Development Of ATP Bioluminescent Method And Its Kit For The Detection Of Bacterial Count In Food

Depending on the advantages of fast response and convenient operation, ATP bioluminescent method has been used to detect bacterial count in food. However, some undeniable faults existed in this method, such as low sensitivity, intolerance to storage of reagent resulting in unstable determination, restrict its applications in fields of food safety, sanitation and environment monitor. In view of the above shortcomings, in this paper, the reaction conditions of ATP bioluminescent method were optimized and the new technology of ATP bioluminescent method was constructed by single factor experiment, double factors optimization experiment. According to the structure and characteristics of firefly luciferase, the stability of enzyme was studied. New enzyme protectants were developed, which made the stability and sensitivity of the ATP bioluminescent detection techniques were greatly improved. In order to determine the most effective ATP extractant, the concentration of the extractant and extraction time, the ATP bioluminescent method was combined with the plate count method. The same conclusions were reached using both methods. When the optimized ATP bioluminescent method were applied to detect the bacterial count in food( liquid, solid, cooked food and fresh food) , the feasibility of this method applied to food was determined. The main research results are as follows:(1) The optimization of ATP bioluminescent reaction conditions: Such conditions were optimized as the concentration of luciferin of 80 mg/L, firefly luciferase of 53 mg/L, Mg²⁺ of 0.25 mM, pH value of 7.2, optimal temperature of 23℃.(2) The stability study of firefly luciferase: The combination agent of BSA, Casein sodium salt, Are octylic acid sodium and Tris buffer was capable of enhancing the stability of the enzyme and the sensitivity of ATP bioluminescent method.The change from the enzyme only saved in freezing conditions to 25℃was realized.When it was saved at 4℃, preservation time could be extended to 7 d and the enzyme activity recovery up to 85.99%.( 3 ) The research of ATP extraction method: According to the selection of intracellular ATP extraction, CTAB was confirmed as the best bacterial cells ATP extractant. Optimum ATP extraction was reached using 0.015% CTAB as the bacterial ATP extractant, the extraction time of 3 minutes and the addition of 0.25%β-cyclodextrin to the reaction system after the extraction of bacterial cells ATP. The accuracy and sensitivity of ATP bioluminescent method were improved.( 4) The appraisal of this method: The detection limit was as high as single bacteria. The coefficient of variation was less than 7% and 8% for intra- and inter-assay precision, respectively. RSD was 4.0%. Recoveries were from 82.2% to 112.4%. It showed that ATP bioluminescent method established in this paper was correct and reliable. The optimized ATP bioluminescent method were applied to detect the bacterial count in apple juice, flour, cooked products, fresh food, and good correlations were reached when compared with the plate count method( R2> 0.96) .