LIU Lanet al: Screening and Identification of Lipase Inhibitor-Producing Strain Research Article 15;pH 7.2~ 试剂 :SIGMA ;Taq dntp: TAKARA ; : 1

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1 兰等 : 脂肪酶抑制剂产生菌的筛选和鉴定兰 123 周培华 2 曾伟 23 陈桂光 2 梁智群 *23 ( ; ; ) : 通过对不同地点采集样品分离纯化得到的不同单菌落再进行发酵培养并以其产物是否对脂肪酶具有抑制活性作为筛选指标从 2013 株野生菌中筛选到一株产脂肪酶抑制活性较高的菌株 Sf16 对菌株 Sf16 的菌落气生菌丝孢子丝及孢子的形态特征和在不同碳源培养基上的生长表现的特征以及一些生理生化方面特性进行观察并对菌株的 DNA 进行提取并扩增 16S rdna 通过对后者序列测定数据库同源搜索和比对进而构建菌株的系统进化树通过形态学和分子生物学手段对产脂肪酶抑制剂菌株 Sf16 进行初步鉴定结果确定其为 Streptomyces sp. : 脂肪酶抑制剂 ;PCR;16SrDNA; 系统发育树 :TQ :A : (2013) Screening and Identification of Lipase Inhibitor-Producing Strain LIU Lan 123 ZHOU Pei-hua 2 ZENG Wei 23 CHEN Gui-guang 2 LIANG Zhi-qun *23 (1. Guangxi Yulin Institute for Food and Drug ControlYulin China;2. College of Life Sciences and TechnologyGuangxi UniversityNanning China;3. Key Laboratory of Subtropical Agricultural Bioresources Conservation and Utilization of GuangxiNanning China) Abstract: We collected many samples from different locationsthen isolated and purified single colony. By fermentationthe products whether have lipase inhibition activity as a screening indicator. We find a good strain Sf16 after we cultured 2013 wild strains. The morphological characteristics of colonyaerial hyphaesporothrix and spores and the growth performance characteristics in different carbon sources medium of strain Sf16as well as some physiological and biochemical characteristics were observed. Extract the DNAamplified and determined the 16SrDNA.Then homology search in databasecompared and constructed the phylogenetic tree. Inhibitor-producing strain Sf16 preliminary identification by means of morphological and molecular biology techniquesthe result is Streptomyces sp. Keywords: lipase inhibitorpcr16srdnathe phylogenetic tree ; : * : (1959 ) zqliang@gxu.edu.cn 貍貗貪

2 LIU Lanet al: Screening and Identification of Lipase Inhibitor-Producing Strain Research Article 15;pH 7.2~ 试剂 :SIGMA ;Taq dntp: TAKARA ; : 仪器 MEITLER : [1] MEITLER ;SPX-250 : ;HVE-50 : HIRAYAMA;SKY-211B : ;OLYMPUS : ;Micropipette :Socorex ;DNA : ;PCR : Biometra ; : ;Thermo Scientific Multiskan Go 50 g : 菌株分离培养 ml EP ; 250 ml [2-5] [6-7] 菌株生长形态学 Sf ; Sf16 Sf16 Sf16 Streptomyces sp 土壤 Sf16 DNA DNA 培养基 ( g/l): 20NaCl 0.5KNO 3 1K 2 HPO 3 3H 2 O 0.5 MgSO 4 7H 3 O 0.5FeSO 4 7H 2 O 0.01K 2 Cr 2 O [8] Sf 菌株 Sf16 的分子生物学 [9] self1492r self27f 16S rdna PCR PCR 1PCR [10] 2 NCBI 貍貙財 Journal of Food Science and Biotechnology Vol.32 No

3 兰等 : 脂肪酶抑制剂产生菌的筛选和鉴定 BLAST 16S rdna 100% 99% 18 16S rdna MEGA4 [11-13] NJ 1 Table 1 Table 2 2 PCR PCR reaction system /μl DNA PCR mmol/l MgCl mmol/l 5 10 mmol/l dntp 0.2 mmol/l 1 10 μmol/l 0.6 μmol/l 3 10 μmol/l 0.6 μmol/l 3 5 U/μL Taq 0.05 U/μL 0.5 ddh 2 O min 2 PCR PCR reaction parameters s s 72 2 min 2.1 Sf16 2~3 4 Table 4 Sf16 Sf min 1 Sf16 (a) (b) (c)(400 ) (d)(400 ) Fig. 1 Aerial hyphae (a) substrate mycelium (b) Spore morphology (c) (400 ) and aerial mycelium morphology (d)(400 ) of Sf16 3 Table 3 Sf16 Culture characteristics of Sf16 in a variety of different medias (ISP) (ISP) Sf16 Physiological and biochemical characteristics of Sf Sf16 DNA 16SrDNA Sf16 DNA 48.5 kb 16S rdna 1.2~1.5 kb 16SrDNA bp lcl DNA 16SrDNA Marker D L L- + + D-+ + D- - H 2 S + D 貍貙貢

4 LIU Lanet al: Screening and Identification of Lipase Inhibitor-Producing Strain Research Article MEGA4 Sf16 16SrDNA 3 Sf16 16S rdna Sf16 Streptomyces sp. 99% Streptomyces sp. 2 Sf16 Fig. 2 Gene by agarose gel electrophoresis of Sf Fig. 3 Sf16 Phylogenetic status of Sf16 Sf16 ; ; Streptomyces sp. : [ 1 ] T Ellrott. Aktuelle medikamentose ansatze in der Adipositastherapie[J]. Deutsche Medizin200017(6): [ 2 ]. [J] (2): WANG Yan -feiguo Guan -xin. Purification and characterization of pancreatic lipase inhibitor derived from rice germ [J]. Science and Technology of CerealsOils and Foods200614(2):32-34.(in Chinese) [ 3 ]. [J] (9): WU Zi -jianwang HongZHANG Jun -qinget al. Effects of grape seeds extracts on pancreatic lipase activity [J]. Food Research and Development200930(9):83-86.(in Chinese) [ 4 ]. [J] (6): 貍貙貣 Journal of Food Science and Biotechnology Vol.32 No

5 兰等 : 脂肪酶抑制剂产生菌的筛选和鉴定 CHEN Xi -pingyang PengZHANG Yang -de. Studies on separation and purification of alkaloid from lotus leaves and inhibition effects of extracts on lipase activity [J]. Research of Agricultural Modernization (6): (in Chinese) [ 5 ]. [J] (6): SHEN LiWANG Xin-liangWANG zhenet al. Progress in research of traditional Chinese medicine and natural products in antiobesity field[j]. Chinese Journal of Pharmaceutical Technology Economics and Managenment20071(6):23-29.(in Chinese) [ 6 ]. [J] (11): ZHONG Wei-hongLU Jing. Screening of lipase inhibitor producing strain [J]. Chinese Journal of Antibiotics200727(11): (in Chinese) [ 7 ]. Lipstatin [J]. 2009(12):5-7. YAN ZhenLI Hai-junZHU Xi-qianget al. Fermentation process optimization of lipstatin[j]. Food and Drug2009(12):5-7. (in Chinese) [ 8 ] E B ShirlingD Gottlieb. Methods for characterization of streptomyces species [J]. International Journal of Systematic Bacteriology196616(3): [ 9 ]. PCR DNA [J] (1): ZHANG Shu-meiZHANG LongDAI Shi-kun. A quick and efficient method for genomic DNA extraction from Actionbacteria[J]. Biotechnology200717(1):39-41.(in Chinese) [10]. [M]. : 2000: [11]. ε- [J] (2): LI ShuCHEN Xu-shengLIAO Li-juanet al. A protocol for screening of ε-polylysine producing strains [J]. Journal of Food Science and Biotechnology201029(2): (in Chinese) [12]. [M]. : [13]. 16S rdna [J] (2): ZHANG MengZHAO Wei-quanYU Xiu-meiet al. Genetic diversity of potato scab pathogens in China based on 16S rdna sequences[j]. Scientia Agricultura Sinica200942(2): (in Chinese) (Trends in Food Science & Technology) - [ ]Kirsi S. MikkonenMaija Tenkanen Sustainable food -packaging materials based on future biorefinery products: Xylans and mannans[j] (12): (EU)No 25/2013 (potassium diacetate) (E262) (E261)E261 E 261(i)- E 261(ii)- [ ]. [EB/OL]. ( ). news/yujing/2013/01/ html 貍貙貤

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus