Study On The Variation Of Quality And Flora Of Freshwater Fish In Yangzhou Region During Cold Storage

Freshwater fish deteriorates rapidly after postmortem as they provide a nutrition rich environment with high water activity, rich protein and a near-neutral pH optimal for bacterial growth. The bacteria contained in fish skin, gill and intestinal grow rapidly after fish’s death, which eventually led to the spoilage of fish. This paper aims to study the quality changes and the volatile compounds of three popular freshwater fish samples (Crucian carp, Bighead carp and Grass carp) during cold storage; combining the analysis of PCR-DGGE and high throughput sequencing methods with volatile compounds, the specific spoilage organisms were revealed; knowledge about the freshwater’s micro-flora shifts and SSO can significantly benefit the microbiological inspections, shelf-life predictions and study for new preservation or production methods. The main research methods and results of this paper are as follows:1. Three popular freshwater fish samples (grass carp, crucian carp, bighead carp) were collected in Yangzhou. Through the measurements of conductivity, pH and TVBN, K value, microbial counts and QIM analysis, the quality changes of freshwater fish during cold storage were described. The results showed that the indices including conductivity, pH and TVB-N, K value, microbial counts and sensory analysis increased significantly across time(p<0.05). Meanwhile, TVB-N, conductivity, K value, microbial counts showed a extremly significant correlation with sensory analysis (p<0.01), pH were significantly correlated with sensory analysis (p<0.05); Of three kinds of freshwater fish, grass carp corrupted the fastest, followed by bighead carp and crucian carp; grass carp kept good quality within 4 days,while crucian carp and bighead carp within 6 days, and all three fishes were determined to deteriorate from every indices.2. Using SPME-GC-MS method to analyze freshwater fish at different storage points,64 volatile compounds were identified, including 11 aldehydes,5 ketones,15 alcohols,6 acids,3 alkenes,8 esters,8 alkanes and 8 others (2 benzene,1 sulfide and 5 amine); little difference was found among different species:40 compounds detected in grass carp,46 compounds in carp samples and 44 compounds in bighead carp. Characteristic odours of fresh water fish samples were mainly some aldehydes and alcohols, among which hexanal and 1-octen-3-ol contributed most; characteristic odours of spoilage were mainly heptanal, hexanol, isoamyl alcohol, 5-methyl-3-heptanol,2-methyl Cyclopentanol, isobutyric acid, acetic acid, ethyl acetate, dimethyl disulfide,2-Formic acid, histamine acid, trimethylamine, among which 2-Formic acid, histamine acid and trimethylamine are metabolites of degradation of enzymes to proteins or amino acids, which is the main source of the stench of corruption;3. PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) technology was applied to reveal the microflora succession and dominant bacteria of freshwater fish during 4℃ storage. The results showed that the bands in DGGE prifile changed significantly and the most bands found in samples at loss of freshness point; UPGMA clustering and PCA diagram shows the bacterial community at fresh point was significantly different with that at middle and spoilage points; 28 bands were obtained from the DGGE profile and 7 genus were identified as Pseudomonas, Aeromonas, Comamonas, Acinetobacter, Morganella and Iodobacter,78.6% of which belonging to the gammaproteobacteria and 21.4% Betaproteobacteria; the dominant bacteria of freshwater fish at fresh point were Aeromonas, Comamonas and Acinetobacter, the bacterial community at middle point were much complex, including Pseudomonas, Aeromonas, Shewanella and Acinetobacter; the genus of Pseudomonas, Aeromonas, Shewanella and Iodobacter were predominant at spoilage point; Pseudomonas and Shewanella increased across storage, while Aeromonas the contrary trend, and Iodobacter and Morganella were only detected at certain points.4. The amplification of 16S rDNA V3 region were combined with high-throughput sequencing technology, aiming to assess and understand the microbial communities in depth of freshwater fish during cold storage. A total of 1091,338 high quality sequences obtained after quality filtering (QF) of raw reads were clustered into 10635 OTUs at 97% identity threshold. The number of OTU fish at fresh point (1462±1640) was higher than that at middle point (573±216) and spoilage point (694±251); the 10635 OTUs comprised 36 phyla and 549 genera; the dominant bacteria of freshwater fish at fresh point were Pseudomonas, Aeromonas, Comamonas, Acinetobacter and Lactococcus; the dominant bacteria at middle point were Pseudomonas, Aeromonas, Shewanella, Psychrobacter, Janthinobacterium and Neisseria; the dominant bacteria at spoilage point were Pseudomonas, Aeromonas, Neisseria, Shewanella, Moraxella, Psychrobacter and Janthinobacterium; Sequencing associated with Pseudomonas and Shewanella made a large contribution to the spoilage of fish, while Aeromonas presenting a relative low contribution; additionally, Neisseria occupied little at fresh point but showed an increasing trend across the whole storage.