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1 5 11 Vol. 5 No Journal of Food Safety and Quality Nov., 2014 陈峥, 刘波 *, 朱育菁, 潘志针 (, ) 摘要 : 目的 Bacillus gibsonii FJAT 方法 Bacillus gibsonii FJAT MassHunter, (MFE), Metlin, 结果 Bacillus gibsonii FJAT-10019, 561, Metlin 15, 90.89, 1.14% 6.522, 结论关键词 : ; ; ; Analysis and testing on leflunomide from Bacillus gibsonii FJAT CHEN Zheng, LIU Bo *, ZHU Yu-jing, PAN Zhi-Zhen (Institute of Agrobiological Resources, Fujian Academy of Agricultural Sciences, Fuzhou , China) ABSTRACT: Objective To analyze and test on leflunomide from Bacillus gibsonii FJAT Methods Extracellular metabolites of Bacillus gibsonii FJAT were analyzed by liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry. Molecular Feature Extraction(MFE)and database retrieval wereapplied by Agilent MassHunter software, for the information overall metabolites. Results Totally 561 compounds were detected, and 15 compounds were identified by library searching of Metlindatabase. The compound of leflunomide with the match score 90.89, and relative content is 1.14%. The retention time of leflunomide is about min, accurate mass is Conclusion This result provided the theory basis for development and utilization of this immunomodulator from Bacillus gibsonii. KEY WORDS: liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry; Bacillus gibsonii; extracellular metabolites; leflunomide 1 引言, [1],, ; ; 基金项目 : ( ) ( )(2013dqd-6) Fund: Supported by the Chinese Special Fund for Agro-scientific Research in the Public Interest( ), The Youth Innovation Fund of FAAS (2013dqd-6). * 通讯作者 :,, fzliubo@163.com *Corresponding author: LIU Bo, Researcher, Fujian Academy of Agricultural Sciences, Fujian , China. fzliubo@163.com

2 11, : 3673, (leflunomide, LEF) A771726,, T B [2] ; NFKB, IL-1 TNF ;, LEF 噁 (Bacillus gibsoniifjat-10019),, [3] S-2,,, 41 kd [4] S-2,,, 39 kd 37 kd 38 kd [5] [6], SL-25, 24 h 7 d 66.7 % 28.9 %,, (LC-QTOF-MS),, 2 材料与方法 2.1 材料 : Bacillus gibsonii FJAT : 1.0 %, 0.5 %, 0.3 %, 1.0 %, 0.5 %, CaCl2 0.5 %, ph 7.0 : TSB : TSB ( BD ), ( JT Baker ), (CNW, ) : Agilent 1260/6520( ); METTLER TOLEDO AL104( ); ZHWY-2102C ; ph Sartorius PB-10( ) 2.2 方法 : LB Bacillus gibsonii FJAT h 20 ml LB, r/min 12 h, 1 % 50 ml TSB, r/min 48 h Bacillus gibsonii FJAT-10019, 4 : 50 ml Bacillus gibsonii FJAT ,, 0.25 μm, GC 4, : Agilent ZORBAX Extend C 18 (2.1 μm 50 mm, 1.8 Micron); : 35 ; : 2 μl; : A=10 mmol/l +, B= ; : 0.2 ml; : B=90 % 0 3 min; B=50 % 3 25 min; B=90 % min; MS : ; : 300, 5 L/min; Nebulizer 30 psig; Capillary voltage 3500 V; Fragmentor 175 V; Skimmer 65.0 V; Mass range m/z : 结果与分析 3.1 吉氏芽胞杆菌 Bacillus gibsonii FJAT 发酵液 LC-MS 物质成分分析 561,, 15, 3 : -3,4,5-2, Plocamene C beta-2,3,4,5,6- Pentachlorocyclohexanol 5- -2,4- Zuccagin Galbeta1-3GalNAcalpha1-3(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcb eta-cer(d18:1/18:0) PE(26:2(5E,9Z)/26:2(5E,9Z)) 546,,

3 表 1 吉氏芽胞杆菌 Bacillus gibsonii FJAT 菌株发酵液成分 Table 1 The composition of Bacillus gibsonii FJAT fermentation broth Cpd Name METLIN METLIN Mass m/z Score RT Vol % 538 Leflunomide( ) Zuccagin Chlorthalidone( ) ,3-Dibromo-1-propanol(2, ) beta-2,3,4,5,6-pentachlorocyclohexanol Galbeta1-3GalNAcalpha1-3(Fucalpha1-2) Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta-Cer (d18:1/18:0) PE(26:2(5E,9Z)/26:2(5E,9Z)) Etidronic acid() Butonate( ) Chloraminophenamide(5- -2,4- ) Chlorfenson( ) Chloraminophenamide( ) Diazoxide( ) Phosphatidylinositol-3,4,5-trisphosphate (PIP3) -3,4, Plocamene C 吉氏芽胞杆菌 Bacillus gibsonii FJAT 发酵液来氟米特成分分析 Bacillus gibsonii FJAT-10019, 15, 90% 1, LEF(N-(4- - ) ), (Score) 90.89, 1.14 %, 1 2, min, , 4,, [M-H] - [M+X] - (X ) [M-H] -, ,, Metlin, 4 讨论, [7],, 7 [8],, Bacillus sp., 9,,

4 11, : (LC-QTOF MS) LC-QTOF MS [9], [10],, Agilent MassHunter, (MFE), Metlin,, 561, 1 Fig. 1 Extracted compound chromatogram(ecc)of leflunomide in sample Fig. 2 2 Extracted ion chromatogram (EIC) of leflunomide in sample

5 Fig. 3 Isotopic peak of leflunomide in sample Fig. 4 4 Mass spectrum of leflunomide in sample 15, : (1) : (LEF), N-(4- ) ), C 12 H 9 F 3 N 2 O 2, [11] ( ), [12] LEF, A ,, [13] LEF : a. T B ; b. ; c. [12] (2) (diazoxide), (etidronic acid) (chlorthalidone),,,, 1.14%, 90.89,

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Microsoft Word 陈峥-二校 4605.doc

Microsoft Word 陈峥-二校 4605.doc 6 11 Vol. 6 No. 11 215 11 Journal of Food Safety and Quality Nov., 215 - 陈峥, 刘波 *, 朱育菁, 潘志针 (, 353) 摘要 : 目的方法 - 31 结果,, (Bacillus cohnii FJAT-117) (Bacillus alcalophilus FJAT-114) (Brevibacillus agri