5 11 Vol. 5 No. 11 2014 11 Journal of Food Safety and Quality Nov., 2014 陈峥, 刘波 *, 朱育菁, 潘志针 (, 350003) 摘要 : 目的 Bacillus gibsonii FJAT-10019 方法 Bacillus gibsonii FJAT-10019 MassHunter, (MFE), Metlin, 结果 Bacillus gibsonii FJAT-10019, 561, Metlin 15, 90.89, 1.14% 6.522, 270.0623 结论 关键词 : ; ; ; Analysis and testing on leflunomide from Bacillus gibsonii FJAT-10019 CHEN Zheng, LIU Bo *, ZHU Yu-jing, PAN Zhi-Zhen (Institute of Agrobiological Resources, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China) ABSTRACT: Objective To analyze and test on leflunomide from Bacillus gibsonii FJAT-10019. Methods Extracellular metabolites of Bacillus gibsonii FJAT-10019 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 6.522 min, accurate mass is 270.0623. 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 : ( ) (201303094)(2013dqd-6) Fund: Supported by the Chinese Special Fund for Agro-scientific Research in the Public Interest(201303094), The Youth Innovation Fund of FAAS (2013dqd-6). * 通讯作者 :,, Email: fzliubo@163.com *Corresponding author: LIU Bo, Researcher, Fujian Academy of Agricultural Sciences, Fujian 350003, China. E-mail: fzliubo@163.com
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- 10019 : 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-10019 24 h 20 ml LB, 37 200 r/min 12 h, 1 % 50 ml TSB, 37 200 r/min 48 h Bacillus gibsonii FJAT-10019, 4 : 50 ml Bacillus gibsonii FJAT- 10019,, 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 % 25 35 min; MS : ; : 300, 5 L/min; Nebulizer 30 psig; Capillary voltage 3500 V; Fragmentor 175 V; Skimmer 65.0 V; Mass range 150 1000 m/z : 112.9855 119.036 301.9981 966.0007 980.0163 1033.9881 1633.9486 1933.9306 2533.8923 3 结果与分析 3.1 吉氏芽胞杆菌 Bacillus gibsonii FJAT- 10019 发酵液 LC-MS 物质成分分析 561,, 15, 3 : -3,4,5-2,3- -1- 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,,
3674 5 表 1 吉氏芽胞杆菌 Bacillus gibsonii FJAT-10019 菌株发酵液成分 Table 1 The composition of Bacillus gibsonii FJAT-10019 fermentation broth Cpd Name METLIN METLIN Mass m/z Score RT Vol % 538 Leflunomide( ) 66762 270.0623 269.055 90.89 6.522 1.14 543 Zuccagin 50018 284.0696 283.0623 67.6 8.909 0.21 535 Chlorthalidone( ) 1747 338.0138 337.0065 56.16 5.5 0.09 384 2,3-Dibromo-1-propanol(2,3- -1- ) 72947 215.8782 214.8709 54.27 1.309 0.3 389 beta-2,3,4,5,6-pentachlorocyclohexanol 71135 269.8948 268.8875 52.79 1.313 0.2 69 Galbeta1-3GalNAcalpha1-3(Fucalpha1-2) Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta-Cer (d18:1/18:0) 54990 1765.966 881.9756 51.63 1.054 0.09 75 PE(26:2(5E,9Z)/26:2(5E,9Z)) 40567 963.7646 480.875 51.46 1.054 0.03 490 Etidronic acid() 2629 205.9738 204.9666 49.51 1.774 0.02 496 Butonate( ) 72726 325.9631 324.9558 49.42 1.857 0.29 400 Chloraminophenamide(5- -2,4- ) 66651 284.9649 283.9576 49.31 1.331 0.12 480 Chlorfenson( ) 72775 301.9563 300.949 48.36 1.675 0.06 296 Chloraminophenamide( ) 66651 284.9654 283.9581 47.18 1.066 0.11 337 Diazoxide( ) 1992 229.9908 228.9835 47.11 1.083 0.87 192 Phosphatidylinositol-3,4,5-trisphosphate (PIP3) -3,4,5 7040 629.9532 628.9459 45.25 1.058 0.02 388 Plocamene C 71568 317.9332 316.9259 45.1 1.313 0.1 3.2 吉氏芽胞杆菌 Bacillus gibsonii FJAT- 10019 发酵液来氟米特成分分析 Bacillus gibsonii FJAT-10019, 15, 90% 1, LEF(N-(4- - )-5- -4- ), (Score) 90.89, 1.14 %, 1 2, 6.522 min, 2.5 10 4 3, 4,, [M-H] - [M+X] - (X ) 269.0550 [M-H] -, 270.0550,, Metlin, 4 讨论, [7],, 7 [8],, Bacillus sp., 9,,
11, : 3675 - (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
3676 5 3 Fig. 3 Isotopic peak of leflunomide in sample Fig. 4 4 Mass spectrum of leflunomide in sample 15, : (1) : (LEF), N-(4- )-5- -4- ), C 12 H 9 F 3 N 2 O 2, [11] ( ), [12] LEF, A-771726,, [13] LEF : a. T B ; b. ; c. [12] (2) (diazoxide), (etidronic acid) (chlorthalidone),,,, 1.14%, 90.89,
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