Microsoft Word 陈峥-二校

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1 6 10 Vol. 6 No Journal of Food Safety and Quality Oct., 2015 / - 陈峥, 刘波 *, 朱育菁, 郑梅霞, 张连宝, 刘国红, 潘志针 (, ) 摘要 : 目的方法 - Metlin 32 结果 (Bacillus cohnii FJAT-10017) (Aneurinibacillus aneurinilyticus FJAT-10004) (Paenibacillus glucanolyticus FJAT-10020) (Brevibacillus agri FJAT-10018) (Bacillus cohnii FJAT-10017), 1.73% 结论 (Bacillus cohnii FJAT-10017) 关键词 : / ; ; Screening of Bacillus strains producing non-protein amino acid named L-Djenkolic acid based on liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry CHEN Zheng, LIU Bo *, ZHU Yu-Jing, ZHENG Mei-Xia, ZHANG Lian-Bao, LIU Guo-Hong, PAN Zhi-Zhen (Institute of Agrobiological Resources, Fujian Academy of Agricultural Sciences, Fuzhou , China) ABSTRACT: Objective To Screen the Bacillus strains that producing L-Djenkolic acid. Methods Extracellular metabolites of 32 Bacillus strains were analyzed by liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF MS) and Metlin database. Results The result showed that 4 Bacillus strains contain L-Djenkolic acid, including Bacillus cohnii FJAT-10017, Aneurinibacillus aneurinilyticus FJAT-10004, Paenibacillus glucanolyticus FJAT and Brevibacillus agri FJAT The content of L-Djenkolic acid was the highest in Bacillus cohnii FJAT-10017, with the relative content of 1.73%. Conclusion Bacillus cohnii FJAT is the high-yield Bacillus strain producing L-Djenkolic acid. This result provided theory basis for development and utilization of active substances from Bacillus. KEY WORDS: liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry; Bacillus cohnii; 基金项目 : ( ) ( ) ( ) (2013dqd-6) (2014JQ-2) (2013GB2C400236) Fund: Supported by National Natural Science Foundation of China ( ), the Chinese Special Fund for Agro-scientific Research in the Public Interest ( ), the Youth Innovation Fund of FAAS (2013dqd-6), the Outstanding Youth Fund of FAAS (2014JQ-2), Agricultural Science and Technology Achievements Transformation Funds Project (2013GB2C400236) * 通讯作者 :,, fzliubo@163.com *Corresponding author: LIU Bo, Researcher, Fujian Academy of Agricultural Sciences, Fujian , China. fzliubo@163.com

2 L-Djenkolic acid 1 引言 (non-protein amino acid) 20 [1], [2], 700 [3], 400, 240, 50, [4] (L-Djenkolic acid, C 7 H 14 N 2 O 4 S 2 ; CAS ) (non-protein amino acid),,, [5-8] [9] [10] [11],,,, [12] [13], ; [14] ; [15], [16],,, (Bacillus cohnii FJAT-10017) (Aneurinibacillus aneurinilyticus FJAT-10004) (Paenibacillus glucanolyticus FJAT-10020) (Brevibacillus agri FJAT-10018),, (LC-QTOF-MS), 32,, 2 材料与方法 2.1 材料 : 32 ( 1) 表 1 Table 1 供试芽胞杆菌菌株 Bacillus strains for test 1 FJAT Aneurinibacillus aneurinilyticus 2 FJAT-8775 Bacillus mycoides 3 FJAT Bacillus funiculus 4 FJAT Bacillus agaradhaerens 5 FJAT Bacillus clarkia 6 FJAT Bacillus safensis 7 FJAT Bacillus cohnii 8 FJAT Bacillus halmapalus 9 FJAT Bacillus macyae 10 FJAT Bacillus methanolicus

3 10, : / 续表 1 11 FJAT Bacillus subtilis subsp. spizizenii 12 FJAT Bacillus subtilis subsp. inaquosorum inaquosorum 13 FJAT Bacillus isronensis 14 FJAT Bacillus taeanensis 15 FJAT Bacillus subtilis subsp. subtilis 16 FJAT Bacillus shackletonii 17 FJAT Bacillus murimartini 18 FJAT Bacillus selenitireducens 19 FJAT Bacillus selenatarsenatis 20 FJAT Bacillus seohaeanensis 21 FJAT Bacillus thermoamylovorans 22 FJAT Bacillus alcalophilus 23 FJAT Bacillus altitudinis 24 FJAT Bacillus hwajinpoensis 25 FJAT Bacillus gibsonii 26 FJAT Brevibacillus agri 27 FJAT Gracilibacillus dipsosauri 28 FJAT Paenibacillus chondroitinus 29 FJAT Paenibacillus glucanolyticus 30 FJAT Psychrobacillus psychrotolerans 31 FJAT Viridibacillus arenosi 32 FJAT Viridibacillus arvi : 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 24 h 20 ml LB, r/min 12 h, 1 % 50 ml TSB, r/min 48 h, 4 : 50 ml,, 0.25 μm, GC 4, : Agilent ZORBAX Extend C 18 (2.1 μm 50 mm, 1.8 μm); : 35 ; : 2 μl; : A=10 mmol/l +, B= ; : 0.2 ml/min; : B=90%(0 3 min); B=50%(3 25 min); B=90%(25 35 min); MS : ; : 300,

4 : 5 L/min; : 30 psig; : 3500 V; : 175 V; Skimmer: 65.0 V; : m/z 3 结果与分析 3.1 产今可豆氨酸芽胞杆菌的筛选 32 4 ( 2), : FJAT FJAT FJAT FJAT FJAT (Score), 99.90% FJAT-10017, 1.73% 3.2 芽胞杆菌来源的今可豆氨酸成分分析 4, , FJAT , 6,,, [M-H] - [M+X] - (X ) [M-H] -, metlin,, 6 Table 2 表 2 产今可豆氨酸芽胞杆菌 Bacillus strains which produce L-Djenkolic acid 1 FJAT FJAT FJAT FJAT Fig. 1 1 FJAT Extracted Ion Chromatogram(EIC)of L-Djenkolic acid in Bacillus cohnii FJAT-10017

5 10, : / Fig. 2 2 FJAT Extracted Ion Chromatogram(EIC)of L-Djenkolic acid in Aneurinibacillus aneurinilyticus FJAT Fig. 3 3 FJAT Extracted Ion Chromatogram(EIC)of L-Djenkolic acid in Paenibacillus glucanolyticus FJAT Fig. 4 4 FJAT Extracted Ion Chromatogram(EIC)of L-Djenkolic acid in Brevibacillus agri FJAT-10018

6 FJAT Fig. 5 Isotopic peak of L-Djenkolic acid in Bacillus cohnii FJAT FJAT Fig. 6 mass spectrum of L-Djenkolic acid in Bacillus cohnii FJAT 讨论, [17-19] [20], [21,22], - (LC-QTOF MS) [17],,, Agilent MassHunter, (MFE), Metlin,, 4 : (1) : Piluk [5,6] M. pudica, ; Laue [7] ; Kim J [8] L- TcyABC, (2) : Piluk [6]

7 10, : / , ; ; (3) : [11] ; Burguière [18] ; (4) : [10] 7 32, 4, (1) : (Bacillus cohnii FJAT-10017); (2) : (Aneurinibacillus aneurinilyticus FJAT-10004); (3) : (Paenibacillus glucanolyticus FJAT-10020); (4) : (Brevibacillus agri FJAT-10018), (Bacillus cohnii FJAT-10017) 1.73%, 92.47, 参考文献 [1],,. [J]., 2000, 20(3): Yan AX, Tian GL, Ye YH. Progress in modification of bioactive peptides with non-protein amino acids and their application in the studies of structure- activity relationship [J]. Chin J Org Chem, 2000, 20(3): [2]. [J]., 2009, (7): Fan ZJ. Research progress of application and function of non-protein amino [J]. Guangdong Sci Tech. 2009, (7): [3],,. [J]., 2003, 3(3): Li Y, Wang X, Yang F. Studies on the function and application of non-protein amino acids [J]. J Shanghai Inst Tech: Nat Sci, 2009, (7): [4]. [J]., 1989, (3): Wu XR. Structure and function of non-protein amino acids [J]. Amino Acids Biotic Res, 1989, (3): [5] Piluk J, Hartel PG, Haines BL. Production of carbon disulfide (CS2) from L-Djenkolic acid in the roots of Mimosa pudica L [J]. Dev Plant Soil Sci, 1998, 82: [6] Piluk J, Hartel PG, Haines BL, et al. Association of carbon disulfide with plants in the Family Fabaceae [J]. J Chem Ecol, 2001, 27(7): [7] Laue P, MacDonald RE. Growth stimulation of Staphylococcus aureus by L-cystine or L-Djenkolic acid [J]. J Bacteriol. 1968, 95(6): [8] Kim J, Senadheera DB, Lévesque CM, et al. TcyR regulates L-cystine uptake via the TcyABC transporter in Streptococcus mutans [J]. FEMS Microbiol Lett, 2012, 328(2): [9] Pepi M, Baldi F. Modulation of chromium (VI) toxicity by organic and inorganic sulfur species in yeasts from industrial wastes [J]. Biometals, 1992, 5(3): [10] Melnikov P, Nascimento VA, Silva AF, et al. Consolo. Structural modeling of djenkolic acid with sulfur replaced by selenium and tellurium [J]. Mol, 2014, 19(4): [11] Elbetieha A, Owais WM, Saadoun I, et al. Effect of glutathione L-cystein and L-Djenkolic acid in the synthesis and mutagenicity of azide metabolite in Bacillus subtilis ATCC 6633 strain [J]. New Microbiol, 1999, 22(4): [12],,,. [J]., 2011, 26(5): Liu GH, Lin YZ, Lin NQ, et al. Study on genus Bacillus classification [J]. Fujian J Agric Sci, 2011, 26(5): [13],,,. FJAT-8754 [J]., 2014, (4): Wang K, Lan JL, Liu B, et al. The Characteristics of cellulase and amylase from B. amyloliquefaciens FJAT-8754 and optimization of fermentation conditions [J]. Fujian J Agric Sci, 2014, (4): [14],. [J]., 1998, 18(4): Li L, Yu ZN. The current situation of bacterial insecticides research and its prospect [J]. J Microbiol, 1998, 18(4): [15]. [J]., 2015, (2): Yuan HW. Effect analysis of Bacillus subtilis two (Live) enteric-coated capsules combined with Bacillus licheniformis capsule in the treatment of antibiotic associated diarrhea [J]. Chin Mod Med, 2015, (2): [16],,.

8 [J]., 2006, (5): Zhang K, Ning DG, Xu WD. Advance on the surface dispaly of recombinant vaccines on subtilis bacillus spores [J]. Microbiol, 2006, (5): [17],,,. LC QTOF MS [J]., 2012, 27 (10): Shi H. Liu B, Chen Z, et al. Metabonomics Analysis of Bacillus based on LC/Q-TOF MS [J]. Fujian J Agric Sci, 2012, 27 (10): [18] Burguière P, Auger S, Hullo MF, et al. Martin-Verstraete I.Three different systems participate in L-cystine uptake in Bacillus subtilis [J]. J Bacteriol, 2004, 186(15): [19] Heinecke JW, Rosen H, Suzuki LA, et al. The role of sulfur-containing amino acids in superoxide production and modification of low density lipoprotein by arterial smooth muscle cells [J]. J Biol Chem, 1987, 262(21): [20] Parry J, Clark DP. Identification of a CysB-regulated gene involved in glutathione transport in Escherichia coli [J]. FEMS Microbiol Lett, 2002, 209(1): [21] Ramírez EC, Whitaker JR. Biochemical characterization of cystine lyase from broccoli (Brassica oleracea var. italica) [J]. J Agric Food Chem, 1999, 47(6): [22] Kunert J. Metabolism of sulfur-containing amino acids in the dermatophyte Microsporum gypseum. I. Neutral amino acids [J]. J Basic Microbiol, 1985, 25(1): [23] Castric PA, Conn EE. Formation of -cyanoalanine by O-acetylserine sulfhydrylase [J]. J Bacteriol, 1971, 108(1): 作者简介 ( 责任编辑 : 白洪健 ) 陈峥, 助理研究员, 主要研究方向为微生物代谢物研究 acerdestiny@163.com 刘波, 研究员, 主要研究方向为微生物生物技术与农业生物药物 fzliubo@163.com

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