Research On Key Techniques For Preparation About Quantitation Reference Materials Of Staphylococcus Aureus

Staphylococcus aureus is a common pathogenic bacteria that cause of bacterial food poisoning,mainly through processing,transportation,marketing and other ways to cause food contamination,seriously affecting human health.Therefore those food that contaminate S.aureus vulnerable before into the market should make rigorous testing.To ensure the testing capacity of the relevant inspectors,standardize the management of reference materials,and to reduce the leak detection and error detection rate in food,we should increase efforts to develop the relevant biological reference materials,so that ensure the test results true and reliable,supply a reference value for the same type of reference materials' preparation and verification in future.This dissertation systematically studied the preparation method of quantitative reference materials for S.aureus.The main contents included the study of the adhesion for S.aureus and the determination of growth curve,the optimization of protective agents and freeze-drying process,the uniformity detection,stability tracking,and assessment of uncertainty of reference material.Based on the viability,the formulation of protective agents for freeze-dried S.aureus was optimized by means of L9(34)orthogonal test in this study,results indicated the optimal formula were trehalose 2%,sodium glutamate 15%,L-cysteine hydrochloride 0.5%,and bovine serum albumin 0.5%.Validation experiments of the formula were also carried out,showing that the protection rate after freeze-drying could reach 96.78%.According to the freeze-drying test and eutectic point determination results,the freeze-drying parameters were systematically investigated.The optimum process was obtained as follows:at precool stage,the products were cooled under-80? for 2.5 h,the temperature of plates was set to-45 ?,then the products were put into the freeze dryer for 0.5 h at low temperature;at sublimation drying stage,the temperature of plates was periodically set,increased from-45 ? to 0?,the total time was 6.5 h;at parsing drying stage,the temperature of plates was increased from 0? to 20 ?,the total time was 8 h,the whole freeze-drying process lasted for 18.5 h.The resulting products exhibit a white loose spongy conglomeration state,residual water percentage of 1.7%?2.7%,and the freeze-drying viability was 97.16%,which was in accordance with the standard.The result of the variance analysis showed that the F value of the reference material was 1.90,which was less than the critical value of 2.04 at the confidence level,and the P value was more than 0.05,indicating that the uniformity was good.Simulation of transport stability test results show that the reference material using ice box for optimal transport stability;long-term stability test results show that the reference material store at-20 ?,after 180 days,the microbial content of raw matrix remain 450 CFU/mL,and the stability standards compliant.The standard uncertainty of the reference material is 38(confidence probability 95%),and the standard value is(620 ± 38)CFU/mL.