Microsoft Word - 5--韦露_news_.doc

Size: px

Start display at page:

Download "Microsoft Word - 5--韦露_news_.doc"

稗扶
5 years ago
Views:

1 一株拮抗菌 C-5 的分离及鉴定 1, 韦露 2, 陈偿 1 1, 2 3, 龙云映, 蔡奕明 (1., ; ; 3., ) 摘要 : 针对目前海参养殖中水产微生态制剂品种单一施用效果不稳定等现状, 作者从地瓜参 (Acaudina molpadioidea) 肠道分离土著肠杆菌 (Enterobacter), 筛选到 1 株具有较好拮抗作用的海洋细菌 C-5, 该菌对副溶血弧菌 (Vibrio parahaemolyticus) 溶藻弧菌(Vibrio alginolyticus) 杀鲑气单胞菌(Aeromonas salmonicida) 等 7 种细菌有拮抗作用 ; 通过形态观察生理生化和 16S rdna 基因序列分析鉴定为霍肠杆菌 (Enterobacter hormaechei); 通过卤虫 (Artemia) 和药敏实验检测该菌具有安全性, 其对卤虫的 72 h 半致死浓度 (LC 50 ) 为 10 8 cfu/ml, 只对 20 种常规抗生素中的 5 种产生耐药性 ; 对 C-5 的发酵条件进行优化, 该菌优化后的发酵条件为 : 温度 30, ph 7.0, 盐度 2% 结果表明, C-5 有望作为一种益生菌应用于海水养殖病害控制关键词 : 霍氏肠杆菌 (Enterobacter hormaechei); 益生菌 ; 分离鉴定中图分类号 : S967 文献标识码 : A 文章编号 : (2015) doi: //hykx (Acaudina molpadioidea) (Holothuriodea), (Molpadida), (Caudinidae), (Acaudina) [1],,,,,, (Vibrio) [2],,,,,,,, [3], [4-5],,, [6] Kozasa [7], (Bacillus toyoi) (Anguilla japonica),,, [8], (Scophthalmus maximus) ; [9], ; [10], (Pseudosciaena crocea) [11],,,, 1 材料与方法 1.1 菌株 : ; : : ( ); (KZCX2-EW-Q215); (2014B ) : (1990-),,,,,, : , luluwei007@163.com;,,, chen.chang@ scsio.ac.cn; : , Marine Sciences / Vol. 39, No. 10 /

2 1.1.2 (Vibrio parahaemolyticus)13150 (Vp13150) (CDC), (Bacillus subtilis) (Staphylococcus aureus) (Chromobacterium. Violaceum) CV026 (Vibrio alginolyticus) (E167) (Aeromonas salmonicida) 199 (A.saL) (Vibrio cholerae) 006, (L1) (Bacillus amyloliquefaciens)l1( L1 ), LB, 实验方法 , [12],, Biolog, 95 C-5 DNA, (27F 1492R) 16S rdna PCR [13], NCBI Blast,, C-5 Sugita [14], Vp13150, L1, C-5, 3, 2 h OD 600, Goulden [15], 30, 12, 5 (C-5 Vp13150) 10 4, 10 5, 10 6, 10 7, 10 8 cfu/ml, 3, 3 d,, (Karber ), lg LC 50 =XK C [P i + P (i+1) ], : XK ; C ; P i P (i+1) LC 50, (K-B), C-5 20, [16] (ph NaCl ) C-5 : ph : NaCl : 0% 1% 2% 3% 4% 5%, 1% r/min 24 h, OD 600, LB ph NaCl, OD 600 [17] 1.3 数据统计 Origin 8.1, SPSS17.0 (ONE-WAY ANVOA), Duncan, 2 结果 2.1 拮抗菌的筛选与抗菌谱 81, 1 C-5, 1,, C-5, 80, L1 表 1 C-5 和 L1 对 7 种指示菌的抗菌活性 Tab.1 Antimicrobial activities of the bacterium C-5 and L1 towards 7 indicator bacteriums C-5 L1 (mm) 13±0.1-12±0.1 - CV026 20±0.1 - E167 16± (A.saL) 21±0.1 - Vibrio parahaemolyticus ±0.2 - Vibrio cholerae ±0.1 - : - 30 / 2015 / 39 / 10

3 2.2 拮抗菌 C-5 的鉴定 C-5 2, C-5,,,, 16S rdna ( 1), (Enterobacter hormaechei) TMPSB-T10 (EU ) 99% (Enterobacter hormaechei), NCBI KJ660958,, C-5 Enterobacter sp. 表 2 C-5 菌株的生化特征 Tab.2 Biochemical analysis of isolated C-5 C-5 C-5 C-5 C D- + D- - D D ph 6 + ph 5 +, - -D D- + D- - N- -D- + N- - -D- + N- -D- - N- - 1% NaCl + 4% NaCl + 8% NaCl - -D- + D- - D- + D D- - L- + L D- - D- - D- - D D D D- - D- - + SV e -L- + L- + L- - L- - L- + L- + L- - L- +, D- + L- - D- + D- + + ; p D- - L D- - L D, L S rdna C-5 Fig.1 Phylogenetic tree of C-5 base on 16S rdna Marine Sciences / Vol. 39, No. 10 /

4 2.3 拮抗菌 C-5 的胞外产物对副溶血弧菌的抑菌作用 2, 0~4 h, ; 5~12 h, ; 12~14 h, ; 16 h, ; 0~10 h, 12 h LC cfu/ml 3 表 3 不同浓度 C-5 和 Vp13150 条件下卤虫的死亡率 Tab.3 Mortalities of Artemia nauplii with different doses of C-5 and Vp13150 C-5 Vp13150 (CFU/mL) 72h (%, mean SD) LC 50 (CFU/mL) ~10 8 ~ C-5 Vp13150 Fig.2 Inhibitory curve of extracellular products of antagonistic bacterium C-5 against V. parahaemolyticus 卤虫毒性实验 (LC 50 ) 72 h LC cfu/ml, C 药敏实验 C , C-5 15, 初步优化拮抗菌 C-5 的发酵条件 ph C-5 ( 3), C-5 ph 7 ph 4~11, C-5 OD 600 ph, ph 7, C-5 OD ; ph 7, OD 600 ; ph (ph=4, 5) (ph=10, 11), C-5 表 4 菌株 C-5 药敏实验结果 Tab.4 Results of antibiotic sensitive test of strain C-5 C-5 (μg/ ) (mm) C-5 (μg / ) (mm) S S R R S R S R S 1 26 S S 2 22 S S R S 5 25 S S S S S : S (d 14), R (d 13) 32 / 2015 / 39 / 10

5 3 ph C-5 Fig.3 Effect of ph on the growth of strain C-5 C-5 ( 4), C-5 20~40, C ~30,, C-5 OD 600, ; 30, C-5 OD 600 (10 ) (45 ), C-5 OD 600, C-5 ( 5), C-5 0~5%, C-5 2% NaCl 0~5%,, C-5 OD 600, 2% 1.462; 2%, C-5 OD C-5 Fig.4 Effect of temperature on the growth of strain C-5 3 讨论,,,,, [18], 5 C-5 Fig.5 Effect of NaCl on the growth of strain C-5,, [19],,,,,,, [20], [21],,,, C-5,,,, C-5,,, C-5,, 10 h, 10 h,, [22],, (LC 50 ) [23] (Artemia), C-5, C-5 72 h LC cfu/ml LC cfu/ml, LC cfu/ml [24] Marine Sciences / Vol. 39, No. 10 /

6 ,, ph [25] C-5, 30, ph 7, 2%NaCl,,,,,,, ( ),, C-5, : [1]. : [M]. :, 1997: [2] Gatesoupe F. The use of probiotics in aquaculture[j]. Aquaculture, 1999, 180(1-2): [3] Balczar J L, Blas I D, Ruiz-Zarzuela I, et al.the role of probiotics in aquaculture[j]. Veterinary Microbiology, 2006, 114(2): [4] Cai Y, Benno Y, Nakase T, et al. Specific probiotic characterization of Weissella hellenica DS-12 isolated from flounder intestine[j]. Journal of General and Applied Microbiology, 1998, 44(5): [5],,,.3 [J]., 2007, 38(1): [6]. [M]. :, 2002: [7] Kozasa M. Toyocerin (Bacillus toyoi) as growth promotor for animal feeding[j]. Microbiologie Aliments Nutrition, 1986, 4: [8],,,. [J]., 2012, 36(2): [9],,,. [J]., 2009, 33(1): [10],,,. [J]., 2004, 28(3): 5-9. [11],. [J]., 2008, 29(6): [12],,,. L 1-9 [J]., 2012, 33(19): [13] Weisburg W G, Barns S M, Pelletier D A, et al. 16s Ribosomal DNA amplification for phylogenetic study[j]. Journal of Bacteriology, 1991, 173(2): [14] Sugita H, Hirose Y, Matsue N, et al. Production of antibacterial substance by Bacillus spp. Strain NM12, an intestinal bacterium of Japanese coastal fish[j]. Aquaculture, 1998, 165(3-4): [15] Goulden E F, Hall M R, Bourne D G, et al. Pathogenicity and infection cycle of Vibrio owensii in larviculture of the ornate spiny lobster (Panulirus ornatus)[j]. Applied and Environmental Microbiology, 2012, 78(8): [16],,,. ( ) [J]., 2012, 36(7): [17],,,. [J]., 2007, 42(6): 1-6. [18],,. PCR-DGGE [J],, 2010, 31(3): [19],,. [J]., 2003, 27(3): [20],,,. [J]., 2014, 1(2): [21] Sugita H, Ohta K, Kuruma A, et al.an antibacterial effect of Lactococcus lactisisolated from the intestinal tract of the Amur catfish, Silurus asotuslinnaeus[j]. Aquaculture Research, 2007, 38(9): [22] Ringo E, Gatesoupe F J. Lactic acid bacteria in fish: a review[j]. Aquaculture, 1998, 160(3-4): [23],,,. [J]., 2012, 33(9): [24] Vinoj G, Vaseeharan B, DavidJayaseelan B, et al. 34 / 2015 / 39 / 10

7 Inhibitory effects of Bacillus licheniformis (DAB1) and Pseudomonas aeruginosa (DAP1) against Vibrio parahaemolyticus isolated from Fenneropenaeus indicus [J]. Aquaculture International, 2013, 21(5): [25],,,. [J]., 2001, 31(2): Isolation and identification of a probiotic strainc-5 WEI Lu 1, 2, CHEN Chang 1, LONG Yun-ying 1, 2, CAI Yi-ming 3 (1. South China Sea Institute of Oceanology Chinese Academy of Science, Guangdong Provincial Key Laboratory of Applied Marine Biology, Guangzhou , China; 2. University of Chinese Academy of Science, Beijing, , China; 3. Jinan University College of Life Science and Technology, Guangzhou , China) Received: Jul., 28, 2014 Key words: Enterobacter hormaechei; probiotic; isolation and identification Abstract: A probiotic strain of native Enterobacter named as C-5 has been isolated from the intestine of acaudina molpadioidea in this study. By observing colony morphology, analyzing physiological and biochemical characters, and sequencing of 16S rdna, C-5 was identified as Enterobacter hormaechei. C-5 showed inhibitory activity against seven pathogens, such as Vibrio parahaemolyticus, Vibrio alginolyticus, Aeromonas salmonicida. The security of C-5 was confirmed by the Artemia challenge test and drug sensitivity tests. The LC 50 of C-5 was found to be 10 8 cfu/ml, and it was resistant to five antibiotics among twenty used. The optimized fermentation condition of C-5 is: temperature 30, ph 7.0, 2% salinity. Our results showed that C-5 could be used as a kind of probiotic applied to aquaculture disease control. ( 本文编辑 : 谭雪静 ) Marine Sciences / Vol. 39, No. 10 /

Microsoft Word - 32--方卫东_new_.doc

Microsoft Word - 32--方卫东_new_.doc 46 5 Vol.46, No.5 2015 9 OCEANOLOGIA ET LIMNOLOGIA SINICA Sep., 2015 FA08 * 1, 2 3 3 3 3 3 3 (1. 361021; 2. 363102; 3. 361005), FA08,, 16S rdna, FA08 (Bacillus licheniformis, GenBank : KM892857) FA08 ph,

Microsoft Word - 5--韦 露_news_.doc

Microsoft Word - 5--韦露_news_.doc