植物乳杆菌JCM 1149与短乳杆菌JCM 1170停喂后罗非鱼抗病力差异机制研究

Probiotics has been increasingly popular as alternatives to fish dietary antibiotics. However, the potential risk of using probiotics deserves more attention. The applicant found the following interesting phenomenon during the probiotics research. Lactobacillus plantarum subsp. plantarum JCM 1149 and Lactobacillus brevis JCM 1170 have the same adherence ability to the gut of tilapia, the same antagonistic activity against Aeromonas hydrophila NJ-1, as well as the same NJ-1 infection resistance efficiency (disease resistance) when fed to tilapia. However, the stop of feeding JCM 1149 leads to the disease resistance collapsing of tilapia, while JCM 1170 doesn’t have this effect, implying that risk exists in JCM 1149 application. This project will make systematic comparison of the effects of JCM 1149 and JCM 1170 on series barriers of tilapia, i.e., the intestinal microbiota barrier, the mucus barrier, the mechanical barrier, the immune barrier, etc, through analysis tools including resistant plates, microbial diversity sequencing analysis, Ussing chamber, transcriptome, metabolome, etc, to clarify the reason and mechanism underlying the phenomenon of disease resistance collapsing following the stop-feeding of JCM 1149 in tilapia. Accordingly, optimized protocols for administration and stop-feeding of JCM 1149 will be established to avoid the risk involved in its utilization. Moreover, another few highly adhering lactobacilli strains will be characterized with similar process, and the risks involved in their stop-feeding will be evaluated. An improved standard based on the gut adherence property will be proposed for selection of fish dietary probiotic lactobacilli.Overall, the results obtained in this study will provide theoretical and technological supports for the promotion of development and application of safe and efficient fish dietary probiotics.

益生菌使用风险需引起重视：植物乳杆菌植物亚种JCM 1149与短乳杆菌JCM 1170饲喂罗非鱼具有同等肠道黏附力、对嗜水气单胞菌NJ-1抑菌力与抗NJ-1感染能力（抗病力），但JCM 1149停喂会诱发鱼体抗病力崩溃，JCM 1170却不会，提示JCM 1149存在使用风险。为探讨该现象的原因和机理，本项目拟结合罗非鱼抗NJ-1感染崩溃模型，借助抗性平板、微生物多样性测序、尤斯灌注室、转录组及代谢组等分析手段，从肠道微生物屏障、黏液屏障、机械屏障及免疫屏障等角度，系统比较这两株菌的相关生物学特性，弄清JCM 1149停喂诱发鱼体抗病力崩溃的原因和机理，研究和建立针对JCM 1149投饲或停喂环节优化的风险规避方案，并对其它几株高黏附力乳杆菌进行类似的特性鉴定及停喂风险评估，提出鱼类饲用益生乳杆菌基于肠道黏附的优选标准，为推进安全、高效鱼类饲用益生菌的开发与应用提供理论依据和技术支撑。

站在水产养殖业角度，我国全面减少乃至禁止抗生素饲用的任务艰巨且迫切。作为水产动物饲用抗生素的重要替代品，益生菌由于生产门槛低、添加成本不高、易推广而在商业化应用中走在前面。农业农村部2013年批准允许水产动物饲用微生物达31种，其中乳酸菌达20种（占全部批准64.5%），乳酸杆菌8种更是其中的重要代表。但申请人前期工作表明乳酸杆菌水产饲用存在巨大风险，本项目旨在机制上予以澄清并提出风险规避方案。.项目率先揭示了传统益生菌在鱼类养殖调控上的潜在问题与风险。首次划分了益生菌鱼类肠道3大粘附类型（黏膜粘附型、黏液粘附型及混合粘附型），指出黏膜型乳杆菌可致损鱼肠黏膜（Scientific Reports，2017通讯），而黏液型乳杆菌停喂后会因迅速流失而诱发鱼体抗病力崩溃（Scientific Reports，2016通讯），明确了传统益生菌在鱼类养殖调控上的潜在问题与风险，并提出鱼类饲用益生菌肠道混合粘附这一优选标准及双（多）菌减量混合使用方案（Scientific Reports，2017通讯）。为引导行业规范，申请人制订了《水产动物饲用乳酸菌筛选标准》与《水产动物饲用芽孢杆菌筛选标准》团体标准。.项目成果直接引领了水产养殖领域在农业农村部政策法规范围内的饲用益生菌的规范化添加。