2株海水鱼源潜在益生菌的分离、鉴定及特性分析

Isolation, identification, and characterization of two potential probiotics from marine fish

关键词:  许氏平鲉  大泷六线鱼  枯草芽孢杆菌  河流漫游球菌  产酶  抑菌

Key words:  Sebastes schlegelii  Hexagrammos otakii  Bacillus subtilis  Vagococcus fluvialis  Enzyme production  Bacteriostasis

摘要:

为筛选用于微生态制剂研发的海水鱼源益生菌，本研究对海捕野生许氏平鲉(Sebastes schlegelii)和大泷六线鱼(Hexagrammos otakii)的消化道内壁黏膜样品进行细菌分离纯化，获得80株可培养细菌。使用选择性培养基对菌株的产酶能力进行测定，选取海水鱼常见病原菌为指示菌测定分离菌株产物的抑菌活性，筛选出2株潜在益生菌TS2和TH8，并进行菌株的生理生化检测、16S rDNA序列分析、生长特性及其对宿主安全性的研究。结果显示，TS2产蛋白酶(protease)、淀粉酶(amylase)和脂肪酶(lipase)，其无菌培养产物可显著抑制鳗弧菌(Vibrio anguillarum)、副溶血弧菌(Vibrio parahaemolyticus)、哈维氏弧菌(Vibrio harvey)和假交替单胞菌(Pseudoalteromonas nigrifaciens)的生长。TH8产蛋白酶和脂肪酶，其无菌培养产物可显著抑制鳗弧菌、溶藻弧菌(Vibrio alginolyticus)、副溶血弧菌、假交替单胞菌、嗜水气单胞菌(Aeromonas hydrophila)、金黄色葡萄球菌(Staphyloccocus aureus)和大肠杆菌(Escherichia coli)的生长。基于细菌生理生化检测和16S rDNA序列比对，鉴定TS2为枯草芽孢杆菌(Bacillus subtilis)，TH8为河流漫游球菌(Vagococcus fluvialis)。TS2在温度为15~40 ℃、NaCl浓度为0~0.08 g/L、pH为5~9时生长较快，6 h进入对数期，26 h后进入稳定期；TH8在温度为20~40 ℃、NaCl浓度为0~0.08 g/L、pH为5~12时生长较快，2 h进入对数期，14 h后进入稳定期。菌株对同源宿主的安全性检测发现，TS2和TH8在108 CFU/mL浓度条件下对同源宿主是相对安全的。筛选的枯草芽孢杆菌TS2、河流漫游球菌TH8产酶能力强，其产物能抑制多种病原菌的生长，具有广温、广盐、耐酸碱、生长速度快等优点，可作为候选菌株开发为微生态制剂，研究结果为其在海水鱼养殖业中的应用提供数据支撑。

Abstract:

Marine fish are rich in minerals (calcium and iron), vitamins (riboflavin and niacin), nutrients that are beneficial for brain (fatty acids), docosahexaenoic acid that prevent cardiovascular and cerebrovascular diseases, and are an important source of protein for humans. In recent years, the nexus between the increased demand of high-quality protein and the decrease in marine fishery resources resulted in the intensification of marine farming. However, this high-density farming has led to various bacterial diseases frequently due to lack of disease control methods, which has caused economic losses in the farming industry and impeded the healthy development of the industry. Therefore, it is particularly important to reduce the rates of bacterial diseases and improve the survival rate of cultured fish. It is of particular concern on how to maintain intestinal health of cultured fish under this farming method. Probiotics, a kind of living microorganisms, are beneficial to the health of the host. In the 1980s, probiotics have been viewed as an environmentally friendly and effective product in aquaculture; they improve the host health by influencing intestinal microbiota and nonspecific immunity to increase disease resistance ability. Moreover, they can be used as an ideal substitute for antibiotics in aquaculture production. Probiotics from Bacillus, lactic acid bacteria, saccharomyces, and nitrobacteria are diverse, and the function of each varies widely. As the typical representative of probiotics, lactic acid bacteria, may inhibit the growth of pathogens by the production of its metabolites, such as lactic acid, acetic acid, peroxide hydrogen, and bacteriocin. Lactic acid bacteria are also able to balance intestinal microecological imbalance and maintain intestinal microbiota balance when the intestinal microbiota contains pathogenic bacteria or the host is treated with antibiotics. Bacillus is an aerobic or facultative anaerobic gram-positive bacteria, which is stable, possesses strong stress resistance and high resurrection rate, and can produce various macromolecules, such as proteases and amylases and thus can improve the digestive function of the host by promoting the absorption of nutrients. Furthermore, as a non-specific immune antigen, Bacillus can improve the immune resistance of the host by stimulation of the components of cells or cell walls. The application of lactic acid bacteria and Bacillus has demonstrated favorable results, but this has been limited in marine fish culture because the non-fish origin of some strains and the different specificity of strains for different fish species or the same growth stage make their application difficult. Thus, it is essential to develop marine fish-derived probiotics, analyze their characteristics, define growth conditions, verify safety effects, and determine dosage and methods for their administer in marine fish culture. In this study, probiotics from marine fish were screened for the development of microbial ecological agents; 80 strains of culturable bacteria were obtained by separating bacteria from the mucosa samples of the digestive tract of wild Sebastes schlegelii and Hexagrammos otakii. The enzyme-producing ability of the strains was determined using a selective culture medium. The common pathogenic bacteria infecting marine fish were selected as indicator bacteria to determine the antibacterial activity of the isolated strains. Two potential probiotics, strains TS2 and TH8, were screened, and their physiological and biochemical identification, 16S rDNA sequence, growth characteristics, and host safety were determined. The results showed that TS2 had the strongest ability to hydrolyze starch, protein, and fat, and its sterile culture products could significantly inhibit the growth of Vibrio anguillarum, V. parahaemolyticus, V. Harvey, and Pseudoalteromonas nigrifaciens. TH8 has the strongest ability to hydrolyze protein and fat, and its sterile culture products could significantly inhibit the growth of V. anguillarum, V. alginolyticus, V. parahaemolyticus, P. nigrifaciens, Aeromonas hydrophila, Staphyloccocus aureus, and Escherichia coli. According to the analysis of the physiological and biochemical characteristics of the bacteria and 16S rDNA sequence alignment analysis, strain TS2 was identified as Bacillus subtilis and strain TH8 as Vagococcus fluvialis. Strain TS2 showed significant growth at 15–40 ℃, sodium chloride concentration of 0–0.08 g/L, and pH of 5–9; it entered the logarithmic phase after 6 h and the stable phase after 26 h. TH8 grew rapidly at 20–40 ℃, sodium chloride concentration of 0–0.08 g/L, and pH of 5–12; it entered the logarithmic phase after 2 h and the stable phase after 14 h. The safety of strains TS2 and TH8 was analyzed on a homologous host, and it was found that the strains were relatively safe for the homologous host at the concentration of 108 CFU/mL. The screened B. subtilis TS2 and V. fluvialis TH8 strains have a strong enzyme production ability and inhibit the growth of various pathogens by their metabolites; they have the following advantages: Wide temperature and salt tolerance, acid and alkali resistance, and fast growth speed. Thus, they could be considered as potential probiotic candidates for the development of microbial pharmaceuticals and can be used in more applications in marine aquaculture.