Enterobacter Sakazakii In Powdered Milk Processing Hazard Analysis And Critical Control Point

Cronobacter sakazakii is gram-negative foodborne pathogen parasitic in animalsand humans that can cause severe neonatal meningitis, bacteremia and necrotizingenterocolitis with more than50%mortality. Infant formula is considered to be themain channel for newborns C.sakazakii infection. C.sakazakii traceability andeffective control in infant formula is main problems to be solved in milk powderfactories for quality assurance.To analyze C.sakazakii possible sources in milk powder factory and to designeffective measures to deal with contamination at different point of processing.Processing points in milk power which C.sakazakii contamination may occure wassampled and C.sakazakii was detected,16SrDNA gene sequence and homology wasanalyzed on C.sakazakii obtained. Survival rate of C.sakazakii from different sourcesat different temperatures as well as different cleaning conditions was investigated. Tocompare gene difference between the four C. sakazakii strains obtained, ERIC-PCRmethods was used to analyze the repeated genome sequence.Results showed that samples from all sectors of the milk plant, a total of23, weredetected by GB and conventional PCR detection method respectically. Bacteria werefound in three samples3of which was C. sakazakii; four strains C.sakazakii E(ATCC29544), C.sakazakii A isolated from infant formula, C.sakazakii H isolated from finepowder recovery pipe, C.sakazakii D separated from the drain were sequenced by16srDNA method. The results showed that C.sakazakii A and C.sakazakii H had thehighest homology. The four C. sakazakii strains were cultivated at4℃,25℃,37℃,44℃,55℃,72℃. Total colony numbers of C. sakazakii unchanged at4℃, had aslightly increase at25℃, increased the fastest at37℃, significantly decreased at55℃, decreased to zero at72℃. The results showed that C. sakazakii had thermalresistance but can not survival at72℃even higher temperatures; the four C.sakazakii strains was suspended in the1.5%sodium hydroxide,1.5%nitric acid,75%ethanol,1.5%detergent,1.5%SDS and1.5%peracetic acid for5min. The results showed that1.5%peracetic acid had the best sterilization effect than1.5%sodium hydroxide,1.5%nitric acid and75%alcohol.Washing powder and1.5%SDS has little effect on C. sakazakii. ERIC-PCR method was used to comparegenome differences of the four strains. Genetic stripe obtained by ERIC-PCR ofC. sakazakii D was different from the other three strains.The conclusions were:(1) The phylogenetic tree of different sources C.sakazakii made by16SrDNAsequence showed C. sakazakii A which from infant formula and C. sakazakii H fromfine powder recovery pipe were in the same phylogenetic tree branch, indicating thatthe highest homology of them. To determine phenotypic properties of four genotypestrains，the four strains were cultured at different temperatures, suspended in differentcleaning agents. The four strains showed differences in the condition. C. sakazakii Hand C. sakazakii D has a higher resistance to heat and cleaning agents. ERIC-PCRshowed that the four C. sakazakii genomic repeats were vary, so the method can beused to trace the source of C. sakazakii.(2) This study showed that72℃water,1.5%sodium hydroxide,1.5%nitric acid,75%ethanol,1.5%peracetic acid can be used to various processing points in milkpower factory to reduce C.sakazakii contamination.