Study On Rapid And Quantitative Detection Method Of Viable Coenzyme Q₁₀ Producing Rhodobacter Sphaeroides

As one of the main microbiology that produce coenzyme Q₁₀,quantitative detection of the number of viable bacteria is of great significance to optimize the fermentation conditions.Plate counting method is classical method for the quantitative detection of Rhodobacter sphaeroides.However,due to the disadvantages of plate counting method,such as complicated procedures,time-consuming and so on,it cannot meet the needs of rapid measurement of Rhodobacter sphaeroides.The aim of this study was to eslablish rapid method for the quantification of viable Rhodobacter sphaeroides.The results are as follows:1.Establishment of plate counting method for Rhodobacter sphaeroides.In this paper,the inoculation method,the amount of inoculation,the type of medium and the culture temperature of the plate counting method are studied.Through the optimization and experimental verification of the plate method,the best plate counting method of Rhodobacter sphaeroides is explored.The results show that the spreading plate method is the best inoculation method for the plate counting method,50?L is the best inoculation amount for the plate counting method,nutrient agar is the best medium for the plate counting method,and 32°C is the best medium for the plate counting method.Optimal culture temperature;through the research and analysis of experimental results,the relative expanded uncertainty of the plate counting method for Rhodobacter sphaeroides is 9.2%(k=2),which is suitable for quantitative measurement of Rhodobacter sphaeroides.2.The establishment of flow cytometric analysis of Rhodobacter sphaeroides.Mark Rhodobacter sphaeroides by using specific fluorescent antibodies to identify Rhodobacter sphaeroides;use nucleic acid dyes to mark dead Rhodobacter sphaeroides to distinguish between dead and live bacteria;then use a flow analyzer to be tested The solution is counted and analyzed to obtain the concentration of viable Rhodobacter sphaeroides with different fluorescence signal intensities.The results show that the established flow cytometry method has good accuracy and specificity.The detection range for the number of viable Rhodobacter sphaeroides is 10⁴?10⁸ CFU/mL,the detection limit of the method can reach 10³ CFU/mL,and the detection time for 40minutes,this method can quickly and quantitatively detect the number of viable Rhodobacter sphaeroides.3.The establishment of the capacitance method of Rhodobacter sphaeroides Since the flow analysis method cannot monitor the change of the number of viable microorganisms in the fermentation process online,this paper prepares the bacterial suspension into different concentrations of the bacterial liquid,uses the capacitance method to detect the capacitance value of the bacterial suspension,and simultaneously uses the flow analysis method to the bacteria suspension is counted for live bacteria to establish the relationship curve between the capacitance value and the number of live bacteria,and the number of live bacteria of the actual sample is calculated according to the standard curve.This method can be used to quickly detect the number of live bacteria in the fermentor;At the same time,methodological verification of the detection method is carried out.The results show that when the number of viable bacteria is10⁷?10¹⁰CFU/mL,there is a good linear relationship between the number of viable Rhodobacter sphaeroides and the capacitance value(R²>0.95),and the minimum detection limit of this method is 7.14×10⁵ CFU/mL,the detection time is 5 min.