Detection of the apr gene in proteolytic psychrotrophic bacteria isolated from refrigerated raw milk

• Published in: International journal of food microbiology Vol. 102; no. 2; pp. 203 - 211
• Main Authors: Martins, Maurilio L., de Araújo, Elza F., Mantovani, Hilário C., Moraes, Célia A., Vanetti, Maria C.D.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15-07-2005; Elsevier
• Subjects: Animals; apr gene; bacterial contamination; Bacterial Typing Techniques; Biological and medical sciences; biomarkers; Catalase - metabolism; collagenase; DNA, Bacterial - analysis; food contamination; Food Contamination - analysis; Food industries; Food microbiology; food spoilage; Fundamental and applied biological sciences. Psychology; Gene Amplification; General aspects; genes; heat stability; Methods of analysis, processing and quality control, regulation, standards; microbial detection; Milk - enzymology; Milk - microbiology; Oxidoreductases - metabolism; Polymerase chain reaction; Polymerase Chain Reaction - methods; Proteolytic psychrotrophs; Pseudomonas - classification; Pseudomonas - enzymology; Pseudomonas - genetics; Pseudomonas - isolation & purification; psychrotrophic bacteria; raw milk; Refrigerated raw milk; Refrigeration; Species Specificity; Temperature; virulence; Polymerase chain reaction; Proteolytic psychrotrophs; apr gene; Refrigerated raw milk; Raw milk; Analysis method; Gene; Dairy product; Bacteria; Control method; Molecular biology; Detection
• ISSN: 0168-1605; 1879-3460
• DOI: 10.1016/j.ijfoodmicro.2004.12.016

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.