Detection of the apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated raw milk Maurilio L. Martins, Elza F. de Arau ´ jo, Hila ´rio C. Mantovani, Ce ´lia A. Moraes, Maria C.D. Vanetti T Department of Microbiology, Federal University of Vic ¸osa, Vic ¸osa, MG, 36570.000, Brazil Received 3 June 2004; received in revised form 31 August 2004; accepted 18 December 2004 Abstract Bacteria of the genus Pseudomonas have been associated with the spoilage of raw milk and dairy products due to the production of thermostable proteolytic enzymes. The apr gene encodes for alkaline metalloprotease in Pseudomonas and other related bacteria. Its presence in psychrotrophic proteolytic bacteria isolated from raw milk collected from cooling tanks was verified. A polymerase chain reaction (PCR) technique was used with degenerate primers. Total DNA from 112 isolates was pooled in different groups and then used as template for the amplification reactions. Controls consisted of DNA extracted from 26 cultures. An expected DNA fragment of 194 bp was detected in groups that contained bacteria identified as Pseudomonas . The PCR product was observed only when DNA from control cultures of Pseudomonas aeruginosa , Pseudomonas fluorescens , Serratia marcescens and Aeromonas hydrophila were used. A detection limit assay indicated that the apr gene could be directly amplified from pasteurized milk inoculated with 10 8 CFU/ml of P. fluorescens . With this method it was possible to detect proteolytic bacteria at 10 5 CFU/ml in reconstituted skim milk powder if cells were recovered for DNA extraction before amplification. D 2005 Elsevier B.V. All rights reserved. Keywords: Proteolytic psychrotrophs; Refrigerated raw milk; Polymerase chain reaction; apr gene 1. Introduction Proteolytic psychrotrophic bacteria are the main microorganisms responsible for spoilage of milk and milk products, due to their ability to produce thermo- stable proteases that hydrolyze casein and decrease the yields and sensory qualities of dairy products (Sørhaug and Stepaniak, 1997). Some bacteria also secrete lecithinases and lipases that can play a significant role in the deterioration of these products (Wiedmann et al., 2000; Dogan and Boor, 2003). Standard plate count procedures are traditionally used to detect psychrotrophic bacteria in milk and 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.12.016 * Corresponding author. Tel.: +55 31 3899 2954; fax: +55 3138992573. E-mail address: mvanetti@ufv.br (M.C.D. Vanetti). International Journal of Food Microbiology 102 (2005) 203– 211 www.elsevier.com/locate/ijfoodmicro