A kind of microbial agent for efficiently degrading tetracycline antibiotics and its application
A technology of tetracyclines and microorganisms, which is applied in the direction of microorganisms, microorganisms, and methods based on microorganisms, can solve the problems of slow plant degradation, high temperature of the pile, and harsh requirements for enzymatic hydrolysis, so as to reduce the degradation of tetracycline antibiotics and Good effect and simple operation
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Embodiment 1
[0027] Isolation, purification and identification of Enterococcus faecium DTC7:
[0028] The applicant obtained it by separating, screening and identifying pig manure from the experimental pig farm of Huazhong Agricultural University, Tianmen, Jingmen and other places. The specific separation and purification method is as follows: Weigh 5g of pig manure sample into a 50ml centrifuge tube, add water to 50ml of shaker at 200r / min43°C for 2h, take 1ml of the supernatant and inoculate them with antibiotic concentration of 20μg / ml containing tetracycline, aureomyces In the LB liquid medium of oxytetracycline and oxytetracycline. After 24 hours, draw 100 μl and inoculate it in LB liquid medium containing the above-mentioned antibiotics at a concentration of 40 μg / ml. Increase the antibiotic concentration by 20 μg / ml for each inoculation according to the above method, and repeat the inoculation until the concentration reaches 200 μg / ml. Take 100 μl of the medium cultured to 200 μg / ...
Embodiment 2
[0034] Enterococcus Faecium (Enterococcus Faecium) DTC7 fermentation culture:
[0035] Resuscitate Enterococcus faecium, absorb 100 μL and inoculate it into 5 mL LB liquid medium, and culture it on a shaking table at 37°C at 200 r / min for 12 hours to the stable stage. The effective bacterial concentration is 2.2×10 by plate colony counting method. 8 cfu / ml.
Embodiment 3
[0037] Degradation of tetracycline antibiotics by Enterococcus faecium DTC7:
[0038] Preparation of Antibiotic-Containing Medium
[0039] Weigh 20g of tryptone, 10g of yeast extract, 10g of sodium chloride, add water to 2000mL, adjust the pH to 8±0.05 with NaOH, and autoclave at 120°C for 20min. After the temperature is cooled down, 0.1 g of tetracycline, chlortetracycline, and oxytetracycline are added therein to prepare a 100 mg / kg antibiotic culture medium.
[0040] Bacterial inoculation
[0041] Resuscitate Enterococcus faecium DTC7, draw 100 μL and inoculate it into 5 mL LB liquid medium, and culture it on a shaker at 200 r / min at 37°C for 12 hours until the stable phase. Take out 100 μL of the culture medium in the stationary phase, inoculate it into 50 mL of LB medium containing 100 mg / kg of antibiotics, and cultivate on a shaker at 43° C. at 200 r / min. Culture media at 6 time points of 1d, 2d, 3d, 4d, 5d, and 6d were collected, and the samples were stored at -40°C in...
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