Sensory characteristics of milk spoilage by Pseudomonas and enumeration of Pseudomonas by QC-PCR.

摘要

Post-pasteurization contamination of milk is most commonly associated with the genus Pseudomonas. Characteristics of spoilage aromas caused by Pseudomonas inoculated in milk were determined. Secondly, enumeration of Pseudomonas was determined by the Quantitative Competitive-Polymerase Chain Reaction (QC-PCR) method. Pseudomonas fluorescens, P. fragi, and P. putida were used for determining spoiled milk aromas. Double steamed (5 min at 72°C, 30 min at 30°C, 10 min at 72°C) whole and skim milks were spiked separately with approximately 1 × 103 CFU/ml of each pseudomonad. Milk samples were stored at 5°C. Growth of Pseudomonas was determined by conducting plate counts every 3 d, while descriptive aroma analysis was conducted weekly. Ten trained panelists replicated the experiments in triplicate. Growth was not different between strains (p 0.05). Spiked milks reached 1 × 107 CFU/ml within 3 d of storage reached 1 × 108 CFU/ml by wk 1. Most milks did not have any noticeable spoilage aromas until wk 2. Fruity aromas predominated treatments with aromas at wk 1, while wk 2 had a preponderance of cheesy and rotten aromas. Barn aromas were noticed at 3 wk storage. Onset and type of spoilage depended on milkfat content and strain of Pseudomonas (p 0.05). A QC-PCR assay was developed to enumerate pseudomonads. A 990 by target sequence of 16S rDNA was amplified using Pseudomonas specific primers (P5F and P5R) and an 876 by competitive sequence was constructed using a composite P5F primer. Target and competitor sequences coamplified at a similar efficiency. Reaction products were separated by gel electrophoresis and band intensities were compared by computerized analysis of digitized images. Extracted DNAs from 13 strains of Pseudomonas broth cultures were enumerated by QC-PCR. The method was able to enumerate at 72% or higher accuracy in contrast with plate counts. Commercially pasteurized milk and nonfat dry milk (NFDM) were determined to have inherently high concentrations (equivalent to ∼5.00 to 6.00 Log cells/ml) of Pseudomonas DNA. This prevents direct prediction of milk shelf-life by the QC-PCR assay developed in this study.