基于高通量测序的牡蛎冷藏过程中微生物群落分析

为揭示牡蛎冷藏过程中微生物群落结构的变化规律,采用高通量测序技术对冷藏0、4、8 d牡蛎的菌群进行分析。结果表明高通量测序可以全面反映牡蛎中菌群的物种组成及丰度信息。牡蛎体附着的细菌以变形菌门(Proteobacteria)、γ-变形菌纲(Gammaproteobacteria)、弧菌目(Vibrionales)为主。在科的分类水平上,主要是弧菌科(Vibrionaceae)、希瓦氏菌科(Shewanellaceae)和交替假单胞菌科(Pseudoalteromonadaceae),其中初始样品以Vibrionaceae为主,比例达36.8%;第4天样品Pseudoalteromonadaceae比例相对较高;第8天样品同样以Pseudoalteromonadaceae为主,而Shewanellaceae比例明显增加,达到19.5%。在属的分类水平上,在牡蛎初始菌群中比例前三位依次为弧菌属(Vibrio)、希瓦氏菌属(Shewanella)和交替假单胞菌属(Pseudoalteromonas),其中Vibrio在冷藏前期比例迅速下降;Shewanella和Pseudoalteromonas在系统进化关系上较为接近,在冷藏后期比例占优,可能在牡蛎腐败过程中起到重要作用。研究结果可以为牡蛎的贮运保鲜策略提供参考。

Microbial flora analysis of oyster during refrigerated storage by high throughput sequencing technology

Abstract: Oysters are perishable food due to their high contents in moisture and nutrients, as well as the bacterium amount and diversity of high level. Microorganisms play a key role in the quality deterioration of shellfish. Therefore, the study on the microbial community has great importance in the freshness-keeping and preservation of aquatic products. To analyze microbial flora is greatly limited for traditional cultivation method because only a small part of bacteria can be cultured. Some molecular biology technologies such as real-time polymerase chain reaction (real-time PCR), denaturing gradient gel electrophoresis (DGGE), and terminal restriction fragment length polymorphism (TRFLP) also have their individual shortcomings. Nowadays, high throughput sequencing technology is emerging and has attracted wide attention of researchers. In this study, high throughput sequencing technology based on Illumina MiSeq 2500 PE250 was applied to investigate the changes in bacteria composition of oysters during refrigerated storage. Results showed that high throughput sequencing technology had great advantages and application prospect in food microbiology. The quality of total bacterial DNA (deoxyribonucleic acid) extraction and amplification of the 16SrDNA-V4 area was satisfied and could meet the needs of database construction. The number of raw/effective sequences was 56 867/39 390, 45 210/30 847 and 39 873/27 603 respectively for the sample stored for 0, 4 and 8 d. The number of OTUs (operational taxonomic units) was 587, 699 and 531 respectively. The rarefaction curve indicated that the numbers of sequences were adequate, and the numbers of OTUs were almost saturated. For the sample stored for 0, 4 and 8 d, the Chao1 index was 587.2, 766.6 and 683.0, the Simpson index was 0.945, 0.897 and 0.856, and the Shannon index was 5.48, 5.51 and 4.34, respectively; besides, the goods coverage was 0.996, 0.995 and 0.993, respectively. The research data showed that the dominant bacteria belonged to Proteobacteria in phylum level, Gammaproteobacteria in class level and Vibrionales in order level. In the family level, Vibrionaceae, Shewanellaceae and Pseudoalteromonadaceae were dominant, however their proportions were different among the oyster samples with different storage time. In the genus level, the proportion of Vibrio (28.3%), Shewanella (10.3%) and Pseudoalteromonas (7.2%) was relatively high in the oyster (stored for 0 d). During ice storage, the proportion of Vibrio decreased significantly when stored for 1 d, and reached 6.4% when stored for 4 d. However, the proportion of Vibrio changed little in the later stage, which was 6.2% when stored for 8 d. The proportion of Shewanella decreased to 4.0% when stored for 4 d and increased since then; when stored for 8 d, their proportion increased to 19.5%. The proportion of Pseudoalteromonas increased from 7.2% (0 d) to 32.2% (8 d), because of their strong adaptability to low temperature. Pseudoalteromonas and Shewanella could cause offensive odor by producing small molecule volatile substances, like NH3, H2S and TMAO. They played important role in the quality deterioration of oysters during cold storage. Therefore, the means that can strategically inhibit the growth and metabolism of Pseudoalteromonas and Shewanella will help to improve the storage quality and extend the shelf life of oysters. This study can provide reference for the microbial ecology research onfresh fishery products, and enrich the basic theory for the freshness keeping of oysters.