Screening Of Tylosin Degrading Bacteria And Harmless Treatment Of Drug Residues

Tylosin is a widely used veterinary drug at present.It produces a large amount of dregs during the fermentation process.The residue contains rich nutrients.However,the residue of tylosin makes it impossible to use.The accumulation of so much dregs has caused huge pollution to the environment.At present,incineration and other disposal methods are commonly used,resulting in serious pollution and waste.The biological treatment process is faced with long fermentation cycles,high disposal costs,complicated processes,and secondary pollution of the environment,which makes the dregs unable to be industrialized.In recent years.The method of microbial degradation has become a hot spot for antibiotic research,This paper deals with the treatment of tylosin dregs by screening for tylosin-degrading bacteria,and analyzes them for harmlessness and application on rape.At the same time,the degradation analysis of tylosin dregs by inorganic reagents was performed,and it was expected to be applied to the amplification treatment of dregs.The main research results of this paper are as follows:（1）11 strains of bacteria were isolated and purified by the dilution gradient method.The 8 strains of probiotics in the laboratory were degraded and cultured at a concentration of 20μg/mL tylosin.Two high-efficiency degrading bacteria were obtained after screening by HPLC.After HPLC determination,two strains of highly efficient degrading bacteria were obtained and the degradation rate reached 100%.They are numbered A4-22 and A2-10,respectively.The A4-22 was identified as Dietzia by 16SrDNA sequencing.The colonies were orange-red,regular and opaque at the edges.The bacteria were short rods and no spores.They were Gram-positive bacteria.The A2-10 was Bacillus licheniformis,with a light brown colony,a protuberant surface,irregular edges,and opaqueness.The bacteria was short rods and spores,and was Gram-positive bacteria.（2）The tylosin residue was degraded by A4-22 and A2-10 bacteria,and determination of tylosin residues by HPLC.The content of tylosin in the original dregs was 1.36 mg/g.After the study showed that degrading 25 g of tylosin dregs with 50 mL of bacteria liquid for 72 h,the A4-22 was degrade the tylosin content of the original dregs to 9.2μg/g,and the A2-10 reduced the content of tylosin in the original drug to 12.41μg/g,and the degradation rate of both bacteria reached 99.9%.In order to further improve the harmless treatment effect and reduce the processing cost,we also carried out the degradation analysis of tylosin dregs with inorganic reagents.Degradation of tylosin dregs at room temperature and 60°C for 12 h was used,and the degradation rates of 10%CaO at room temperature,5%H₂SO₄ at 60°C,and 10%CaO all reached 100%.（3）Due to the current lack of criteria for the determination of antibiotic residues in dregs,in order to investigate the harmlessness of A4-22,A2-10,and degradation products of inorganic agents and the biological toxicity of dregs,we have established a set of evaluation methods based on OD effects.First,ethanol was used as a solvent,0.1%ethanol addition and 0.1μg/mL of tylosin was determined.The degradation effect of the residue was evaluated by OD analysis.The results showed that the inhibition rate of A4-22 bacteria was 93.7%,and the inhibition rate of A2-10 bacteria reached 96.58%.Inorganic reagent is not suitable for OD effect evaluation because of its own characteristics.（4）The transformation products of A4-22 and A2-10 bacteria were studied by HPLC and mass spectrometry.It was initially determined that A4-22 bacteria converted tylosin A to tylosin D,and A2-10 bacteria completely converted tylosin A to small molecules.（5）Finally,we performed agronomic evaluation of the degraded dregs on rape.By measuring the length of the ground,the length of the ground,the dry weight of the ground,the dry weight of the underground,the diameter of the soil,the thickness of the leaves,and the seven indexes of the fibrous roots.Dregs have certain promoting effect on rapeseed.After treatment with A4-22 and A2-10,the effect of dregs fertilizer is more significant.Among them,the C condition of A2-10 is 43.44%higher than that of dregs in ground dry weight,and the 135.02%increase in underground dry weight is higher than dregs and44.74%in excavated diameter.At the same time,we carried out the determination of tylosin residues in rape.After testing its residue below the HPLC detection limit,it is far below the national standard limit for vegetable pesticide residues.In conclusion,through the screening tylosin efficient degradation of bacteria and the tylosin residues were degraded with inorganic reagents in this paper,.It has a good degradation effect and provides a certain guiding significance for the microbial degradation of antibiotics.