Microsoft Word - 4--张喜昌_news_.doc

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1 刺参肠道微生物组成分析及产酶溶血性试验张喜昌 1, 费世洲 2, 常亚青 3, 刘小林 2 2, 王高学 (1., ; 2., ; 3., ) 摘要 : 对野生和人工养殖刺参的肠壁及内容物中的菌群数量种类组成进行了研究 ; 并结合产酶试验和溶血性试验, 对刺参肠道益生菌做了初步的体外筛选结果表明, 野生刺参肠壁及内容物中的细菌数量分别为 (3.30 ± 0.41) 10 7 cfu/g (6.39 ± 0.32) 10 7 cfu/g, 养殖刺参肠壁及内容物中的细菌数量分别为 (2.83 ± 0.31) 10 7 cfu/g (5.67 ± 0.53) 10 7 cfu/g 野生刺参肠道优势菌为弧菌属(Vibrio), 次优势菌为假单胞菌属 (Pseudomonas) 和希瓦氏菌属 (Shewanella); 养殖刺参肠道优势菌为弧菌属 (Vibrio), 次优势菌为假单胞菌属 (Pseudomonas) 在 224 株细菌中, 共有 160 株细菌具有产酶能力, 所占比例为 71.43%, 其中具产蛋白酶淀粉酶脂肪酶能力菌株分别为 114 株 114 株 108 株, 所占比例分别为 50.89% 50.89% 48.21% 99 株细菌中有 23 株具有溶血性, 所占比例为 23.23% 综合分析实验数据, 确定 6 株细菌作为刺参肠道潜在益生菌, 菌株代号分别为 HS1(Pseudomonas) HS5(Bacillus) HS7(Shewanella) HS8(Vibrio) HS10(Vibrio) HS11(Vibrio) 关键词 : 刺参 (Apostichopus japonicus); 肠道 ; 菌群 ; 产酶 ; 溶血性中图分类号 : S 文献标识码 : A 文章编号 : (2014) doi: /hykx , (Echinodermata), (Holothurioidea) 20,,,,,, :,,,,,,, (Apostichopus japonicus) 55 %,, 1.85 %,,, B 1, B 2, B 6, A, D, E,, Mn, Fe, Zn, Co, Se [1-2],,,,, [3],,, [4],, [5] ( ) ( ),, : 1 ; 2 ; 3 : ; : : (2012GA651002); 863 (2012- AA10A412); ( ) : (1965-),,,,, zhangxczhangxc@163.com;,,,, wanggaoxue@126.com 24 / 2014 / 38 / 5

2 ,, Gate [6],,,,,,,, (99 ),, 6, 1 材料与方法 1.1 材料 30, , 40.6 g ± 0.5 g, 10.1 cm ± 1.0 cm;, 32.1 g ± 0.5 g, 7.6 cm ± 1.0 cm 1.2 肠道菌的培养分离和纯化 4,, Sawabe [7], 75 %,,,, 0.9 %,,,, 5 ml 10 6,, 100 μl 2216E, 3, 28 5 d 30~300, 40, 2216, 细菌鉴定 (Bioteke) DNA, 16S rdna, PCR 1 %,, NCBI Blast, 16S rdna, Clustal W, MEGA 5.0 Kimura 2 Parameter Distance neighbour joining, 1000 Bootstraps, 1.4 产酶试验 224,, h [8] [9] : : 10 g, 1 g, 16 g, ml; ph 7.4; min : 10 g, 5 g, 1 g, 16 g, ml; ph 7.4; min : 10 g, ml, CaCl 2 7H 2 O 0.1 g, 9 g, ml; ph 7.4; min 1.5 溶血性试验 25, 99, 25, 48 h,,, 2 结果 2.1 刺参肠道细菌数量 4 Marine Sciences / Vol. 38, No. 5 /

3 30~300,, ( = (5 10 )/ ) ( 1) 1, (2.83~6.39) 10 7 cfu/g, 表 1 各组肠道细菌含量统计 ( 10 7 cfu/g ) Tab.1 Content analysis of gut bacteria in different groups ( 10 7 cfu/g ) 2.83 ± ± ± ± 刺参肠道细菌组成, %,,,, 2, 1~ 6 2,,, 2.3 产酶试验和溶血性试验, 224, 表 2 刺参肠道菌群组成比例 (%) Tab.2 Proportion of gut bacteria of Apostichopus japonicas (%) Microflora Vibrio Pseudomonas Shewanella Agarivorans Aeromonas Bacillus Y Y Y Y Y : ; Y, 1%. Fig. 1 1 Phylogenetic tree of representative strain for Vibrio among gut bacteria of Apostichopus japonicus Fig. 2 2 Phylogenetic tree of representative strain for Pseudomonas among gut bacteria of Apostichopus japonicus 26 / 2014 / 38 / 5

4 Fig. 3 3 Phylogenetic tree of representative strain for Shewanella among gut bacteria of Apostichopus japonicus Fig. 4 4 Phylogenetic tree of representative strain for Agarivorans among gut bacteria of Apostichopus japonicus Fig. 5 5 Phylogenetic tree of representative strain for Aeromonas among gut bacteria of Apostichopus japonicus Fig. 6 6 Phylogenetic tree of representative strain for Bacillus among gut bacteria of Apostichopus japonicus 24 ; 45, 44, 39 ; 6, 1, , %, , % % %, 99 23, %, 99 39, 39 8, % Marine Sciences / Vol. 38, No. 5 /

5 2.4 刺参肠道潜在益生菌菌株的确定, 6,,, HS5(Bacillus) 3 表 3 刺参肠道潜在益生菌菌株信息 Tab.3 Information of intestinal potential probiotic strains of Apostichopus japonicus HS1 (Pseudomonas) HS5 (Bacillus) HS7 (Shewanella) HS8 (Vibrio) HS10 (Vibrio) HS11 (Vibrio) : 3 讨论,,,,, [3-5],,, [6] ;,, ~ cfu/g, [10] [11],, [12],,,,, 50 %,, [11], [12], [13-15],, [16],,,,,,, [17-19], [9],,,,, [19],,,,, [9],,,, [17-19], [20-22] [20] 1,,,, 28 / 2014 / 38 / 5

6 ,,, [21-22],,, 99,,,,, : [1] Chen Jiaxin. Overview of sea cucumber farming and sea ranching practices in China[EB]. Beche-de-mer Information Bulletin, 2003, (18): [2] Zhang C Y, Wang Y G, Rong X J, et al. Natural resources, cultule and problems of sea cucumber worldwide[j]. Marine Fisheries Research, 2004, (25): [3]. [M]. :, [4] Nomoto K. Prevention of infections by probiotics [J]. Journal of Bioscience and Bioengineering, 2005, 100: [5] Gatesoupe F J. The use of probiotics in aquaculture[j]. Aquaculture, 1999, 180: [6] Gatesoupe F J, Lesel R. An environmental approach to intestinal microflora in fish [J]. Cahiera Agricultures, 1998, 7(1): [7] Sawabe T, Oda Y, Shiomi Y, et al. Alginate degradation by bacteria isolated from the gut of sea urchins and abalones[j]. Microbial Ecology, 1995, 30: [8],. [M]. :, [9],,,. [J]., 2008, (03): [10],,,. [J]., 2012, 4: [11], 騳. [J]., 1989, 4: [12],,,. [J]., 2009, 27: [13] Dang H Y, Lovell C R.Bacterial primary colonization and early succession on surfaces in marine waters as determined by amplified rrna gene restriction analysis and sequence analysis of 16S rrna genes [J]. Applied and Environmental Microbiology, 2000, 66: [14] Kim D, Baik K S, Kim M S, et al. Shewanella haliotis sp. nov., isolated from the gut microflora of abalone, Haliotis discus hannai[j]. International Journal of Systematic and Evolutionary Microbiology, 2007, 57: [15] Chan P S, Baik K S, Kim M S, et al. Shewanella marina sp. nov., isolated from seawater[j]. International Journal of Systematic and Evolutionary Microbiology, 2009, 59: [16] Spanggaard B, Huber I, Nielsen J, et al. The microflora of rainbow trout intestine: a comparis on of traditional and molecular identification[j]. Aquaculture, 2000, 182: [17]. [M]. :, [18],,. [M]. :, [19],,,. [J]., 2007, 5: [20],,,. [J]., 2007, 6: [21] Ascencio F, Wadström T. Effect of Aeromonas proteases on the binding of Aeromonas hydrophila strains to connective tissue proteins[j]. Microbies, 1999, 66: [22] Howard S P, Buckley J T. Activation of the hole-forming toxin aerolysin by extracellular processing[j]. Bacteriol 1985, 163, 1: Marine Sciences / Vol. 38, No. 5 /

7 Microflora in digestive tract of Apostichopus japonicus and enzyme producing and hemolytic analysis ZHANG Xi-chang 1, FEI Shi-zhou 2, CHANG Ya-qing 3, LIU Xiao-lin 2, WANG Gao-xue 2 (1. Dalian Haibao Fishery Company Ltd, Lüshun , China; 2. College of Animal Science, Northwest A & F University, Yangling , China; 3. Key Laboratory of Mariculture Stock Inhancement in North China s Sea, Ministry of Agricultures, Dalian Ocean University, Dalian , China) Received: Apr., 10, 2013 Key words: Apostichopus japonicus; intestinal tract; microflora; enzyme producing; hemolytic Abstract: Microflora in the intestinal tract and on the intestinal wall of both cultured and wild Apostichopus japonicus was studied in this paper. The screening for probiotics was performed based on enzyme producing and hemolytic analysis. The results showed that the number of bacteria in the intestinal wall and tract of wild Apostichopus japonicus was (3.30 ± 0.41) 10 7 cfu/g and (6.39 ± 0.32) 10 7 cfu/g, respectively. The number of bacteria in the intestinal wall and tract of cultured group was (2.83 ± 0.31) 10 7 cfu/g and (5.67 ± 0.53) 10 7 cfu/g, respectively. The dominant species in the intestinal tract of wild group was Vibrio and the Pseudomonas and Shewanella were the secondary dominant species. The dominant species in the cultured group was Vibrio and Pseudomonas. In 224 strains of bacteria, a total of 160 strains of bacteria produced enzyme with a ratio of %. Among these bacteria, 114 strains could produce protease, 114 strains could produce amylase, and 108 strains could produce lipase. The percentages were %, %, and %, respectively. A total of 23 strains of bacteria could produce hemolytic toxin in 99 strains of bacteria, which accounts for 23.23% of the total bacterial population. Through the comprehensive analysis of test data, we selected 6 strains of bacteria as intestinal potential probiotic strains of Apostichopus japonicus, which were HS1(Pseudomonas), HS5(Bacillus), HS7(Shewanella), HS8(Vibrio), HS10(Vibrio), and HS11(Vibrio) respectively. ( 本文编辑 : 康亦兼 ) 30 / 2014 / 38 / 5

Microsoft Word - 2-冷晓飞_news_.doc

Microsoft Word - 2-冷晓飞_news_.doc 皱纹盘鲍肠道菌群组成及 PCR-DGGE 指纹图谱分析 1, 2, 2, 2, 2, 1, 3 (1., 116045; 2., 712100; 3., 116023) 摘要 : 对养殖和野生皱纹盘鲍 (Haliotis discus hannai Ino ) 不同个体间肠道菌群数量和种类组成进行了研究, 并采用 PCR-DGGE 指纹技术对比分析了二者肠道优势菌群的差异研究结果表明, 养殖和野生