| China has been the largest aquaculture country in the world over the past decade.However,due to the relative backwardness of cold chain technology,a large part of aquatic products have become corrupted during transport and storage,causing great economic losses.Pseudomonas is the dominant spoilage bacteria of aquatic products in the aerobic refrigeration process.It can still grow under low temperature conditions.What's more,Pseudomonas has a strong ability to decompose protein and fat,producing ammonia and other corrupt products.Therefore,the corruption of aquatic products can be delayed by inhibiting the growth and reproduction of Pseudomonas.Bacteriophage has host specificity,no toxic or side effects on the tissue structure other than the host,and it is safe and efficient.The application of bacteriophage in food storage is an effective method against spoilage bacteria.Up to now,there have been successful cases of using bacteriophages in food storage.In this study,Pseudomonas P-181 was identified.And the taint ability of Pseudomonas P-181 in sterile fish juice was tested.Then,a virulent phage PrH-181 of Pseudomonas P-181 was isolated by double-layer plate method,from 70 sewage and soil samples collected from different provinces.And the morphological and biochemical characteristics of the phage were identified.Then the antibacterial activity of the bacteriophage in different substrates,temperatures and pH conditions was studied,respectively.Finally,the antibacterial ability of the bacteriophage against the spoilage of large yellow croaker under aerobic refrigeration at 4? was studied.The experimental results are as follows:1.According to the 16S rRNA sequence analysis and physicochemical properties of Pseudomonas P-181,it was identified as Pseudomonas helleri.The growth activity of Pseudomonas helleri at different temperatures and salt concentrations showed that it could grow well at 4~30? and 0-3%NaCl concentration,and could not grow at 37? and 6%NaCl concentration,which indicated that it was a psychrotrophic bacterium,not a halophilic bacterium.2.The bacteriophage PrH-181 has an icosahedral structure,insensitive to chloroform,and its nucleic acid type was identified as RNA.PrH-181 was identified as a Leviviridae virus phage according to the ICTV classification criteria for the virus.The optimal multiplicity of infection(MOI)of bacteriophage PrH-181 is 0.01.The one-step growth curve showed that the incubation period was 20 min,the outbreak period was 180 min,and the burst size was 584.3.When the bacteriophage PrH-181 titer was greater than 10~3 pfu/mL,it showed significant antibacterial activity in the LB liquid medium,or sterilized fish juice.The higher the titer was,the better the antibacterial effect was.The bacteriophage PrH-181 was tested at different temperatures and pH conditions.The results indicated that the bacteriophage PrH-181 maintained a high survival rate in a water bath at 70? within 60min,showing good heat resistance.The optimum growth pH was 6.0~7.0,the survival rate was as high as 94.1 to 98.8%at pH 6.0 to 9.0,and the antibacterial activity in the alkaline environment was stronger than that in the acidic environment.4.Uder 4? aerobic refrigerated conditions,the decaying process of large yellow croaker was inhibited when the bacteriophage PrH-181 with different titers was applied it.The control group began to corrupt on the 4th day,the phage-treated group with 10~4pfu/mL phage began to decay on the 8th day,and phage-treated groups with 10~6 pfu/mL and 10~8 pfu/mL began to decay on the 12th day.It can be inferred that the preservation effect of bacteriophage PrH-181 on the large yellow croaker is obvious,and the higher the titer is,the better the preservation effect is. |
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