| Food safety is becoming more and more important around the world,especially some incidents of food safety caused by food-borne pathogens frequently occur.For some industries of soybean products,the GB2712-2014 Food Safety National Standard has been issued by relevant state departments to effectively control their food hygiene and safety of bean products.However,there is no official requirement for the total number of bacterial colonies in soybean products but limited requirements for only coliform bacteria.In fact,because toufu is ready-to-eat food,GB/T22106,NY/T1052 is used for evaluating and analyzing the hyiene and safety of tofu.Therefore,the total number of colonies needs to be tested.The conventional method is the national standard method:GB4789.2-2016 Food Safety National Standard Food Microbiological Examination of the Total Number of Bacteria.The method adopts the plate agar counting method to detect and count the total number of bacterial colonies,but the disadvantages of this methods,such as large workload,long working cycle and complex steps,and so on.Therefore,there is an urgent need for a fast,accurate and efficient method to detect and count the total number of colonies.This paper adopts the flow cytometry to creat the method of detecting the total number of colonies in soybean products,using fluorescent dye PI and SYTO to dye the particles of samples.Furthermore,this method can distinguish impuritise and the active and inactive bacteria,and the counting result of total number of bacterial colonies can be learned by final test.The result show that test limit and test time of are 10~2 cell/μL,2 min respectively and it has high stability and repeatability.In the first part,due to the small number of colonies in tofu,the particulate in toufu may affect the stability of the test,so it is necessary to carry out sample pretreatment to reduce interference.It includes the effect of dilution on sample preparation and dilution optimization,pre-incubation time on pretreatment results and different initial proportions of tofu pretreatment optimization.The results show that the gradient dilution stability is good,the best pre-incubation time is 1 h,and the best initial ratio of soybean products is 25 g+225 mL.In the second part,different combinations of fluorescent dyes have a great influence on the detection results.It includes the influence and difference of different fluorescent dye combinations on the detection results,and the comparison of the dyeing effects and results of different dyes in this experiment.The results show that the best dye combination is PI+SYTO,the best dye volume ratio is PI:SYTO~?24 is 5μL+5μL(1:1)The third part is about the establishment of flow cytometry to detect the total number of colonies in soybean products.Escherichia coli and Staphylococcus aureus were used to simulate sample detection.The results show that the flow cytometry detection method can be applied to the research object of the concentration of sample particle over or equal to 10~4/g(mL),the concentration of the sample should be controlled as much as possible between 10~5 and 10~7 CFU/mL during the actual test.The results show that the best loading concentration of fluorescent dye PI is 5μg/mL,and the best loading concentration of fluorescent dye SYTO~?24 is 0.05 mmoL/mL,the best time for incubation is 5 min,the best loading speed is 30μL/min,and 120seconds for data reading time.Further compared the difference between the automatic door setting and the manual door setting in the flow detection method,it is finally known that there is no significant difference between the two setting methods in the counting.The numerical difference is less than an order of magnitude,so both door-to-door methods can be used in the experiment.After comparing the flow cytometry method with the national standard method,it can be known that there is a significant linear relationship between the flow detection method and the national standard method in the range of 10~3 to 10~7 CFU/mL.Parallel experiments were carried out on the same type of samples,the results show that the cv%was less than 5%.In summary,a method for the detection of the total number of colonies in soy products by flow cytometry was established.Furthermore,the sample pretreatment and the selection of fluorescent dye combinations were discussed,and then carried out research.The results show that flow cytometry is a fast,accurate,high efficiency detection method.Flow cytometry will have broad prospects in applied research field of food microbiology in the future. |
