Study On The Effect Of Shewanella Biofilm On The Decay Of Large Yellow Croaker Under Low Temperature Modified Atmosphere Packaging

Shewanella putrefaciens（S.putrefaciens）is a Marine gram-negative bacterium commonly found in water and soil,which dominates the spoilage of aquatic animals or protein-rich foods（e.g.large yellow croaker）.In order to resist the harsh environment of the outside,most bacteria usually exist in the form of biofilms.The tolerance of biofilm is usually stronger than planktonic,which makes it difficult to be removed,thus causing great harm to the food processing industry.As a relatively mature food preservation technology,modified atmosphere packaging has been widely used in all kinds of food packaging that can extend the shelf life.However,foods in modified atmosphere packaging have the potential to form biofilms when contaminated by microorganisms.At present,there are few studies on the formation of biofilms by putrefying bacteria on a modified atmosphere packaging plastic films.Therefore,in this paper,the biofilm ability of S.putrefaciens on the packaging plastic film was evaluated using quantitatively and qualitatively methods,respectively.Furthermore,the relationship between the biofilm of S.putrefaciens and the putrefaction of large yellow croaker was investigated.Finally,the control effect of modified atmosphere packaging on the biofilm developed by S.putrefaciens was also studied.The research contents and conclusions of this paper are as follows:（1）Evaluation the biofilm formation ability of S.putrefaciens in culture medium.Firstly,the formation of the biofilm by S.putrefaciens was studied by crystal violet quantification method,and then the three-dimensional structure of the biofilm was characterized by scanning electron microscopy and laser confocal scanning microscope.The results showed that S.putrefaciens had the strongest biofilm-forming ability under Luria-Bertani medium（LB,p H=7）,the initial bacteria concentration of 5.2×10⁷ CFU/m L and Na Cl mass fraction of 1.5%.Under this condition,when the bacteria were cultured at 30^oC and 4^oC for 120 h,the amount of biofilm reached a peak after 48 h and 60 h,respectively.At this time,the OD₆₀₀ was 0.635 and 1.014,respectively.It is obvious that the maximum amount of biofilms at 4°C were significant higher than that at 30°C.In addition,the image of SEM revealed that the internal structures of mature biofilms are staggered with each other and present a multi-layer network.At the same time,it was observed that bacteria began to adhere after 24 h,polysaccharide secretion increased after48 h,and a three-dimensional biofilm formed using laser confocal microscope.（2）Study on the biofilm formation of S.putrefaciens and the putrefaction effect on the large yellow croaker at low temperature.The dynamic process of the biofilms by S.putrefaciens was studied in large yellow croaker juice and fish pieces under low temperature conditions,respectively.By measuring the protease activity of the biofilm formed by the bacterium and its ability to decompose the protein of large yellow croaker,the putrefaction of the biofilm was proved.The results showed that a large number of biofilms could also be formed in the food system with the maximum OD₆₀₀ value of 1.877.In addition,the measurement of the protease activity of the biofilm and the analysis results of SDS-page also proved that the protease activity of biofilm is higher than that of planktonic bacteria.（3）Explored the control effect of modified atmosphere packaging on the biofilm developed by S.putrefaciens.The inhibitory effect of MAP on the biofilm was evaluated by measuring the amount of biofilm and its protease activity under modified atmosphere packaging conditions.The results show that the MAP can significantly inhibit the formation of biofilms in the medium or food system in the early stage of culture（12-36 h）.With the extension of the cultivation time（48-120 h）,the amount of biofilm will gradually increase,and the maximum value OD₆₀₀ can reach 0.953.In addition,MAP also has a certain inhibitory effect on the protease activity of the biofilm,making the hydrolysis loop of the protease shrink by 1/3.Furthermore,the autoaggregation and hydrophobicity of S.putrefaciens were also significantly increased at 56.74%and 54.87%,respectively.