Residual Titer Detection Of Representative Antibiotics Bacterial Residues And Preliminary Research Of Biopharmaceutical Residue Resources Utilization

With the production of antibiotics increasing, the problem of antibiotics mycelium treatment has become a serious problem which led to limiting the development of the pharmaceutical industry. Due to the antibiotic residues in antibiotics mycelium may cause antibiotic-resistance, antibiotics mycelium has been listed as dangerous waste. Choose oxytetracycline and bacitracin as representative, the high performance liquid chromatography method was developed for the determination of antibiotics bacterial residues by exploring the chromatographic conditions(chromatographic column, column temperature, mobile phase, wavelength) and pretreatment conditions(extraction, purification).Represented by bacitracin mycelium, explore the feasibility of antibiotic mycelium used as feed additives.For oxytetracycline mycelium, oxytetracycline were extracted with methanol/ice acetic acid, using 50 mg GCB, 50 mg C18 and 1mg Na SO4 as agent for purifing samples, chromatographic conditions : PLRP-S chromatographic column, column temperature is 30°C, mobile phase is methanol/0.05mol/L oxalic acid, wavelength is 355 nm. In result, oxytetracycline is linear within the range of 0.1~1000mg/L, the recovery rate is 86.86~97.33%, RSD is 2.45~4.38%, the LOD and LOQ are respectively 0.0506 and 0.1533, the method is simple and effective. On this basis, establish the detection method of E-OTC, α-OTC and β-OTC, which are three degradation products of oxytetracycline. E-OTC and OTC are isomers, structures and properties are similar, so the way of extraction and purification is the same as OTC. Chromatographic conditions for detection of E-OTC: PLRP-S chromatographic column, mobile phase is acetonitrile/0.2% formic acid water, wavelength is 270 nm. α-OTC and β-OTC are isomers, structures and properties are similar, so they can be extracted and detected together. Choose ice acetic acid/methanol0.02mol/LNa2 EDTA as extraction agent, Waters Sep-Pak C18 as solid phase extraction column, methanol 、ultrapure water、Na2EDTA solution activated the column, 5ml ultrapure water leaching, 2ml of methanol and 1ml ethyl acetate elution target, chromatographic conditions: PLRP-S chromatographic column, methanol/acetonitrile/p H=8 phosphate buffer solution as mobile phase, wavelength is 254 nm. In result, the recovery of E-OTC, α-OTC and β-OTC were 75.98~84.73%, 59.22~76.76% and 63.91~72.57%, RSD were 2.23~4.26%, 2.12~4.85% and 2.79~4.85%, the LOD and LOQ were 0.0658~0.0774 and 0.1990~0.0658 respectively.For bacitracin mycelium, bacitracin A were extracted by methanol/0.1% formic acid water, add acetonitrile in addition to protein, n-hexane excepting the fat twice, then purified by Oasis HLB solid-phase extraction column, 5ml methanol and 5ml ultrapure water for activation, 3ml ultrapure water leaching, 3ml methanol for elution, chromatographic conditions: Aglient TC-C18 chromatographic column, the mobile phase was 0.1% formic acid acetonitrile/0.1% formic acid water, column temperature 30°C, detection wavelength was 254 nm. After the examination, bacitracin A correlation coefficient was 0.9997 within 0.1~1000 mg/L, had good linear, recovery of bacitracin A was 87.89 ~ 97.33%, RSD was 3.05% ~ 5.18%. Finally explores the feasibility of bacitracin mycelium used as feed additives. Analyzing the composition of bacitracin mycelium, conventional nutrients content in the bacitracin mycelium and the amino acid content is compared with several conventional feed, shows that nutrients in bacitracin mycelium reaching the level of nutrients in feed, and toxic heavy metals content in the bacitracin mycelium are below the maximum allowed, meaning bacitracin mycelium without toxicity. On this basis, design and optimize the bacitracin mycelium preparation process of feed additives. Finally, the resistant genes in bacitracin mycelium were tested, bcr B gene was detected, although bcr B gene existing alone is not sufficient to confer bacitracin resistance, there still exists certain risk, we can kill bacitracin-resistant bacteria by pressure and heat,then the bacitracin-resistant genes can be destroyed.