Study On The Application Of Fast Counting Method For Streptococcus Thermophilus Based On Flow Cytometry

Liquid milk products have always been popular because of their advantages such as easy digestion and absorption,good quality and low price,and convenient eating.In recent years,with the in-depth study of human physiological functions of lactic acid bacteria and the perfection of fermented milk production technology,the proportion of fermented dairy products in liquid dairy products has become increase,and the market size is expected to reach 190.6 billion yuan by the end of 2020.Accounting for 50% of the entire liquid milk market.In order to adapt to market development and ensure the quality of fermented milk products,government departments have also issued a series of standards on fermented milk products at the national level.In accordance with the relevant provisions of the Food Safety Law and its implementing regulations,the Ministry of Health has formulated the "List of Strains Available for Food".The "Bacterial Species Available for Food" contains 35 species of ten species of genus Bifidobacterium,Bifidobacterium adolescent,Bifidobacterium bifidus,Streptococcus thermophilus,Lactobacillus acidophilus,etc.Bacteria can be used in general food;Nine strains of two genus bacteria can be used in foods for infants and young children;11 kinds of fungal bacteria and 10 kinds of probiotic bacteria in three genus can be used for health food.According to the relevant provisions of GB 19302-2010 "National Standard for Food Safety Fermented Milk",the number of lactic acid bacteria in fermented milk products shall not be less than 106CFU/m L(g).For the count of lactic acid bacteria,the current national standard is used: GB 4789.35-2016 National Food Safety Standard Food Microbiological Inspection Lactic Acid Bacteria Inspection.However,because this method uses the plate culture method,it can only accurately count Lactobacillus,Bifidobacterium and Streptococcus.Counting of 35 species of bacteria.Moreover,the culture method has limitations such as unstable counting results,long time to obtain results,heavy workload,and only counting live bacteria.Therefore,based on the above background,the first part of this article first investigated the types of lactic acid bacteria in common milk and dairy products in the market.The results showed that the highest proportion of Streptococcus thermophilus reached 29%,Lactobacillus bulgaricus and Lactobacillus acidophilus accounted for 25% and 13% respectively.Follow-up experiments selected S.thermophilus as the research object.A rapid counting method for Streptococcus thermophilus based on flow cytometry was established and standard operating specifications were established.The amount of dye working solution,incubation time,injection speed,loading time,threshold and gain were determined by orthogonal experiments Parameters and other parameters to establish theoretical basis and operating standards for subsequent actual sample detection experiments.The second part mainly evaluates the rapid detection method based on the established standard operating methods.The linear range,detection limit,limit of quantification,relative accuracy,uncertainty,repeatability,reproducibility,and comparison between laboratories of the method are evaluated.And other technical indicators for systematic evaluation and research to ensure that the test results made by this method are accurate and reliable,and to establish release standards for the various testing needs of various types of enterprises.The third part is based on flow cytometry to count lactic acid bacteria in commercially available samples.Ten batches of commercially available fermentation strains are selected from the group consisting of Streptococcus thermophilus milk and dairy product starter,daily chemical products,etc.Check it out.First,through microbial diversity sequencing analysis,the types of lactic acid bacteria contained in the product were identified,and then the flow cytometry method that had been evaluated was used to count and measure the lactic acid bacteria.In the process of real sample detection,there are still some steps with strong repetitive and mechanical operations.The fourth part of this research further develops and builds a quickly detect cloud platform based on the original method,so that this method can be more easily applied in more scenarios.The aim is to realize the online detection of the Internet + food rapid detection method,and to meet the needs of the promotion and display of the method for detecting the number and activity of lactic acid bacteria by flow cytometry.All in all,this paper establishes a set of evaluation methods for rapid flow detection methods,verifies and evaluates the rapid counting method of lactic acid bacteria based on flow cytometry,and constructs a standard operating procedure for detection and a platform for automatic flow chart reading programs.The application of the technique in the food field has laid a theoretical foundation.