Investigation On Bacterial Contamination Of Red Swamp Crayfish And Its Control Technology

PurposeIn order to reduce the occurrence of red swamp crayfish related foodborne diseases,it is necessary to find an effective method to control the initial bacteria number of red swamp crayfish,so as to avoid the occurrence of related food hygiene problems,and to provide a theoretical basis for the development of red swamp crayfish antibacterial cleaning agent to ensure the hygienic quality of crayfish related products.MethodsThe bacterial diversity in whole crayfishes and tail muscle,as well as on the surface of crayfish from Nanjing,Suqian and Suzhou were investigated using the traditional culture-dependent method and metagenomic analysis in this study.Using SAEW,AEW,0.5%,1%,2%PLA,0.5%PLA+SAEW and 1%PLA+SAEW treated planktonic bacteria and biofilms of Citrobacter freundii C6,Aeromonas rivipollensis A5,Klebsiella oxytoca K6 and Vibrio parahemolyticus CICC21617,which had strong biofilm formation ability,for 5,10,20,30,60 min,respectively.Explored the bacteriostatic effect of sanitizers by colony count,measuring extracellular ATP content determination,SEM,CLSM,ATR-FTIR.The optimal treatment time was selected to wash crayfish with oscillations,the changes of V.parahemolyticus CICC21617 colony number on the surface of crayfish and colony number of different patrs of crayfish were recorded,and the species abundance changes of samples were compared by metagenomic sequencingResults(1)The results showed that the initial microbial load in crayfish were large,which were all more than 6(lg(CFU/g)).The species abundance obtained by metagenomic sequencing method was higher than that obtained by traditional culture medium identification method,mainly including Flavobacterium,Acinetobacter,Chryseobacterium,Aeromonas,Pseudomonas and Shewanella.It was found by principal component analysis that the species abundance of the whole crayfish was similar to the surface.The main contaminating bacteria of crayfish are Citrobacter,Klebsiella and Aeromonas.(2)The biofilm formation ability of C.freundii C6,A.rivipollensis A5 and K.oxytoca K6 were strong,which biofilms were mature after cultured for 3 d.The tolerance of bacteria in biofilm to bacteriostatic agents was higher than that of planktonic bacteria.The membrane structure of planktonic bacteria treated with 1%PLA+SAEW was more wrinkled and damaged than that of single bacteriostatic agent.The red fluorescence intensity of thallus was higher,accompanied by the phenomenon of thallus self-aggregation and agglomeration,and intracellular ATP leaked out.When 1%PLA+SAEW was applied for 30 min,the exopolysaccharide content of biofilm was reduced,bacteria in biofilms were wrinkled and showed bright red fluorescence,intracellular ATP leaked out,and the peak position of ATR-FTIR shifted.(3)The biofilm formation abilities of V.parahemolyticus CICC21617 was strong,which biofilm was mature after cultured for 3 d.The tolerance of bacteria in V.parahemolyticus CICC21617 biofilm to bacteriostatic agent was significantly higher than that of planktonic bacteria.When 1%PLA+SAEW was applied for 30 min,the biofilm structure was destroyed and bacteria in the biofilm were inactivated.The biofilm showed red fluorescence and the surface material structure changed.And V.parahemolyticus CICC21617 successfully attached to the surface of crayfish can be completely inactivated.(4)The cleaning effect of different sanitizers on the surface of crayfish was significant,1%PLA+SAEW effectively reduced the surface bacteria by 3.95(lg(CFU/g)).At the same time,the species abundance of dominant genera such as Flavobacterium,Aeromonas Shewanella decreased.The relative abundance of some minor bacteria genera such as Bacteroidetes and Pseudomonas increased.ConclusionThe species similarity between crayfish and surface is high,and the initial bacteria number is mainly related to the water environment.The activity of bacteria in the biofilm of contaminated bacteria was higher than that of planktonic bacteria.The compound of phenyllactic acid and electrolytic water can effectively destroy the cell membrane of the bacteria so that its contents are released and thus inactivated.The antibacterial effects of PLA and SAEW on biofilms were mainly due to dissolution of exopolysaccharide and change of substance composition on the surface of biofilm.The effect of 1%PLA+SAEW for 30 min can effectively reduce the number of bacteria on the surface of crayfish,kill V.parahemolyticus CICC21617 inoculated on the surface of crayfish,and change its species diversity.It is an effective cleaning and bacteria reduction method.