Burkholderia sp. and application thereof

Abstract

The application provides a Burkholderia strain BC11-1, which has the ability to degrade F-2 toxin and has the function of inhibiting Fusarium fungi. The growth and metabolism of the strain can inhibit F-2 toxin-producing harmful microorganisms, so that the toxin-producing capacity of the harmful microorganisms is reduced, and the strain can also degrade the existing F-2 toxin, thereby realizing the synchronous management of the source and stock of F-2 toxin. Further, the strain also has the ability to inhibit aflatoxin-producing harmful microorganisms and degrade aflatoxin B1. Thus, by using a single strain, the two types of harmful microorganisms with the highest content and the most harm in feed can be inhibited and the related toxins can be degraded, thereby improving the safety of feed prevention and treatment technology. At the same time, the strain can be put into use before feed production and circulation, so that the strain can continuously play the roles of inhibiting bacteria and detoxifying, and is beneficial to the maintenance of feed nutritional components.

Description

Technical Field

[0001] This application relates to the field of microbial control technology, and in particular to a strain of Burkholderia that degrades zearalenone and also has antibacterial effects; it also relates to a biocontrol agent made from this strain, and the application of the biocontrol agent in feed detoxification. Background Technology

[0002] my country is a major livestock producer with a high demand for feed. However, due to the nutritional components of feed, contamination by harmful microorganisms such as molds is prevalent in all stages of feed preparation, storage, and transportation. Harmful microorganisms contaminate feed not only by consuming nutrients through their own metabolism but also by producing toxins during their reproduction and metabolism. Common toxins found in feed include aflatoxin, ochratoxin, ergot toxicant, vomitoxin, and zearalenone. Zearalenone (F-2 toxin) is distinguished from other toxins because it is an endogenous hormone found in grains such as wheat and soybeans.

[0003] F-2 toxin is one of the most widespread and toxic mycotoxins, causing extensive contamination in grain cultivation and its byproducts worldwide. F-2 toxin has estrogen-like effects, easily leading to abortion or stillbirth in livestock. It can also accumulate in livestock and poultry and be passed on to consumers, causing acute or chronic poisoning in animals or humans.

[0004] To remove F-2 toxin, various approaches have been explored, including physical control, chemical degradation, and biological control. Among these, biological detoxification is more specific than the other two approaches, effectively preserving the nutritional components of feed. Currently, biological methods for treating F-2 toxin mainly focus on enzyme preparations, such as Chinese invention patent CN101903527B; the strains involved are primarily Bacillus and Bacillus strains, such as Chinese invention patents CN102406100B and CN102181376B. Existing research demonstrates that the microorganisms in the feed system and the interactions between microorganisms and toxins constitute a complex system. Current technologies all require additional processing before feed use and cannot be applied to the preparation, storage, and transportation of feed.

[0005] As mentioned earlier, the production of F-2 toxin involves two pathways: the metabolism of harmful microorganisms and accumulation within plants. F-2 toxin exhibits strong temperature tolerance, remaining undecomposed at 120°C for 4 hours. Therefore, existing biological control methods relying solely on antibacterial action are ineffective in eliminating F-2 toxin from feed. Summary of the Invention

[0006] This application provides a strain of Burkholderia contaminans, which was screened by the inventors in the natural environment. The strain number is BC11-1, and it was deposited on February 22, 2022, at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 24417. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] First, strain BC11-1 has the ability to degrade F-2 toxin, and its growth and metabolism can be used to achieve biological control of F-2 toxin.

[0008] Secondly, strain BC11-1 possesses the ability to both inhibit Fusarium fungi and degrade F-2 toxin. The growth and metabolism of strain BC11-1, on the one hand, inhibits the normal metabolism of Fusarium fungi, the main harmful microorganisms producing F-2 toxin, thus reducing their toxin-producing capacity; on the other hand, it also degrades existing F-2 toxin, simultaneously controlling both the source and the existing quantity of F-2 toxin.

[0009] Furthermore, strain BC11-1 also possesses the ability to inhibit aflatoxin-producing harmful microorganisms and degrade aflatoxin B1. Existing research has demonstrated the existence of interactions between microorganisms in feed systems and between microorganisms and toxins. Strain BC11-1's inhibition of the two most abundant and harmful types of microorganisms in feed and the degradation of their toxins, achieving multiple functions through a single strain, helps reduce the variety of agents required for overall feed disease control and further improves the safety of feed disease control technologies.

[0010] Since strain BC11-1 has both antibacterial and detoxifying abilities, it can be added to feed production and distribution in the early stages to allow it to continuously exert its antibacterial and detoxifying effects, which is beneficial for maintaining the nutritional components of the feed. At the same time, no additional treatment is required when using the feed, thus maintaining the normal feed usage process.

[0011] This application also provides the whole culture medium of strain BC11-1, the application of its extracellular metabolites as biocontrol agents, and methods for preparing the aforementioned biocontrol agents.

[0012] The BC11-1 whole-culture biocontrol agent contains strain BC11-1 and its extracellular metabolites. The specific preparation method includes: activating strain BC11-1 on LB solid medium, culturing it on LB liquid medium to obtain a seed culture, and then preparing a bacterial suspension with a concentration of not less than 1×10⁻⁶ from the seed culture. 8 Total culture medium of cfu / mL.

[0013] To facilitate preservation and use, the above-mentioned whole culture medium can also be freeze-dried to prepare a powdered biocontrol agent, with the total concentration of viable bacteria per gram of powder controlled at 1×10⁻⁶. 11 CFU.

[0014] To improve the safety of biocontrol agents, a biocontrol agent based on the extracellular metabolites of strain BC11-1 can also be prepared. The specific preparation method is to centrifuge the aforementioned whole culture medium at low temperature, and then filter the supernatant through a microporous membrane to remove bacteria.

[0015] One embodiment of this application discloses the use of the above-mentioned biocontrol agent to soak feed for sterilization and detoxification.

[0016] As mentioned earlier, F-2 toxin is an endogenous hormone in some plants, and it accumulates within the plants. Although the amount of toxin accumulated in plants is not large, it still poses a significant threat to livestock such as cattle that require large amounts of feed and ruminate. Another embodiment of this application discloses the application of soaking plants in the aforementioned biocontrol agent for detoxification. Detoxification of plants can further improve feed safety.

[0017] This application also provides a method for cultivating strain BC11-1 to obtain higher F-2 toxin degradation efficiency. Specifically, when cultivating strain BC11-1 seed culture, an additional carbon source is added to LB liquid medium. The carbon source is one or more of glycerol, sucrose, and mannitol. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this application or the prior art, the drawings used in the embodiments or the description of the technology will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This study demonstrates the growth-inhibiting effect of the BC11-1 strain whole culture broth biocontrol agent disclosed in Example 2 of this application on a variety of harmful microorganisms, wherein CK is a control experiment conducted using sterile water;

[0020] In the picture,

[0021] Culture media 1-5 were inoculated with *Sclerotium rolfsii*, *Botrytis cinerea*, *Fusarium incarnatum*, *Aspergillus flavus*, and *Phomopsis asparagi*, respectively.

[0022] Culture media 7-10 were inoculated with Fusarium graminearum, Rhizoctonia solani, Alternaria tenuissima, and Curvularia oryzae, respectively.

[0023] Figure 2 This demonstrates the effect of the BC11-1 strain whole culture solution biocontrol agent disclosed in Example 2 of this application on plant detoxification.

[0024] Figure 3 To illustrate the degradation effect of the biocontrol agent obtained by adding different carbon sources to the seed culture in Example 3 of this application on F-2 toxin.

[0025] Figure 4 This paper demonstrates the inhibitory effect of the exfoliated metabolite biocontrol agent of strain BC11-1 in Example 5 of this application on Aspergillus flavus and Fusarium graminearum in feed. CK is a control experiment conducted using sterile water. Detailed Implementation

[0026] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the embodiments of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0027] The embodiments of this application will now be described in detail with reference to the accompanying drawings.

[0028] Example 1: Strain BC11-1

[0029] The strain BC11-1 was isolated by the applicant from forest soil in Hejiang County, Luzhou City, Sichuan Province in 2021. The specific isolation steps are as follows:

[0030] Disperse the sample with sterile water at a volume-to-mass ratio of (30-50):1, incubate at 28°C with shaking for at least 3 hours, then dilute and spread onto LB solid medium, and incubate until single colonies grow. Strand purification yields pure strain BC11-1, which is then cultured in LB liquid medium with the addition of 20% glycerol and stored at -20°C for later use.

[0031] Whole-genome DNA was extracted from BC11-1, and PCR amplification was performed using universal primers for 16S rRNA. The amplified product was sequenced, and the 16S rRNA sequence of BC11-1 is shown in SEQ ID No. 1. Based on morphological and physiological / biochemical identification, strain BC11-1 was confirmed as *Burkholderia contaminans*. Currently, strain BC11-1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 22, 2022, with accession number CGMCC No. 24417.

[0032] Example 2: Preparation and application of biocontrol agents in whole culture medium

[0033] (1) Preparation of the whole culture medium biocontrol agent of strain BC11-1, the specific steps are as follows:

[0034] S1. Activation

[0035] Inoculate strain BC11-1 onto LB solid medium and incubate at 28°C for 2-3 days;

[0036] S2. Seed liquid

[0037] A single colony was picked from the activated culture medium and transferred to LB liquid medium. The culture was shaken at 28°C for 2 days to obtain the seed culture of strain BC11-1.

[0038] S3. Whole-culture medium biological control agent

[0039] Inoculate the seed culture into fresh LB liquid medium at a ratio of 1:100, and incubate at 28°C with shaking until the bacterial concentration is not less than 1×10⁻⁶. 8 cfu / mL.

[0040] (2) The specific steps for verifying the antibacterial effect of the above-mentioned whole culture medium biocontrol agent are as follows:

[0041] T1. Fungal culture

[0042] Common harmful microorganisms in feed and environment were selected, activated and cultured on PDA plate medium at 28℃ for no less than 4 days, and then the bacterial blocks were taken by punching holes.

[0043] T2. Antibacterial culture

[0044] Inoculate the collected bacterial blocks around the perimeter of a new PDA plate culture medium, with the bacterial blocks spaced 6 cm apart; punch holes in the center of the new PDA plate culture medium and inject the above-mentioned whole culture medium biocontrol agent (5 mm hole corresponds to 50 μL of whole culture medium biocontrol agent); incubate at 28℃ for 5 days.

[0045] T3. Antibacterial effect

[0046] Sterile water was used as a blank control for the whole culture medium biocontrol agent.

[0047] like Figure 1 As shown, the whole-culture medium biocontrol agent exhibited significant inhibitory effects on the growth of fungi such as Sclerotium rolfsii, Botrytis cinerea, Fusarium incarnatum, Aspergillus flavus, Phopsis asparagi, Fusarium graminearum, Rhizoctonia solani, Alternaria tenuissima, and Curvularia oryzae.

[0048] (3) The specific steps for verifying the detoxification effect of the above-mentioned whole culture medium biocontrol agent are as follows:

[0049] E1. Additive toxins

[0050] Strain strain BC11-1 was inoculated onto LB solid medium and cultured at 28°C for 3 days. Single colonies were then picked and inoculated into LB liquid medium containing both F-2 toxin standard and aflatoxin B1 standard. The concentration of F-2 toxin was controlled at 100 μg / L and the concentration of aflatoxin B1 was controlled at 10 μg / L. LB liquid medium without strain BC1 and with added toxins served as a control.

[0051] E2. Degradation Treatment and Effect Verification

[0052] After incubation at 28℃ with shaking for 2 days, the content of the target toxin in each experimental group was detected by liquid chromatography. The results are shown in the table below:

[0053]

[0054] To test the tolerance of strain BC11-1 to a toxic environment, this example selected the initial stage of seed culture preparation when strain BC11-1 had not yet formed a bacterial community. As shown in the table above, on the second day after adding the toxin, the contents of F-2 toxin and aflatoxin B1 in the culture medium decreased compared to the control group, and then remained stable. In the culture medium inoculated with strain BC11-1, the decrease in the concentrations of F-2 toxin and aflatoxin B1 on the second day after adding the toxin was significantly greater than that in the control group. By the tenth day of culture, aflatoxin B1 had been completely degraded, and F-2 toxin had been degraded by more than 95%. This demonstrates that strain BC11-1 has a good ability to tolerate high-concentration toxic environments and can grow and metabolize normally in such environments to remove F-2 toxin and aflatoxin B1.

[0055] (4) Application of whole culture medium biocontrol agents in plant detoxification

[0056] P1. Toxin soaking

[0057] Sterile water and whole culture medium biocontrol agent were mixed at a volume ratio of 5:1, and aflatoxin B1 was added to adjust the concentration to 10 μg / L. After standing at 28℃ for 24 h, 50 rice seeds that had been disinfected with carbendazim and washed with clean water were added to the mixture. After standing at room temperature for 24 h, the seeds were germinated and the germination rate was recorded.

[0058] P 2. Effect Verification

[0059] The treated seeds were cultured at 28℃ for 8 days, such as Figure 2 As shown, the germination rate of rice seeds treated with aflatoxin B1 was only 3%, while the germination rate of seeds treated with the whole culture solution of strain BC11-1 as a biocontrol agent was 65%.

[0060] This proves that the whole culture solution of strain BC11-1 can be used for plant detoxification, and the detoxified plants can grow and develop normally.

[0061] Example 3

[0062] In this embodiment, the culture medium for strain BC11-1 was studied. It was found that adding an additional carbon source to the LB broth during seed culture improved the degradation efficiency of F-2 toxin by strain BC11-1 in the whole culture medium. The specific seed culture medium formula was as follows: LB liquid as the base medium, with the addition of 0.5% glycerol, mannitol, maltose, sucrose, and glucose.

[0063] The verification methods specifically include:

[0064] Strain BC11-1 was inoculated into LB agar plates and activated at 28°C for 3 days. LB liquid was used as the basal medium, with different carbon sources added (0.5% glycerol, mannitol, sucrose, glucose, etc.). Single colonies were picked using an inoculation loop and inoculated into fresh 50 mL of LB liquid medium with different carbon sources, and fermented at 28°C for 3 days.

[0065] Simultaneously, 6 μL of zearalenone standard with a stock solution concentration of 100 μg / mL was added to 20g of animal feed (purchased from the market). Subsequently, 10 mL of fermentation broth with different carbon sources was added to each feed sample, with the uninoculated sample serving as a positive control. The zearalenone content in the feed was determined according to YT2071-2011 "Determination of Aflatoxin, Zearalenone and T-2 Toxin in Feed - Liquid Chromatography-Tandem Mass Spectrometry".

[0066] The effects of fungal toxin degradation are as follows: Figure 3 As shown, glycerol, sucrose, and mannitol were selected as additional carbon sources and added to the LB broth used for seed culture, which is beneficial to improving the degradation efficiency of F-2 toxin by the biocontrol agent of strain BC11-1 in the whole culture medium.

[0067] Example 4

[0068] To facilitate the preservation of the biocontrol agent from the whole culture medium of strain BC11-1, the whole culture medium biocontrol agent can be centrifuged, the bacterial cells collected, and then freeze-dried biocontrol agent prepared using existing freeze-drying technology.

[0069] Although strain BC11-1 belongs to the genus Burkholderia, it is not a currently known pathogenic species. To demonstrate the safety of strain BC11-1, acute and long-term toxicity tests were conducted in mice.

[0070] Mice were randomly divided into a control group and a treatment group. The control group received 5000 mg / kg of lyophilized biocontrol agent of strain BC11-1 (hereinafter referred to as BC). The control group received the corresponding volume of physiological saline. In the acute test, BC was administered once by gavage. After 72 hours, general manifestations, body weight, blood glucose, major organ indices (organ weight to body weight ratio), and histopathological analysis were performed. In the long-term toxicity test, BC was administered by gavage once daily for one week, and the above indicators were observed after 30 days. The results are shown in the table below:

[0071] Table 1. Effects of long-term BC administration on mouse body weight ( n=6)

[0072]

[0073] Table 2 Effects of BC on blood glucose in mice ( n=6)

[0074]

[0075] Table 3. Effects of acute BC administration on organ indices in mice ( n=6)

[0076]

[0077] Table 4. Effects of long-term BC administration on organ indices in mice ( n=6)

[0078]

[0079] Acute toxicity tests showed that BC had no significant effect on the general condition, blood glucose, body weight, or major organ indices of mice. Histopathological results showed that BC administration had some effect on the alveolar and glomerular structures, but no significant toxic histopathological changes were found in the heart, liver, and spleen.

[0080] Long-term toxicity tests showed that BC had no significant effect on the general condition, blood glucose, body weight, and major organ indices of mice. Histopathological results showed that the liver of the BC-treated group exhibited pathological changes, while no significant toxic-induced histopathological changes were found in the heart, spleen, lungs, and kidneys.

[0081] Example 5: Preparation and application of extracellular metabolic biocontrol agents

[0082] (1) Preparation of extracellular metabolic biocontrol agents for strain BC11-1, the specific steps are as follows:

[0083] The whole culture medium of the biocontrol agent obtained in Example 2 was centrifuged, and the supernatant was collected and filtered through 0.22 μm micropores to remove bacterial cells.

[0084] The specific steps for using extracellular metabolic biocontrol agents in feed detoxification treatment are as follows:

[0085] P1. Soaking

[0086] After sterilizing the purchased grain feed, it was divided into multiple groups and placed in sterilized glass bottles. Extracellular metabolic biocontrol agent of strain BC11-1 was added to the feed at a mass-to-volume ratio of 2:1. After soaking for 24 hours, Aspergillus flavus conidia and Fusarium graminearum mycelia cultured on PDA plates were picked up with an inoculation loop and inoculated into the glass bottles containing the grain feed. The bottles were then incubated at 28°C for 7 days. A treatment with added sterile water served as a blank control.

[0087] P2. Verification of antibacterial effect

[0088] like Figure 4 As shown, the mold rate in the blank control was 100%, while the mold rate in grains treated with extracellular metabolic biocontrol agents was about 10%.

[0089] P3. Verification of toxin degradation effect

[0090] The content of the target toxins in each experimental group was determined by liquid chromatography. The aflatoxin B1 content in cereals treated with extracellular metabolic biocontrol agents was 0.13 μg / kg, and the zearalenone content was 4.37 μg / kg. The aflatoxin B1 content in the control group that was not treated with extracellular metabolic biocontrol agents was 150.13 μg / kg, and the zearalenone content was 1900 μg / kg.

Claims

1. A strain of Burkholderia, characterized in that, for Burkholderia contaminans Strain BC11-1 was deposited at the China General Microbiological Culture Collection Center on February 22, 2022, with accession number CGMCC No. 24417.

2. The application of Burkholderia as described in claim 1 for degrading zearalenone.

3. The application of Burkholderia as described in claim 1 to inhibit Fusarium fungi and degrade zearalenone, wherein the Fusarium fungi are Fusarium rubrum (…). Fusarium incarnatum ) and Fusarium graminearum ( Fusarium graminearum ).

4. The application of Burkholderia as described in claim 1, which simultaneously inhibits fungi and degrades aflatoxin and zearalenone, wherein the fungus is Sclerotium sclerotiorum (… Sclerotium rolfsii Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinerea Fusarium rosenbergii ( ), Fusarium incarnatum Aspergillus flavus Aspergillus flavus ), Pseudostem mold ( Phomopsis asparagi Fusarium graminearum ( ), Fusarium graminearum Rhizoctonia solani ( ) Rhizoctonia solani Alternaria ( Alternaria tenuissima ), Curvularia rice ( Curvularia oryzae One or more of the following.

5. A biological control agent, characterized in that, The supernatant is obtained by centrifuging the whole culture medium of Burkholderia as described in claim 1 or by filtering the supernatant through a microporous membrane to remove bacteria.

6. A biological control agent according to claim 5, characterized in that, The complete culture medium of Burkholderia was prepared by the following steps: S1. The Burkholderia bacillus described in claim 1 was activated using LB solid medium to obtain activated bacteria; S2. The activated bacteria obtained in step S1 are cultured in LB liquid medium to obtain seed culture; S3. The bacterial culture prepared using the seed culture from step S2 has a concentration of not less than 1×10⁻⁶. 8 Fermentation broth with cfu / mL.

7. The biological control agent according to claim 6, characterized in that, Step S2 uses LB liquid + 0.5% carbon source as the culture medium, wherein the carbon source is any one of glycerol, sucrose, and mannitol.

8. A biological control agent according to claim 5, characterized in that, It is prepared by the following steps: T1. The Burkholderia bacillus described in claim 1 was activated using LB solid medium to obtain activated bacteria; T2. The activated bacteria obtained in step T1 are cultured in LB liquid medium to obtain seed culture; T3. Prepare bacterial culture with a concentration of not less than 1×10⁻⁶ using the seed culture from step T2. 8 Fermentation broth with cfu / mL; T4. Centrifuge the fermentation broth obtained in step T3, collect the bacterial cells, and freeze-dry to obtain a total viable cell concentration of 1×10⁻⁶ per gram. 11 CFU powder, wherein the powder is a whole culture broth biocontrol agent of Burkholderia; Alternatively, the fermentation broth obtained by centrifugation step T3 can be used to collect the supernatant and filter it through micropores to prepare the extracellular metabolite biocontrol agent of Burkholderia.

9. The biocontrol agent according to any one of claims 5-8 is used for the detoxification of zearalenone and aflatoxin in feed.

10. The biocontrol agent according to any one of claims 5-8 is used for the detoxification of aflatoxin in plants.

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