Biomass Quantification Method Of Single Strain In Compound Probiotics

With the rapid development of the probiotic industry,the determination of biomass in probiotic products has become a hot research topic in the industry.The number of live bacteria in probiotic products is not only an important parameter for evaluating products,but also an important indicator for measuring their efficacy.Traditional counting methods are time-consuming and labor-intensive,and it is difficult to distinguish the specific addition amount of each strain in composite probiotic products and the number of live bacteria in each strain during storage.Therefore,this article focuses on the study of Lacticaseibacillus casei Zhang and Lactiplantibacillus plantarum P-8,and establishes a linear model between the two methods by combining plate counting,flow cytometry,and microdroplet digital PCR(dd PCR),The quantification of the live count,live cell count,and total cell count of Lacticaseibacillus casei Zhang or Lactiplantibacillus plantarum P-8 in composite probiotic products can be achieved through dd PCR alone.The main results are as follows:(1)Compared with the other three DNA extraction methods,the improved Tiangen reagent box method has smaller errors and stable results.Therefore,the improved Tiangen reagent box method was chosen as the DNA extraction method for this study.(2)The primer specificity of the selected Lacticaseibacillus casei Zhang and Lactiplantibacillus plantarum P-8 was good,and the optimal annealing temperatures were 53℃and 54℃,respectively.(3)By optimizing the treatment conditions of PMA,the optimal concentration of Lacticaseibacillus casei Zhang was obtained to be 35μg/m L,dark incubation time of 10minutes,exposure time of 15 minutes,the optimal treatment concentration for Lactiplantibacillus plantarum P-8 is 40μg/m L,dark incubation time of 10 minutes,exposure time of 20 minutes.(4)Establish a single strain quantitative model by correcting the quantitative results of the three methods.Among them,the PMA-dd PCR and plate counting results were linearly fitted to obtain the linear equations for the number of viable strains of Lacticaseibacillus casei Zhang and Lactiplantibacillus plantarum P-8,respectively:y=0.838x+3.6163(R~2=0.9924),y=1.6821x-1.2738(R~2=0.9951);Linear fitting of PMA-dd PCR and flow cytometry results resulted in linear equations for the number of live cells of Lacticaseibacillus casei Zhang and Lactiplantibacillus plantarum P-8,respectively:y=0.8439x+3.678(R~2=0.9925),y=1.6744x-1.1789(R~2=0.9919);By fitting the results of dd PCR and flow cytometry,the linear equations for the total cell count of Lacticaseibacillus casei Zhang and Lactiplantibacillus plantarum P-8 were obtained:y=0.758x+3.678(R~2=0.9971),y=0.5583x-6.972(R~2=0.9920).And through the verification of manually mixed bacterial powder,it was found that there was no significant difference between the quantitative results and the actual addition ratio(P>0.05),proving that the model has high accuracy.(5)Further applying the quantitative models of Lacticaseibacillus casei Zhang and Lactiplantibacillus plantarum P-8 to the detection of composite probiotic products,it was found that the biomass quantitative results were basically consistent with the added amount indicated on the label.The quantitative model method of Lacticaseibacillus casei Zhang and Lactiplantibacillus plantarum P-8 constructed in this study can quickly and accurately detect the biomass of a single strain in composite probiotic products,without relying on cultivation technology,breaking through the shortcomings of current quantitative detection of composite probiotics,and meeting the trend of future microbial rapid detection.It can provide technical support for quality control and deep development of probiotic and metabiotic products,It also provides a new solution for the quantitative detection standards of microorganisms.