| The protein content of freshwater fish is high, so after the death, spoilage process happens rapidly; microorganism is the main factor of spoilage. Freshwater fish gills, body surface, and intestinal canal carry a large number of bacteria, which can easily lead to spoilage of fish after death. Fishes may be carrying different bacteria, because the living waters, fishing seasons and fish species are different. Therefore, in this paper, we collected different kinds of freshwater fish samples from different regions and analyzed the bacteria flora and the change of micro flora during storage at low temperature. The growth parameters between different strains and spoilage characteristics also were studied in the process of spoilage, which provide theoretical basis on the inhibition of spoilage bacteria in freshwater fishes during cold storage. The main research methods and results are as follows:(1) The samples (grass carp, bighead, and crucian carp) were collected from Hunan Yueyang and Jiangsu Yangzhou.143 strains of cold resistant bacteria were isolated,83 strains of which were isolated from samples of Hunan Yueyang, and the rest 60 strains were isolated from Jiangsu Yangzhou samples. Results of Gram staining showed that 121 strains were G". accounting for 84.6%, bacterial shapes were spherical, rod-shaped and ball rod el al. By morphological observation, physiological and biochemical analysis, Rep-PCR fingerprinting and 16S rDNA sequencing identification, we found that 143 cold resistant isolates were divided into seven genera, namely:Flavobacterium, Shewanella, Acinetobacter, Aeromonas, Psychrobacter, Pseudomonas and Comamonas. Among them, Shewanella strains were the most, including 66 strains, accounted for 36.25% in samples of Hunan Yueyang, followed by Acinetobacter and Pseudomonas, accounted for 25% and 13.75% respectively; Jiangsu Yangzhou samples contained 58.73 high percent of Shewanella strains, followed by Flavobacterium, accounting for 23.81% of the isolated bacteria. We studied flora changes on three species of freshwater fish in the process of cold storage, and found the Shewanella and Pseudomonas was the dominant spoilage bacteria at the end of shelf life.(2) 29 typical representative strains of different species were selected to measure growth curve under the condition of 30℃, there were differences in growth parameters between different genera, the lag phase of most strains were in 2-6 h, and grew rapidly in 6-20 h, went into the stable phase at around 30-35 h. Then 13 strains of psychrotolerants, which were obvious different in growth rate at 30℃, were chose to measure its growth curve at 5℃. In the conition of low temperature, bacteria grew more slowly than under high temperature. The lag phase is longer, and went into the stable period at about 225 h. The strains which had high growth rate under the condition of high temperature, the growth rate were also high under low temperature.(3) The fish blocks were sterilized two minutes with 1.25 g/L hydrogen peroxide, and we simulated the pollution with a concentration of 106 cfu/L single bacteria suspension. The sensory changes, total bacteria, TVB-N value and pH value were determined during storage process, Results showed that sterile fish blocks inoculated with Shewanella putrefaciens and Pseudomonas reached to the high quality life at about 48 h during cold storage. At the same time, the blocks began to smell bad, fish tissue became loose and elasticity decreased. At 96 h to 120 h, the fish began to turn green, the smell is obvious. The beginning of storage, pH decreased, with the decomposition of protein in fish, and release the alkaline substances such as amines, pH value began to rise. The maximum growth rates of Shewanella and Pseudomonas were higher than other strains, which were respectively 0.065 h-1 and 0.0635 h-1; yield factor (YTVB-N/CFU) of Shewanella was 1.47×10-8 mg TVB-N/CFU, which was bigger than Pseudomonas.(4) In this paper, the volatile compounds of grass carp in different conditions were detected by the HS-SPME-GC-MS, the results showed that there were 29,23,25,20,25 kinds of volatile compounds were detected in fresh fish and which were inoculated with Shewanella putrefaciens, Pseudomonas, Acinetobacter and Psychrobacter, but the volatile compounds were diverse. The Zierone, Nitro methane,2-hydroxy propyl amide, Ethyl acetate and 2-nitro ethanol were the highest in volatile components under different conditions, the percents were 8.3%, 28.21%,17.5%,37.7% and 17.05% respectively, therefore, the characteristic odors in fish were different. |
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