Establishment Of Listeria Growth Prediction Model And The Phage Bio-control In Boiled-salted Duck

Listeria is a kind of gram-positive pathogenic bacteria that can cause listeriosis in animals and human,among which L.monocytogenes(Lm)is the most important food borne zoonotic pathogenic bacteria.Ingestion of the food contaminated Lm by mistake can lead to meningitis,septicemia and abortion among those people with low immunity,such as newborns,the elderly and pregnant woman.It has the characteristics of high hospitalization rate(80%)and mortality rate(more than 20～30%),which is a serious threat to human health.In this study,the contamination of Lm in boiled-salted duck was monitored,the growth prediction model of Lm in boiled-salted duck was established,bio-control effect of Lm in boiled-salted duck was analyzed by phage.The non-coding RNA RliB defective strain further clarified the interaction between phage and Lm,which provided reference for the effective application of phage in the biological control of ready-to-eat food(1)Collecting boiled-salted duck samples to analyze the contamination status of Lm.Determining the growth of boiled-salted ducks at 4,10,16,25℃,and selecting four primary models(Gompertz,Logistic,Richards and MMF models)to fit the data,comparing the decision coefficient(R2)and mean square error(MSE)of each model to determine the most suitable primary model,and establish a second-level model related to temperature according to the fitting parameters.The results showed that the detection rate of Lm in boiled-salted duck was 8%(8/100).Gompertz model is the most suitable primary model for the growth of Lm.Under 4℃,Lm entered the logarithmic growth period after 22.05 h,with a long lag period,but almost no lag period at 25℃;When the temperature was increased from 4℃ to 10℃,the growth rate increases from 0.02 1g(CFU/g)·h-1 to 0.05 1g(CFU/g)·h-1,indicating that temperature has a great influence on the growth of Lm.Using Ratkowsky square root model and quadratic polynomial model to build the second model R2 are 0.98 and 0.99 respectively;Evaluating the mathematical parameters of the second model,the deviation factors(Bf)are 0.99 and 1.01,respectively and the accuracy factors(Ar)are 1.01 and 1.08,respectively.The results show that the growth prediction model can simulate the growth of Lm in boiled-salted duck,and can provide reference for food safety and quantitative risk assessment of boiled-salted duck.(2)The bio-control of phage in boiled-salted duck was determined by the natural antimicrobial characteristics.Single phage vB-LmoM-SH3-3 and mixed phage SNG(vB-LmoM-SH3-3、vB-LmoM-NJ05、LipG2-5)were used in boiled-salted ducks to determine the inhibition effect on Lm.In the boiled-salted ducks treated with single phage and SNG at 4℃ for 24 h,Lm decreased by 2.92 lgCFU/g and 2.62 lgCFU/g respectively,and the two groups of phages still showed good inhibition effect at 144 h.At 16℃,the number of Lm decreased by 3.74 lgCFU/g(p<0.05)and 1.92 lgCFU/g after 4 h,respectively At 56 h,the inhibition effect of single phage was always better than that of mixed phage,and the maximum reduction of Lm was 4 log.At 25℃ for 4 h,the number of Lm decreased by 3.34 lgCFU/g(p<0.05)and 0.81 lgCFU/g with the single and SNG groups respectively.At 12 h,the inhibition effect of single phage was better,and the number of bacteria decreased by 4.5 log at most.In conclusion,the antimicrobial effect of single phage was better than that of mixed phage SNG at 16℃ and 25℃.However,both single phage and SNG show great inhibition effect on boiled-salted duck under 4℃(3)Phage can effectively inhibit the contamination of Lm,but its interaction with Lm is significantly different,so the characteristics of the interaction between phage and Lm can provide technical reference for the application of biological preservation.A non coding RNA RliB defective strain,Lm3-22-ΔRliB,was constructed by homologous recombination.The growth characteristics,cell invasion,adhesion and intracellular proliferation of Lm3-22-ΔRliB were analyzed.The sensitivity and efficiency of plaque formation(EOP)were determined by the interaction with phage.The results showed that the deficiency of RliB did not affect its biochemical and hemolytic properties.At 37℃,the growth was slower than that of wild type,and the logarithmic growth period was delayed about 1.5 h.Compared with wild-type Lm,the adhesion rate of Lm3-22-ΔRliB with macrophage RAW264.7 decreased significantly(9.37%),and the invasiveness decreased slightly(1.33%).However,the EOP of the defective strain increased significantly(2 log),and the sensitivity to phage reaction increased.In conclusion,the deficiency of Rlib does not directly affect the basic characteristics of Lm but the interaction with phage has increased,which provides a reference for the future application of phage.