刺参养殖池塘中一株植物乳杆菌的分离及其生物学特性

Screening and Characteristic Analysis of Lactobacillus plantarum from Ponds for Sea Cucumber Farming

中文关键词: 刺参  腐皮综合征  灿烂弧菌  假交替单孢菌  植物乳杆菌  抑菌活性

英文关键词: Apostichopus japonicus  Skin ulcer syndrome  Vibrio splendidus  Pseudoalteromonas nigrifaciens  Lactobacillus plantarum  Antagonistic activity

中文摘要:

      2017年5月，从山东东营刺参(Apostichopus japonicus)养殖池塘底泥中分离获得49株乳酸菌。以刺参“腐皮综合征”的2株致病菌[灿烂弧菌(Vibrio splendidus)和假交替单胞菌(Pseudoalteromonas nigrifaciens)]为指示菌进行拮抗作用实验，获得1株具有显著抑菌活性的乳酸菌CLY-5，对该菌株进行了生理生化实验、16S rDNA序列分析、生长特性及其对刺参的安全性研究。结果显示，菌株CLY-5对灿烂弧菌和假交替单胞菌具有较好的抑制作用，且菌株的胞内产物与胞外产物均具有抑菌效果，抑菌圈分别为20 、23 mm和27 、38 mm，胞外产物的拮抗作用优于胞内产物。利用该菌对刺参进行高浓度浸浴测试其安全性，浓度为1×109、1×108和1×107 CFU/ml，实验期间刺参状态良好，无死亡现象。16S rDNA序列分析表明，CLY-5与植物乳杆菌(Lactobacillus plantarum NR117813.1)的相似性为100%，初步鉴定该菌株为植物乳杆菌。菌株CLY-5在30℃~44℃、pH 6~8范围内生长较快，20 h进入对数生长期，28~32 h达到生长高峰。筛选的植物乳杆菌具有良好的抑菌能力，且其生长特性适应刺参池塘的养殖环境，为刺参疾病的生态防控及乳酸菌资源的开发提供应用数据参考。

英文摘要:

      Forty-nine lactic acid bacteria strains were isolated from sediment samples from cultured ponds of sea cucumbers (Apostichopus japonicus) in Dongying, Shandong Province, in May 2017. First, antagonistic bacterial experiments were conducted using Vibrio splendidus and Pseudoalteromonas nigrifaciens, which are the main pathogenic bacteria of “skin ulcer syndrome” for sea cucumbers, and a lactic acid bacteria CLY-5, which has demonstrated significant bacteriostatic activity. Next, physiological and biochemical experiments and a similarity analysis of 16S rDNA sequences as well as the growth character of CLY-5 were carried out. A safety experiment for sea cucumbers was also performed. The results revealed that CLY-5 strain successfully inhibited V. splendidus and P. nigrifaciens. Meanwhile, the strain’s intracellular and extracellular products effectively inhibited V. splendidus and P. nigrifaciens, as demonstrated by inhibition zone diameters of 20 mm, 23 mm, and 27 mm, 38 mm, respectively, with extracellular products showing superior performance compared with intracellular products. Dipping bath concentrations were set to 1×109, 1×108, and 1×107 CFU/ml to test the safety of CLY-5 for sea cucumbers. During the experiment, all sea cucumbers were in good conditions. Based on the 16S rDNA sequence analysis, strain CLY-5 was determined to be identical to Lactobacillus plantarum NR117813.1. In addition, strain CLY-5 exhibited superior growth at 30℃~44℃ and salinities of 6 to 8. Moreover, CLY-5 came into a logarithmic growth phase after 20 h of cultivation and reached peak growth after 28~32 h. In summary, strain CLY-5 could be used to reduce the occurrence of sea cucumber disease and is also suitable for pond culture environment. These results provide a basis for ecological prevention strategies in disease control, and should prove useful in the development and utilization of lactic acid bacteria.