| P. fluorescens is responsible for the highest depredation of milk because of its capacity to synthesize extracellular lipase and protease which hydrolyze milk fat and proteins,which leads to bitter flavor and gelation of milk that decrease the yields and sensory qualities of dairy products. Standard plate count procedures are traditionally used to detect P. fluorescens, but they are time consuming and do not permit to rapidly evaluate the potential of depredation. So, there is a need for rapid, accurate, and sensitive methods for the detection of P. fluorescens.The polymerase chain reaction(PCR) technique is a powerful tool for the analysis of large number of samples, because of its simplicity, versatility, sensitivity, and reproducibility. Previous studies have indicated that PCR might be useful for detection of psychrotrophic bacteria. However, currently PCR-based detection technology of Pseudomonas fluorescens in foods is still at the exploratory stage and there are limited species-specific gene targets which are lack of systematic evaluation. In this research, 10 pairs of primers were designed which targeted at gyr B gene and apr gene respectively using bioinformatic analysis. And we selected two pairs of primers(gyr and apr) based on its gyr B gene and apr gene. Among all the bacterial strains used in this research, duplex PCR amplified two specific products(384 bp and 194 bp) from the strains of Pseudomonas fluorescens, but not from the negatives. The specifity of our assays was 16.9 fg/μL for pure DNA and 1.8 CFU/m L for UHT milk with 24 h enrichment. So, the methods described in our study can be adopted to detect Pseudomonas fluorescens in dairy product samples with higher sensitivity and specificity.Extracellular proteolytic protease of Pseudomonads could hydrolyze casein and decrease the yields and sensory qualities of dairy products that leads to bitter flavor and gelation of milk. Traditional methods based on skimmed milk plate detection could only qualitatively detect hydrolysis vitality of extracellular proteolytic protease of Pseudomonas. In this study, we took azo-casein as a substrate, randomly selected out of the 29 psychrotrophic Pseudomonas cells from the Shanghai area ranch and made a quantitative detection of hydrolysis ability and heat resistance of extracellular proteolytic protease of these Pseudomonas, and also assessed the potential of corruption for different kinds of Pseudomonas. P. fluorescens was found to be the most frequent according to our Molecular phylogeny test in species level based on rpo B gene sequencing. The results showed that 14 strains of them belonged to Pseudomonas fluorescens. We also did a quantitative detection of proteolytic vitality for all Pseudomonas, at 6 ℃ and 30 ℃ culture conditions respectively. The detection results showed that average proteolytic vigor size of Pseudomonas was 3.46 DAh-1m L-1 at 6 ℃ while 4.57 DAh-1ml-1 at 30 ℃. It means that average protease activity(6.425 DAh-1m L-1) of Pseudomonas fluorescens was higher than other strains, and these protease also had good heat resistance. It confirming that P.fluorescens had greater potential for corruption than others. and it also indicated the necessity to establish a double PCR method for detection of Pseudomonas fluorescens.In summary, Pseudomonas fluorescens is responsible for the highest depredation rates of milk, the duplex PCR system which established in this study can compensate for the sufficient of single PCR technology and detect Pseudomonas fluorescens in dairy products efficiently and specifically, It can be applied to monitoring of actual food safety and it is worth to be extended. |
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