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刘吉丹, 樊英, 刘洪军, 唐学玺, 王晓璐, 王友红, 叶海斌, 盖春蕾. 斑节对虾(非洲群体)肠道中哈维氏弧菌拮抗菌的筛选、鉴定及生理特性分析[J]. 水生生物学报, 2022, 46(12): 1876-1887. DOI: 10.7541/2022.2021.0286
引用本文: 刘吉丹, 樊英, 刘洪军, 唐学玺, 王晓璐, 王友红, 叶海斌, 盖春蕾. 斑节对虾(非洲群体)肠道中哈维氏弧菌拮抗菌的筛选、鉴定及生理特性分析[J]. 水生生物学报, 2022, 46(12): 1876-1887. DOI: 10.7541/2022.2021.0286
LIU Ji-Dan, FAN Ying, LIU Hong-Jun, TANG Xue-Xi, WANG Xiao-Lu, WANG You-Hong, YE Hai-Bin, GAI Chun-Lei. SCREENING, IDENTIFICATION AND PHYSIOLOGICAL CHARACTERISTICS OF INTESTINAL BACTERIA AGAINST VIBRIO HARVEYI FROM PENAEUS MONODON[J]. ACTA HYDROBIOLOGICA SINICA, 2022, 46(12): 1876-1887. DOI: 10.7541/2022.2021.0286
Citation: LIU Ji-Dan, FAN Ying, LIU Hong-Jun, TANG Xue-Xi, WANG Xiao-Lu, WANG You-Hong, YE Hai-Bin, GAI Chun-Lei. SCREENING, IDENTIFICATION AND PHYSIOLOGICAL CHARACTERISTICS OF INTESTINAL BACTERIA AGAINST VIBRIO HARVEYI FROM PENAEUS MONODON[J]. ACTA HYDROBIOLOGICA SINICA, 2022, 46(12): 1876-1887. DOI: 10.7541/2022.2021.0286

斑节对虾(非洲群体)肠道中哈维氏弧菌拮抗菌的筛选、鉴定及生理特性分析

SCREENING, IDENTIFICATION AND PHYSIOLOGICAL CHARACTERISTICS OF INTESTINAL BACTERIA AGAINST VIBRIO HARVEYI FROM PENAEUS MONODON

  • 摘要: 基于斑节对虾(非洲群体)(Penaeus monodon)肠道菌的生理生化特性, 为解决斑节对虾急性肝胰腺坏死综合征(AHPND)提供新思路, 文章分离纯化健康斑节对虾肠道中的优势菌株; 以致病性哈维氏弧菌为指示菌, 用牛津杯法从纯化菌株中筛选拮抗菌; 通过生理生化特征、Biolog系统鉴定及16S rDNA技术综合方法对菌株进行鉴定; 通过药敏实验, 评价拮抗菌株的安全性; 绘制高敏拮抗菌株的生长曲线, 得出其生长特性; 通过测定其产酶活性, 形成对其益生机制的初步推断。从斑节对虾肠道中共分离得到14株的优势土著菌, 分别标记为P.m-1、P.m-2······P.m-14, 经过拮抗实验得到了3株拮抗菌(P.m-1、P.m-9和P.m-13)。经鉴定P.m-1、P.m-9和P.m-13分别为枯草芽孢杆菌(Bacillus subtilis)、屎肠球菌(Enterococcus faecium)和溶藻弧菌(Vibrio alginolyticus)。药敏实验结果表明, 3株拮抗菌均不属于耐药菌, P.m-1对药物高度敏感。生长特征实验表明: P.m-1在2h进入对数期, 10h达到生长高峰, 菌体密度可达1.14×109 cfu/mL, 表现出了强劲的生命力。经过蛋白酶活性测定, P.m-1具有较强的产酶能力。优势菌P.m-1具有成为益生菌的潜力, 后续将作为功能性饲料添加剂或水质调节剂进行应用, 为AHPND进行生物防治提供新思路。

     

    Abstract: Penaeus monodon (African group) is a new cultured prawn species, which has good development prospects. At present, aquaculture diseases are seriously affecting the health of prawn farming. Based on physiological characteristics of intestinal bacteria in P. monodon, the experimental study was carried out to provide new treatments for acute hepatopancreas necrosis syndrome (AHPND). The dominant intestinal strains in healthy P. monodon were isolated and purified. Using pathogenic Vibrio harveyi as indicator bacteria, antagonistic bacteria were screened from the strains by the Oxford cup method. To identify these antagonistic bacteria, high-throughput sequencing technology was used as the main method. Biolog microbe identification system was also used to identify the strains. These two methods were supplemented by biochemical characteristic tests. To evaluate the safety of antagonistic strains, the drug susceptibility test was carried out in the study. The basic drug resistance information of intestinal bacteria was obtained by this test. According to the drug resistance information, antagonistic strains with high drug sensitivity were selected. The growth curve of the hypersensitive antagonistic strain was plotted to show its growth characteristics. The activity of extracellular enzymes of potential probiotics was calculated. As a result, sixteen dominant indigenous strains were isolated from the intestinal tract of P. monodon. The three antagonistic strains which were named as P.m-1, P.m-9, P.m-13. P.m-1, P.m-9 and P.m-13 were identified as Bacillus subtilis, Enterococcus faecium and Vibrio alginolyticus, respectively. The results of drug sensitivity tests showed that they were not intestinal drug-resistant bacteria, and P.m-1 was highly sensitive to drugs. P.m-1 entered the exponential growth stage from the 2nd hour, and reached the growth peak at the 10th hour. The maximum concentration of P.m-1 was 1.14×109 cfu/mL, which indicated that P.m-1 had the strong vitality. After protease activity determination, P.m-1 was considered as a strain that had the strong protease production capacity, which could provide a basis for digestion or purification of the environment. P.m-1 has the potential to become probiotics, which could contribute to the prevention and treatment of acute hepatopancreas necrosis syndrome. The experiment would provide the theoretical basis for functional feed additives from the perspective of intestinal microecology.

     

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