CN118147020A - Non-decarboxylated Leucol bacteria and application thereof in inhibition of zearalenone - Google Patents
Non-decarboxylated Leucol bacteria and application thereof in inhibition of zearalenone Download PDFInfo
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- CN118147020A CN118147020A CN202410578519.1A CN202410578519A CN118147020A CN 118147020 A CN118147020 A CN 118147020A CN 202410578519 A CN202410578519 A CN 202410578519A CN 118147020 A CN118147020 A CN 118147020A
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- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 title claims abstract description 37
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 241000894006 Bacteria Species 0.000 title claims abstract description 21
- 230000005764 inhibitory process Effects 0.000 title description 6
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 title description 2
- 241001647841 Leclercia adecarboxylata Species 0.000 claims abstract description 11
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 230000012010 growth Effects 0.000 abstract description 6
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 23
- 240000008042 Zea mays Species 0.000 description 20
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 20
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 20
- 235000005822 corn Nutrition 0.000 description 20
- 239000002609 medium Substances 0.000 description 15
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- 239000007788 liquid Substances 0.000 description 13
- 241000233866 Fungi Species 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 235000013339 cereals Nutrition 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 230000015556 catabolic process Effects 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 239000002245 particle Substances 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
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- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 239000006059 cover glass Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 2
- 241000223218 Fusarium Species 0.000 description 2
- 241000906158 Leclercia sp. Species 0.000 description 2
- 241000192132 Leuconostoc Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000004820 blood count Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
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- 238000012360 testing method Methods 0.000 description 2
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- 241000589291 Acinetobacter Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 240000001592 Amaranthus caudatus Species 0.000 description 1
- 235000009328 Amaranthus caudatus Nutrition 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000179039 Paenibacillus Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
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- 210000004027 cell Anatomy 0.000 description 1
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- 230000000911 decarboxylating effect Effects 0.000 description 1
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- 238000002360 preparation method Methods 0.000 description 1
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- 231100000765 toxin Toxicity 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses non-decarboxylated luxuriant bacteria and application thereof for inhibiting zearalenone, belonging to the technical field of microbial agents, wherein the non-decarboxylated luxuriant bacteria SGLH-11 are classified and named as non-decarboxylated luxuriant bacteria LECLERCIA ADECARBOXYLATA, and are preserved in China center for type culture collection (CCTCC NO), the preservation date is 2024, 1 month and 4 days: m2024020; the non-decarboxylated Lecanis can inhibit the growth of the corncob and the generation of spores thereof, the antibacterial rate of the corncob and the generation of the corncob can reach 55.88%, and the non-decarboxylated Lecanis can also be used for inhibiting the generation of the corncob and the generation of the corncob.
Description
Technical Field
The invention relates to the technical field of microbial agents, in particular to non-decarboxylated luxuriant bacteria and application of the non-decarboxylated luxuriant bacteria in inhibition of zearalenone.
Background
The corn ear kernel rot is mainly harmful to the corn ear, and is generally developed from the top or the base of the corn ear, so that large pieces or the whole corn ear is rotten, the bracts of the disease ear are bonded with the corn ear, 1 light purple to light pink mold layer is generated between the bracts, the top of the corn ear turns pink, and red to grey white hyphae are generated between the corn kernels. Not only does ear kernel rot cause loss of corn yield, but also various toxins including Zearalenone (ZEN), fumonisins, etc. are produced in the corn kernel.
Zearalenone (ZEN), also known as F-2 toxin, is ubiquitous in plants and can be detected in both cereal and agricultural by-products. Zearalenone is an estrogenic mycotoxin produced by multiple Fusarium spp, and poses serious health and agricultural hazards. Recent studies have shown that zearalenone has genotoxicity and immunotoxicity and also has a certain effect on tumorigenesis.
The biodegradation method is a detoxification method for adsorbing and degrading the zearalenone by utilizing microorganisms, has the characteristics of safety, environment friendliness, high efficiency, strong specificity and high detoxification rate, does not influence the nutritional value of grains, and becomes a hotspot for the degradation research of the zearalenone. Microorganisms which have been found to be ZEN detoxified include bacteria such as Bacillus, lactobacillus, pseudomonas, acinetobacter, etc., fungi such as Aspergillus, paenibacillus, yeast, etc.
The non-decarboxylated luxuriant bacteria (Leclerciaadecarboxylata) belongs to the genus non-decarboxylated luxuriant bacteria, is a gram-negative bacillus with power of enterobacteriaceae, is usually derived from environment and animals, is found in 1962, has similar biochemical characteristics to escherichia coli, and has not been reported before on biocontrol effect on corn ear grain rot bacteria.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides non-decarboxylated luxuriant bacteria and application for inhibiting zearalenone, which can be used for inhibiting the growth of the corn ear grain rot fungi and the generation of spores thereof and can also be used for inhibiting the zearalenone generated by the corn ear grain rot fungi.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
The non-decarboxylated Lecanis bacteria are classified and named as non-decarboxylated Lecanis bacteria LECLERCIA ADECARBOXYLATA, and are preserved in China center for type culture Collection, with addresses of China university of Wuhan, and with a preservation date of 2024, 1 month and 4 days, and a preservation number of CCTCC NO: m2024020.
The non-decarboxylated Lecanis bacteria are yellow, round and convex, the edges are neat, the surfaces are smooth and moist, and the diameter is 2-3mm.
Use of the above non-decarboxylating luxuriant bacteria for inhibiting zearalenone.
Compared with the prior art, the invention has the beneficial effects that:
(1) The non-decarboxylated Leuconostoc SGLH-11 can effectively inhibit the growth of the corncob grain rot fungi, and the antibacterial rate of the corncob grain rot fungi can reach 55.88%;
(2) The non-decarboxylated Leuconostoc SGLH-11 can effectively inhibit the generation of the corn ear particle rot fungi spores, and the inhibition rate of spores can reach 100%;
(3) The non-decarboxylated Lecanis SGLH-11 can effectively degrade zearalenone produced by the corncob and grain rot fungi, and after the zearalenone is mixed with the corncob and grain rot fungi for 9 days, the degradation rate of ZEN can reach 72.68%.
Detailed Description
Specific embodiments of the present invention will now be described in order to provide a clearer understanding of the technical features, objects and effects of the present invention.
EXAMPLE 1 preparation of SGLH-11 Strain
1. Collecting a sample: 1 part of fresh soil sample of humus under the forest is collected from a shou forest sea ecological show garden.
2. And (3) separating and purifying: 1g of collected fresh soil sample is taken, 10mL of sterile water is added, the mixture is fully vibrated to obtain suspension, 100 mu L of suspension is sucked and coated in a solid NA culture medium (peptone 10g, beef extract 5g, sodium chloride 5g, agar 15g and water 1000 mL) plate by using a coater, the culture is carried out for 24 hours at a constant temperature of 37 ℃, typical bacterial colonies (bacterial colonies on the NA culture medium plate are yellow, round, convex, neat in edge and smooth in surface and moist and have a diameter of 2-3 mm) are selected, and 3 times of streaking separation are carried out by using an inoculating loop to obtain SGLH-11, SGLH-62, SGLH-91, SGLH-105 and SGLH-23 pure culture strains.
Example 2 growth inhibition of mycelium of F.corncob YUFU-02
1. Separating and purifying corn ear and grain rot fungi: selecting corn ear grain rot samples with typical symptoms, picking hyphae, inoculating into PDA culture medium, culturing 4 pieces per dish, and observing in a constant temperature oven at 28 ℃. After hyphae germinate, a typical colony is selected and transferred for 3 times, a puncher with the inner diameter of 0.5cm is used for punching a bacterial cake from the edge of the colony after 7d, and the bacterial cake is transferred to a PDA flat plate for purification culture, and is marked as YUFU-02. Strains with dense hypha growth, regular colony and no pollution on the PDA plate are stored in a 1.8mL centrifuge tube and are stored in a refrigerator at 4 ℃.
The corn ear particle rot germ grows vigorously on the PDA culture medium, cotton is initially white, and gradually turns to earthy yellow, and the culture medium pigment is amaranth to brownish red; the conidium is sickle-shaped and morphologically identified as fusarium.
2. Inoculating: the inhibition of the growth of the corn ear particle rot fungi YUFU-02 by the strains SGLH-11, SGLH-62, SGLH-91, SGLH-105 and SGLH-23 obtained in the example 1 was measured by a crisscross method, specifically, 6 PDA plate culture mediums were prepared, each PDA plate culture medium was perforated by a crisscross method, four holes were perforated, the distance from the center point to the holes was 2.5cm, corn ear particle rot fungi YUFU-02 cakes (5 mm) were inoculated in the centers of the plates, SGLH-11 bacterial solutions, SGLH-62 bacterial solutions, SGLH-91 bacterial solutions, SGLH-105 bacterial solutions and SGLH-23 bacterial solutions (the bacterial concentration in the bacterial solutions was 2.15X10 8/mL) were inoculated in the four holes of the 5 PDA plate culture mediums, respectively, and the other 1 PDA plate culture medium was used as a blank control, and no bacterial solution was inoculated. After 5d, measuring the diameter of YUFU-02 bacterial cakes by using a crisscross method, performing 3 repeated tests, averaging the diameters, and calculating the bacteriostasis rate.
3. Analysis of results: the obtained antibacterial rate results are shown in the following table, and the antibacterial rate of the strain SGLH-11 is highest and is more than 50%, so that the strain SGLH-11 can effectively inhibit the hypha growth of YUFU-02.
Example 3 identification of SGLH-11 Strain
1. Morphological identification: strain SGLH-11 is gram-negative bacillus, grows well on NA culture medium plates, and has yellow, round and convex colonies, regular edges, smooth and moist surfaces and diameters of 2-3mm.
2.16S rDNA sequence identification: the method comprises the steps of taking extracted genome DNA of a strain as a template, carrying out PCR amplification by using 27F (with a sequence shown as SEQ ID NO. 1) and 1492R (with a sequence shown as SEQ ID NO. 2) as primers, detecting a PCR product by using a 1.0% agarose gel electrophoresis method, preliminarily judging the size of a target fragment to be detected according to the relative position of a Marker, and obtaining DNA fragments with lengths between 750 and 1500 bp, wherein the length of the DNA fragments obtained by the PCR amplification is similar to the length of the target fragment of bacterial 16S rDNA, and the sequence of the 16S rDNA is shown as SEQ ID NO. 3. The amplified PCR product was sent to China large gene company for DNA sequence determination, and the determined base sequence was subjected to BLAST alignment in GenBank, and by alignment with Leclerciaadecarboxylata gene sequence （MG254798.1（Leclercia sp. strain T3196-2）、KT260889.1（Leclerciaadecarboxylata strain RCB677）、LT899940.1（Leclerciaadecarboxylata partial 16S rRNA gene, strain HAMBI 1696）、MH071320.1（Leclerciaadecarboxylata strain G9TT3）、MT133354.1（Leclerciaadecarboxylata strain LSRBMoFPIKRGCFTRI47）、OR054217.1（Leclercia sp. strain H-S-2））, the homology was >99.9%, SGLH-11 could be identified as non-decarboxylated Leucocalyxa (Leclerciaadecarboxylata).
The SGLH-11 strain screened by the invention is classified and named as non-decarboxylated luxuriant bacteria LECLERCIA ADECARBOXYLATA, and is preserved in China center for type culture Collection, with the address of China university of Wuhan, the preservation date of 2024, 1 month and 4 days, and the preservation number of CCTCC NO: m2024020.
EXAMPLE 4 SGLH-11 Strain growth inhibition of YUFU-02 spores
1. PDA liquid medium was prepared: taking 2 PDA liquid culture mediums, wherein one is used as a treatment culture medium, the other is used as a control culture medium, and adding 100 mu L SGLH-11 bacterial solutions (the bacterial concentration in the bacterial solutions is 2.15X10 8/mL) and one YUFU-02 (5 mm) bacterial cake obtained in example 2 into the treatment culture medium; only one YUFU-02 (5 mm) cake obtained in example 2 was added to the control medium. The treatment medium and the control medium were shake-cultured at 28℃and sampled after 1d, 3d, 5d, 7d, and 9d, respectively, and the number of spores was counted by the hemocytometer method.
2. And (3) result statistics: recording and calculating YUFU-02 spores in the treatment culture medium and the control culture medium by adopting a blood cell counting method, covering a cover glass on a counting chamber of a blood cell counting plate, sucking sample liquid which is fully and uniformly mixed by vibration by using a liquid transfer device, dripping the sample liquid on the edge of the cover glass, allowing bacterial liquid to infiltrate into the counting chamber from a gap between the cover glass and the counting plate, standing for a moment, and starting counting after bacterial cells naturally settle and stabilize. 2 replicates were run, 3 replicates were run for each run, and the average was taken.
The results of the number of YUFU-02 spores in the shaking culture 1d, 3d, 5d, 7d, 9d post-treatment medium and control medium recorded by the hemocytometer method are shown in the following table, and it can be seen from the following table that the number of conidia gradually increases with the prolonged shaking culture time in the control medium without SGLH-11 bacteria solution. The number of conidium in the treated culture medium added with SGLH-11 bacterial liquid is 0, and the inhibition rate reaches 100%. It is demonstrated that SGLH-11 can effectively inhibit the generation of the spores of the corn ear putrescence YUFU-02.
EXAMPLE 5 SGLH-11 degradation of ZEN by Strain
The ZEN content of the treated medium and the control medium after 1d, 3d, 5d, 7d, and 9d shaking culture obtained in example 4 was measured using Zearalenone (ZEN) rapid assay card (Wuhan Zhi Biotechnology Co., ltd.). The method comprises the following steps:
Taking 100 mu L of sample liquid from a treatment medium or a control medium by using a liquid transfer device, adding 100 mu L of extracting solution (absolute ethyl alcohol: distilled water=1:1), carrying out high-speed vortex for 3min and centrifuging for 3min at 4000rpm to obtain a treated sample liquid to be detected; before the test, the used Zearalenone (ZEN) rapid detection card and the processed sample liquid to be tested are restored to the room temperature; taking out a Zearalenone (ZEN) rapid detection card, sucking 50 mu L of the treated sample liquid to be detected by a pipette, and vertically adding the sample liquid into a sample adding hole; the Zearalenone (ZEN) rapid detection card is placed in an immunity quantitative rapid measuring instrument (opened 15min in advance), the 'incubation detection' is selected, the dilution number is 1, and the quantitative result is read after the instrument is automatically set for 10 min.
As a result, it was found that the amount of ZEN produced by YUFU-02 bacteria gradually increased with the increase of the shaking culture time in the control medium to which the SGLH-11 bacteria solution had not been added. However, in the treatment medium containing SGLH-11 bacterial liquid, the difference between the ZEN content in the control medium and the ZEN content in the treatment medium gradually expands along with the extension of the shaking culture time, and the degradation rate reaches 72.68% at 9d, which indicates that the SGLH-11 bacterial strain can effectively reduce the generation of ZEN in the corn ear particle rot fungi YUFU-02.
As can be seen from the results of examples 2 and 4 to 5, SGLH-11 strain acts as a degradation agent for ZEN by inhibiting the hyphal growth, sporulation and direct degradation of zearalenone by YUFU-02 bacteria.
Claims (2)
1. The non-decarboxylated luxury bacteria SGLH-11 are characterized in that the non-decarboxylated luxury bacteria are classified and named as non-decarboxylated luxury bacteria LECLERCIA ADECARBOXYLATA, and are preserved in China center for type culture Collection, the preservation date is 2024, 1 month and 4 days, and the preservation number is CCTCC NO: m2024020.
2. Use of the non-decarboxylated luxuriant bacteria SGLH-11 according to claim 1 for inhibiting zearalenone.
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