两株海水鱼肠道芽孢杆菌的分离鉴定及特性分析
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摘要
为获取具有益生特性的芽孢杆菌(Bacillus)应用于养殖生产,从野生蓝点马鲛(Scomberomorus niphonius)和许氏平鲉(Sebastes schlegelii)肠道内分离筛选出两株芽孢杆菌,分别命名为YB1和YH2,其革兰氏染色均呈阳性,基于形态学观察、生理生化特征分析及16S rDNA序列比对,鉴定YB1为蜡样芽胞杆菌(Bacillus cereus YB1),YH2为壁芽孢杆菌(Bacillus muralis YH2)。分析了温度、pH和NaCl含量(w/v%)对YB1和YH2生长的影响,选取蛋白酶、淀粉酶和脂肪酶特性培养基检测了YB1和YH2的产酶特性,并通过腹腔注射的方式测定了YB1和YH2对许氏平鲉和大菱鲆(Scophthalmus maximus)的安全性。结果显示:YB1适宜的生长条件为温度15～35℃、pH 5～9、NaCl含量0～2%,最适生长条件为温度35℃、pH 7、NaCl含量0;YH2适宜的生长条件为温度15～45℃、pH 5～9、NaCl含量1%～3%,最适生长条件为温度30℃、pH 5、NaCl含量2%;YB1和YH2均具有产淀粉酶和蛋白酶的特性;YB1在浓度≤1×10~8 cfu·mL~(-1)时对大菱鲆和许氏平鲉均是相对安全的,YH2在浓度≤1×10~8 cfu·mL~(-1)时对大菱鲆是相对安全的。研究表明:YB1和YH2具有作为有益菌应用于海水鱼养殖的潜力,可作为候选菌株开发为芽孢杆菌微生态制剂。
With the characteristics of anti-stress, producing spores and highly active digestive enzymes, Bacillus, a heterotrophic bacterium, which may improve the activity of intestinal digestive enzymes, inhibit the growth and reproduction of harmful bacteria and regulate the internal environment balance after colonized in the intestinal tract of marine fish, could promote the growth of fish and prevent the occurrence of diseases. Isolation of Bacillus from the intestinal tract of marine fish and its application in marine fish culture carries great implication for healthy development of the industry. To screen the probiotic bacillus for farming application of marine fish, the study was carried out. Two Bacillus strains Bacillus cereus YB1 and Bacillus muralis YH2 isolated from the intestines of wild Scomberomorus niphonius and Sebastes schlegelii were identified by morphological observation, physiological and biochemical characteristic analyses, and 16 S rDNA sequencing. The effects of pH, temperature, and NaCl concentration(w/v%) on the growth of strains YB1 and YH2 were analyzed. Some characteristic mediums(proteases, amylases and lipases)were used to measure the enzyme-producing activity of the two strains. Meanwhile, animal safety of strains YB1 and YH2 were tested by intraperitoneal injections to S. schlegelii and Scophthalmus maximus. Results show that YB1 can survive in temperature range of 15～35°C, pH 5～9 and the NaCl content of 0～2%, best at 35℃, pH 7, NaCl content 0. YH2 can survive in temperature range of 15～45℃, pH 5～9, NaCl content 1%～3%, best at 30℃, pH 5, NaCl content 2%. Both YB1 and YH2 have the characteristics of producing amylase and protease. YB1 is relatively safe to the growth and survival of S. schlegelii and S. maximus within 1×10~8 cfu·mL~(-1) exposure, and YH2 is relatively safe to the growth and survival of S. maximus within 1×10~8 cfu·mL~(-1) exposure. Therefore, YB1 and YH2 could be considered as potential probiotic candidates for marine fish farming, and more applications shall be developed in marine aquaculture.
With the characteristics of anti-stress, producing spores and highly active digestive enzymes, Bacillus, a heterotrophic bacterium, which may improve the activity of intestinal digestive enzymes, inhibit the growth and reproduction of harmful bacteria and regulate the internal environment balance after colonized in the intestinal tract of marine fish, could promote the growth of fish and prevent the occurrence of diseases. Isolation of Bacillus from the intestinal tract of marine fish and its application in marine fish culture carries great implication for healthy development of the industry. To screen the probiotic bacillus for farming application of marine fish, the study was carried out. Two Bacillus strains Bacillus cereus YB1 and Bacillus muralis YH2 isolated from the intestines of wild Scomberomorus niphonius and Sebastes schlegelii were identified by morphological observation, physiological and biochemical characteristic analyses, and 16 S rDNA sequencing. The effects of pH, temperature, and NaCl concentration(w/v%) on the growth of strains YB1 and YH2 were analyzed. Some characteristic mediums(proteases, amylases and lipases)were used to measure the enzyme-producing activity of the two strains. Meanwhile, animal safety of strains YB1 and YH2 were tested by intraperitoneal injections to S. schlegelii and Scophthalmus maximus. Results show that YB1 can survive in temperature range of 15～35°C, pH 5～9 and the NaCl content of 0～2%, best at 35℃, pH 7, NaCl content 0. YH2 can survive in temperature range of 15～45℃, pH 5～9, NaCl content 1%～3%, best at 30℃, pH 5, NaCl content 2%. Both YB1 and YH2 have the characteristics of producing amylase and protease. YB1 is relatively safe to the growth and survival of S. schlegelii and S. maximus within 1×10~8 cfu·mL~(-1) exposure, and YH2 is relatively safe to the growth and survival of S. maximus within 1×10~8 cfu·mL~(-1) exposure. Therefore, YB1 and YH2 could be considered as potential probiotic candidates for marine fish farming, and more applications shall be developed in marine aquaculture.
引文
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[6] 于广宝,张德胜.微生态制剂对提高漠斑牙鲆育苗成活率的作用[J].海洋渔业,2009,31(2):167-172.YU G B,ZHANG D S.The role of microecological modulator in the improvement of seed rearing survival rate of southern flounder Paralichthys lethostigma[J].Marine Fisheries,2009,31(2):167-172.
[7] 曹煜成,李卓佳,杨莺莺,等.地衣芽孢杆菌De株对黄鳍鲷生长及其养殖池塘主要环境因子的影响[J].南方水产,2010,6(3):1-6.CAO Y C,LI Z J,YANG Y Y,et al.Effects of Bacillus licheniform strain De on growth of Sparus latus and main environmental factors in aquaculture pond[J].South China Fisheries Science,2010,6(3):1-6.
[8] SUN Y Z,YANG H L,MA R L,et al.Probiotic applications of two dominant gut Bacillus strains with antagonistic activity improved the growth performance and immune responses of grouper Epinephelus coioides[J].Fish & Shellfish Immunology,2010,29(5):803-809.
[9] 陶荣霞.17株芽孢杆菌产酶特性及益生特性的研究[D].武汉:华中农业大学,2012.TAO R X,Study on characteristics of enzyme production and probiotics by 17 Bacillus strain[D].Wuhan:Huazhong Agricultural University,2012.
[10] 布坎南,吉本斯.伯杰细菌鉴定手册[M].北京:科学出版社,1984:729-731.BUCHANAN R E,GIBBONS N E.Bergey’s manual of systemaic bacteriology[M].Beijing:Science Press,1984:729-731.
[11] 东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:45-65.DONG X Z,CAI M Y.Manual for systematic identification of common bacteria[M].Beijing:Science Press,2001:45-65.
[12] 王腾腾,韦秀梅,常城,等.一株许氏平鲉(Sebastes schlegelii)肠道乳酸菌的分离鉴定及特性分析[J].海洋与湖沼,2017,48(1):94-100.WANG T T,WEI X M,CHANG C,et al.Identification and characteristics of a lactic acid bacteria associated from intestine of black rockfish Sebates schlegelii[J].Oceanologia Et Limnologia Sinica,2017,48(1):94-100.
[13] EI-RHMAN A M A,KHATTAB Y A E,SHALABY A M E.Micrococcus luteus and Pseudomonas species as probiotics for promoting the growth performance and health of Nile tilapia,Oreochromis niloticus.[J].Fish & Shellfish Immunology,2009,27(2):175-180.
[14] 闫法军,田相利,董双林,等.刺参养殖池塘降解有机污染物常、低温芽孢杆菌的分离筛选[J].中国海洋大学学报(自然科学版),2013,43(6):17-24.YAN F J,TIAN X L,DONG S L,et al.Isolation and selection of normal and low temperature Bacillus degrading organic pollutants in sea cucumber culturing ponds[J].Periodical of Ocean University of China(Natural Science),2013,43(6):17-24.
[15] 陈跃.海洋源益生芽孢杆菌的筛选、鉴定及应用研究[D].南京:南京农业大学,2014.CHEN Y.Screening,identification and applied research of marine probiotic Bacillus spp.[D].Nanjing:Nanjing Agricultural University,2014.
[16] 王金燕,李彬,王印庚,等.刺参养殖池塘一株贝莱斯芽孢杆菌的分离及其生理特性[J].中国水产科学,2018,25(3):567-575.WANG J Y,LI B,WANG Y G,et al.Screening and characteristic analysis of Bacillus velezensis from sea cucumber(Apostichopus japonicus)ponds[J].Journal of Fishery Sciences of China,2018,25(3):567-575.
[17] 杜佗,李彬,王印庚,等.刺参(Apostichopus japonicus)大水面养殖池塘环境中优势益生菌筛选及其特性分析[J].渔业科学进展,2017,38(3):180-187.DU T,LI B,WANG Y G,et al.Screening and characteristic analysis of potential probiotics from large water ponds used for sea cucumber(Apostichopus japonicus)farming[J].Progress in Fishery Sciences,2017,38(3) :180-187.
[18] 刘文亮,许华,唐杨,等.饲料中补充蜡样芽孢杆菌(Bacillus cereus)生物膜对凡纳滨对虾(Litopenaeus vannamei)生长、抗病力及其肠道微生物组成的影响[J].渔业科学进展,2017,38(4):87-95.LIU W L,XU H,TANG Y,et al.The effect of diet with Bacillus cereus biofilm on the growth rate,disease resistance and intestinal microflora of Litopenaeus vannamei[J].Progress in Fishery Sciences,2017,38(4):87-95.
[19] 刘晓勇,张颖,齐茜,等.枯草芽孢杆菌对杂交鲟幼鱼生长性能、消化酶活性及非特异性免疫的影响[J].中国水产科学,2011,18(6):1315-1320.LIU X Y,ZHANG Y,QI Q,et al.Effects of Bacillus subtilis on growth,digestive enzyme activity,and non-specific immunity in hybrid sturgeon(Acipenser baeri♂×Acipenser schrenkii ♀)juveniles[J].Journal of Fishery Sciences of China,2011,18(6):1315-1320.
[20] 马如龙.5株斜带石斑鱼肠道原籍菌的益生作用研究[D].南京:南京农业大学,2010.MA R L.Effect of probiotics on growth performance,digestive enzyme activtie,immunity and gut microbiota of Epimiphelus coioides[D].Nanjing:Nanjing Agricultural University,2010.
[21] LIU C H,CHIU C H,WANG S W,et al.Dietary administration of the probiotic,Bacillus subtilis E20,enhances the growth,innate immune responses,and disease resistance of the grouper,Epinephelus coioides [J].Fish & Shellfish Immunology,2012,33(4):699-706.
[22] WANG Y G,LEE K L,NAJIAH M,et al.A new bacterial white spot syndrome(BWSS)in cultured tiger shrimp Penaeus monodon and its comparison with white spot syndrome(WSS)caused by virus[J].Diseases of Aquatic Organisms,2000,41(1):9-18.
[23] 胡宗云,宋文华,张涛,等.黄颡鱼“红头病”病原菌蜡状芽孢杆菌分离鉴定及药敏分析[J].水产学杂志,2016,29(4):33-37.HU Z Y,SONG W H,ZHANG T,et al.Identification and drug sensitivity test of pathogen Bacillus cereus from yellow catfish(Pelteobagrus fulvidraco)with red-head disease[J].Chinese Journal of Fisheries,2016,29(4):33-37.
[24] 吕利群,刘丽玲,刘浩,等.一株芽孢杆菌用作水霉病防治的研究[J].渔业现代化,2010,37(4):31-34.LV L Q,LIU L L,LIU H,et al.The potential of a Bacillus strain as the biological source of pharmaceutical compound against saprolegeniasis[J].Fishery Modernization,2010,37(4):31-34.
[25] 巴翠玉,张林波,张培军,等.2株枯草芽孢杆菌的分离鉴定及特性研究[J].华南农业大学学报,2017,38(3):46-51.BA C Y,ZHANG L B,ZHANG P J,et al.Isolation,identification and characterization of two strains of Bacillus subtilis[J].Journal of South China Agricultural University,2017,38(3):46-51.
[2] 吴秀红,张新中,周永灿,等.关于芽孢杆菌(Bacillus)应用概述[J].现代渔业信息,2007(7):16-18.WU X H,ZHANG X Z,ZHOU Y C,et al.Outline of related Bacillus spp.[J].Modern Fisheris Information,2007(7):16-18.
[3] 姚清华,王宝珍,郭清雄,等.枯草芽孢杆菌对黑仔期菲律宾鳗鲡生长、体成分和消化酶活性的影响[J].中国食品学报,2016,16(12):177-184.YAO Q H,WANG B Z,GUO Q X,et al.Effects of Bacillus subtilis on growth performance,body composition and digestive enzymes activity of Philippine eel black fry,Anguill marmorata[J].Journal of Chinese Institute of Food Science and Technology,2016,16(12):177-184.
[4] 黄灵,李小梅,舒琥,等.饲料中添加不同益生菌对虎龙斑生长、免疫力及抗氧化能力的影响[J].饲料工业,2016,37(22):15-20.HUANG L,LI X M,SHU H,et al.Effects of dietary probiotics on growth,immune and antioxidant abilities of hybrid grouper(Epinephelus fuscoguttatus ♀×E.lanceolatus ♂)[J].Feed Industry,2016,37(22):15-20.
[5] CHAVES-POZO E,MU?OZ P,LóPEZ-MU?OZ A,et al.Early innate immune response and redistribution of inflammatory cells in the bony fish gilthead seabream experimentally infected with Vibrio anguillarum[J].Cell Tissue Research,2005,320(1):61-68.
[6] 于广宝,张德胜.微生态制剂对提高漠斑牙鲆育苗成活率的作用[J].海洋渔业,2009,31(2):167-172.YU G B,ZHANG D S.The role of microecological modulator in the improvement of seed rearing survival rate of southern flounder Paralichthys lethostigma[J].Marine Fisheries,2009,31(2):167-172.
[7] 曹煜成,李卓佳,杨莺莺,等.地衣芽孢杆菌De株对黄鳍鲷生长及其养殖池塘主要环境因子的影响[J].南方水产,2010,6(3):1-6.CAO Y C,LI Z J,YANG Y Y,et al.Effects of Bacillus licheniform strain De on growth of Sparus latus and main environmental factors in aquaculture pond[J].South China Fisheries Science,2010,6(3):1-6.
[8] SUN Y Z,YANG H L,MA R L,et al.Probiotic applications of two dominant gut Bacillus strains with antagonistic activity improved the growth performance and immune responses of grouper Epinephelus coioides[J].Fish & Shellfish Immunology,2010,29(5):803-809.
[9] 陶荣霞.17株芽孢杆菌产酶特性及益生特性的研究[D].武汉:华中农业大学,2012.TAO R X,Study on characteristics of enzyme production and probiotics by 17 Bacillus strain[D].Wuhan:Huazhong Agricultural University,2012.
[10] 布坎南,吉本斯.伯杰细菌鉴定手册[M].北京:科学出版社,1984:729-731.BUCHANAN R E,GIBBONS N E.Bergey’s manual of systemaic bacteriology[M].Beijing:Science Press,1984:729-731.
[11] 东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:45-65.DONG X Z,CAI M Y.Manual for systematic identification of common bacteria[M].Beijing:Science Press,2001:45-65.
[12] 王腾腾,韦秀梅,常城,等.一株许氏平鲉(Sebastes schlegelii)肠道乳酸菌的分离鉴定及特性分析[J].海洋与湖沼,2017,48(1):94-100.WANG T T,WEI X M,CHANG C,et al.Identification and characteristics of a lactic acid bacteria associated from intestine of black rockfish Sebates schlegelii[J].Oceanologia Et Limnologia Sinica,2017,48(1):94-100.
[13] EI-RHMAN A M A,KHATTAB Y A E,SHALABY A M E.Micrococcus luteus and Pseudomonas species as probiotics for promoting the growth performance and health of Nile tilapia,Oreochromis niloticus.[J].Fish & Shellfish Immunology,2009,27(2):175-180.
[14] 闫法军,田相利,董双林,等.刺参养殖池塘降解有机污染物常、低温芽孢杆菌的分离筛选[J].中国海洋大学学报(自然科学版),2013,43(6):17-24.YAN F J,TIAN X L,DONG S L,et al.Isolation and selection of normal and low temperature Bacillus degrading organic pollutants in sea cucumber culturing ponds[J].Periodical of Ocean University of China(Natural Science),2013,43(6):17-24.
[15] 陈跃.海洋源益生芽孢杆菌的筛选、鉴定及应用研究[D].南京:南京农业大学,2014.CHEN Y.Screening,identification and applied research of marine probiotic Bacillus spp.[D].Nanjing:Nanjing Agricultural University,2014.
[16] 王金燕,李彬,王印庚,等.刺参养殖池塘一株贝莱斯芽孢杆菌的分离及其生理特性[J].中国水产科学,2018,25(3):567-575.WANG J Y,LI B,WANG Y G,et al.Screening and characteristic analysis of Bacillus velezensis from sea cucumber(Apostichopus japonicus)ponds[J].Journal of Fishery Sciences of China,2018,25(3):567-575.
[17] 杜佗,李彬,王印庚,等.刺参(Apostichopus japonicus)大水面养殖池塘环境中优势益生菌筛选及其特性分析[J].渔业科学进展,2017,38(3):180-187.DU T,LI B,WANG Y G,et al.Screening and characteristic analysis of potential probiotics from large water ponds used for sea cucumber(Apostichopus japonicus)farming[J].Progress in Fishery Sciences,2017,38(3) :180-187.
[18] 刘文亮,许华,唐杨,等.饲料中补充蜡样芽孢杆菌(Bacillus cereus)生物膜对凡纳滨对虾(Litopenaeus vannamei)生长、抗病力及其肠道微生物组成的影响[J].渔业科学进展,2017,38(4):87-95.LIU W L,XU H,TANG Y,et al.The effect of diet with Bacillus cereus biofilm on the growth rate,disease resistance and intestinal microflora of Litopenaeus vannamei[J].Progress in Fishery Sciences,2017,38(4):87-95.
[19] 刘晓勇,张颖,齐茜,等.枯草芽孢杆菌对杂交鲟幼鱼生长性能、消化酶活性及非特异性免疫的影响[J].中国水产科学,2011,18(6):1315-1320.LIU X Y,ZHANG Y,QI Q,et al.Effects of Bacillus subtilis on growth,digestive enzyme activity,and non-specific immunity in hybrid sturgeon(Acipenser baeri♂×Acipenser schrenkii ♀)juveniles[J].Journal of Fishery Sciences of China,2011,18(6):1315-1320.
[20] 马如龙.5株斜带石斑鱼肠道原籍菌的益生作用研究[D].南京:南京农业大学,2010.MA R L.Effect of probiotics on growth performance,digestive enzyme activtie,immunity and gut microbiota of Epimiphelus coioides[D].Nanjing:Nanjing Agricultural University,2010.
[21] LIU C H,CHIU C H,WANG S W,et al.Dietary administration of the probiotic,Bacillus subtilis E20,enhances the growth,innate immune responses,and disease resistance of the grouper,Epinephelus coioides [J].Fish & Shellfish Immunology,2012,33(4):699-706.
[22] WANG Y G,LEE K L,NAJIAH M,et al.A new bacterial white spot syndrome(BWSS)in cultured tiger shrimp Penaeus monodon and its comparison with white spot syndrome(WSS)caused by virus[J].Diseases of Aquatic Organisms,2000,41(1):9-18.
[23] 胡宗云,宋文华,张涛,等.黄颡鱼“红头病”病原菌蜡状芽孢杆菌分离鉴定及药敏分析[J].水产学杂志,2016,29(4):33-37.HU Z Y,SONG W H,ZHANG T,et al.Identification and drug sensitivity test of pathogen Bacillus cereus from yellow catfish(Pelteobagrus fulvidraco)with red-head disease[J].Chinese Journal of Fisheries,2016,29(4):33-37.
[24] 吕利群,刘丽玲,刘浩,等.一株芽孢杆菌用作水霉病防治的研究[J].渔业现代化,2010,37(4):31-34.LV L Q,LIU L L,LIU H,et al.The potential of a Bacillus strain as the biological source of pharmaceutical compound against saprolegeniasis[J].Fishery Modernization,2010,37(4):31-34.
[25] 巴翠玉,张林波,张培军,等.2株枯草芽孢杆菌的分离鉴定及特性研究[J].华南农业大学学报,2017,38(3):46-51.BA C Y,ZHANG L B,ZHANG P J,et al.Isolation,identification and characterization of two strains of Bacillus subtilis[J].Journal of South China Agricultural University,2017,38(3):46-51.
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