Oxytetracycline degrading bacterium under high-concentration oxytetracycline as well as preparation method and application of oxytetracycline degrading bacterium
By screening and providing an oleyroid degradation strain C4 that can efficiently degrade oleyroid in a high-concentration oleyroidin environment, the shortcomings of the degradation of high-concentration oleyroidin in the prior art are solved, and the efficient degradation effect of oleyroidin is achieved, providing an effective solution for the treatment of high-concentration oleyroidin contamination in the environment.
Patent Information
- Application Number
- CN202311849816.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-29
- Publication Date
- 2025-07-01
AI Technical Summary
The existing technology mainly studies the degradation of low-concentration oleyroid dysfunction, and rarely pays attention to the degradation of high-concentration oleyroid dysfunction. High-concentration oleyroid dysfunction will inhibit the physiological activities of microorganisms and it is difficult to screen out efficient degradation bacteria.
It provides a oleyroid degrading bacteria under high concentration of oleyroid dysproutin. The specific strain number is C4, which can efficiently degrade oleyroid dysproutin in a high concentration of 1000 mg/L-2000 mg/L. The strain was prepared into a bacterial suspension by specific culture methods for degrading oleracycin.
This strain can achieve an 85.1% degradation rate of oleyroid dysfunction at 25℃ and 180rpm for 8 days, significantly improving the degradation efficiency of high concentration of oleyroid dysfunction and providing scientific basis for the degradation treatment of high concentration of oleyroid dysfunction contamination.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of environmental pollutant treatment, and particularly relates to an oxytetracycline-degrading bacterium under high-concentration oxytetracycline, a preparation method and an application thereof. Background Art
[0002] Due to the large and frequent use of antibiotics, the problem of bacterial drug resistance caused by antibiotics has begun to attract people's attention in recent years. During the use of antibiotics, resistant strains with tolerance are induced, so that the effective dose of antibiotics to kill bacteria is continuously increasing. More and more experiments have proved that the increase and spread of environmental pathogenic bacteria drug resistance will pose a potential threat to human public health. Tetracycline antibiotics are a class of broad-spectrum antibiotics produced by actinomycetes, including oxytetracycline, chlortetracycline and tetracycline, etc., and their structures all contain a tetraphenyl basic skeleton. Among them, oxytetracycline is one of the most clinically used and widely used antibiotics, occupies an important position in the drug market, and is also a drug additive used more in China. Oxytetracycline is stable in nature and is not easily degraded in the environment. It can still exist in the environment for a long time after being excreted, thus causing drug residues in the environment. And the production and use of tetracycline in China are very large, so the treatment of oxytetracycline by urban sewage treatment plants has crucial practical significance. Microbial degradation has the advantages of low cost, high efficiency, no secondary pollution, good ecological restoration, etc., and has been applied in many aspects. Therefore, screening out oxytetracycline-degrading bacteria is of great significance for treating oxytetracycline wastewater.
[0003] At present, most studies only focus on the degradation of oxytetracycline at low concentrations, and there are few studies on the degradation of oxytetracycline under high-concentration conditions. However, high-concentration oxytetracycline will inhibit the physiological activities of microorganisms. Therefore, screening out highly efficient degrading bacteria under high-concentration oxytetracycline is a major breakthrough. Summary of the Invention
[0004] The purpose of the present invention is to provide an oxytetracycline-degrading bacterium under high-concentration oxytetracycline, a preparation method and an application thereof, aiming to solve the technical problem that most existing studies only focus on the degradation of oxytetracycline at low concentrations, and there are few studies on the degradation of oxytetracycline under high-concentration conditions. However, high-concentration oxytetracycline will inhibit the physiological activities of microorganisms. Therefore, a fungus capable of degrading oxytetracycline in a high-concentration oxytetracycline environment is provided to achieve the degradation of high-concentration oxytetracycline.
[0005] To achieve the above technical purpose, the technical solution adopted by the present invention is as follows:
[0006] The present invention provides an oxytetracycline-degrading bacterium under high-concentration oxytetracycline. The strain number of the oxytetracycline-degrading bacterium is C4, which was deposited at the China General Microbiological Culture Collection Center on July 22, 2021, with the deposit number CGMCC No. 22940.
[0007] The present invention also provides an application of an oxytetracycline-degrading bacterium under high-concentration oxytetracycline, which is the application of the oxytetracycline-degrading bacterium in degrading oxytetracycline.
[0008] Among them, the application of the oxytetracycline-degrading bacterium is in degrading oxytetracycline with a concentration of 1000 mg / L - 2000 mg / L.
[0009] The present invention also provides an oxytetracycline-degrading bacterium preparation under high-concentration oxytetracycline, and the oxytetracycline-degrading bacterium preparation contains the oxytetracycline-degrading bacterium.
[0010] Among them, the oxytetracycline-degrading bacterium preparation is one or several of a culture solution, a culture concentrate, and a culture separation solution containing the oxytetracycline-degrading bacterium.
[0011] The present invention also provides an application of an oxytetracycline-degrading bacterium preparation under high-concentration oxytetracycline, which is the application of the oxytetracycline-degrading bacterium preparation in degrading oxytetracycline.
[0012] Among them, the application of the oxytetracycline-degrading bacterium preparation is in degrading oxytetracycline with a concentration of 1000 mg / L - 2000 mg / L.
[0013] The present invention also provides a preparation method of an oxytetracycline-degrading bacterium under high-concentration oxytetracycline, including the following steps:
[0014] Inoculate the mold preserved on the slant into a liquid PDA medium, and perform constant-temperature shaking culture at 25°C and 180 r / min for 36 h to obtain an activated seed solution;
[0015] Inoculate the seed solution into a fresh medium according to an inoculation amount of 1%, and continue to perform enlarged culture at 25°C and 180 r / min for 36 h;
[0016] Then centrifuge at 8000 r / min for 5 min, discard the supernatant and collect the thallus, wash it 2 - 3 times with sterile water, and finally prepare a 1 g / L bacterial suspension with sterile water to complete the preparation of the oxytetracycline-degrading bacterium or the oxytetracycline-degrading bacterium preparation.
[0017] Among them, the formula of the PDA liquid medium is: 1.0 L of potato extract, 20 g of glucose, without adjusting the pH additionally, and sterilize at 121°C for 30 min.
[0018] Among them, the preparation method of the potato extract: Take 200 g of peeled potatoes, cut them into small pieces, add 1.0 L of water and boil for 30 min, filter out the potato pieces, and make up the filtrate to 1.0 L.
[0019] A oxytetracycline-degrading bacterium under high-concentration oxytetracycline, a preparation method and an application thereof. The oxytetracycline-degrading strain of the present invention has a high degradation efficiency for oxytetracycline, can tolerate a high concentration of oxytetracycline of 2000 mg / L, can use oxytetracycline as the sole carbon source for growth and reproduction, and degrade oxytetracycline. Under laboratory conditions, with an initial oxytetracycline content of 1000 mg / L, the degradation rate of this bacterium can reach 85.1% at 25 °C and 180 rpm in 8 days. This invention provides a scientific basis for removing high-concentration oxytetracycline in the environment. This strain can be used for the degradation treatment of high-concentration oxytetracycline pollution. The application objects can include wastewater residues, water environment or soil containing high-concentration oxytetracycline pollutants, etc., which can reduce the harm caused by antibiotics to the ecological environment safety. Moreover, compared with methods such as physical adsorption and chemical degradation, using this method to treat antibiotic pollution has the advantages of energy conservation and environmental protection, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The present invention can be further illustrated by the non-limiting embodiments given in the drawings.
[0021] Figure 1 It is the HPLC quantitative detection result of the degradation of oxytetracycline by strain C4 under 1000 mg / L oxytetracycline provided by the embodiment of the present invention.
[0022] Figure 2 It is the HPLC quantitative detection result of the degradation of oxytetracycline by strain C4 under 1500 mg / L oxytetracycline provided by the embodiment of the present invention.
[0023] Figure 3 It is the HPLC quantitative detection result of the degradation of oxytetracycline by strain C4 under 2000 mg / L oxytetracycline provided by the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0024] In order to enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be further described below in conjunction with the drawings and embodiments.
[0025] The reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
[0026] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.
[0027] The high-performance liquid chromatography determination method of the present invention includes:
[0028] Quantitatively analyzing the concentration of oxytetracycline by high-performance liquid chromatography series.
[0029] CAPCELL PAK MG II C18 chromatographic column (8.0 μm, 250×4.6 mm), column temperature was 25 °C, mobile phase was methanol and 0.5% formic acid solution, with a ratio of 60:40 (V:V), detection wavelength was 355 nm, and injection volume was 10 μL.
[0030] Example 1: Isolation and Identification of Oxytetracycline - Degrading Bacteria
[0031] 1. Experimental Materials
[0032] Enrichment medium: sodium chloride 5 g, beef extract 3 g, peptone 10 g, distilled water 1000 mL, pH 7.0 - 7.2.
[0033] For solid medium, add agar powder 20 g / L.
[0034] Inorganic salt medium: (NH4)2SO4 1 g, MgSO4·7H2O 0.1 g, KH2PO4 3 g, K2HPO4·3H2O 7 g, sodium citrate 0.5 g, distilled water 1000 mL, pH 7.0 - 7.2.
[0035] Before the experiment, the above media were placed in a high - pressure steam sterilizer and sterilized at 121 °C for 30 min.
[0036] 2. Isolation and Identification of Strains
[0037] (1) Screening and Domestication of Oxytetracycline - Degrading Bacteria:
[0038] Weigh 5 g of soil from a livestock and poultry farm and add it to 250 mL of enrichment medium containing 500 mg / L of oxytetracycline. Incubate it in the dark at 15 °C and 160 r / min on a shaker for 7 d; take 5 mL of the enrichment culture solution and add it to a new enrichment medium and continue culturing under the above conditions, and then transfer again. Each time, the concentration of the antibiotic added to the enrichment medium is increased by 100 mg / L; repeat the above steps of transfer culture until the antibiotic concentration in the enrichment medium reaches 1000 mg / L.
[0039] After domestication, dilute the bacterial culture solution in gradients of 10 - 1 to 10 - 6. Take 200 μL of the culture solutions of the 10 - 4, 10 - 5, and 10 - 6 dilution gradients respectively and spread - plate inoculate them onto the solid nutrient medium, and incubate them in an incubator at 30 °C for 24 - 36 h.
[0040] Number the grown colonies and pick them out separately for further streak - plate isolation culture until a purified single colony is obtained.
[0041] After that, inoculate each single colony into a PDA medium containing oxytetracycline for degradation experiments to investigate the degradation ability of each bacterium to oxytetracycline.
[0042] Finally, a strain with the best degradation ability and stable growth performance was selected as the target strain, numbered C4.
[0043] (2) Identification of oxytetracycline-degrading bacteria:
[0044] Preparation and observation of the microscope sample of the strain: Drop 1 drop of sterile water in the middle of a clean glass slide. Use a sterilized and cooled inoculation loop to pick up a small amount of bacteria C4 and mix it well with the water droplet on the glass slide, then spread it into a uniform thin layer on the glass slide; let it dry naturally; according to the microscope operation steps, first observe under the low-power microscope, and after finding a suitable field of view, further observe the cell morphology of the bacteria under the high-power microscope.
[0045] Results of the morphological and physiological-biochemical identification of the strain: The morphological appearance of yeast C4 growing on the plate is as Figure 1 shown, and the microscope imaging diagram is as Figure 2 shown. The colony is green, opaque, round, with a rough and filamentous surface; under the microscope, the bacterial cells are filamentous and relatively large.
[0046] Molecular identification of the strain: The molecular identification of bacteria C4 was entrusted to the Guangdong Institute of Microbiology. After DNA extraction, PCR amplification, sequence sequencing and sequence comparison, the ITS sequencing sequence of bacteria C4 has the highest homology with Sakaguchia cladiensis.
[0047] Based on the above morphological identification and molecular identification results, the bacteria C4 screened in the present invention was identified as yeast Sakaguchia cladiensis, and was deposited in the Guangdong Provincial Culture Collection Center of Microorganisms on January 26, 2018, with the deposit number: GDMCC NO: 60319; the deposit address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.
[0048] The oxytetracycline-degrading bacteria resistant to high concentrations of oxytetracycline have a high tolerance to oxytetracycline, and the tolerance concentration exceeds 2000 mg / L.
[0049] The application of the oxytetracycline-degrading bacteria C4 in the degradation treatment of high-concentration oxytetracycline or in improving the degradation and removal rate of oxytetracycline, as well as its application in treating water environment and soil polluted by high-concentration oxytetracycline, shall be within the protection scope of the present invention.
[0050] The oxytetracycline-degrading bacteria preparation is a bacterial suspension of the oxytetracycline-degrading bacteria C4 strain.
[0051] The bacterial suspension is prepared after the activation culture of the fungal strain. Specifically, the oxytetracycline-degrading bacteria C4 strain is cultured and activated in PDA medium or LB medium, the precipitate is collected by centrifugation, and after washing, it is diluted and prepared.
[0052] The preparation method of the bacterial suspension is as follows: inoculate the mold preserved on the slant into the liquid PDA medium, and perform constant-temperature shaking culture at 25°C and 180 r / min for 36 h to obtain an activated seed solution; inoculate the seed solution into the fresh medium at an inoculation amount of 1%, and continue to perform enlarged culture at 25°C and 180 r / min for 36 h; then centrifuge at 8000 r / min for 5 min, discard the supernatant to collect the bacterial cells, wash them 2-3 times with sterile water, and finally prepare a 1 g / L bacterial suspension with sterile water.
[0053] The formula of the PDA liquid medium is: 1.0 L of potato extract, 20 g of glucose, no need to adjust the pH additionally, and sterilize at 121°C for 30 min.
[0054] The preparation method of the potato extract: take 200 g of peeled potatoes, cut them into small pieces, add 1.0 L of water and boil for 30 min, filter out the potato pieces, and make up the filtrate to 1.0 L.
[0055] Example 2: Verification of the degradation effect of C4 under high concentration of oxytetracycline
[0056] After culturing C4 in the PDA medium at 25°C and 180 rpm for 24 h, inoculate it into the PDA medium containing 1000 mg, 1500 mg, and 2000 mg of oxytetracycline per liter at an inoculation amount of 5% respectively, and perform light-shielding shaking culture for 10 d. Set three parallels in each group, and use the one without bacteria as the blank control. Filter the obtained culture through a 0.45 μm water-based filter membrane and detect the residual concentration of oxytetracycline by machine.
[0057] The results show that when the initial content of oxytetracycline is 1000 mg / L, the degradation rate of this bacterium can reach 85.1% in 10 d under the conditions of 25°C and 180 rpm; when the initial content of oxytetracycline is 1500 mg / L, the degradation rate can reach 67.2% in 10 d under the conditions of 25°C and 180 rpm; when the initial content of oxytetracycline is 2000 mg / L, the degradation rate can reach 60.2% in 10 d under the conditions of 25°C and 180 rpm; the degradation rate of the blank control is 51.2%.
[0058] The above embodiments only exemplarily illustrate the principles and effects of the present invention, rather than limiting the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.
Claims
1. An oxytetracycline-degrading bacterium under high-concentration oxytetracycline, characterized in that, The strain number of the oxytetracycline-degrading bacterium is C4, which was deposited at the China General Microbiological Culture Collection Center on July 22, 2021, with the accession number CGMCC No. 22940.
2. Application of oxytetracycline-degrading bacteria under high-concentration oxytetracycline, characterized in that, Application of the oxytetracycline-degrading bacterium in degrading oxytetracycline.
3. The application of the oxytetracycline-degrading bacterium under high-concentration oxytetracycline according to claim 2, characterized in that, Application of the oxytetracycline-degrading bacterium in degrading oxytetracycline with a concentration of 1000 mg / L - 2000 mg / L.
4. An oxytetracycline-degrading bacteria preparation under high-concentration oxytetracycline, characterized in that, The oxytetracycline-degrading bacterium preparation contains the oxytetracycline-degrading bacterium.
5. The oxytetracycline-degrading bacteria preparation under high-concentration oxytetracycline as claimed in claim 4, wherein, The oxytetracycline-degrading bacterium preparation is one or several of the culture solution, culture concentrate, and culture separation solution containing the oxytetracycline-degrading bacterium.
6. Application of oxytetracycline degrading bacteria preparation under high concentration of oxytetracycline, characterized in that, Application of the oxytetracycline-degrading bacterium preparation in degrading oxytetracycline.
7. Use of the oxytetracycline-degrading bacterial agent under high-concentration oxytetracycline as claimed in claim 6, characterized in that, Application of the oxytetracycline-degrading bacterium preparation in degrading oxytetracycline with a concentration of 1000 mg / L - 2000 mg / L.
8. A preparation method of oxytetracycline-degrading bacteria under high-concentration oxytetracycline, which is applied to the oxytetracycline-degrading bacteria or the oxytetracycline-degrading bacteria preparation under high-concentration oxytetracycline described in claim 1 or claim 4, and is characterized in that, Including the following steps: Inoculate the mold preserved on the slant into the liquid PDA medium, and incubate it at 25 °C and 180 r / min with constant shaking for 36 h to obtain an activated seed solution. Inoculate the seed solution into the fresh medium at an inoculation amount of 1%, and continue to expand the culture at 25 °C and 180 r / min for 36 h. Then centrifuge at 8000 r / min for 5 min, discard the supernatant and collect the cells, wash them 2 - 3 times with sterile water, and finally prepare a 1 g / L cell suspension with sterile water to complete the preparation of the oxytetracycline-degrading bacterium or the oxytetracycline-degrading bacterium preparation.
9. The preparation method of oxytetracycline-degrading bacteria under high-concentration oxytetracycline as claimed in claim 8, characterized in that, The formula of the PDA liquid medium is: 1.0 L of potato extract, 20 g of glucose, no need to adjust the pH additionally, and sterilize at 121 °C for 30 min.
10. The preparation method of oxytetracycline-degrading bacteria under high-concentration oxytetracycline as claimed in claim 9, characterized in that, The preparation method of the potato extract: Take 200 g of peeled potatoes, cut them into small pieces, add 1.0 L of water and boil for 30 min, filter out the potato pieces, and make up the filtrate to 1.0 L.