Accurate quantification on the change of some live pathogenic microbial flora in fermented feed

Real-time monitoring of harmful bacteria in the process of feed fermentation is an important step to control and optimise feed quality. Real-time quantitative polymerase chain reaction (qPCR) can effectively determine the total amount of pathogenic bacteria but does not distinguish between active and inactive bacteria. In this work, propidium monoazide dyes (PMA) were combined with qPCR technology to explore the changes of microflora during the fermentation of artificially contaminated feed. The results indicated that the Cycle Threshold (Ct) values of sample detected by PMA-qPCR were all higher than the results of qPCR. PMA could effectively nullify the influence of dead bacteria in the detection of viable bacteria. It was found that the growth of Escherichia coli O157:H7, Shigella flexneri, and Staphylococcus aureus were inhibited during the fermentation process of soybean meal feed with the mortality rates were 99.89, 95.07 and 97.73%, respectively. However, the growth of beneficial bacteria were not affected by the presence of harmful bacteria after 60 days of storage. These results suggest that anaerobically fermented feed is a promising substitute for antibiotics in animal feed and using PMA-qPCR for real-time monitoring of harmful bacteria in fermented feed will further improve the safety risk assessment of fermented feed.