Real-Time PCR Detection of Paenibacillus spp. in Raw Milk To Predict Shelf Life Performance of Pasteurized Fluid Milk Products

Abstract

ABSTRACTPsychrotolerant sporeformers, specificallyPaenibacillusspp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. WhilePaenibacillusspp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number ofPaenibacillusspp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detectPaenibacillusspp. in fluid milk and to discriminate betweenPaenibacillusand other closely related spore-forming bacteria. Specificity was confirmed using 16Paenibacillusand 17Bacillusisolates. All 16Paenibacillusisolates were detected with a mean cycle threshold (CT) of 19.14 ± 0.54. While 14/17Bacillusisolates showed no signal (CT> 40), 3Bacillusisolates showed very weak positive signals (CT= 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 101CFU/ml using total genomic DNA extracted from raw milk samples inoculated withPaenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection ofPaenibacillus. Heat-treated milk samples wherePaenibacillus(≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection ofPaenibacillusthat has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.