| Dairy is riched in protein, amino acids, minerals and other nutrients and presents with delicious taste and easy digestion, which is highly praised and appreciated by consumers. However, the quality and safety of dairy products seriously restrict the development of dairy industry in China. How to ensure the safety and quality of dairy products has become a common focus for production enterprises and consumers. Microbial contamination is one of the main factors, which can cause metamorphism, corruption or bulge of dairy. And some pathogenic microorganism may lead to food poisoning, posing a threat to consumers’ health. Conventional detection method is time consuming can’t be applied to the modern production mode of dairy processing enterprise. Therefore, establishment of a fast microorganism detection method in dairy attracts much attention.In this study, we aimed to establish a rapid and sensitive method to detect the total viable bacteria in pasteurised milk and yeast in fermented milk respectively, using a method of PMA combining with q PCR to quantify the DNA of live microorganism in dairy samples. Firstly, 500 pasteurization milk and fermented milk samples were collected to separate the contaminated bacteria and yeast, and the obtained microorganisms were identified by morphological observation, molecular biology and biochemistry tests. Then the standard curves of the logarithm of bacteria and yeast concentration vs the corresponding Ct values were established after optimization of the PMA treatment conditions for different strains and quantitative analysis by q PCR. Furthermore, the computed tomography(Ct) ranges of the bacteria in pasteurized milk or the yeast in fermented milk were determined according to GB19302-2010 and GB19645-2010. Finally, the practical pasteurized milk and fermented milk samples were simultaneously detected by PMA-q PCR, q PCR and national standard methods so as to validate the accuracy of the PMA-q PCR method established in this study. The results showed the contaminated bacteria in pasteurized milk included Aerococcus viridans, Yokenella regensburgei, Escherichia coli, Hafnia alvei, Grignard Lactococcus, Staphylococcus intermedius, Kocuria kristinae, Streptococcus sanguinis, Cronobacter sakazakii, Staphylococcus and haemolyticus, while the contaminated yeast in fermented milk were K.marxianus, P. kudriavzevii and S.cerevisiae. The best treatment of pasteurized milk samples was 15 μg/m L PMA with 10 min of dark incubation and 15 min of exposure. The best treatment of fermented milk samples was 15 μg/m L PMA with 10 min of dark incubation and exposure. The Ct range of the bacteria at a concentration of 105 CFU/m L in pasteurized milk was 27.24~30.85; while it was 33.97~35.24 with yeast concentration at102 CFU/m L in fermented milk.The test results of PMA–q PCR and the national standard method for viable bacteria in pasteurized milk with the coincidence rate is more than 93.3%. The test results of PMA–q PCR and the national standard method for viable yeast in fermented milk with the coincidence rate is more than 86.6%, and detection time was about 4 h. This method can judge whether processed dairy products qualified quickly, and give the information feedback for detection in time. It apply to the field evaluation in the production line of dairy processing enterprises. |
