枯草芽孢杆菌（Bacillus subtilis）对刺参腐皮综合病致病菌的拮抗作用以及对水质的影响
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摘要
刺参(Apostichopus japonicus)属于棘皮动物门(Echinodermata)海参纲(Holothuroidea)楯手目(Aspidochirota)刺参科(Stichopodidae)仿刺参属(Apostichopus)。刺参含有丰富的胶原成分和蛋白聚糖,还含有大量的黏蛋白及多种氨基酸。最近有研究证明刺参含有防衰老的酸性粘多糖(硫酸软骨素),具有较广的抗肿瘤及抗凝血的作用,并具有增强机体免疫功能的作用。
20世纪80年以来,刺参养殖业迅速发展,与此同时,养殖面积的迅速扩张以及集约化养殖方式下的不规范操作,养殖过程中相继出现一系列病害问题,给刺参养殖业带来了巨大的损失。
刺参“腐皮综合病”因其高传染性、高死亡率成为刺参养殖病害研究的重点。其发病特征一般为:感染初期刺参口部先出现感染症状,表现为围口膜松弛,触手对外界刺激反应迟钝,继而出现排脏现象;感染中期刺参身体收缩、僵直,口腹部出现小面积溃疡,形成蓝白色小斑点,逐渐丧失摄食能力;感染末期刺参的病灶变大,溃疡处增多,表皮腐烂面积变大,最后导致刺参死亡,溶化成鼻涕状胶体。
我们从养殖池内中分离得到一株菌Bs-1,通过测定Bs-1对C、N利用能力等30多项生理生化指标,结合表型特征,确定Bs-1为枯草芽孢杆菌(Bacillus subtilis)。
该菌生长迅速,通过PDB培养基的发酵培养,枯草芽孢杆菌Bacillus subtilis(Bs-1)能产生抗菌物质,该发酵液能抑制分离自刺参腐皮综合病病灶部位的致病菌C6、TB的生长;所产生的抗菌物质在一定范围内对热、酸碱、变性剂和蛋白酶K均有较好的稳定性。进一步尝试用Bs-1的含菌培养液来控制养殖海水中C6、TB的数量,结果表明每升海水中加入3-4ml培养液(浓度为1×108CFU·ml~(-1))已能达到较好的抑菌效果;
应用Bs-1对养殖水体中各项水质因子进行控制和处理,实验接种菌液量为4、7、10ml(1×108CFU·ml~(-1))。结果表明:枯草芽孢杆菌能显著降低养殖试验池NH_3-N,NO_2~--N, COD等污染物浓度。使用效果表明:在投加枯草芽孢杆菌菌液后,水质各项指标均优于未投加菌液的对照池,NH_3-N,NO_2-N, COD污染物的最高去除率分别是:94.5%,90.3%和68.3%,明显改善养殖水体水质条件,因此,Bs-1有望开发成良好的益生菌剂。
Apostichopus japonica belongs to Echinodermata, Holothuroidea, Aspidochirota, Stichopodidae, Apostichopus. Apostichopus japonica is well know for its rich in Chondroitin sulfate, acidic mucopolysaccharides and collagen, and effect of enhancing human’s immune system.
Since 1980s, rearing industry of Apostichopus japonica is booming. At the same time, a series of diseases on rearing industry have emerged and bring enormous losses on rearing industry.
Reared Apostichopus japonica in China suffers from a kind of skin ulceration disease, which has caused severe economic loss in recent years. The course of this disease is often very short, usually several days. The infection involves a head shaking and viscera expelling at early time, and is followed with mouth and cloacal opening tumidity, then the body of the diseased individuals will shrink and titanic together with white or blue lesion spots on the body wall. In the later period, these small lesions grow more and bigger to large ulceration and finally causes the death of individuals. This disease is highly infectious, and can often cause all individuals reared in the same pool die in a very short time.
A Bacillus strain was isolated from sea water, To identify the BS-1 strains, 30 assays of physiological and biochemical determination were done. And according to the previous studies,which established the position of the two strains in bacterial phylogeny through spore stain,Bs-1 were identified as Bacillus subtilis.
Marine-derived Bacillus subtilis (Bs-1)grown in PDB medium produces a antimicrobial substance which can inhibit the growth of severa1 marine bacteria isolated from sea cucumber. 1ike C6、TB.The production characteristics of the antimicrobial substance and the antimicrobia1 activity were studied. The results showed that Bs-1 grew rapidly. The results also showed that the substance was stable to heat, pH, denature reagent and proteinase to some extent. Further use of the culture supernatant of Bs-1 to control the C6、TB population demonstrated that the best result could be obtained when 3-4 ml supernatant per liter was added to the water.
In application experiment,4、7、10mL per cubic meter of the Bs-1 broth culture was inoculated into fish pond water. The result showed the application of bacillus subtilis could efficiently decompose pollutants and the organic substances in the fish pond water. The concentrations of NH_3-N,NO_2-N, COD were reduced significantly comparing with the non-inoculating control. The highest remove rate of NH_3-N,NO_2-N, COD were 94.5%, 90.3% and 68.3%,respectively. Thus,Bs-1 could probably be developed to be an efficient probiotic agent.
20世纪80年以来,刺参养殖业迅速发展,与此同时,养殖面积的迅速扩张以及集约化养殖方式下的不规范操作,养殖过程中相继出现一系列病害问题,给刺参养殖业带来了巨大的损失。
刺参“腐皮综合病”因其高传染性、高死亡率成为刺参养殖病害研究的重点。其发病特征一般为:感染初期刺参口部先出现感染症状,表现为围口膜松弛,触手对外界刺激反应迟钝,继而出现排脏现象;感染中期刺参身体收缩、僵直,口腹部出现小面积溃疡,形成蓝白色小斑点,逐渐丧失摄食能力;感染末期刺参的病灶变大,溃疡处增多,表皮腐烂面积变大,最后导致刺参死亡,溶化成鼻涕状胶体。
我们从养殖池内中分离得到一株菌Bs-1,通过测定Bs-1对C、N利用能力等30多项生理生化指标,结合表型特征,确定Bs-1为枯草芽孢杆菌(Bacillus subtilis)。
该菌生长迅速,通过PDB培养基的发酵培养,枯草芽孢杆菌Bacillus subtilis(Bs-1)能产生抗菌物质,该发酵液能抑制分离自刺参腐皮综合病病灶部位的致病菌C6、TB的生长;所产生的抗菌物质在一定范围内对热、酸碱、变性剂和蛋白酶K均有较好的稳定性。进一步尝试用Bs-1的含菌培养液来控制养殖海水中C6、TB的数量,结果表明每升海水中加入3-4ml培养液(浓度为1×108CFU·ml~(-1))已能达到较好的抑菌效果;
应用Bs-1对养殖水体中各项水质因子进行控制和处理,实验接种菌液量为4、7、10ml(1×108CFU·ml~(-1))。结果表明:枯草芽孢杆菌能显著降低养殖试验池NH_3-N,NO_2~--N, COD等污染物浓度。使用效果表明:在投加枯草芽孢杆菌菌液后,水质各项指标均优于未投加菌液的对照池,NH_3-N,NO_2-N, COD污染物的最高去除率分别是:94.5%,90.3%和68.3%,明显改善养殖水体水质条件,因此,Bs-1有望开发成良好的益生菌剂。
Apostichopus japonica belongs to Echinodermata, Holothuroidea, Aspidochirota, Stichopodidae, Apostichopus. Apostichopus japonica is well know for its rich in Chondroitin sulfate, acidic mucopolysaccharides and collagen, and effect of enhancing human’s immune system.
Since 1980s, rearing industry of Apostichopus japonica is booming. At the same time, a series of diseases on rearing industry have emerged and bring enormous losses on rearing industry.
Reared Apostichopus japonica in China suffers from a kind of skin ulceration disease, which has caused severe economic loss in recent years. The course of this disease is often very short, usually several days. The infection involves a head shaking and viscera expelling at early time, and is followed with mouth and cloacal opening tumidity, then the body of the diseased individuals will shrink and titanic together with white or blue lesion spots on the body wall. In the later period, these small lesions grow more and bigger to large ulceration and finally causes the death of individuals. This disease is highly infectious, and can often cause all individuals reared in the same pool die in a very short time.
A Bacillus strain was isolated from sea water, To identify the BS-1 strains, 30 assays of physiological and biochemical determination were done. And according to the previous studies,which established the position of the two strains in bacterial phylogeny through spore stain,Bs-1 were identified as Bacillus subtilis.
Marine-derived Bacillus subtilis (Bs-1)grown in PDB medium produces a antimicrobial substance which can inhibit the growth of severa1 marine bacteria isolated from sea cucumber. 1ike C6、TB.The production characteristics of the antimicrobial substance and the antimicrobia1 activity were studied. The results showed that Bs-1 grew rapidly. The results also showed that the substance was stable to heat, pH, denature reagent and proteinase to some extent. Further use of the culture supernatant of Bs-1 to control the C6、TB population demonstrated that the best result could be obtained when 3-4 ml supernatant per liter was added to the water.
In application experiment,4、7、10mL per cubic meter of the Bs-1 broth culture was inoculated into fish pond water. The result showed the application of bacillus subtilis could efficiently decompose pollutants and the organic substances in the fish pond water. The concentrations of NH_3-N,NO_2-N, COD were reduced significantly comparing with the non-inoculating control. The highest remove rate of NH_3-N,NO_2-N, COD were 94.5%, 90.3% and 68.3%,respectively. Thus,Bs-1 could probably be developed to be an efficient probiotic agent.
引文
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