罗非鱼无乳链球菌拮抗株的筛选及特性研究
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摘要
自吉富罗非鱼养殖池塘水体、底泥和鱼体肠道中筛选无乳链球菌拮抗株。对其中1株抑菌能力最强的菌株LT3-1进行溶血性、形态、生理生化、16S rRNA基因序列测定,并进一步对其抑菌活性与发酵时间的关系、抑菌物质对温度的耐受性、产酶性能、降解亚硝酸盐性能、药物敏感性、安全性试验、攻毒试验进行研究。鉴定结果表明,菌株LT3-1为枯草芽孢杆菌,无溶血性。液体发酵16 h时,抑菌活性最强,抑菌圈平均直径为(25.30±0.57) mm。随温度的升高抑菌活性下降,但仍然存在,说明抑菌物质有耐高温特性。枯草芽孢杆菌LT3-1具有水解蛋白、淀粉和纤维素的能力。亚硝酸盐降解试验表明,枯草芽孢杆菌LT3-1菌液密度为10~5 cfu/mL时,6 h即可将亚硝酸盐质量浓度由2.5 mg/L降至0.03 mg/L,降解率达98.8%(P<0.05)。枯草芽孢杆菌LT3-1对检测的9种抗生素均敏感,罗非鱼接种密度为10~7 cfu/mL的枯草芽孢杆菌LT3-1菌液200μL后,仍然存活且未有异常表现。动物攻毒试验表明,添加0.05%枯草芽孢杆菌LT3-1菌粉的试验组罗非鱼存活率提高20%,显著高于对照组(P<0.05)。综上所述,筛选自吉富罗非鱼肠道的枯草芽孢杆菌LT3-1能有效拮抗罗非鱼无乳链球菌,可以作为防治当地罗非鱼无乳链球菌病的良好菌种资源。
In this study, one of Bacillus strains with an antagonistic effect on Streptococcus agalactiae, the strain LT3-1 with strongest antagonistic effect, screened from pond water, sediment and intestinal tract of GIFT tilapia Oreochromis niloticus was identified by hemolytic, morphological, physiological and biochemical characteristics, and 16 S rRNA gene sequencing. The relationship between antibacterial activity and fermentation time, tolerance of antibacterial substance to temperature, ability of enzyme production and nitrite degradation, drug sensitivity, safety and infection were also investigated in the strain LT3-1. Results showed that LT3-1 was identified as Bacillus subtilis without hemolysis and with the best antagonistic effect, the strongest antibacterial activity, with average inhibition diameter of 25.30 mm in 16 h fermentation. With the increasing temperature, antibacterial activity of LT3-1 was observed, even though decreased, indicating that antibacterial substance of LT3-1 can withstand high temperature. LT3-1 hydrolyzed casein, starch and cellulose, degrade nitrite from 2.5 mg/L to 0.03 mg/L within 6 h at 10~5 cfu/mL(P<0.05), showing good ability of water purification. Besides, it was sensitive to nine kinds of antibiotics tested, and the safety test indicated that the tilapia injected with 10~7 cfu/mL LT3-1 were alive and no showed abnormal symptoms. The infection test revealed that there was 20% higher survival rate in the tilapia in 0.05% addition of LT3-1 than that in the control group(P<0.05). Therefore, Bacillus subtilis LT3-1 has an obvious antagonistic effect on S. agalactiae in tilapia and can be an excellent candidate for the safe and effective control of S. agalactiae.
In this study, one of Bacillus strains with an antagonistic effect on Streptococcus agalactiae, the strain LT3-1 with strongest antagonistic effect, screened from pond water, sediment and intestinal tract of GIFT tilapia Oreochromis niloticus was identified by hemolytic, morphological, physiological and biochemical characteristics, and 16 S rRNA gene sequencing. The relationship between antibacterial activity and fermentation time, tolerance of antibacterial substance to temperature, ability of enzyme production and nitrite degradation, drug sensitivity, safety and infection were also investigated in the strain LT3-1. Results showed that LT3-1 was identified as Bacillus subtilis without hemolysis and with the best antagonistic effect, the strongest antibacterial activity, with average inhibition diameter of 25.30 mm in 16 h fermentation. With the increasing temperature, antibacterial activity of LT3-1 was observed, even though decreased, indicating that antibacterial substance of LT3-1 can withstand high temperature. LT3-1 hydrolyzed casein, starch and cellulose, degrade nitrite from 2.5 mg/L to 0.03 mg/L within 6 h at 10~5 cfu/mL(P<0.05), showing good ability of water purification. Besides, it was sensitive to nine kinds of antibiotics tested, and the safety test indicated that the tilapia injected with 10~7 cfu/mL LT3-1 were alive and no showed abnormal symptoms. The infection test revealed that there was 20% higher survival rate in the tilapia in 0.05% addition of LT3-1 than that in the control group(P<0.05). Therefore, Bacillus subtilis LT3-1 has an obvious antagonistic effect on S. agalactiae in tilapia and can be an excellent candidate for the safe and effective control of S. agalactiae.
引文
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[20] 柴俊,郤翠仙,张以芳.乳酸杆菌主要代谢产物种类及其特性[J].食品工业科技,2007,28(8):257-260.
[21] 连燕萍.解淀粉芽孢杆菌MG-3抗菌物质的分离纯化及其应用[D].福州:福州大学,2016.
[22] 张欢,丛丽娜,侯英敏,等.海洋枯草芽孢杆菌HS-A38产直链脂肽活性物质的初步检测[J].海洋科学,2011,35(8):37-42.
[23] 马树瑞,丛丽娜,孙蕾,等.产直链脂肽类抗菌物质枯草芽孢杆菌HS-A38的生物学特性[J].大连工业大学学报,2016,35(3):177-180.
[24] 刘佳欣,丛丽娜,李成,等.枯草芽孢杆菌HS-A38产抗菌脂肽Bacillomycin D的组分分析及鉴定[J].工业微生物,2017,47(2):11-17.
[25] Anadon A,Maria R M,Maria A M.Probiotics for animal nutrition in the European Union-Regulation and safety assessment[J].Regulatory Toxicology and Pharmacology,2006,45(1):91-95.
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[27] 刘勇,张勇,张和平.世界益生菌安全性评价方法[J].中国食品学报,2011,11(6):141-151.
[28] 王晓琳,马甡,单洪伟.两株潜在益生菌安全性评价及其对凡纳滨对虾免疫力和抗WSSV能力影响[J].中国海洋大学学报:自然科学版,2016,46(10):39-47.
[2] 邓永强,汪开毓.鱼类无乳链球菌病的研究进展[J].中国畜牧兽医,2016,43(9):2490-2495.
[3] 李凌波,黄天聚.罗非鱼病例分析及用药选择[J].海洋与渔业技术,2014(10):50-51.
[4] 赵光军.拮抗菌的筛选及其对无乳链球菌粘附罗非鱼粘液的拮抗作用研究[D].海口:海南大学,2014.
[5] 李仕成.拮抗罗非鱼无乳链球菌的芽孢杆菌筛选与研究[D].长沙:湖南农业大学,2016.
[6] 汪攀,易敢峰,孙自博,等.一株凝结芽孢杆菌的分离鉴定及益生性研究[J].中国畜牧兽医,2017,44(4):1195-1202.
[7] 陈辉.海豚链球菌高效拮抗菌的筛选、特性分析及其生态效应的研究[D] .长沙:湖南农业大学,2010.
[8] Yamashita M M,Pereira S A,Cardoso L,et al.Probiotic dietary supplementation in Nile tilapia as prophylaxis against streptococcosis[J].Aquaculture Nutrition,2017,23(6):1235-1243.
[9] 刘观斌,王淼,卢迈新.一株能抑制罗非鱼源无乳链球菌的反硝化芽孢杆菌的筛选鉴定[J].中国水产科学,2016,23(1):207-217.
[10] Wing K N,Young C K,Nicholas R,et al.Effects of dietary probiotics on the growth and feeding efficiency of red hybrid tilapia,Oreochromis sp.,and subsequent resistance to Streptococcus agalactiae[J].Journal of Applied Aquaculture,2014,26(1):22-31.
[11] 许本宏,林俊芳,叶志伟,等.带鱼肠道中芽孢杆菌的分离鉴定及其发酵液抗菌性质研究[J].水产科学,2018,37(2):193-200.
[12] 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.
[13] Paul D V,George M G,Dorothy J,et al.Bergery′s manual of systematic bacteriology(2)[M].Athens,USA:Springer,2009.
[14] 田珂,贾爱卿,陈善真,等.罗非鱼无乳链球菌生物学特性与致病力研究[J].中国畜牧兽医,2013,40(6):203-207.
[15] 黄艳华,彭亚,刘杰.罗非鱼致病性无乳链球菌的分离鉴定及药敏试验[J].南方农业学报,2014,45(3):498-504.
[16] 张德锋,刘礼辉,任燕,等.我国罗非鱼源新型无乳链球菌的分离、鉴定及其分子特征[J].中国水产科学,2015,22(5):1044-1054.
[17] 王蓓,黎源,陈贺,等.我国华南地区罗非鱼源无乳链球菌分子流行病学研究[J].水产科学,2014,33(12):791-799.
[18] 刘树彬,王新锐,林壮其,等.枯草芽孢杆菌HAINUP40水质净化作用的研究[J].水产科学,2018,37(2):159-166.
[19] 李雪莉,王超,虞德夫,等.猪源乳酸菌的分离、鉴定及其生物学特性[J].微生物学报,2017,57(12):1879-1887.
[20] 柴俊,郤翠仙,张以芳.乳酸杆菌主要代谢产物种类及其特性[J].食品工业科技,2007,28(8):257-260.
[21] 连燕萍.解淀粉芽孢杆菌MG-3抗菌物质的分离纯化及其应用[D].福州:福州大学,2016.
[22] 张欢,丛丽娜,侯英敏,等.海洋枯草芽孢杆菌HS-A38产直链脂肽活性物质的初步检测[J].海洋科学,2011,35(8):37-42.
[23] 马树瑞,丛丽娜,孙蕾,等.产直链脂肽类抗菌物质枯草芽孢杆菌HS-A38的生物学特性[J].大连工业大学学报,2016,35(3):177-180.
[24] 刘佳欣,丛丽娜,李成,等.枯草芽孢杆菌HS-A38产抗菌脂肽Bacillomycin D的组分分析及鉴定[J].工业微生物,2017,47(2):11-17.
[25] Anadon A,Maria R M,Maria A M.Probiotics for animal nutrition in the European Union-Regulation and safety assessment[J].Regulatory Toxicology and Pharmacology,2006,45(1):91-95.
[26] 冯媛媛,乔琳,姚宏明,等.益生菌安全性和有效性评价方法的研究进展[J].中国畜牧兽医,2017,44(7):2022-2032.
[27] 刘勇,张勇,张和平.世界益生菌安全性评价方法[J].中国食品学报,2011,11(6):141-151.
[28] 王晓琳,马甡,单洪伟.两株潜在益生菌安全性评价及其对凡纳滨对虾免疫力和抗WSSV能力影响[J].中国海洋大学学报:自然科学版,2016,46(10):39-47.