CN118207102B - Antibiotic-degrading bacterium GXJ-SJ9, biological agent and application - Google Patents
Antibiotic-degrading bacterium GXJ-SJ9, biological agent and application Download PDFInfo
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- CN118207102B CN118207102B CN202410629090.4A CN202410629090A CN118207102B CN 118207102 B CN118207102 B CN 118207102B CN 202410629090 A CN202410629090 A CN 202410629090A CN 118207102 B CN118207102 B CN 118207102B
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- 239000003124 biologic agent Substances 0.000 title claims abstract description 25
- 241000894006 Bacteria Species 0.000 title claims abstract description 19
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- 229960000740 enrofloxacin Drugs 0.000 claims abstract description 32
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 229940088710 antibiotic agent Drugs 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
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- 238000001514 detection method Methods 0.000 description 6
- 150000002500 ions Chemical class 0.000 description 6
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- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
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- 240000008415 Lactuca sativa Species 0.000 description 1
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- 241001215623 Talaromyces cellulolyticus Species 0.000 description 1
- 241001207467 Talaromyces sp. Species 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention relates to the technical field of microorganisms, in particular to an antibiotic degrading bacterium GXJ-SJ9, a biological agent and application thereof. The classification of the antibiotic-degrading bacteria GXJ-SJ9 is named as blue fungus Talaromyces sp, and is preserved in 2024 and 03 month 19 to China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation address of North Star Xway No. 1, no. 3, and a preservation number of CGMCC No.41125 in the Korean region North Star of Beijing city. The antibiotic-degrading bacterium GXJ-SJ9 provided by the invention is separated from strawberry rhizosphere soil with a large amount of applied organic fertilizer, and is obtained through enrichment culture, multiple screening, separation and purification, and has the antibiotic-degrading effects of tetracycline, enrofloxacin, tylosin and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to an antibiotic degrading bacterium GXJ-SJ9, a biological agent and application thereof.
Background
In recent years, researchers have detected a variety of drug resistant strains/resistance genes from leafs (such as lettuce), tubers (such as radishes), fruits (such as tomatoes), vegetables and fruits (such as strawberries), almost all known antibiotic categories are contained, and the problem of antibiotic pollution in soil-plant systems is gradually paid attention to and studied, and becomes an important research topic in the field of environmental pollution.
At present, many microorganisms with good restoration effect on soil antibiotic residues are found, but little research on degradation of antibiotics by Talaromyces (blue fungus) strains is still carried out. The invention patent application CN 117551557A discloses a strain TLT1 for soil and water pollution remediation, and the strain TLT1 can be used for well degrading enrofloxacin and metallic zinc ions in water and soil. However, the variety of antibiotics existing in the soil is often more than one, the strain TLT1 cannot degrade other common antibiotics such as tetracycline, tylosin and the like, and even the activity of the strain TLT1 can be inhibited by other antibiotics in the soil, so that the degradation effect of enrofloxacin is affected. More importantly, antibiotics are absorbed by plants in the soil and further enriched, resulting in the transmission of resistance genes, whereas the strain TLT1 does not have the function of reducing the enrichment of antibiotics in plant fruits.
Disclosure of Invention
Aiming at the technical problem of lacking a cyanobacteria strain capable of degrading various antibiotics, the invention provides an antibiotic degrading strain GXJ-SJ9, a biological agent and application, wherein the antibiotic degrading strain GXJ-SJ9 is separated from strawberry rhizosphere soil with a large amount of applied organic fertilizer, and is a strain with antibiotic degrading effects of tetracycline, enrofloxacin, tylosin and the like, which is obtained through enrichment culture, multiple screening, separation and purification.
In a first aspect, the present invention provides an antibiotic-degrading bacterium GXJ-SJ9, which is classified and named as blue fungus Talaromyces sp, and has been deposited in the general microbiological center of the China Committee for culture Collection of microorganisms at 19 of 2024, wherein the deposited address is number 3 of the west way 1, the sunny region North Star of the Beijing city, and the deposited number is CGMCC No.41125.
In a second aspect, the invention provides an application of the antibiotic-degrading bacterium GXJ-SJ9 in degrading tetracycline, enrofloxacin or/and tylosin in soil.
In a third aspect, the invention provides an application of the antibiotic-degrading bacterium GXJ-SJ9 in reducing enrichment of tetracycline, enrofloxacin or/and tylosin in plant fruits in soil.
In a fourth aspect, the present invention provides a biological agent comprising spores of the antibiotic-degrading bacterium GXJ-SJ9 described above.
Further, the preparation method of the biological agent comprises the following steps:
culturing alternately with PDA culture medium illumination and darkness at 28deg.C for 10 days to give spore, and resuspending spore with PBS buffer (sterilized, pH=7.4).
Further, the concentration of the antibiotic-degrading bacteria GXJ-SJ9 spores in the biological microbial inoculum is 10 6-107 CFU/mL.
In a fifth aspect, the invention provides the use of a biological agent as described above for degrading tetracycline, enrofloxacin or/and tylosin in soil.
In a sixth aspect, the invention also provides an application of the biological agent in reducing enrichment of tetracycline, enrofloxacin or/and tylosin in plant fruits in soil.
The invention has the beneficial effects that:
the antibiotic-degrading bacterium GXJ-SJ9 provided by the invention is an antibiotic-degrading bacterium screened from rhizosphere microorganisms of healthy strawberry plants, belongs to indigenous flora, has unique advantages in colonisation and biosafety, can degrade tetracycline, enrofloxacin and tylosin efficiently, and simultaneously reduces enrichment of the tetracycline, enrofloxacin and tylosin in plant fruits.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is a photograph showing colony morphology of the antibiotic-degrading bacterium GXJ-SJ9 of the present invention.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
The preparation method of the 0.1mol/L Na 2 EDTA-McIlvaine buffer solution used in the specific embodiment of the invention comprises the following steps:
accurately weighing 21.01g of citric acid and 28.41g of disodium hydrogen phosphate, and respectively fixing the volume to 1000mL to prepare 0.1mol/L citric acid solution and 0.2mol/L disodium hydrogen phosphate solution. Mixing 1000mL and 625mL of the two solutions uniformly, regulating the pH value to 4.0+/-0.05, preparing a McIlvaine buffer solution, weighing 60.5g of disodium ethylenediamine tetraacetate, adding the solution into the McIlvaine buffer solution, mixing uniformly, and regulating the pH value to 4.0 and 6.0 for standby.
Example 1
In 2022, 6 months, weighing 5g of strawberry rhizosphere soil collected in a strawberry planting greenhouse in urban area Tang Wangzhen of Jinan, shandong, adding 30mL of sterilized physiological saline, standing for 30min after vortex oscillation, taking 2mL of supernatant, respectively adding 50mL of PDB culture medium containing 20mg/L tetracycline, enrofloxacin or tylosin, and culturing in a shaking table at 28 ℃ in a dark place for 3 days; 2mL of culture solution is added into a new PDB culture medium, and the culture is continued according to the culture conditions, the culture is transferred every 3 days, the concentration of antibiotics added into the PDB culture medium is increased by 20mg/L each time, and the concentration of tetracycline, enrofloxacin or tylosin in the PDB culture medium reaches 200mg/L. The culture solution is uniformly coated on a PDA culture medium after being diluted in a gradient way, the morphological characteristics of the bacterial colonies are observed, single bacterial colonies with different bacterial colony morphologies are picked for further separation and purification, and the glycerol tube is used for preservation.
The individual single strains isolated were isolated at 1:100 volume ratio is respectively inoculated into a screening culture medium containing 200mg/L tetracycline, enrofloxacin or tylosin, and after shaking culture for 7 days at 28 ℃ in a shaking table in a dark place, the corresponding residual concentration of antibiotics is quantitatively detected by using a high performance liquid chromatography-triple quadrupole mass spectrometer, and the degradation rate is calculated.
Wherein, the detection conditions of the tetracycline and the enrofloxacin are as follows:
Mass spectrometry instrument: waters Xevo TQ-XS; ion source: an electrospray ion source; scanning mode: scanning positive ions; the detection mode is as follows: monitoring multiple reactions; atomization temperature: 350 ℃; ionization voltage: 2kV; atomizing air flow rate: 700L/h.
TABLE 1 Mass Spectrometry detection conditions for Tetracycline and enrofloxacin
Liquid chromatographic column: ACQUITY UPLC BEH C18 a 18; pore diameter: 1.7 μm; column specification: 3.0 x 100 mm.
TABLE 2 gradient elution procedure for tetracycline and enrofloxacin
The detection conditions for tylosin are as follows:
Mass spectrometry instrument: waters Xevo TQ-XS; ion source: an electrospray ion source; scanning mode: scanning positive ions; the detection mode is as follows: monitoring multiple reactions; atomization temperature: 350 ℃; ionization voltage: 2kV; atomizing air flow rate: 700L/h.
TABLE 3 Mass Spectrometry detection conditions for tylosin
Liquid chromatographic column: ACQUITY UPLC BEH C18 a 18; pore diameter: 1.7 μm; column specification: 3.0 x 100 mm.
TABLE 4 gradient elution procedure for tylosin
According to analysis and comparison, the strain GXJ-SJ9 can be enriched in the enrichment culture process of three antibiotics of tetracycline, enrofloxacin and tylosin, has degradation effects on the tetracycline, enrofloxacin and tylosin, and has degradation rates of 57.2 percent (tetracycline), 37.8 percent (enrofloxacin) and 94.4 percent (tylosin) after 5 days of culture, and the degradation rates of 66.4 percent (tetracycline), 41.3 percent (enrofloxacin) and 97.7 percent (tylosin) after 7 days of culture.
The strain GXJ-SJ9 was grown for 96 hours at 28℃on PDA medium, and the colony morphology was as shown in FIG. 1. The ITS gene sequence (SEQ ID No. 1) of the strain GXJ-SJ9 is:
TGATATGCTTAAGTTCAGCGGGTAACTCCTACCTGATCCGAGGTCAACCGTGGTAAAAACATGGTGGTGACCAACCCCCGCAGGTCCTTCCCGAGCGAGTGACAGAGCCCCATACGCTCGAGGACCAGACGGACGTCGCCGCTGCCTTTCGGGCAGGTCCCCGGGGGGACCACACCCAACACACAAGCCGTGCTTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCATTGTTGAAAGTTTTGACAATTTTCATAGTACTCAGACAGCCCATCTTCATCAGGGTTCACAGAGCGCTTCGGCGGGCGCGGGCCCGGGGACGTGCGTCCCCCGGCGACCAGGTGGCCCCGGTGGGCCCGCCAAAGCAACAGGTGTATAGAGACAAGGGTGGGAGGTTGGGCCGCGAGGGCCCGCACTCGGTAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTACACTTT.
The strain GXJ-SJ9 is identified as Talaromyces cellulolyticus, and is preserved to the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the 19 th day of 2024, the preservation address is North Star Xway No. 1, 3 in the Korean area of Beijing, the preservation number is CGMCC No.41125, and the strain is classified and named as blue fungus Talaromyces sp.
Example 2
Culturing strain GXJ-SJ9 at 28deg.C with PDA culture medium alternately in light and dark for 10 days to give spore, and suspending spore with PBS buffer (sterilizing, pH=7.4) until spore concentration is 10 6 CFU/mL.
Example 3
Taking healthy strawberry cultivation soil, exogenously adding tetracycline, enrofloxacin and tylosin until the concentration of each antibiotic reaches 100mg/kg, and according to the following steps of 1:100 volume ratio the biological agent of example 2 was applied, the ambient temperature was maintained at 18-25 ℃ during the experiment, samples were taken on days 3, 5, 7 respectively, the soil samples were placed in a freeze-dryer for dehydration and then ground thoroughly with a ball mill, and passed through a 60 mesh (pore size 0.25 mm) standard soil screen. Accurately weighing 5.00g of the sieved soil sample, placing the soil sample into a 50mL centrifuge tube, adding 18mL of methanol (analytically pure) and 2mL of Na 2 EDTA-McIlvaine buffer solution (0.1 mol/L, pH=6.0), swirling for 30s, oscillating for 10min, carrying out ultrasonic treatment for 20min and centrifugation for 5min at 10000r/min, transferring the supernatant into a new centrifuge tube, extracting twice continuously, and combining the two supernatants. Nitrogen was purged to 10mL at 40℃and diluted to 30mL with Na 2 EDTA-McIlvaine buffer (0.1 mol/L, pH=6.0), the HLB solid phase extraction column was activated with 6mL methanol, 6mL Na 2 EDTA-McIlvaine buffer (0.1 mol/L, pH=4.0) in sequence, and the sample was subjected to column enrichment at a flow rate of 1mL/min. The HLB column was dried by rinsing with 10mL of ultrapure water and vacuum filtration. 8mL of methanol (chromatographic purity) was added to the mixture, and the eluate was collected in a 10mL centrifuge tube. The eluate was slowly purged to near dryness with nitrogen at 40℃and was filtered through a 0.22 μm filter with acetonitrile to a volume of 2.0mL, and the soil was quantitatively assayed for tetracycline, enrofloxacin or tylosin by the assay conditions described in example 1. And (5) taking a soil sample without the biological agent as a control, and calculating the degradation efficiency.
According to calculation, the degradation rates of the tetracycline, the enrofloxacin and the tylosin in soil are respectively 15.5%, 9.4% and 31.3% in 3 days, 21.7% in 5 days, 15.3% in 55.7% in 7 days and 57.5% in 40.4% in 7 days.
Example 4
In the fruit setting period of healthy strawberries, tetracycline, enrofloxacin and tylosin are externally added into strawberry cultivation soil until the concentration of each antibiotic reaches 100mg/kg, the biological microbial inoculum of the example 2 is applied according to the volume ratio of 1:100, plant samples are respectively collected on days 3, 5 and 7, repeatedly washed by tap water and ultrapure water, wiped clean by paper towels, pre-frozen at the temperature of minus 20 ℃ and then placed in a freeze dryer for 48 hours. Accurately weighing 0.5g to 50mL of freeze-dried fruits, adding 18mL of acetonitrile (analytically pure) and 2mL of Na 2 EDTA-McIlvaine buffer solution (0.1 mol/L, pH=6.0), vortexing for 30s, oscillating for 10min, carrying out ultrasonic treatment for 20min, centrifuging for 5min at 10000r/min, transferring the supernatant into a new centrifuge tube, extracting twice continuously, and combining the supernatants of the two times. 10mL of n-hexane was added, the mixture was shaken for 5 minutes, centrifuged at 8000r/min for 5 minutes, and the lower layer solution was removed. Nitrogen was purged to 10mL at 40℃and diluted to 30mL with Na 2 EDTA-McIlvaine buffer (0.1 mol/L, pH=6.0), the HLB solid phase extraction column was activated with 6mL methanol, 6mL Na 2 EDTA-McIlvaine buffer (0.1 mol/L, pH=4.0) in sequence, and the sample was subjected to column enrichment at a flow rate of 1mL/min. The HLB column was dried by rinsing with 10mL of ultrapure water and vacuum filtration. 8mL of methanol (chromatographic purity) was added to the mixture, and the eluate was collected in a 10mL centrifuge tube. The eluate was slowly purged to near dryness with nitrogen at 40℃and was filtered through a 0.22 μm filter with acetonitrile to a volume of 2.0mL, and the strawberry fruit was quantitatively analyzed for tetracycline, enrofloxacin or tylosin content by the machine according to the test conditions in example 1. The degradation efficiency was calculated using the strawberry fruit sample without the biological agent as a control.
After 3 days, the contents of tetracycline, enrofloxacin and tylosin in the strawberry fruits added with the biological agent are respectively reduced by 77.2%, 53.8% and 87.1% compared with the strawberry fruits which are not treated by the biological agent; after 5 days, the contents of tetracycline, enrofloxacin and tylosin in the strawberry fruits added with the biological agent are respectively reduced by 68.4 percent, 62.3 percent and 55.7 percent compared with the strawberry fruits which are not treated by the biological agent; after 7 days, the tetracycline, enrofloxacin and tylosin contents of the strawberry fruits added with the biological agent are respectively reduced by 75.3%, 50.1% and 60.2% compared with the strawberry fruits which are not treated by the biological agent.
Although the present invention has been described in detail by way of preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (8)
1. The antibiotic degrading bacterium GXJ-SJ9 is characterized in that the strain is classified and named as blue fungus Talaromyces sp, and is preserved in 2024, 03 and 19 days to China general microbiological culture Collection center with a preservation address of North Star Xway No. 1, no. 3 in the Korean region North Star of Beijing city and a preservation number of CGMCC No.41125.
2. Use of an antibiotic-degrading bacterium GXJ-SJ9 according to claim 1, for degrading tetracycline, enrofloxacin or/and tylosin in soil.
3. Use of an antibiotic-degrading bacterium GXJ-SJ9 according to claim 1, for reducing the enrichment of strawberry fruits with tetracycline, enrofloxacin or/and tylosin in soil.
4. A biological agent comprising spores of the antibiotic-degrading bacterium GXJ-SJ9 of claim 1.
5. The biological agent as defined in claim 4, wherein the preparation method of the biological agent comprises the following steps:
Culturing alternately with PDA culture medium illumination and darkness at 28deg.C for 10 days to give spore, and resuspending spore with PBS buffer solution.
6. The biological agent according to claim 4, wherein the concentration of the antibiotic-degrading bacteria GXJ-SJ9 spores in the biological agent is 10 6-107 CFU/mL.
7. Use of the biological agent of claim 4 for degrading tetracycline, enrofloxacin or/and tylosin in soil.
8. Use of the biological agent of claim 4 to reduce the enrichment of strawberry fruits with tetracycline, enrofloxacin or/and tylosin in the soil.
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| CN111356366A (en) * | 2017-11-17 | 2020-06-30 | 诺华丝国际股份有限公司 | Compositions and methods for increasing dietary phosphorus and calcium availability in animals |
| CN117551557A (en) * | 2023-11-03 | 2024-02-13 | 中国科学院沈阳应用生态研究所 | A low-temperature-tolerant multifunctional bacterium and its application in soil and water pollution remediation |
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| CN111356366A (en) * | 2017-11-17 | 2020-06-30 | 诺华丝国际股份有限公司 | Compositions and methods for increasing dietary phosphorus and calcium availability in animals |
| CN117551557A (en) * | 2023-11-03 | 2024-02-13 | 中国科学院沈阳应用生态研究所 | A low-temperature-tolerant multifunctional bacterium and its application in soil and water pollution remediation |
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