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研究生:范凱希
研究生(外文):Catherine A. Franje
論文名稱:以毛細管電泳、紫外光吸收及抗菌活性評估Amphenicol類抗生素熱穩定性之研究
論文名稱(外文):Heat Stability of Florfenicol, Thiamphenicol and Chloramphenicol Characterized by Changes in Electropherographic Profiles, Ultraviolet Spectrometry and Antimicrobial Activity
指導教授:周濟眾
指導教授(外文):Chi-Chung Chou
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
畢業學年度:97
語文別:英文
論文頁數:93
中文關鍵詞:氯黴素類抗生素熱穩定性毛細管電泳最小抑菌濃度協同活性作用
外文關鍵詞:amphenicolsheat stabilitycapillary electrophoresisminimum inhibitory concentrationsynergistic activity
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殘留在食物中的熱穩定獸藥可能會對消費者造成潛在危害,然而目前對抗生素之熱穩定性之研究卻不多,抗生素經過加熱降解後其結構改變與其抗菌活性間之關係亦未有完整的研究。本論文將Amphenicol類氟甲砜黴素(florfenicol,FF)、甲砜黴素(thiamphenicol,TAP)與氯黴素(chloramphenicol,CAP)三種抗生素分別於水中、硼酸鈉緩衝液與雞肉中,經100℃加熱30分鐘、1小時與2小時後,以毛細管電泳分析其降解產物並以E. coli及 S. aureus兩株菌種進行最小抑菌濃度試驗(MIC)。結果顯示Amphenicol類抗生素在水中加熱後降解率並不高(小於5%,以FF為例)且經過2小時加熱後藥物之抑菌活性並無明顯改變。然而在硼酸鈉緩衝液中加熱後FF降解率可達98%且產生四個新的波峰,MIC也增加至4倍。此外,雞肉中的抗生素加熱結果顯示其對Amphenicol類抗生素並不具有減低降解的效果。GC/MS結果發現FF加熱後可降解成TAP。但是Amphenicol類抗生素加熱後結構之降解程度不一定與MIC之上升呈正相關。因此,除了降解產物有可能仍具抗菌活性外,Amphenicol間協同抗菌作用亦可能存在。實驗顯示併用低達1/32MIC之FF與1/2MIC之TAP仍足以抑制細菌生長,進一步證實此一推測之可能性。總而言之,Amphenicol類抗生素降解後之產物仍可能具抗菌活性故無法排除衍生抗藥性菌之疑慮。因此,以100℃加熱殘留在食物中的Amphenicol類抗生素並無法使藥物完全降解,因而不宜假設安全。
Heat stable veterinary drug residues present in food are undesirable due to its potential harmful consequences to the consumers. Very limited information is available on heat stability of amphenicols under cooking treatments and most found studies were reports on chloramphenicol (CAP). To our knowledge, the relationship between the structural degradation of antibiotics after heating and their antimicrobial activity has not been well investigated. Florfenicol (FF), thiamphenicol (TAP), and CAP were heated at 100 °C in water, sodium borate buffer and chicken meat for 30 min, 1 h and 2 h. Amphenicol reductions were evaluated through capillary electrophoresis (CE) and minimum inhibitory concentration (MIC) test against E. coli and S. aureus. Results showed that amphenicols were minimally degraded at <5% (FF), and without significant changes on its antimicrobial activities after 2 h of heating in water. However, amphenicols boiled in borate buffer were extensively degraded up to 98% (FF), produced 4 new peaks (FF), and further increased its MIC up to 8-folds (FF), suggesting that compounds present in the buffer might have facilitated the degradation of amphenicols. Amphenicols boiled in meat showed higher reductions relative to water up to 81% (FF), suggesting the possible absence of protective effects of chicken meat on amphenicols. However, possible production of microbiologically active degradation product must not be disregarded. CE and GC/MS results showed that TAP was produced after heating of FF. This unfavorable transformation of structure was further supported by the possible synergistic activity between FF and TAP. The combination of FF at 1/32 of its initial concentration and 1/2 of TAP was found to inhibit the growth of test bacteria. The possible production of TAP as one of the breakdown products of FF is relevant to public food safety. Therefore, it cannot be assumed that heating of amphenicol residues in food and thus its degradation products are always safe.
Abstract i
List of Figures vii
List of Tables x
List of Appendices xi
Preface xiii
Chapter 1 Introduction 1
1.1 Amphenicol residues in food 1
1.2 Amphenicols 2
1.2.1 Chemical structure 2
1.2.2 Pharmacokinetics and metabolites of amphenicols 4
1.2.3 Toxicity of amphenicols 7
1.3 Heat stability of amphenicols 11
1.4 Evaluation of residual amphenicol heat stability 14
1.4.1 Antimicrobial assay 14
1.4.2 Capillary electrophoresis 15
1.4.3 Ames test 17
Chapter 2 Materials and methods 19
2.1 Study design 19
2.2 Equipment 20
2.3 Methods 22
2.3.1 Sample preparation in water and in sodium borate buffer 22
2.3.2 Sample preparation in meat and extraction method 22
2.3.3 Thermal treatments 23
2.3.4 Buffer preparation: 50 mM sodium borate + 25 mM SDS, Ph 9 23
2.3.5 CE method 24
2.3.6 Evaluation of peaks and UV spectra 24
2.3.7 Determination of MIC on amphenicols 25
Chapter 3 Results 30
3.1 Heat stability of amphenicols in water 30
3.2 Heat stability of amphenicols in sodium borate buffer 34
3.3 Heat stability of amphenicols in chicken meat 40
3.4 Suspected production of thiamphenicol after heating of florfenicol 45
3.5 Synergistic activity among amphenicols 48
Chapter 4 Discussion 51
4.1 Heat stability of amphenicols 51
4.1.1 Heat stability in water 52
4.1.2 Heat stability in sodium borate buffer 54
4.1.3 Heat stability in chicken meat 55
4.2 Production of suspected TAP upon heating of FF 56
4.3 Synergistic activity among amphenicols 60
Chapter 5 Conclusions 62
References 63
Appendices 78
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