Flow Cytometry-based Rapid Counting Method Study And Activity Determination Of Lactic Acid Bacteria

In GB 19302-2010 National Food Safety Standard-Fermented Milk,the number of lactic acid bacteria has been limited to more than or equal to10~6CFU/mL in order to control the quality and safety.GB 4789.35-2016 National Food Safety Standards for Food Microbiology Testing-Lactic Acid Bacteria Testing is the main method based on MRS agar plate counting,however,it still has a lot of limitation such as time consuming and unstable.So a quick and accurate method is needed in lactic acid bacteria counting.In this article,a method based on flow cytometric to test the viable and unviable lactic acid bacteria staining with SYTO?24 and propidium iodide(PI)in starter,fermented milk beverage and fermented milk was built.In the first part,Lactobacillus bulgaricus(LB.)and Streptococcus thermophiles(S)were chosen as the researching objects.The growth curve showed that the lag period of these two bacteria was 5hours and 6 hours,respectively,much higher than the time consumed in flow cytometry analyzing and during this period of time the number of bacteria will not increase.In the second part,the counting method based on flow cytometry was constructed,the method was applied to the samples,in which the bacteria should be greater than or equal to 10~5CFU/mL(g).The loading concentration of the sample should be controlled at 10~2-10~3cells/?L.Orthogonal analysis showed that 5?g/mL PI and 0.05?M SYTO?24 is the best concentration dying,the sample should be incubated for 3 minutes at 37?.During the FCM analysis,the voltage and fluorescence channel threshold were optimized.Finally the sample should be run at the medium speed(30?L/min)for 180s,the results of flow cytometry and plate counting method showed that there was no significant difference at P<0.01.Flow cytometry sensitivity was 10~2 cells/?L and the CV value is 2.4%In the third part,different methods were used to get non-viable bacteria,and the result showed that 70?10min was the best condition for it is commonly used in industrial production.Through the MoFlo XDP sorting system,the viable and non-viable bacteria were separated and plate counting was taken for verification.The total number of active bacteria was in the same range between two methods and there was no significant colony on the non-active bacteria plate.Finally,observed by fluorescence microscope,red fluorescence was found in non-viable bacteria and green fluorescence was in viable bacteria.In the last part,11 starter cultures,10 fermented milk beverages and 17fermented milk products were tested by FCM and compared with the current national standard method.Compared with the plate counting method,it has some advantages such as short time-consuming and it can get the number of viable and non-viable bacteria.In summary,this paper constructed a rapid counting method of lactic acid bacteria based on flow cytometry,which laid the theoretical foundation for the application of flow cytometry in food industry.