将传统的微生物生理生化鉴定技术与16S rDNA分子生物学方法相结合,并通过系统发育树分析,对冷藏带鱼贮藏期间的主要细菌组成比例进行研究。样品在(4±1)℃贮藏,分别于不同贮藏时间取样,对各个阶段的微生物菌落进行分离纯化、形态观察和部分生理生化鉴定,对最终得到的细菌纯培养物直接提取DNA,作16SrDNA PCR扩增,产物经测序后于NCBI与已知序列进行相似性比对,最终得到13株具有典型特征的纯菌株。结果得出,冷藏带鱼在货架期终点时,其主要微生物的种类与所占比例依次为:腐败希瓦氏菌(Shewanella putrefaciens 34.7%)、荧光假单胞菌(Pseudomonas fluorescens 14.5%)、松鼠葡萄球菌(Staphylococcus sciuri 10.2%)、嗜水气单胞菌(Aeromonas hydrophila 8.2%)、弧菌(Vibrio rumoiensis 6.1%)、恶臭假单胞菌(Pseudomonas putida6.1%)、绿色气球菌(Aerococcus viridans 4.1%)、金黄色葡萄球菌(Staphyloccocus aureus 4.1%)、蜡样芽孢杆菌(Bacillus cereus 4.0%)、铜绿假单胞菌(Pseudomonas aeruginosa 2.0%)、嗜冷杆菌(Psychrobacter sp.2.0%)、成团肠杆菌(Enterobacter agglomerans 2.0%)、约氏不动杆菌(Acinetobacter johnsonii 2.0%),其中腐败希瓦氏菌为带鱼冷藏期间的主要优势腐败菌,假单胞菌在带鱼冷藏过程中的所占比例大小依次为荧光假单胞菌>恶臭假单胞菌>铜绿假单胞菌。

In this research,traditional microbiological method combined with molecular technology based on 16S rDNA were used to isolate and identify the dominate spoilage bacteria on cutlassfish(Trichiurus haumeda) under the cold storage,which were analyzed by phylogenetic tree.The products were sampled at different times during the cold storage(4±1)℃,the microbial colonies were isolated and classified by morphological and biochemical tests,then the final pure culture of bacteria DNA were directly extracted and subjected to PCR to amplify the special region of the 16S rDNA,follow by sequencing which were aligned to 16S rDNA gene fragments available from NCBI to find the closest known relatives to this partial 16S rDNA sequences.The result showed that there were mainly 13 typical bacterial strains of Trichiutus haumeda.And their proportions at the end of shelf life were: Shewanella putrefaciens 34.7%,Pseudomonas fluorescens 14.5%,Staphylococcus sciuri 10.2%,Aeromonas hydrophila 8.2%,Vibrio rumoiensis 6.1%,Pseudomonas putida 6.1%,Aerococcus viridans 4.1%,Staphyloccocus aureus 4.1%,Bacillus cereus 4.0%,Pseudomonas aeruginosa 2.0%,Psychrobacter sp.2.0%,Enterobacter agglomerans 2.0%,Acinetobacter johnsonii 2.0%,and respectively.Among them,Shewanella putrefaciens(34.7%) was the Specific Spoilage Organism(SSO),the proportion of Pseudomonas sp.in descending order were Pseudomonas fluorescens,Pseudomonas putida and Pseudomonas aeruginosa.