Application Of Bacillus Cereus Phage And Cocktail Preparation In Food

Bacillus cereus is a facultative anaerobic food borne opportunistic pathogen,which can cause nausea,vomiting,abdominal distension,abdominal pain and diarrhea.The food poisoning caused by Bacillus cereus ranks third in China.Because spores have strong tolerance,traditional food bacteriostatic methods often can not eliminate the pollution of Bacillus cereus.Therefore,it is of great significance to develop effective and safe prevention and control methods and accurate and rapid quantitative detection methods for this bacterium in the food industry.Phage has the advantages of strong specificity,low separation cost and high safety.It is expected to become an ideal means to prevent and control such foodborne pathogens.In this thesis,the basic biological characteristics of three Bacillus cereus bacteriophages preserved in the laboratory were studied.On this basis,phage cocktail preparations were prepared,and the effects of three bacteriophages alone and cocktail preparations on inhibiting the formation of Bacillus cereus biofilm and preventing and controlling Bacillus cereus in milk,fresh pork and rice were evaluated.The experimental results are as follows:Bacteriophages LY3,LY32 and LY3W can form clear and transparent plaque,and LY3W plaque has halo.The optimal infection complex of bacteriophages LY3,LY32 and LY3W were 0.00001,0.001 and 1 respectively,and the phage titers were 4.24×10¹⁰PFU/ml4.4×10¹⁰PFU/ml,1.24×10¹⁰PFU/ml.The one-step growth curveshowed that the incubation period of LY3 was 10min,the cracking period was 40min,and the average cracking amount was 28 PFU/cell,the incubation period of LY32 was 10min,the cracking period was 80min,the average cracking amount was 89 PFU/cell,the incubation period of LY3W was 20min,the cracking period was 60min,and the average cracking amount was 71PFU/cell.The results of environmental tolerance test showed that the three phages had strong temperature and acid-base tolerance,and could maintain high activity in the range of 30-50℃and p H3-9.The sequencing results showed that the genome sizes of LY3,LY32 and LY3W were 46341bp,48648bp and 25255bp respectively.None of the three phages contained virulence genes and transferable drug resistance genes.Good biological characteristics are the basis of phage application.The above results show that the three phages have application potential and safe biological basis.The inhibition of biofilm formation by three bacteriophages alone and cocktail preparation showed that the inhibition of biofilm formation by bacteriophage cocktail preparation was stronger than that by any single bacteriophage,and LY3 was the strongest among the single bacteriophages;The bacteriostatic effect of Artificially Polluted milk,fresh pork and rice showed that:The use of three bacteriophages alone and mixed cocktails can effectively reduce the number of host bacteria.The sterilization effect of LY32 and LY3 is significantly stronger than LY3W.The sterilization effect of bacteriophage mixed cocktails is better than any single bacteriophage and can effectively prevent the production of anti bacteriophage strains.In the three foods,phages showed the characteristics that the greater the MOI,the higher the potency.After phage treatment,compared with 4℃,the number of bacteria decreased more at 25℃,but low temperature at 4℃and phage could form a fence effect,and the ability of bacterial regeneration was significantly weakened.To sum up,the bacteriophage and phage cocktail preparations used in this study can carry out good biological control of Bacillus cereus pollution in food.It is a promising green and natural food antibacterial agent.