Two tylosin degrading bacteria and application thereof in compost

Abstract

The invention discloses a tylosin degrading strain and application thereof in compost, and belongs to the technical field of microbial degradation of antibiotics. According to the experiment, two strains of tylosin degrading bacteria are separated from pig manure, and the two strains of tylosin degrading bacteria are identified as Sphingobacterium kyonggiense TYL-1 and Empedobacter falsenii TYL2. The obtained tylosin degrading bacterium TYL2 is separated from pig manure of a pig farm, and the obtained strain can be directly used for degrading tylosin in wastewater and compost of the pig farm.

Description

Technical Field

[0001] This invention belongs to the field of microbial antibiotic degradation, specifically involving a tylosin-degrading bacterium and its application in composting. Background Technology

[0002] Tylosin, also known as Tylenol, is a class of weakly basic antibiotics produced by Streptomyces. Its molecule contains a fourteen- to sixteen-membered lactone structure. Figure 1 Tylosin, also known as a macrolide antibiotic, binds to the 23S rRNA of the 50S subunit of bacterial ribosomes, inhibiting protein synthesis and thus exerting its antibacterial effect. Tylosin has antibacterial activity against Gram-positive bacteria, anaerobic bacteria, and mycoplasma. Because of its growth-promoting effects on animals, it is widely used in veterinary drugs and feed additives.

[0003] With the rapid development of my country's aquaculture industry, the consumption of tylosin has increased year by year. By the end of 2024, the total domestic tylosin production capacity was approximately 21,700 tons (including 2,000 tons from overseas), and this year, after capacity expansion, it will reach 28,000 tons. This increase in production capacity and consumption directly leads to a large amount of tylosin residues in the environment, such as in wastewater discharged by manufacturers, especially manure and sewage from livestock farms. Manure from livestock farms is mainly composted into organic fertilizer, which ultimately flows into farmland. If the compost contains high concentrations of antibiotics, its microbial composition will contain many antibiotic resistance genes (ARGs), affecting the farmland ecosystem. Therefore, finding a strain that can rapidly and efficiently degrade tylosin is an urgent problem to be solved.

[0004] Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to provide a strain that rapidly and efficiently degrades tylosin, thereby solving the problem of tylosin residue in pig manure compost.

[0006] To achieve the above objectives, this invention screened two tylosin-degrading bacteria, *Sphingobacterium kyonggiense* TYL-1 and *Empedobacter falsenii* TYL2, from fresh pig manure, and evaluated their antibiotic degradation rates under different conditions. Based on this, when these bacteria were inoculated into pig manure compost, the degradation rate of tylosin in the compost reached 95%. Attached Figure Description

[0007] Figure 1These are strains TYL-1 (Sphingobacterium kyonggiense) and TYL-2 (Empedobacter falsenii) from Example 1;

[0008] Figure 2 This is the HPLC chromatogram of tylosin in Example 2;

[0009] Figure 3 This refers to the effect of temperature on the degradation rate of tylosin in Example 3;

[0010] Figure 4 This refers to the effect of salinity on the degradation of tylosin by TYL-2 in Example 3;

[0011] Figure 5 This refers to the effect of TYL-2 on the degradation rate of tylosin at different degradation times in Example 3;

[0012] Figure 6 This describes the degradation effect of tylosin in simulated compost by inoculating TYL-1 and TYL-2 in Example 3. Detailed Implementation

[0013] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0014] Unless otherwise specified, the materials, reagents and equipment used in this invention are commercially available.

[0015] Example 1: Screening of Tylosin-Degrading Strains

[0016] Take 3g of fresh pig manure and add it to 100ml of enrichment medium (as shown in Table 1). Incubate on a shaker at 30℃ and 150r / min for 1 day. Serially dilute the culture with sterile water and spread the diluted solutions onto solid screening medium (as shown in Table 2). Incubate at 30℃. After 2 days of incubation, observe the microbial growth on the surface of the medium. Select strains with good growth, suspend them in a sterile room, dilute them, spread them onto solid screening medium, and further purify them to obtain pure strains. The pure strains were inoculated into inorganic salt liquid culture medium (as shown in Table 3) and cultured on a shaker at 30℃ and 150 r / min for 2 days. 1 ml of the culture broth was taken, 1 ml of methanol was added, and ultrasonic extraction was performed. The extract was centrifuged, the supernatant was collected, and filtered through a 0.22 μm organic membrane. The tylosin content was analyzed by HPLC, and the degradation rate was calculated. The two strains with the highest degradation rates were screened and identified by 16S DNA detection as *Sphingobacterium kyonggiense* TYL-1 and *Empedobacter falsenii* TYL2. Figure 2).

[0017] Table 1 Liquid enrichment culture medium

[0018]

[0019] Table 2 Solid Screening Culture Media

[0020]

[0021] Table 3 Inorganic Salt Screening Culture Medium

[0022]

[0023] Example 2: Analytical Method for Tylosin

[0024] The analytical method for tylosin in fermentation broth was as follows: Analysis was performed using a Waters Alliance-e2695-2489 high-performance liquid chromatograph with an evaporative light scattering detector (ELSD). The stationary phase was a Unitary C18 column (5 μm 100A 4.6 × 250 nm). The mobile phase consisted of 0.5% formic acid aqueous solution and acetonitrile, with gradient elution from 0 to 15 min, increasing the acetonitrile concentration from 20% to 70% at a flow rate of 1.0 mL / min. The column temperature was 30 °C. The ELSD drift tube temperature was 85 °C, and the carrier gas, high-purity nitrogen, flowed at a rate of 2.0 L / min.

[0025] Prepare a stock solution of tylosin standard and dilute it serially to different concentrations. Perform HPLC analysis on the standards of different concentrations to obtain the peak area. Then, plot a standard curve of concentration versus peak area: y = 5E + 06x - 144439.

[0026] Example 3: Degradation of tylosin by strains under different conditions

[0027] Two bacterial strains were inoculated into inorganic salt culture medium at 50 mg / L each and cultured on shakers at different temperatures for 48 hours. The concentrations of the three antibiotics were monitored by HPLC-ELSD to investigate the effect of temperature on antibiotic degradation by the strains. The results are as follows: Figure 3 As shown, the degradation rate of tylosin by both strains was best at 37℃, followed by 30℃. The degradation rate decreased significantly with increasing temperature. At 30℃, there was no significant difference in the degradation rate of tylosin by the two strains. At 37℃, strain TYL-2 showed slightly better degradation performance, reaching a degradation rate of 60%. At 50℃, TYL-2 showed almost no degradation ability for tylosin.

[0028] Different concentrations of NaCl were added to an inorganic salt medium, and TYL-2 culture medium was inoculated and cultured in a shaker at 37°C to investigate the effect of salinity on the degradation rate of macrolide antibiotics. Results are as follows: Figure 4 As shown, the growth of TYL-2 and its degradation of antibiotics are most sensitive to salinity. With increasing salinity, its degradation rate of tylosin decreases linearly, reaching only 20% at salinities of 2% and 2.5%.

[0029] To investigate the change in tylosin degradation rate by TYL-2 over time, TYL-2 was inoculated into an inorganic salt medium with 50 mg / L of antibiotic and cultured in a shaker at 37°C. The effect of culture time on the degradation rate of tylosin was then examined. The results are as follows: Figure 5 As shown, the degradation rate of antibiotics by each strain gradually increased with the extension of culture time. After 96 hours of culture, the degradation rate of tylosin and antibiotics approached 90%.

[0030] Example 4: Effect of TYL-1 and TYL-2 combined on tylosin degradation rate in simulated composting

[0031] Feces from piglets fed with tylosin-contaminated feed were mixed with dry sawdust at a ratio of 10:1 to achieve a C / N ratio of 27. A small amount of water was added to adjust the moisture content to 60%. TYL-1 and TYL-2 bacterial cultures (equal parts TYL-1 and TYL-2) cultured in LB medium at an inoculation rate of 0.25‰ were inoculated and composted in a 30°C incubator for 8 days. The temperature was then programmed to gradually increase to 60°C from day 9 to 13, maintain a high temperature of 60°C from day 14 to 27, and gradually decrease to 30°C from day 28 to 38. Samples were taken at different time points and freeze-dried.

[0032] Take 0.15 g of the freeze-dried sample into a 50 mL polytetrafluoroethylene centrifuge tube, add 20 mL of antibiotic extraction buffer [EDTA-SPB / acetonitrile (V:V, 1:1) + Mg(NO3)2-NH3·H2O (V:V, 96:4), 3+1] and extract three times. The extract is then enriched using an HLB microextraction column, and the eluent is concentrated by nitrogen blowing and analyzed by LC-MS. Results are as follows: Figure 6 As shown, after 8 days of simulated composting, the degradation rate of tylosin reached 87%, and then slowly increased. After 36 days of composting, the degradation rate reached 96%. This shows that adding TYL-1 and TYL-2 bacterial solutions to compost can significantly reduce the concentration of tylosin in the compost.

[0033] The above specific embodiments are intended to further illustrate the content of the present invention, but should not be considered as limiting the scope of protection of the present invention. Any non-substantial modifications or adjustments made by any party based on the present invention and the provided technical teachings should be covered within the scope of protection of the present invention.

Claims

1. Two strains of tylosin-degrading bacteria were identified as... Sphingobacterium kyonggiense TYL-1 and Pseudocerobacterium ( Empedobacter falsenii )TYL-2.

2. A microbial agent containing the tylosin-degrading bacteria as described in claim 1.

3. The application of the tylosin-degrading bacteria according to claim 1 or the bacterial agent according to claim 2 in the degradation of tylosin.

4. The application of the tylosin-degrading bacteria according to claim 1 or the microbial agent according to claim 2 in the removal of tylosin-containing compost.

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