelength: fluorescence detector, excitation wavelength 335nm, emission wavelength 440nm;

[0055] Derivatization solution: 2.0 g o-phthalaldehyde was dissolved in 20 mL methanol, diluted to 500 mL with 0.1 mol / L borax solution, and 500 μL 2-mercaptoethanol was added;

[0056] Derivatization liquid flow rate: 0.4 mL / min;

[0057] Column temperature: 40°C;

[0058] Reactor temperature: 40 °C;

[0059] Injection volume: 50 μL.

[0060] 2. Degradation of fumonisins by Marine Bacillus Fum-5

[0061] (1) Inoculate the marine bacillus Fum-5 into LB liquid culture medium and culture at 30°C and 200 rpm for 48 h to prepare a bacterial solution;

[0062] 10 mg FB 1 Standard, 1mg FB 1 +FB 2 Mixed standard and 1mg FB 3 The standard was dissolved in 10 mL of 50% (v / v) acetonitrile to obtain a concentration of 1000 mg / L FB 1 Solution, 100mg / L FB 1 +FB 2 Mixed standard solution and 100mg / L FB 3 Solution;

[0063] Take 1 mL of bacterial solution and place it in a 2 mL centrifuge tube. Add 10 μL of the above three different fumonisin standard solutions to the tubes respectively. After sufficient oscillation and mixing, incubate at 30°C, 140 rpm for 8 h, centrifuge at 12000 rpm for 10 min and collect the supernatant to obtain the experimental group solution.

[0064] Take 1 mL of LB liquid culture medium and place it in a 2 mL centrifuge tube. Add 10 μL of the above three different fumonisin standard solutions to the tube. After sufficient oscillation and mixing, incubate at 30°C, 140 rpm for 24 h, centrifuge at 12000 rpm for 10 min, and collect the supernatant to obtain the control group solution.

[0065] The experimental group solution and the control group solution were used as the test solution, and the following operations were performed: ① Take 0.8 mL of the supernatant, add 4 times the volume of PBS solution and mix well, take 1 mL of the mixed liquid, and load it onto the immunoaffinity column, controlling the flow rate at 1-3 mL / min; use 10 mL of PBS solution to elute the immunoaffinity column at the same flow rate; ② Use 1 mL of methanol-acetic acid solution (98+2) to elute the immunoaffinity column 3 times, respectively, and collect the eluate; blow dry with nitrogen at 55°C, add 1 mL of acetonitrile-water (1+1) to dissolve the precipitate, and vortex for 30 s; pass through a 0.2 μm PTFE filter membrane, collect the liquid in a 2 mL injection vial, and obtain the sample solution; ③ Take the sample solution obtained in step ② and detect it by HPLC (pre-column chemical derivatization).

[0066] (2) Fumonisin degradation rate (%) = (remaining fumonisin content in the control group - remaining fumonisin content in the experimental group) / remaining fumonisin content in the control group × 100.

[0067] (3) The results showed that:

[0068] The experiment was repeated four times and the results were averaged.

[0069] like Figure 4 As shown, A is FB 1 Standard (FB 1 The retention time is 5.801min); B is the control group (FB 1 Retention time is 5.300min); C is the experimental group (FB 1 Retention time: 5.276 min).

[0070] Residual FB in the control group 1 The content is 6.13mg / L.

[0071] Residual FB in the experimental group 1 The content is 0.057mg / L.

[0072] The results showed that Fum-5 of Marine Bacillus could inhibit FB 1It has a good degradation effect, with a degradation rate of 99.1%.

[0073] No FB was detected in the experimental group 2 and FB 3 The residues indicate that Fum-5 of Marine Bacillus is effective against FB 2 and FB 3 The degradation effect is better, and the degradation rate reaches 100%.

[0074] The degradation rates are shown in the following table:

[0075] <![CDATA[FB 1 ]]> <![CDATA[FB 2 ]]> <![CDATA[FB 3 ]]> Degradation rate 99.1％ 100％ 100％

[0076] Example 4 Bacterial agent for degrading fumonisin and preparation method thereof

[0077] The preparation method of the bacterial agent for degrading fumonisin comprises the following steps:

[0078] (1) The strain Fum-5 preserved in 15% glycerol was inoculated on LB solid medium by streak plate method and then cultured at 30°C for 36-48h;

[0079] (2) The activated strain in step (1) was inoculated into LB liquid culture medium and cultured at 30° C. and 200 rpm for 48 h to obtain a liquid bacterial agent.

[0080] Furthermore, the liquid bacterial agent is centrifuged to obtain bacterial cells, and 6% w / v trehalose (the mass ratio of 6% w / v trehalose to bacterial cells is 1:1) is added for re-suspending. The air-dried rice bran is passed through a 40-mesh sieve and sterilized by high pressure wet heat at 121°C. The bacterial suspension is mixed with the rice bran, the mass ratio of the bacterial suspension to the rice bran is 1:1, and the mixture is evenly spread in a 9 cm culture dish, placed in a vacuum freeze dryer and freeze-dried by vacuum freezing to obtain a solid bacterial agent.

[0081] In other embodiments, other physiologically acceptable carriers can be used to mix with the solid bacterial agent, and the physiologically acceptable carrier is selected from one or more of maltodextrin, milk powder, cyclodextrin, sucrose, starch, wheat, wheat bran, rice, limestone, talc or montmorillonite.

[0082] According to the test, the concentration of the protective agent trehalose in the range of 2% to 10% can achieve a survival rate of more than 90% of the strains. Protective agents such as oligoxylose, skim milk powder, sucrose, etc. can also be used, and a mixture of the above protective agents can also be used.

[0083] Example 5 Application of Degrading Fumonisin

[0084] After the corn silage is opened, the liquid bacterial agent or solid bacterial agent in Example 4 is added at a ratio of 10 g / t, and the material is piled for 2-3 days after stirring to achieve the purpose of degrading fumonisins in the feed. After mixing, the temperature of the pile is monitored and maintained at about 30°. If the temperature is too low, the volume of the pile should be appropriately increased. If the temperature is too high, the material should be properly turned or stirred.

[0085] After testing, the total content of fumonisin B1, fumonisin B2 and fumonisin B3 after stacking was less than 2 mg / kg.

[0086] This article uses specific examples to illustrate the principles and implementation methods of this application. The above is only a preferred embodiment of this application and is not intended to limit this application. For those skilled in the art, this application may have various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this application should be included in the scope of protection of this application.

Claims

1. Pelagibacterium sp.Fum-5, whose 16S rDNA sequence is shown in SEQ ID NO: 1, is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration with a deposit number of CGMCC No.26300.

2. The marine bacillus Pelagibacterium sp.Fum-5 according to claim 1, characterized in that The marine bacillus Pelagibacterium sp.Fum-5 grows on LB solid culture medium, and the colonies are milky white and opaque, with smooth surfaces and flat edges, and single colonies are round.

3. A bacterial agent for degrading fumonisins, characterized in that: The active ingredient of the bacterial agent is Pelagibacterium sp.Fum-5, and the preservation number of Pelagibacterium sp.Fum-5 is CGMCC No.26300.

4. The bacterial agent for degrading fumonisins according to claim 3, characterized in that: The bacterial agent is a liquid bacterial agent or a solid bacterial agent.

5. The method for preparing the bacterial agent for degrading fumonisin according to claim 3, characterized in that The following steps are involved: (1) activating Pelagibacterium sp. Fum-5 with a deposit number of CGMCC No. 26300 on LB solid medium; (2) The colony activated in step (1) was inoculated into LB liquid culture medium and cultured until the concentration reached 10 8 CFU / mL, and liquid bacterial agent was prepared.

6. The method according to claim 5, wherein the culture conditions of step (2) are 27-33°C and 180-220rpm.

7. The method according to claim 5, characterized in that The method further comprises (3) centrifuging the liquid bacterial agent to obtain bacterial cells, resuspending the bacterial cells with a freeze-drying protective agent to obtain a bacterial suspension, wherein the mass ratio of the freeze-drying protective agent to the bacterial cells is 1:1, mixing the bacterial suspension with a physiologically acceptable carrier, wherein the mass ratio of the bacterial suspension to the carrier is 1:1, and placing the mixture in a vacuum freeze dryer for freeze-drying using a vacuum freezing method to obtain a freeze-dried powder.

8. The method according to claim 7, characterized in that The lyoprotectant is selected from one or more of trehalose, oligoxylose, skim milk powder, and sucrose; the physiologically acceptable carrier is selected from one or more of maltodextrin, milk powder, cyclodextrin, sucrose, starch, wheat, wheat bran, rice, rice bran, limestone, talc, or montmorillonite.

9. A method for degrading fumonisins, comprising adding the marine bacillus Pelagibacterium sp. Fum-5 of claim 1, or the bacterial agent of claim 3, or the bacterial agent prepared by the method of claim 5 to a sample containing fumonisins.

10. The method according to claim 9, characterized in that The degradation reaction takes place at room temperature.

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